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Full text of "Annual report : National Cancer Institute (U.S.)"

Division of 



CANCER 
TREATMENT 



1985 Annual Report Volume I 
October 1, 1984-September 30, 1985 



LIBRARY 

APR 1 1 1990 
National Institutes of Health 



U.S. DEPARTMENT 

OF HEALTH 

AND HUMAN SERVICES 

National 
Institutes of 
Health 

National 

Cancer 

Institute 

Bethesda, 
Maryland 20892 

AC/ 

Nationat Cancer Institute 



{PC 



NATIONAL CANCER INSTITUTE 
ANNUAL REPORT 
October 1, 1984 through September 30, 1985 
CONTENTS 
DIVISION OF CANCER TREATMENT Page 

Director-OD 
Director's Report 1 

Publications 6 

Code for Division of Cancer Treatment Stratification 7 

Table I Analysis of Contract Activities for FY85 9 

Table II Analysis of Contracts by Activity for FY85 11 

Table III Description of Contracts 29 

ASSOCIATE DIRECTOR FOR DEVELOPMENTAL THERAPEUTICS 

Summary Report 71 

Drug Synthesis and Chemistry Branch - DS&CB 

Summary Report 87 

Publications 91 

Project Report 

CM-07101-10 Computer Methods for Drug Preselection Based 92 

on Structure Activity 

Natural Products Branch - NPB 

Summary Report 93 

Publications 99 

Drug Evaluation Branch - DEB 

Summary Report " 101 

Publications 104 

Animal Genetics and Production Branch - AG&PB 

Summary Report 109 



Page 



Pharmaceutical Resources Branch - PRB 



Summary Report 113 

Publications 116 

Project Report 

CM-03584-13 Research in the Development of Dosage Forms 118 

of New Antitumor Drugs 

Toxicology Branch - TB 

Summary Report 123 

Publications 125 

Information Technology Branch - ITB 

Summary Report 127 

Publications 131 

Extramural Research and Resources Branch - ER&RB 

Summary Report 133 

Laboratory of Biological Chemistry - LBC 

Summary Report 137 

Project Reports 

CM-06162-01 Pharmacology of Antitumor Agents 142 

CM-06163-01 Pharmacologic Aspects of Nucleotide Metabolism 149 

CM-06164-01 Inhibitors of Phospholipid Metabolism as 154 

Potential Chemotherapeutic Agents 

CM-06165-01 Stable Isotope Studies of de novo Pyrimidine 157 

and Purine Pathways 

CM-06166-01 Macromolecular Interactions of Vinca Alkaloids 161 

CM-061 67-01 Inhibition of Myristoylati on-Dependent Oncogene 164 

Mediated Cellular Transformation 

CM-06168-01 Cellular Pharmacology of Interferon and Double- 168 

stranded RNA 

CM-06169-01 Cellular and Molecular Pharmacology of Phospholipid 172 

and Diacylglycerol Analogs 



Page 



Laboratory of Biological Chemistry - LBC (cont'd) 



CM-07109-09 Molecular and Cellular Pharmacology of 176 

Nucleoside Analogs 

CM-071 56-02 Differentiation of Human Leukemia Cells 183 

Laboratory of Experimental Therapeutics and Metabolism - LETM 

Summary Report 187 

Publications 190 

Pharmacology and Toxicology Section - LTEM 

Project Reports 

CM-07140-03 BCNU-induced Pulmonary Fibrosis 193 

CM-07160-02 In Vitro Models for Evaluating Nephrotoxicity 197 

CM-071 61 -02 In Vivo Studies on the Toxicity of Alkylfurans 201 

CM-07162-02 Role of Arachidonic Acid Metabolism in Human 205 

Lung Cancer 

CM-071 63-02 Xenobiotic Metabolism by Prostaglandin 209 

Endoperoxide Synthetase (PES) 

CM-071 68-01 Biochemical Toxicology of the Nitrosoureas 213 

and the Thiol Modulating Agent, BSO 

CM-07169-01 Tetraplatin Toxicity: Comparative Nephrotoxicity 217 

of Platinum Anticancer Agents 

CM-07171-01 Thio-Promoted Lipid Peroxidation in Kidney 221 

Microsomes 

CM-071 72-01 Role for Reactive Oxyradicals and Lipid Peroxidation 223 

in Anthracycline Cytotoxicity 

CM-071 73-01 Arachidonic Acid Metabolism in Human Lung 226 

Adenocarcinoma 

CM-07174-01 Arachidonic Acid in Pulmonary Large Cell Carcinoma 230 

Pathology and Ultrastructural Oncology Section - LETM 

Project Reports 

CM-071 52-02 Studies on Clara Cell Mediated Lung Carcinogenesis 234 

In Vivo 

CM-071 53-02 Biology of Human Lung Cancer Cell Lines In Vitro 238 



Page 



Pathology and UUrastructural Oncology Section - LETM (cont'd) 



CM-071 54-02 Isolation and Selective Growth of Rodent 242 

Lung Cell Types ln_ Vitro 

CM-07175-01 UUrastructural Classification of Human Lung 246 

Cancers and Cell Lines 

Laboratory of Pharmacology and Experimental Therapeutics - LPET 

Summary Report 249 

Publications 251 

Medicinal Chemistry Section - LPET 

Project Reports 

CM-03580-16 Chemical Research in the Development of New 256 

Anticancer Drugs 

CM-03581-16 The Analytical Chemistry of New Anticancer Drugs 262 

CM-07167-01 Enzyme Inhibitors as Potential Anticancer Drugs 270 

Biochemical Pharmacology Section - LPET 

Project Reports 

CM-071 02-10 Tubulin Structure and Microtubule Formation 276 

as Sites for Pharmacologic Attack 

CM-071 04-10 L-Phenyl alanine Mustard Cytotoxicity and Therapy 281 

CM-071 19-06 Studies on the Biochemical Toxicology of Oncolytic 284 

Platinum Compounds 

CM-071 20-06 Role of Drug Metabolism in Modulating Toxicological 288 

Responses 

CM-071 22-05 Biochemical and Pharmacologic Studies with 291 

Oncolytic Nucleosides 

CM-071 29-04 Copper and Its Chelates in Cytotoxicity, 294 

Chemotherapy and Melanoma Promotion 

CM-07164-01 Differential Competition between Metabolites 297 

and Cytotoxic Analogs 

CM-071 65-01 The Sulfhydryl Group in Cancer Cell Growth 300 

and in Chemotherapy 

CM-071 66-01 Biochemical and Pharmacologic Studies with 303 

Oncolytic Barbituric Acid Derivatives 



Page 

Laboratory of Tumor Cell Biology - LTCB 

Summary Report 307 

Project Reports 

CM-06n7-13 Molecular and Physiological Control Mechanisms 310 

in Normal and Neoplastic Cells 

CM-071 48-02 Cellular Biological Studies on T-Cell 332 

Malignancies and Lymphomas 

CM-07149-02 Molecular Biological Studies on HTLV and Oncogenes 333 

CM-071 50-02 Seroepidemiological Studies on Human T-Lympho- 335 

tropic Retroviruses 

Laboratory of Molecular Pharmacology - LMPH 

Summary Report 337 

Project Reports 

CM-061 40-09 Regulation of Hi stone Biosynthesis 342 

CM-06150-04 Protein-associated DNA Strand Breaks as Indicator 347 

of Topoisomerase II Inhibition 

CM-06160-02 Mechanism of Action of DNA Crosslinking Agents 352 

CM-06161-02 Topoisomerase II as Target of Action of Anticancer 357 

Drugs 

CM-06170-01 Isolation of Human Variant Histone Genes 363 

CM-061 71 -01 Chromatin Synthesis and the Control of Cell 366 

Proliferation 

CM-061 72-01 Base Sequence Selective DNA Alkylating Reactions 369 

ASSOCIATE DIRECTOR FOR CANCER THERAPY EVALUATION 

Summary Report 373 

Publications 382 

Biometric Research Branch - BR 

Summary Report 385 

Project Report 

CM-06308-14 Biometric Research Branch 393 



Page 
Clinical Investigations Branch - CIB 

Summary Report 394 

Investigational Drug Branch - ID 
Summary Report 414 

ASSOCIATE DIRECTOR FOR RADIATION RESEARCH 
Summary Report 427 

Diagnostic Imaging Research Branch - DIRB 

Summary Report 438 

Radiotherapy Development Branch - RDB 
Summary Report 443 

ASSOCIATE DIRECTOR FOR CLINICAL ONCOLOGY 

Summary Report 449 

Project Reports 

CM-07200-03 Pathogenic Human Retroviruses 473 

Biostatistics and Data Management Section - COP 

Project Report 

CM-07202-02 Biostatistics and Data Management Section 489 

Clinical Pharmacology Branch - CPB 

Summary Report 501 

Project Reports 

CM-06513-09 Molecular Pharmacology of Antitumor Agents 504 

CM-06515-05 The Biochemistry of the Adriamycin-Iron Complexes 507 

CM-06516-04 Drug Resistance in Human Tumor Cells 510 

CM-06518-04 Pharmacokinetics 514 

CM-06519-02 Non-Invasive Studies of Metabolism Using 518 

Nuclear Magnetic Resonance Methods 

CM-06520-02 Magnetic Resonance Imaging Applied to Cancer 522 



Page 



Clinical Pharmacology Branch - CPB (cont'd) 



CM-06521-02 Conformations and Interactions of Nucleic Acids, 526 

Proteins and Drugs in Solution 

CM-06522-02 Enzymatic Mechanisms Protecting Cells Against 532 

Free Radical Damage 

CM-06523-01 Metabolism, Irreversible Binding and Mechanism 535 

of Action of Etoposide {VP-16, 213) to Cellular 
Macromolecules 

Medicine Branch - MB 

Project Reports 

CM-03403-20 Clinical Trials and Miscellaneous Clinical 537 

Investigations 

CM-06119-16 Cytogenetic Studies 552 

CM-06700-12 Clinical Program in Breast Cancer 556 

CM-06702-10 Mechanisms of Hormone Dependence of Human Malignancy 560 

CM-06708-06 Genetic Regulation of the Immune Response 563 

CM-06709-05 Mechanisms of Drug Resistance 569 

CM-06710-03 Transforming Genes and Retroviruses in Human 574 

Malignancy 

CM-067n-01 Functional and Molecular Biology of T Lymphocytes 577 

NCI-Navy Medical Oncology Branch - NMOB 

Summary Report 583 

Project Reports 

CM-03024-16 Clinical Trials and Other Clinical Investigations 586 

CM-06575-10 Laboratory Investigation of Tumor Cell Biology 600 

CM-06578-02 Structure, Expression of Peptide Hormone Genes 602 

in Human Small Cell Lung Carcinoma 

CM-06579-02 Chromosomal Abnormalities that Highlight Regions 604 

of Differentiated Activity 

CM-06580-02 Control of Differentiation by Oncogene Expression 606 

in Erythroleukemia Cell Lines 



Page 



NCI-Navy Medical Oncology Branch - NMOB (cont'd) 



CM-06581-02 Molecular Genetics of B-lymphocyte Development 607 

and Transformation 

CM-06582-01 Growth Factors for Human Lung Cancer 614 

CM-06583-01 Expression of Histocompatability Antigens on 615 

Lung Cancer 

CM-06584-01 Immunohistochemical Study of Marker Expression 616 

on Lung Cancers 

CM-06585-01 Culture and Characterization of Human Myeloma 617 

CM-06586-01 Studies of Human Immunoglobulin Gene Structure 618 

CM-06587-01 RNA-DNA In Situ Hybridization of Tissue Sections 619 

CM-06588-01 The Role of Glycosyltransferase in Development 620 

and Malignancy 

CM-06589-01 Establishment and Characterization of Human Lung 622 

Cancer Cell Lines 

CM-06590-01 Growth and Characterization of Pulmonary Endocrine 623 

Cells 

Pediatric Branch - PB 

Summary Report 625 

Project Reports 

CM-06830-15 Infectious Complications of Malignancy: Prevention, 630 

Diagnosis and Therapy 

CM-06840-10 Treatment of Acute Leukemia 653 

CM-06880-08 Clinical Pharmacology 660 

CM-06890-06 Lymphoma Biology and Epstein-Barr Virus 669 

CM-06811-03 Controlled Trial of Adjuvant Chemotherapy in 674 

the Treatment of Osteosarcoma 

CM-06813-03 Molecular Biology of Pediatric Tumors 677 

CM-06814-03 Biology and Treatment of Pediatric Soft Tissue 683 

and Ewing's Sarcomas 

CM-06815-03 The Investigation and Treatment of Patients with 688 

Non-Hodgkin's Lymphoma 



Page 



Pediatric Branch - PB (cont'd) 



CM-06816-02 Studies of the Nature, Measurement and Management 692 

of Pain in Children with Cancer 

Radiation Oncology Branch - ROB 

Summary Report 699 

Project Reports 

CM-00650-30 Service Radiation Therapy 703 

CM-00684-30 Nonclinical Irradiation Services 705 

CM-06310-06 Surgery versus Radiation Therapy in Treatment of 707 

Primary Breast Cancer 

CM-06320-06 Response of Mammalian Cells to Chemotherapy Drugs 710 

CM-06321-06 Radiosensitization of Aerated and Hypoxic 712 

Mammalian Cells 

CM-06329-05 Clinical Radiation Physics Service 715 

CM-06330-05 Extension of a 3-D Dose Field Model 718 

CM-06331-05 Computer-assisted 3-D Radiation Treatment Planning 721 

CM-06333-05 Dosimetry of Total Skin Electron Irradiation 724 

CM-06348-04 Interactive Linear-Source Brachytherapy 726 

Dosimetry Program 

CM-06349-04 Relationship of Cellular Redox State and 728 

Thermotolerance 

CM-06351-03 Response of Mammalian Cells to Halogenated 730 

Pyrimidines 

CM-06352-03 Relaxation Agents for NMR Diagnostic Imaging 733 

CM-06353-03 Metal Chelate Conjugated Monoclonal Antibodies 736 

for Tumor Diagnosis and Therapy 

CM-06354-03 Iron-57 Nuclear Magnetic Resonance: A New Tool x741 

for Biomedical Research 

CM-06355-03 Total Skin Electron Beam Radiation for AIDS 744 

Associated Kaposi's Sarcoma 

CM-06356-02 Treatment of Malignant Brain Tumors with 747 

Interstitial Radiotherapy 



Page 

Radiation Oncology Branch - ROB (cont'd) 

CM-06357-02 Clinical Studies on Intraoperative Radiation Therapy 750 

CM-06358-.02 Effects of -Irradiation on Cells and Their 754 

Constituents 

CM-06359-02 A Phase I Study of lododeoxyuridine (NSC39661) 759 

Given as an Intravenous Infusion 

CM-06360-02 Radionuclide Generators to Produce Alpha-Emitters 762 

CM-06363-02 DNA Damage by Alkylating Agents and Their Repair 765 

in Tumor Cells 

CM-06364-02 Measurement DNA Base Damage Produced by X-rays in 767 

Human Cells 

CM-06365-02 RNA Transcripts Induced by Hyperthermia in 769 

Rodent Cells 

CM-06366-02 Nuclear Magnetic Resonance Studies on Mammalian Cells 771 

CM-06367-02 Mechanisms of Radioprotection 773 

CM-06369-02 Radiation Characteristics of a 22 MeV Medical 775 

Microtron 

CM-06370-01 Optimization of Treatment Planning for Brain Implants 777 

CM-06371-01 Modification of the Cross Beam Fluence Rate 780 

Distribution in Electron Beams 

CM-06372-01 Extension of the Net Fractional Depth Dose for 783 

Inhomogeneity Correction 

CM-06373-01 Radiation Therapy Treatment Planning Optimization 785 

CM-06374-01 Effect of Radioprotectors and Radiosensitizers on 787 

DNA Damage Produced by X-rays 

CM-06375-01 DNA Damage in X-irradiated Cells Treated with 789 

Halogenated Pyrimi dines 

CM-06376-01 Repair Recombination in UV-sensitive CHO Cells 791 

Surgery Branch - SB 

Summary Report 733 

Project Reports 

CM-03800-15 Surgical Consultants and Collaborative Research 803 

Involving Surgical Services at the NIH 



Page 

Surgery Branch - SB (cont'd) 

CM-03801-15 Clinical Studies in Cancer Surgery 810 

CM-03811-11 The Immunotherapy of Animal and Human Sarcomas 820 

CM-06652-09 Studies of Immune Regulation 825 

CM-06654-08 Studies in Malignant Disease 827 

CM-06655-05 Factors Influencing Host Cellular and Humoral 829 

Immune Responses to Neoplasia 

CM-06657-03 Studies in Cancer Cachexia 831 

CM-06658-03 Studies of the Pineal Gland Hormone Melatonin 833 

and Estrogen Receptor Activity 

CM-06659-03 Studies of Urologic Malignancy 835 

CM-06660-02 The Study of Immune Adjuvants in Rodent 837 

Tumor Models 

CM-06661-02 Immunologic Studies in Patients with Cancer 839 

ASSOCIATE DIRECTOR FOR BIOLOGICAL RESPONSE MODIFIERS 

Summary Report °^'' 

Biological Resources Branch - BRB 

Summary Report ^^' 

Biological Therapeutics Branch - BTB 

Summary Report ^^^ 

Clinical Investigations Section - BTB 

Project Reports 

CM-09200-05 Phase I Trials of Recombinant and Nonrecombinant 902 

Interferons in Cancer Patients 

CM-09233-04 Trials of Recombinant Leukocyte Interferon in 906 

Patients with Lymphoproliferative Disorders 

CM-09235-04 Phase I Trials of Antitumor Monoclonal Antibodies 911 

in Patients with Cancer 

CM-09276-02 Phase I Trial of Poly ICLC in Cancer Patients 917 

CM-09278-02 Gene Expression Studies of Human Monocytes: 920 

Potential Clinical Applications 



Page 



Clinical Investigations Section - BTB (cont'd) 



CM-09279-02 Phase I Trials of Interleukin 2 in Patients 926 

with Cancer 

CM-09280-02 Characterization of Elutriator-Purif ied Human 930 

Monocytes: Clinical Applications 

CM-09281-01 The Synthesis and Evaluation of Drug-Monoclonal 938 

Antibody Conjugates 

Monoclonal Antibody/Hybridoma Section - BTB 

Project Reports 

CM-09226-05 Preclinical Evaluation of Immunoconjugates Against 943 

Established Tumors and Matastases 

Natural Immunity Section - BTB 

Project Reports 

CM-09228-05 Further Characterization of Natural Killer (NK) 949 

Cells in the Rat 

CM-09246-17 Characteristics, Regulation and In Vivo Relevance 954 

of NK Cells 

CM-09247-05 Natural Cell-Mediated Immunity in Man: Studies 960 

of Fresh LGL 

CM-09255-03 Role of NK Cells in the Control of Metastatic 967 

Spread and Growth 

CM-09256-03 Natural Cell-Mediated Immunity in Man: In Vitro 971 

Activated and Cultured LGL 

CM-09257-03 Functional Activity of Large Granular Lympho- 976 

cytes in Rats 

CM-09259-03 Characterization and Differentiation of NK Cells 981 

and Lymphocyte Subsets 

CM-09262-03 Antitumor Effects of NK Cells, rIL2-Stimulated 987 

Lymphocytes, and Macrophages in Mice 

CM-09275-02 Effect of Mutagen Treatment on the Immunogenic 994 

Properties of Tumor Cells 

CM-09282-01 Potential Differentiation Capacity of Thymocytes 999 



Page 



Immunopharmacology Section - BTB 



CM-06146-08 Cellular Regulation by Immune Modifiers and Chemo- 1004 

therapy in the Tumor-Bearing Host 

Laboratory of Molecular Immunoregulation - LMI 

Summary Report 1013 

Lymphokines Section - LMI 

Project Reports 

CM-09251-03 Studies of Hematopoietic and Tumor Cell Growth 1019 

Factors 

CM-09254-03 Intracellular Signals Mediating the Growth and 1024 

Differentiation of Cells Modulated by Lymphoid- 
Derived Growth Factors 

CM-09264-03 Regulation of Normal and Neoplastic T-lympocyte 1028 

and Hematopoietic Cell Proliferation 

Immunobiology Section - LMI 

Project Reports 

CM-09216-05 Response of Macrophage-Monocytes to BRM: 1034 

Mechanisms & Pharmacological Modulation 

CM-09260-03 Role of Interleukin 1 in Immunity and Inflammation 1039 

CM-09283-01 Analysis of the Genetic Control and Effects of 1046 

Interferon- Gene Expression 

CM-09284-01 Molecular Cloning of Rat Cytolysin 1050 

CM-09285-01 Studies on the Regulation of IL-1 Gene Expression 1053 

and the Molecular Events Occurring Upon IL-1 
Treatment of Response Thymocytes 

CM-09286-01 Receptors of Human Interferon- and Interferon 1056 

on Peripheral Blood Cells 

CM-09287-01 Interleukin 1: Mechanisms of Production/ 1060 

Anti -tumor Effects 



ANNUAL REPORT 

DIVISION OF CANCER TREATMENT 

October 1, 1984 through September 30, 1985 



The Division of Cancer Treatment (DCT) is the organizational component of the 
National Cancer Institute (NCI) responsible for tne ofcVfciO|ji.ieriu cii.d tvaluation of 
new therapies for the control and cure of cancer. The program is carried out in 
intramural laboratories and clinics as well as through grant-contract and coopera- 
tive agreement-supported projects throughout the nation and the world. Major 
emphasis in the treatment research program is on optimal and integrated use of all 
modalities of therapy, including chemotherapy, surgery, radiotherapy, hyperther- 
mia, biological response modifiers, and immunotherapy, used both individually and 
in combination. 

The Division has five major programs: the Developmental Therapeutics Program 
(DTP), the Cancer Therapy Evaluation Program (CTEP), the Radiation Research Pro- 
gram (RRP), the Clinical Oncology Program (COP), and the Biological Response 
Modifiers Program (BRMP). Each of these programs has a separate and distinct 
responsibility, encompassing research on the development and identification of 
new agents or methods for the treatment of cancer; their evaluation in preclinical 
tumor systems; the testing and evaluation of the safety of new agents or methods 
in preparation of investigational new drug applications (INDs) for submission 
to the Food and Drug Administration for approval to initiate clinical trials- 
the conduct of clinical trials at the earliest level (Phase I); the conduct of 
disease-specific clinical trials (Phase II); the conduct of clinical trials com- 
paring new and standard treatments (Phase III); and the evaluation of combined 
modalities of therapy (Phase IV). 

For scientific and administrative direction, the Division relies heavily on the 
advice of its Board of Scientific Counselors, whose membership and affiliations 
are shown in Table I. The Board represents outstanding extramural scientific 
talents in the fields of surgical oncology, immunology, and radiotherapy. These 
represent the areas of clinical science particularly relevant to the research 
mission of the Division. 

The Division Director relies on the Board of Scientific Counselors not only for 
scientific and administrative advice but also for fiscal direction. For example, 
all new concepts for contracts and Requests for Application (RFAs) must meet with 
Board approval. In addition. Board members serve as chairpersons and members of 
regular site visits to intramural labs. The Board's counsel is invaluable in 
assuring that the best intramural labs receive appropriate support while areas of 
lower scientific priority can be de-emphasized. 

Personnel and Organization 

The DCT is operationally divided in the five major components of treatment program 
priorities. Each program is headed by an Associate Director who is responsible 
for the overall direction of the science within that program. A current organiza- 
tional chart, shown in the following pages, reflects changes that have occurred 
during the past year. Restructuring within the DTP consolidated the Laboratory of 

1 



TABLE I. 



OCT BOARD OF SCIENTIFIC COUNSELORS 



Name 



Affiliation 



Term of Appointment 



Dr. Samuel A. Wells, Jr. 

Dr. Dani P. Bolognesi 

Dr. David G. Bragg 

Dr. Paul Calabresi 

Dr. Mortimer M. El kind 

Dr. Karen K. Fu 

Dr. James Goldie 

Dr. I. David Goldman 

Dr. Leon Goodman 

Dr. Robert L. Goodman 

Dr. Susan B. Horwitz 

Dr. John H. Kersey 

Dr. Rodrigue Mortel 

Dr. Carol S. Portlock 

Dr. Efraim ^^acker 

Dr. Alan S. Rosenthal 



Washington University 
School of Medicine 


1982- 


■1986 


Duke University Medical 
Center 


1982- 


-1986 


University of Utah 
Medical Center 


1982- 


-1985 


Roger Williams General 
Hospital, Brown University 


1982- 


■1986 


Colorado State University 


1982- 


■1986 


University of California, 
San Francisco 


1983- 


■1985 


A. Maxwell Evans Cancer 
Control Agency of 
British Columbia 


1982- 


-1985 


Medical College of Virginia 


1982- 


■1986 


University of Rhode Island 


1982- 


■1986 


University of Pennsylvania 


1984- 


•1987 


Albert Einstein College 
of Medicine 


1982- 


-1986 


University of Minnesota 
Hospitals 


1984- 


-1985 


Milton S. Hershey Medical 
Center, Pennsylvania 
State University 


1983- 


■1985 


Yale University School 
of Medicine 


1982- 


-1985 


Cornell University 


1982- 


■1986 


Merck Sharp and Dohme 
Research Labs. 


1984- 


■1986 



Experimental Therapeutics and Metabolism, Laboratory of Chemical Pharmacology, 
and Laboratory of Medicinal Chemistry into the Laboratory of Biological Chemistry 
and the Laboratory of Pharmacology and Experimental Therapeutics. In addition, 
there were several changes in personnel: 

A. Office of the Director (OD) 

• Dr. Arnold Welch, Acting Deputy Director, left the OD to assume responsibil- 
ities coordinating the newly established National Cooperative Drug Discovery 
Groups within the Developmental Therapeutics Program. 



• Dr. Gregory Curt assumed responsibilities as Deputy Director. 

• Dr. Marcia Browne transferred from the COP to become Special Assistant to 
the Director for Clinical Affairs. 

• Dr. Eddie Reed transferred from the DTP to become Special Assistant to the 
Director for Science. 

• Dorothy Tisevich assumed responsibilities as Deputy Administrative Officer, 
OD, OCT. 

B. Biological Response Modifiers Program (BRMP) 

• Dr. Dan L. Longo left the COP to assume new responsibilities as the Associate 
Director, BRMP. 

• Dr. Ronald Herberman left as Acting Associate Director, BRMP, and Chief of 
the Biological Therapeutics Branch to become head of the Cancer Center at 
the University of Pittsburgh. 

• Dr. Carl Pinsky was recruited as Chief of the Biological Resources Branch. 

• Dr. Freddie Hoffman left the Clinical Investigations Branch, CTEP, to become 
Head of the Clinical Trials Section, Biological Resources Branch. 

• Gordon Cecil became the new Administrative Officer for BRMP. 

C. Cancer Therapy Evaluation Program (CTEP) 

• Dr. Brigid Leventhal spent a sabbatical year from Johns Hopkins University 
as Special Assistant to the Associate Director, CTEP. 

• Dr. Edward T. Jacobs retired from government service. Drs. Silvia Marsoni, 
Mercedes Lassus, Elihu Estey, and Elizabeth Read left the CTEP for positions 
elsewhere, to be replaced by Drs. Bruce Cheson, Hoo Chun, Gisele Sarosy, and 
F. Andrew Dorr; Drs. Michael Stewart, and J. Michael Hamilton also joined 
the staff. 

• Dr. Michael Hawkins was recruited from the University of Wisconsin as Head, 
Biologies Section, Investigational Drug Branch, and Dr. Marianne Roper 
joined the staff of the Biologies Section. 

• Dr. Lawrence Rubinstein joined the staff of the Biometric Research Branch. 

• Drs. Allison Martin and Stacey Nerenstone joined the CTEP staff after fin- 
ishing clinical fellowship training in the Medicine Branch, COP. 

D. Clinical Oncology Program (COP) 

• Dr. Neil Rosen weis appointed a Senior Investigator in the Medicine Branch. 

• Kathy Russell became the new Administrative Officer for COP. 



E. RADIATION RESEARCH PROGRAM (RRP) 

• Robert Morton was recruited from the Food and Drug Administration to join 
the Radiotherapy Department. 

• Barbara Vermillion became the new Administrative Officer for RRP. 

International Treatment Research 

International treatment research activities of the Division are coordinated 
through the Office of the Director. Dr. Michael Friedman, Chief of the Clinical 
Investigations Branch, CTEP, has continued to coordinate the Japanese-American 
Bilateral Agreement. Two meetings were held during the past year, the first in 
Tokyo (Workshop on Biochemical Modulation of Chemotherapy and Immunotherapy) and 
the second in Hawaii (New Drug and Biological Response Modifier Development). 
The Italian and French bilateral agreements were administered by Dr. Gregory Curt. 
The French met with American colleagues in Bethesda this year to exchange informa- 
tion on current concepts in bone marrow transplantation. The proceedings of this 
meeting will be published in CANCER TREATMENT SYMPOSIUM. The Italian-American 
Bilateral Agreement met in Venice to exchange information on drug resistance, 
differentiating agents, and the quality of life following cancer therapy. Admin- 
istration of the agreements with the People's Republic of China, Egypt, and Poland 
were assumed by Dr. Marcia Browne. 

Most importantly, the agreements continued to sponsor individual exchanges of 
scientists on a laboratory-to-laboratory basis to enhance international exchange 
of reagents, techniques, and ideas. 

Cooperative relationships with European cancer researchers and drug industries 
have been further strengthened by the Division's relationship with the EORTC 
(European Organization for Research on Treatment of Cancer). This interaction 
continues to have both preclinical and clinical components. In order to identify 
new agents useful in the treatment of cancer, drugs collected in Western Europe 
are screened at the Institut Jules Bordet in accordance with NCI protocols. In 
addition, important clinical programs are sponsored by this agreement. The EORTC 
has become a major testing ground for Phase I and II agents. Continued exchange 
will allow the expedited access of the best new anticancer drugs on both sides of 
the Atlantic. 

The EORTC and NCI are also sponsoring a new training program that will allow 
postdoctoral fellows from Western Europe and Israel as well as American colleagues 
to spend significant research time in the laboratories of like-minded scientists. 
Candidates will spend three to five years in sponsoring exchange laboratories 
before returning to their home institutions. 

Scientific Accomplishments 

I. Training Programs 

In addition to the EORTC training program, the Office of the Director implemented 
a traineeship program in oncology nursing. This initiative was implemented by 
Dr. Marcia Browne in close collaboration with the Nursing Department of the Clin- 
ical Center, NIH. The purpose of this new nine-month training program is to 
prepare recent baccalaurate nursing graduates to become skilled oncology nurse 



practitioners through a unique program emphasizing both practical and theoretical 
aspects of cancer nursing. This represents the first program of its type in the 
United States to recognize the central role of the nurse as a team member in the 
treatment of cancer patients. It is expected that this program will expand and 
diversify to include a research fellowship for oncology nurses interested in the 
design and implementation of clinical trials. Training will include trial design, 
biostatistics, biochemistry, pharmacology, and immunology. It is hoped that this 
program will serve as a model for similar initiatives throughout NIH and the 
nation. 

In addition, the Office of the Director coordinated a new fellowship program in 
biotechnology. This program, established under the training authority of the 
National Cancer Act, will allow American citizens to pursue exciting new oppor- 
tunities in molecular biology, genetic engineering, immunology, and molecular 
pharmacology. 

II. Surgical Oncology 

Recognizing the importance of the surgeon in the treatment of cancer patients and 
realizing a shortage in academically trained research-oriented surgical oncol- 
ogists, the Office of the Director has worked with Dr. Fred Avis, Section Head 
for Surgical Oncology, and the Division of Cancer Prevention and Control to estab- 
lish a new program for the development of a cadre of cancer surgeons with broad 
expertise in cancer biology and treatment. 

The first of these initiatives was the establishment of the Clinical Investigator 
Award (K08), a prei.iiere support iiiechanisin for the provision of long-term basic and 
clinical research training for M.D.s. Surgical oncology is targeted for special 
priority for K08 awards. 

Most recently, Drs. Fred Avis and Barney Lepovetsky of the DCT Cancer Training 
Branch have collaborated in the establishment of a Surgical Oncology Training 
Program that provides maximal flexibility within the existing National Service 
Research Awards mechanism to allow surgeons meaningful subspecialty training in 
surgical oncology research. 

In order to improve surgical representation on study section rosters, the OD 
distributed detailed information on NCI initiatives in surgical oncology, the 
NCI grants process, and the importance of review to the entire membership of the 
Society of Surgical Oncology. Finally, in order to follow the progress of these 
initiatives, surgical oncology has been designated a Cancer Activity, which will 
allow accurate tracking of the budget in this area of cancer treatment. 

III. Unproven Methods of Cancer Treatment 

Dr. Curt was appointed to the Unproven Methods Committee of the American Cance# 
Society. In that capacity, he v/orked with extramural investigators, the Centers 
for Disease Control, and the Pan American Health Organization to investigate the 
practice of Dr. Lawrence Burton, who had treated 3,000 cancer patients with immuno- 
augmentative therapy, an unproven cancer treatment, at his clinic in Freeport, 
Grand Bahamas. In addition to determining that this treatment was without biolog- 
ical activity, it was discovered that treatment materials were contaminated with 
hepatitis B and HTLV-III antibodies. This information led to the closing of the 
clinic in July 1985. 



IV. HTLV-III 

It has been conclusively demonstrated that HTLV-III is the etiologic agent of 
acquired immunodeficiency syndrome (AIDS). The Office of the Director was 
instrumental in competing and commercializing the EL ISA assay for the screening 
of HTLV-III in blood products. The massive screening program that resulted 
protected the nation's blood resources from iatrogenic spread of the disease. 

V. Special Initiatives 

Working together with the Comprehensive Minority Biomedical Program of the Divi- 
sion of Extramural Activities, the Office of the Director established a minority 
supplement program for the Clinical Cooperative Groups. These awards will improve 
access of minority patients to state-of-the-art clinical trials. This initiative 
is considered particularly important as survival statistics for minority patients 
with cancer have continued to lag behind the steadily improving survival for 
American whites. 

The Office of the Director, DCT, worked with the NIAID and the NHLBI to formulate 
a technology assessment conference for the Office of Medical Applications of 
Research that recommended deferring establishment of a national registry for 
unrelated bone marrow transplantation. 

Detailed preclinical and clinical research accompl ishments will be described under 
each of the Division's program annual reports. 

Publ ications 

Chabner, B.A., Curt, G.A. and Hubbard, S.M.: Surgical oncology research develop- 
ment: the perspective of the National Cancer Institute. Cancer Treat. Rep. 68: 
825-829, 1984. 

Chabner, B.A., Wittes, R., Hoth, D. and Hubbard, S.: Investigational trials of 
anticancer drugs: establishing safeguards for experimentation. Public Health Rep. 
99: 355-360, 1984. 

Chabner, B.A., Fine, R.L., Allegra, C.J., Yeh, G.C. and Curt, G.A.: Cancer chemo- 
therapy: progress and expectations, 1984. Cancer 54: 2599-2608, 1984. 

Chabner, B.A.: The evolution of cancer chemotherapy. Hospital Practice 20: 115- 
127, 1985. 



Division of Cancer Treatment Stratification 

Code Description 

PDD Preclinical Drug Development Program - 

IXX Stage I - Acquisition of Materials 

2A1 Stage II - Basic Screen - Determination of anti-tumor 

activity of new agents. 
2B1 - Develop acceptable experimental formulation. 

232 - Verification screen - Detailed evaluation of new 

agents (dose, route and schedule dependency). 
2B3 - Procurement of sufficient amounts of new agents 

for preclinical studies. 

3X1 Stage III - Toxicology and pharmacology in animals. 

3X2 - Production and formulation for clinical trials. 

PBR Preclinical Basic Research - 

PBS Cellular/Subcellular Studies - Includes biochemistry, biological 

response modifiers, blood products, cell biology, 
cell kinetics, therapeutic nutrition, immunobiology, 
markers, molecular biology, radiobiology, transfusion 
research, hyperthermia, combined modalities, radiation 
modifiers, radiation immunology, radiation physics, 
comparative pharmacology, experimental therapy, 
mechanism of drug action, synthetic and natural 
products, and data processing. 

PTS Treatment Studies - Independent treatments, combined modality 

therapy, radiation physics, radiation 
equipment development, nuclear medicine 
and data processing. 

CTR Clinical Trials Research - 

CTl Phase I Clinical Trials* - Initial clinical evaluation of new 

drugs, clinical pharmacology. 

CT2 Phase II Clinical Trials* - Allocation for specific disease- 
oriented resources to study 
whatever chemotherapy (single 
agents or combinations) or com- 
bined modality regimens have 
highest priority for initial 
efficacy evaluation. 



CT3 Phase III Clinical Trials* - Allocation for specific disease- 
oriented resources to study 
whatever chemotherapy or combined 
modality regimens have highest 
priority for efficacy evaluation 
in a controlled clinical setting. 

CT4 Phase IV Clinical Trials* - Allocation for specific disease- 
oriented resources to evaluate 
the combined modality approach 
to the initial therapeutic attack 
on local or regional disease in 
an attempt to increase the number 
of patients with a long disease- 
free period. 

CT5 Statistics, Data Processing, and Other Clinical Trials Research 

CSR Clinical Trials Supportive Research - Includes special pharmacology/ 

toxicology, cell kinetics, markers, 
blood products, transfusion research, 
protected environment, hyperthermia, 
nutrition, statistics, and data 
processing. 

MGT Program Management - Includes administration, dissemination of 

information to the medical and scientific 
community. 

♦Supportive care used as ancillary therapy should be prorated among the 
phases of clinical trials using such resources. 



TABLE I 
ANALYSIS OF CONTRACT ACTIVITIES FOR FY85 



PDD PRECLINICAL DRUG DEVELOPMENT PROGRAM 
STAGE I 

IXX ACQUISITION OF MATERIALS 
STAGE IIA 

2A1 DETERMINATION OF ANTI-TUMOR ACTIVITY 
STAGE I IB 

281 EXPERIMENTAL FORMULATION DEVELOPMENT 

2B2 DETAILED EVALUATION OF NEW AGENTS 

2B3 PROCURE. OF AGENTSS FOR PRECLIN. STUDIES 

STAGE III 

3X1 TOXICOLOGY & PHARM. IN LARGE ANIMALS 
3X2 PROD. & FORM. FOR CLINICAL TRIALS 

PBR PRECLINICAL BASIC RESEARCH 

PBS CELLULAR/SUBCELLULAR STUDIES 
PTS TREATMENT STUDIES 

CTR CLINICAL TRIALS RESEARCH 

CTl PHASE I CLINICAL TRIALS 
CT2 PHASE II CLINICAL TRIALS 
CT3 PHASE III CLINICAL TRIALS 

CT5 OTHER CLINICAL TRIALS RESEARCH 

CSR CLINICAL TRIALS SUPPORTIVE RESEARCH 

STATISTICS, DATA PROCESSING, AND OTHER CLINICAL 
TRIALS RESEARCH 

MGT PROGRAM MANAGEMENT 

TOTAL 



ANNUAL LEVEL 

34,357,909 
8,011,385 

9,722,009 

8,537,885 

702,696 
3,561,410 
4,273,779 

8,086,630 

2,684,609 
5,402,021 

4,226,524 

1,427,422 
2,799,102 

9,792,311 

3,035,358 

1,433,494 

465,165 

4,858,294 

2,464.842 



PERCENT 

63.42 
14.79 

17.95 

15.76 

1.30 
6.57 
7.89 

14.92 

4.95 
9.97 

7.80 

2.63 
5.17 

18.09 

5.61 

2.65 

.^ 

8.97 

4.55 





Table II 
ANALYSIS OF CONTRACTS BY ACTIVITY 



AREA 



D.T.P. 

D.T.P. 

D.T.P. 

D.T.P. 

D.T.P. 

D.T.P. 

I.S.D.T. 

D.T.P. 

A. P. 

A.P. 

A. P. 

B.R.M.P. 

D.T.P. 

D.T.P. 

D.T.P. 

D.T.P. 

A.P. 

A.P. 

B.R.M.P. 

D.T.P. 

D.T.P. 

A.P. 

D.T.P. 

B.R.M.P. 

A.P. 

A.P. 

A.P. 

A.P. 

A.P. 

A.P. 

D.T.P. 

B.R.M.P. 

A.P. 

A.P. 

I.S.D.T. 

A.P. 

A.P. 

R.R.P. 



ACTIVITY 



CONTRACT 



FOR FISCAL YEAR 1985 
AS OF 06/30/85 



STAGE I AQUISITION OF MATERIALS: 

Alabama, University of N CM2757100 

Alabama, University of N CM3763105 

Alabama, University of N CM3763106 

Alabama, Univeristy of N CM3763107 

Alabama, University of N CM3763108 

Biotech Research Laboratories, Inc. N CM3755800 

Biotech Research Laboratories, Inc. N CM3760900 

Bristol Laboratories N CM3755600 

Charles River Breeding Labs. N CM3762600 

Charles River Breeding Labs. N CM8721200 

Charles River Breeding Labs. N CM9722900 

Chemical Coupling of MoAb RFP57718000 

Dyanamac Corporation N CM3756300 

Fein Marquart N CM4768300 

Flow Laboratories, Inc. N CM2750500 

Georgia Institute of Technology N CM2751700 

Harlan Sprague Dawley, Inc. N CM3762300 

Harlan Sprague Dawley, Inc. N CM2391100 

Harlan Sprague Dawley, Inc. N CM2391100 

Illinois, University of N CM3751300 

Institute of Microbial Chemistry N CM4759300 
Leo Goodwin Inst, for Cancer Research N CM3762500 

Maxima Corporation N CM4762800 

Meloy Laboratories, Inc. N CM4765000 

Microbiological Associates N CM9728700 

Missouri, University of N CM2753400 

Missouri, University of N CM8715700 

Murphy Breeding Labs., Inc. N CM3022900 

Northwestern University N CM3753800 

Papanicolaou Cancer Research Inst. N CM8723000 

Polysciences, Inc. N CM3755700 

Production of Hybridomas RFP57720 

Program Resources, Inc. N C02391000 

Simonsen Laboratories N CM3762400 

Small Business Administration N CM3760900 

Southern Animal Farms N CM5772700 

Southern Animal Farms N CM9724500 

SRI International N CM4761100 



DOLLAR 
LEVEL 



129,181 

30,648 

17,984 

12,126 

12,808 

40,434 

302,933 

364,560 

593,050 

5,600 

13,362 

400,000 

111,454 

15,175 

280,071 

149,236 

104,066 

750,000 

99,825 

113,380 

130,000 

69,000 

76,839 

203,612 

70,144 

16,028 

34,632 

1,835 

25,676 

14,895 

217,923 

175,000 

810,280 

191,129 

128,829 

32,305 

4,800 

210,934 



11 



ANALYSIS OF CONTRACTS BY ACTIVITY 





ACTIVITY FOR FISCAL YEAR 1985 




AS OF 06/30/85 


AREA 


CONTRACT 




STAGE I ACQUISITION OF MATERIALS: 


D.T.P. 


Starks Associates, Inc. 


D.T.P. 


Starks Associates, Inc. 


D.T.P. 


State University of New York 


A.P. 


Taconic Farms 


A.P. 


Taconic Farms 


A.P. 


Texas A & M Research Foundation 


D.T.P. 


VSE Corporation 


D.T.P. 


Warner Lambert 



N CM47 58800 
N CM4760800 
N CM2757000 
N CM3022600 
N CM5773000 
N CM3753600 
N CM5765400 
N CM3761400 



TOTAL 



DOLLAR 
LEVEL 



627,257 

528,026 

190,797 

225 

14,226 

2.691 

191,999 

496,410 

$8,011,385 



12 



ANALYSIS OF CONTRACTS BY ACTIVITY 



ACTIVITY 



FOR FISCAL YEAR 1985 
AS OF 06/30/85 



AREA 



D.T.P. 

D.T.P. 

D.T.P. 

D.T.P. 

D.T.P. 

A. P. 

A. P. 

A. P. 

A.P. 

A. P. 

D.T.P. 

A.P. 

B.R.M.P. 

A.P. 

A.P. 

D.T.P. 

D.T.P. 

A.P. 

B.R.M.P. 

D.T.P. 

D.T.P. 

A.P. 

A.P. 

A.P. 

R.R.P. 

A.P. 

A.P. 

B.R.M.P. 

A.P. 

B.R.M.P. 

A.P. 

A.P. 

A.P. 

A.P. 

D.T.P. 

D.T.P. 

R.R.P. 



CONTRACT 

STAGE II BASIC SCREEN: 

Arizona, University of 

Battel le Memorial Institute 

Biotech Research Laboratories, Inc. 

Bristol Laboratories 

California, University of 

Charles River Breeding Labs. 

Charles River Breeding Labs. 

Charles River Breeding Labs. 

Charles River Breeding Labs. 

Charles River Breeding Labs. 

Fein Marquart Assoc. 

Harlan Sprague Dawley, Inc. 

Harlan Sprague Dawley, Inc. 

Harlan Sprague Dawley, Inc. 

Harlan Sprague Dawley, Inc. 

IIT Research Institute 

Institute of Microbial Chemistry 

Leo Goodwin Inst, for Cancer Research 

Lymphokine Screen 

Mason Research Institute/EG & G 

Mayo Foundation 

Microbiological Associates 

Missouri, University of 

Missouri, University of 

Northern California Cancer Program 

Northwestern University 

Papanicolaou Cancer Research Institute 

Preclinical Assessment of MoAb 

Program Resources, Inc. 

Program Resources, Inc. 

Simonsen Laboratories 

Simonsen Laboratories 

Southern Animal Farms 

Southern Animal Farms 

Southern Research Institute 

Southern Research Institute 

SRI International 





DOLLAR 


# 


LEVEL 


N CM5766200 


258,781 


N CM4764800 


456,719 


N CM3755800 


132,855 


N CM3755600 


64,334 


N CM5771000 


187,079 


N CM3762600 


1,451,950 


N CM3752600 


52,500 


N CM5765300 


97,788 


N CM8721200 


10,400 


N CM9722900 


36,745 


N CM4768300 


65,758 


N CM2391100 


750,000 


N CM2391100 


140,000 


N CM3762300 


438,564 


N CM5772800 


132,638 


N CM4756700 


408,923 


N CM4759300 


130,000 


N CM3762500 


192,000 


RFP57717000 


150,000 


N CM4764700 


331,068 


N CM5771100 


216,015 


N CM9728700 


192,896 


N CM2753400 


44,076 


N CM8715700 


95,237 


N CM3757800 


247,984 


N CM1736300 


70,608 


N CM8723000 


40,960 


RFP57719000 


500,000 


N C02391000 


206,880 


N C02391000 


137,100 


N CM3762400 


527,881 


N CM5772900 


213,243 


N CM5772700 


88,839 


N CM9724500 


13,200 


N CM3755200 


157,506 


N CM4764600 


538,223 


N CM4761100 


210,934 



13 



ANALYSIS OF CONTRACTS BY ACTIVITY 



AREA 



ACTIVITY 



CONTRACT 



FOR FISCAL YEAR 1985 
AS OF 06/30/85 



DOLLAR 
LEVEL 



STAGE II BASIC SCREEN: 

A. P. Taconic Farms 

A. P. Texas A & M Research Foundation 

D.T.P. VSE Corporation 

D.T.P. Warner Lambert 



N CM5773000 
N CM3753600 
N CM5765400 
N CM3761400 



TOTAL 



39,121 

29,602 

576,000 

87,602 

$9,722,009 



14 



ANALYSIS OF CONTRACTS BY ACTIVITY 



ACTIVITY FOR FISCAL YEAR 1985 

AS OF 06/30/85 

DOLLAR 

AREA CONTRACT # LEVEL 

STAGE II FORMULATION: 

D.T.P. Ben Venue Laboratories, Inc. N CM2750800 126,994 

D.T.P. Iowa, University of N CM3756100 69,629 

D.T.P. Iowa, University of N CM4759401 19,610 

D.T.P. Kansas, University of N CM3756200 199,548 

D.T.P. Kentucky, University of N CM3762000 120,446 

I.S.D.T Litton Bionetics, Inc. N CM4765100 166,469 

TOTAL $702,696 



15 



ANALYSIS OF CONTRACTS BY ACTIVITY 



AREA 



ACTIVITY 



CONTRACT 



FOR FISCAL YEAR 1985 
AS OF 06/30/85 



STAGE II VERIFICATION SCREEN: 

D.T.P. Battelle Memorial Institute 

D.T.P. Biotech Research Laboratories, Inc. 

D.T.P. California, University of 

A.P. Charles River Breeding Labs. 

A. P. Charles River Breeding Labs. 

D.T.P. Fein Marquart Associates 

D.T.P. Fungal Fermentation 

A.P. Harlan Sprague Dawley, Inc. 

D.T.P. IIT Research Institute 

A.P. Leo Goodwin Institute for Cancer 

Research 

D.T.P. Mason Research Institute/EG & G 

B.R.M.P. Meloy Laboratories, Inc. 

A.P. Microbiological Associates 

A.P. Missouri, University of 

A.P. Missouri, University of 

A.P. Murphy Breeding Labs., Inc. 

A.P. Northwestern University 

A.P. Papanicolaou Cancer Research Institute 

A.P. Program Resources Incorporated 

A.P. Simonsen Laboratories 

A.P. Southern Animal Farms 

A.P. Southern Animal Farms 

D.T.P. Southern Research Institute 

D.T.P. Southern Research Institute 

D.T.P. Southern Research Institute 

A.P. Taconic Farms 

A.P. Texas A & M Research Foundation 

D.T.P. VSE, Corporation 

TOTAL 





DOLLAR 


# 


LEVEL 


N CM4764800 


205,192 


N CM3755800 


19,255 


N CM3756800 


128,833 


N CM3752600 


52,500 


N CM9722900 


16,703 


N CM4768300 


20,233 


RFPT5769200 


250,000 


N CM3762300 


200,698 


N CM4756700 


183,719 


N CM3762500 


39,000 


N CM4764700 


331,067 


N CM4765000 


203,612 


N CM9728700 


87,680 


N CM2753400 


20,034 


N CM8715700 


43,289 


N CM3022900 


1,836 


N CM3753800 


32,095 


N CM8723000 


18,619 


C02391002P 


517,200 


N CM3762400 


191,130 


N CM5772700 


40,382 


N CM9724500 


6,000 


N CM4758100 


301,725 


N CM4761500 


180,177 


N CM4764600 


241,810 


N CM5773000 


17,783 


N CM3753600 


18,838 


N CM5765400 


192,000 



3,561,410 



16 



ACTIVITY 



ANALYSIS OF CONTRACTS BY ACTIVITY 



FOR FISCAL YEAR 1985 





AS OF 06/30/i 


iS 




DOLLAR 


AREA 


CONTRACT 




# 


LEVEL 




STAGE II PROCUREMENT OF PRECLINICAL 


MATERIAL: 




D.T.P. 


Aerojet Strategic Propulsion Co. 


N 


CM4758400 


202,115 


D.T.P. 


Aldrich Chemical Company, Inc. 


N 


CM4758500 


102,961 


D.T.P. 


Ash Stevens, Inc. 


N 


CM4758600 


274,000 


I.S.D.T. 


Biotech Research Laboratory, Inc. 


N 


CM3759000 


123,630 


D.T.P. 


Flow Laboratories, Inc. 


N 


CM2750500 


140,036 


I.S.D.T. 


Litton Bionetics, Inc. 


N 


CM0572400 


400,000 


I.S.D.T. 


Litton Bionetics, Inc. 


N 


CM3757500 


230,683 


I.S.D.T. 


Litton Bionetics, Inc. 


N 


CM3758200 


128,824 


D.T.P. 


Midwest Research Institute 


N 


CM3755500 


82,525 


D.T.P. 


Midwest Research Institute 


N 


CM3763404 


41,729 


D.T.P. 


Monsanto Research Corporation 


N 


CM2751600 


58,891 


D.T.P. 


Pharm-Eco 


N 


CM4758700 


166,543 


D.T.P. 


Program Resources, Inc. 


N 


C02391000C 


1,249,423 


A.P. 


Program Resources, Inc. 


N 


C02391001P 


189,640 


D.T.P. 


Raylo Chemicals Limited 


N 


CM3763504 


12,092 


D.T.P. 


Research Triangle Institute 


N 


CM2751500 


208,006 


D.T.P. 


Research Triangle Institute 


N 


CM3761900 


65,068 


D.T.P. 


Research Triangle Institute 


N 


CM3763605 


15,321 


D.T.P. 


SISA, Inc. 


N 


CM3763705 


21,117 


D.T.P. 


SISA, Inc. 


N 


CM3763706 


15,493 


B.R.M.P. 


Sloan-Kettering Institute for 
Cancer Research 


N 


CM2560000 


55,000 


D.T.P. 


Southern Florida, University of 


N 


CM3763904 


13,477 


D.T.P. 


Southern Florida, University of 


N 


CM3763905 


10,975 


D.T.P. 


Southern Research Institute 


N 


CM3763805 


22,562 


D.T.P. 


Southern Research Institute 


N 


CM3763806 


30,564 


D.T.P. 


Southwest Foundation for Research 
Education 


& N 


CM3764002 


38,411 


13^ T. P. 


Stanford Research Institute 


N 


CM3761800 


97,174 


D.T.P. 


, Stanford Research Institute 


N 


CM3764105 


15,428 


D.T.P. 


Stanford Research Institute 


N 


CM3764106 


23,868 


D.T.P. 


Starks Associates, Inc. 


N 


CM3764203 


13,674 


D.T.P. 


Starks Associates, Inc. 


N 


CM3764204 


23,873 


D.T.P. 


Starks Associates, Inc. 


N 


CM3764205 


7,935 


D.T.P. 


Starks Associates, Inc. 


N 


CM3764206 


10,256 


D.T.P. 


Warner Lambert 


N 
TOTAL 


CM4758900 
< 


182,485 
f 4,273,779 



17 



ANALYSIS OF CONTRACTS BY ACTIVITY 





ACTIVITY FOR FISCAL YEAR 191 


35 






AS OF 06/30/85 




DOLLAR 


AREA 


CONTRACT 
STAGE III PHARMACOLOGY/TOXICOLOGY: 


# 


LEVEL 


D.T.P. 


Battel le Memorial Institute 


N CM1736500 


1,889,989 


D.T.P. 


Institute Jules Bordet 


N CM5764500 


150,212 


D.T.P. 


Master Agreement for Preclinical 
Pharmacology 


RFPT1736400 


500,000 


D.T.P. 


Mayo Foundation 


N CM3760103 


60,043 


D.T.P. 


Mayo Foundation 


N CM3760104 


81,674 


A.P. 


Texas A&M Research Foundation 


N CM3753600 


2,691 




TOTAL 




$ 2.684,609 



18 



ANALYSIS OF CONTRACTS BY ACTIVITY 



ACTIVITY 
CONTRACT 



FOR FISCAL YEAR 1985 
AS OF 06/30/85 



DOLLAR 

AREA CONTRACT # LEVEL 

STAGE III PROD. AND FORM. FOR CLINICAL TRIALS: 

D.T.P. Aerojet Strategic Propulsion Co. N CM4758400 202,114 

D.T.P. Aldrich Chemical Company, Inc. N CM4758500 191,212 

D.T.P. Arizona, University of N CM3762100 127,554 

D.T.P. Ash Stevens, Inc. N CM4758600 274,000 

D.T.P. Ben Venue Laboratories, Inc. N CM2750800 1,606,941 

D.T.P. Ben Venue Laboratories, Inc. N CM5759500 286,539 

D.T.P. Flow Laboratories, Inc. N CM1739800 325,000 

D.T.P. Flow Laboratories, Inc. N CM2750500 46,678 

D.T.P. Georgia, University of N CM2740100 173,058 

D.T.P. Iowa, University of N CM3756100 278,515 

D.T.P. Iowa, University of N CM4759401 78,442 

D.T.P. Midwest Research Institute N CM3755500 330,102 

D.T.P. Monsanto Reserach Corporation N CM2751600 431,868 

D.T.P. Pharm-Eco N CM4758700 166,543 

D.T.P. Research Triangle Institute N CM2751500 52,001 

D.T.P. Research Triangle Institute N CM3761900 260,274 

D.T.P. Stanford Research Institute N CM3761800 388,696 

D.T.P. Warner Lambert N CM4758900 182,484 

TOTAL $ 5,402,021 



19 



ANALYSIS OF CONTRACTS BY ACTIVITY 



ACTIVITY FOR FISCAL YEAR 1985 

AS OF 06/30/85 DOLLAR 

AREA CONTRACT # LEVEL 

PRECLIN. BASIC RES. - BIOLOGICAL STUDIES 

B.R.M.P. Iowa, Univeristy of N CM3757900 138,115 

I.S.D.T. Litton Bionetics, Inc. N CM3757500 230,682 

I.S.D.T. Litton Bionetics, Inc. N CM2561600 579,507 

CO. P. Litton Bionetics, Inc. N CM4765700 54,892 

CO. P. Litton Bionetics, Inc. N CM4764900 176,035 

CO. P. Meloy Laboratories, Inc. N CM5768800 210,691 

CO. P. Veterans Administration Y CM3025600 37,500 
Medical Center 

TOTAL $ 1,427,422 



20 



ANALYSIS OF CONTRACTS BY ACTIVITY 





ACTIVITY FOR FISCAL YEAR 1985 










AS OF 06/30/85 






DOLLAR 


AREA 


CONTRACT 
PRECLIN. BASIC RES. - TREATMENT STUDIES: 




# 


LEVEL 


R.R.P. 


Arizona 


N 


CM1752200 


122,415 


R.R.P. 


Energy, Department of-Lawerence 
Berkeley Lab 


Y 


CM2011000 


91,024 


CO. P. 


Litton Bionetics, Inc. 


N 


CM4764900 


71,902 


R.R.P. 


Mass. Inst. Tech. 


N 


CM2752500 


59.707 


R.R.P. 


Massachusetts General Hospital 


N 


CM2753200 


129,349 


R.R.P. 


Massachusetts General Hospital 


N 


CM4761600 


106,398 


R.R.P. 


Memorial Hospital for Cancer & 
Allied Diseases 


N 


CM4769500 


127,780 


C.O.P. 


Orkand Corporation 


N 


CM3601000 


345,975 


R.R.P. 


Pennsylvania, University of 


N 


CM2752900 


59,794 


R.R.P. 


Pennsylvania, University of 


N 


CM4769700 


112,426 


R.R.P. 


Sloan-Kettering 


N 


CM3756500 


366,006 


O.T.P. 


Southern Research Institute 


N 


CM47 58000 


611,630 


D.T.P. 


Southern Research Institute 


N 


CM3755200 


128,868 


R.R.P. 


Stanford University 


N 


CM1748000 


80,421 


R.R.P. 


Texas, University of. 


N 


CM2753100 


101,100 


R.R.P. 


Texas University 


N 


CM1752400 


93,686 


R.R.P. 


Utah University 


N 


CM1752300 


74,250 


R.R.P. 


Washington, University of 


N 


CM4769600 


116,371 



TOTAL 



$ 2,799,102 



21 



ANALYSIS OF CONTRACTS BY ACTIVITY 



AREA 



D.T.P. 

C.T.E.P. 

C.T.E.P. 

O.D. 

B.R.M.P. 

A. P. 

D.T.P. 

C.T.E.P. 

C.T.E.P. 

O.D. 



ACTIVITY 
CONTRACT 
PROGRAM MANAGEMENT: 



FOR FISCAL YEAR 1985 
AS OF 06/30/85 



Battel le Memorial Institute 

Emmes Corporation 

Information Management Services, Inc. 

JWK International Corporation 

Koba, Inc. 

National Academy of Sciences 

ORI, Inc. 

Social & Scientific Systems, Inc. 

Social & Scientific Systems, Inc. 

Technical Resources, Inc. 

TOTAL 



N CM1736500 
N CM1737100 
N CM2751000 
N CM2560200 
N CM4766100 
N CM5764400 
N CM3759100 
N CM3757600 
N CM2560600 
N CM5765800 



DOLLAR 
LEVEL 



809,995 
243,301 
300,000 

50,000 
369,137 

34,000 
101,458 
939,918 

90,000 
179,348 

$ 3,117,157 



22 



ANALYSIS OF CONTRACTS BY ACTIVITY 



AREA 



ACTIVITY 
CONTRACT 



FOR FISCAL YEAR 1985 
AS OF 06/30/85 



PHASE I CLINICAL TRIALS: 

R.R.P. Arizona, University of 

R.R.P. Howard University 

C.T.E.P. Johns Hopkins University 

C.T.E.P. Johns Hopkins University 

C.T.E.P. Maryland, University of 

C.T.E.P. Maryland, University of 

R.R.P. Massachusetts General Hospital 

R.R.P. Massachusetts Institute of Technology 

R.R.P. Mayo Foundation 

C.T.E.P. Mayo Foundation 

C.T.E.P. Mayo Foundation 

C.T.E.P. Memorial Hospital for Cancer & 

Allied Diseases 
C.T.E.P. Memorial Hospital for Cancer & 

Allied Diseases 
C.T.E.P. Ohio State University Research 

Foundation 
C.T.E.P. Ohio State University Research 

Foundation 
C.T.E.P. Ohio State University Research 

Foundation 
B.R.M.P. Phase I Clinical Trials 
R.R.P. Stanford University 
R.R.P. Texas, University of 
C.T.E.P. Texas, University of 
C.T.E.P. Texas, University of. Health 

Science Center 
C.T.E.P. Texas, University of. System 

Cancer CTR/MDA 
C.T.E.P. Texas, Universtiy of. System 

Cancer CTR/MDA 
R.R.P. Utah, University of 
C.T.E.P. Wayne State University 
C.T.E.P. Wisconsin, Univeristy of 
C.T.E.P. Wisconsin, University of 

TOTAL 





DOLLAR 


# 


LEVEL 


N CM1752200 


122,416 


N CM2754300 





N CM2750900 


70,333 


N CM5773800 


133,731 


N CM2754100 


78,120 


N CM5773400 


145,030 


N CM1748100 





N CM2752500 


59,854 


N CM2752800 


59,005 


N CM2754800 


94,270 


N CM5773300 


144,780 


N CM2754600 


76,974 


N CM5773200 


172,096 


N CM27 54000 


65,105 


N CM4762200 


121,535 


N CM5773600 


126,347 


RFP37613000 


600,000 


N CM1748000 


80,422 


N CM1752400 


93,687 


N CM5773700 


157,802 


N CM2754200 


72,027 


N CM2755000 


53,621 


N CM57739 


134,044 


N CM1752300 


74,250 


N CM2755100 


76,351 


N CM2754900 


96,992 


N CM5773500 


126,566 




$3,035,358 



23 



ANALYSIS OF CONTRACTS BY ACTIVITY 





ACTIVITY FOR FISCAL YEAR 19 


85 








AS OF 06/30/85 






DOLLAR 


AREA 


CONTRACT 
PHASE II CLINICAL TRIALS 




# 


LEVEL 


R.R.P. 


Health Research Institute 


N 


CM9731100 


43,699 


R.R.P. 


Howard University 


N 


CM2754300 





C.T.E.P. 


Maryland, University of 


N 


CM5773400 


98.590 


R.R.P. 


Massachusetts General Hospital 


N 


CM1748100 





R.R.P. 


Mayo Foundation 


N 


CM2752800 


59,005 


C.T.E.P. 


Mayo Foundation 


N 


CM5773300 


85,619 


C.T.E.P. 


Memorial Hospital for Cancer & 
Allied Diseases 


N 


CM0733700 


70,011 


C.T.E.P. 


Memorial Hospital for Cancer & 
Allied Diseases 


N 


CM5773200 


103,622 


C.T.E.P. 


Michigan, University of 


N 


CM0740500 


171,562 


B.R.M.P. 


Phase II Clinical Trials 


RFP37613000 


597,000 


R.R.P. 


Southern California, University of 


N 


CM2748300 


46,091 


C.T.E.P. 


Texas, University of, System 
Cancer CTR/MDA 


N 


CM5773900 


90,795 


C.T.E.P. 


Wayne State University 


N 


CM0740400 


67,500 






TOTAL 


1,433,494 



24 



AREA 



C.T.E.P. 
C.T.E.P. 
C.T.E.P. 

C.T.E.P. 

C.T.E.P. 
C.T.E.P. 
C.T.E.P. 

C.T.E.P. 



ACTIVITY 



ANALYSIS OF CONTRACTS BY ACTIVITY 

FOR FISCAL YEAR 1985 
AS OF 06/30/85 



CONTRACT 

PHASE III CLINICAL TRIALS 

Maryland, University of 

Mayo Foundation 

Memorial Hospital for Cancer 

& Allied Diseases 
Memorial Hospital for Cancer 

& Allied Diseases 
Michigan, University of 
Pan American Health Organization 
Texas, University of. System 

Cancer CTR/MDA 
Wayne State University 



# 


LEVEL 


N CM5773400 
N CM5773300 
N CM0733700 


10,955 
9,513 
7,779 


N CM5773200 


11,514 


N CM0740500 
N CM2739100 
N CM5773900 


19,062 

388,764 

10,078 


N CM0740400 


7,500 


TOTAL 


465,165 



25 



ANALYSIS OF CONTRACTS BY ACTIVITY 



AREA 



R.R.P. 

R.R.P. 

R.R.P. 

R.R.P. 

R.R.P. 

R.R.P. 

C.T.E.P. 

R.R.P. 

R.R.P. 



ACTIVITY 



CONTRACT 



FOR FISCAL YEAR 1985 
AS OF 06/30/85 



CLINICAL TRIALS - OTHER RESEARCH 

Bowman Gray School of Medicine 
California, University of 
California, University of (SF) 
Cleveland Clinic Foundation 
Duke University 
Fox Chase Cancer Center 
Istituto Nazionale Tumori 
Massachusetts General 
Washington, University of 





DOLLAR 


# 


LEVEL 


N CM4756400 


197,741 


N CM9731500 


1,279,150 


N CM4768400 


243,844 


N CM4768500 


286,548 


N CM4768600 


210,891 


N CM9731400 


1,277,210 


N CM0733800 


145,000 


N CM4768700 


259,089 


N CM9728200 


958,821 


Total 


4,858,294 



26 



AREA 



R.R.P. 
R.R.P. 
R.R.P. 

C.T.E.P. 
C.T.E.P. 
C.T.E.P. 
C.T.E.P. 

O.D. 

C.T.E.P. 



ACTIVITY 
CONTRACT 



ANALYSIS OF CONTRACTS BY ACTIVITY 

FOR FISCAL YEAR 1985 
AS OF 6/30/85 



CLINICAL TRIALS SUPPORTIVE RESEARCH 

Allegheny-Singer Research Corporation 
Bureau of Radiological Health, FDA 
Development of Dos i mi try Standards 

for Neutron Therapy 
Emmes Corporation 
Emmes Corporation 

Information Management Services, Inc. 
Information Management Services, Inc. 

Foundation 
Japanese Foundation for Cancer 

Research 
Theradex Systems, Inc. 



# 


DOLLAR 
LEVEL 


N CM3751200 
Y CM2010700 
RFPT8500300 


367,320 

80,700 

120,000 


N CM1737100 
N CM8719300 
N CP3100900 
N CM2751000 


243,302 

277,597 

30,000 

300,000 


N CM3601100 


19,000 


N CM3755300 


1,026,923 


Total 


2,464,842 



27 



ANALYSIS OF CONTRACTS BY ACTIVITY 



AREA 



D.T.P. 
CO. P. 
D.T.P. 
CO. P. 
R.R.P. 



ACTIVITY 



CONTRACT 



FOR FISCAL YEAR 1985 
AS OF 6/30/85 



# 



DOLLAR 
LEVEL 



STATISTICS, DATA PROCESSING, AND OTHER CLINICAL TRIAL RESOURCES 



Chemical Abstract Services 
Litton Bionetics, Inc. 
Maintenance of NCI/DIS 
Meloy Laboratories, Inc. 
Orkand Corporation 



N CM4770900 
N CM4765700 
RFPT4768900 
N CM5768800 
N CM3757200 



94,217 

6,099 

90,000 

23,410 





Total 



213,726 



28 



TABLE III 

DESCRIPTION OF CONTRACTS 

IN THE 

DIVISION OF CANCER TREATMENT 

AEROJET STRATEGIC PROPULSION COMPANY (N01-CM4-7584) 

This service preparative contract provides for the resynthesis of a variety 
of compounds required for clinical or preclinical evaluation. The compounds 
prepared are not readily available on the open market or from the original 
supplier in the amounts and/or quality required. The major effort (approxi- 
mately 90%) of this contract is devoted to the preparation of large quantities 
of materials, in the multikilogram range, requiring pilot plant facilities. 

ALABAMA, UNIVERSITY OF (N01-CM2-7571) 

This project is one of the three contracts whose objectives are to design and 
synthesize the following: (1) congeners of lead compounds to enhance the 
activity or broaden the antitumor spectrum; (2) "pro-drugs" that are chemically 
altered transport forms of compounds to modify both biological and pharmaceu- 
tical properties, such as (a) improved bio-availability by increasing aqueous 
solubility; (b) increased compound stability; and (3) compounds related to 
products of natural origin and other heterocycles with improved antitumor 
activity and decreased toxicity. These modifications include partial struc- 
tures, structural analogs and novel heterocycles. 

ALABAMA, UNIVERSITY OF (N01-CM3-7631) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program 
for development. Unique compounds of interest emerging from the literature 
that cannot be obtained in sufficient quantities from the original investi- 
gators are synthesized by this mechanism. This mechanism is also used for 
the resynthesis of a limited number of panel compounds, and compounds of 
interest to intramural scientists. Potential radiosensitizers and radio- 
protectors are also synthesized in support of the Radiation Research Program. 

ALDRICH CHEMICAL COMPANY, INC. (N01-CM4-7585) 

This service preparative contract provides for the resynthesis of a variety 
of compounds required for clinical or preclinical evaluation. The compounds 
prepared are not readily available on the open market or from the original 
supplier in the amounts and/or quality required. About 30% of the effort of 
this contract is devoted to the preparation of large quantities of materials, 
in the multikilogram range, requiring pilot plant facilities. 



29 



ALLEGHENY-SINGER RESEARCH CORPORATION (N01-CM3-7512) 

This contract is expected to develop the criteria, guidelines and proce- 
dures for the proper use of the equipment representing the major heat 
generating modalities (radiofrequency, microwave, and ultrasound) and the 
ancillary equipment necessary for the treatment of cancer with heat. The 
criteria and guidelines developed will be utilized to initiate a quality 
assurance and assessment program in hyperthennia. The contractor is 
expected to implement and conduct such a program in the latter years of the 
contract. 

ARIZONA. UNIVERSITY OF (NOl-CMl-7522) 

This contract is for the Phase I evaluation of equipment for the hyperthermic 
treatment of cancer in conjunction with radiotherapy, chemotherapy, surgery, 
or immunotherapy. Five contractors are collaborating in a working group in 
conjunction with the National Center for Devices and Radiological Health to 
develop guidelines for the use of heat generating and thermometry equipment 
to facilitate the development of Phase II and III clinical studies for the 
adjuvant treatment of deeply seated tumors with heat. 

ARIZONA, UNIVERSITY OF (N01-CM3-7621) 

Difficult dosage form development projects are assigned to this contractor 
for evaluation. The contractor has particular expertise with cosolvent 
approaches to improve drug solubility. Several novel solvents are being 
evaluated in an attempt to improve the options available to formulate poorly 
water soluble and unstable compounds. This contractor is also responsible 
for the preparation of pilot batches and chemical analysis of these novel 
formulations. 

ARIZONA, UNIVERSITY OF (N01-CM5-7662) 

This is one of three contracts devoted to the application of a human tumor 
colony forming assay (HTCFA) to drug screening. Contract efforts have focused 
on screening compounds inactive in the standard in vivo pre-screen (murine P388 
leukemia) to establish the potential of the HTCFA to identify compounds not 
detected by the in vivo screen. Materials active against fresh human tumor 
specimens are also tested in a P388 cell line colony forming assay, developed 
under this project for direct comparison with the HTCFA. Screening has been 
initiated on more than 300 compounds and several have been identified as anti- 
tumor drug leads. Activity in the HTCFA, P388 colony forming assay, in vivo 
tumor panel , and degree of structural novelty, are used to determine which 
are recommended to the Decision Network Committee for development to clinical 
trial. To date three compounds have been accepted into the formal drug 
development program. 

ARMED FORCES INSTITUTE OF PATHOLOGY (Y01-CM3-0118) 

This Interagency Agreement provided processing and pathologic examinations 
of all testicular tumor tissue slides and blocks submitted by the Intergroup 
Stage I and II Testicular Protocol to the Testicular Tumor Study Group of 
the Extramural Clinical Trials Program. This Interagency Agreement expired 
January, 1985. 

30 



ARTHUR D. LITTLE, INC. (N01-CM3-7596) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism wil 1 enable the 
prompt solicitation and award of projects to be conducted in this area from a 
pool of highly qualified contractors. The contractor is currently working on 
a task to characterize the preclinical pharmacology of Deoxyspergualin, 
NSC 356894. 

ASH STEVENS. INC. (N01-CM4-7586) 

This service preparative contract provides for the resynthesis of a variety 
of compounds required for clinical or preclinical evaluation. The compounds 
prepared are not readily available from the original supplier or commercial 
sources in the amounts and/or quality required. About 60% of the effort of . 
this contract is devoted to the preparation of large quantities of materials, 
in the multikilogram range, requiring pilot plant facilities. 

BANNER GELATIN PRODUCTS CORPORATION (NOl-CMl-7402) 

The objectives of this contract are to provide facilities and capabilities 
for the development and production of soft gelatin capsules containing 
investigational anticancer agents. The contractor is responsible for 
conformity to FDA Current Good Manufacturing Practices and for completing 
all required analytical testing on each product prepared. All products 
are packaged, labeled, and shipped to the National Cancer Institute for 
subsequent redistribution to clinical investigators. 

BATTELLE MEMORIAL INSTITUTE (NOl-CMl-7365) 

This service type Prime Contract with Battel le Memorial Institute is for 
supervision of subcontractors carrying out the toxicologic evaluation of 
potential oncolytic agents, biologic response modifiers, and other modalities. 
Through the Prime Contract mechanism, preclinical toxicologic studies of agents 
under consideration for potential clinical use are handled under a single 
management -type contract. The work scope under this contract is comprised of 
four tasks as follows: Task I -Full Protocol Studies; Task II - High Priority 
Toxicity Studies {i.e., any portion of the Protocol of the Toxicology Branch); 
Task III Specific Organ Toxicity Testing and Task IV Automation of toxicity 
data, anomaly detection, scheduling of studies, and financial management. 

BATTELLE MEMORIAL INSTITUTE (N01-CM4-7648) 

This contract is for the in vivo screening of new materials in the P388 leu- 
kemia pre-screen and for TFe secondary testing of materials of interest to 
the Developmental Therapeutics Program. The secondary testing may consist 
of testing in both a panel of murine tumor models and a human xenograft tumor 
model. Testing is conducted at a level of approximately 20,000 L1210 equiva- 
lents per year. Special studies for the detailed evaluation of compounds of 
Program interest, as well as studies with new tumor panel systems, are con- 
ducted upon the request of the Project Officer. 

31 



BEN VENUE LABORATORIES. INC. (N01-CM2-7508) 

This resource contract provides for the development and production of 
parenteral clinical dosage forms of anticancer agents. The contractor 
has the capacity for preparing production batches of liquid-filled and 
lyophilized sterile products. Specifically, the contractor performs the 
following services: (1) formulation development of parenteral products; 
(2) production of sterile products; and (3) quality assurance testing of 
finished products. All products are packaged, labeled, and shipped to 
the National Cancer Institute for subsequent redistribution to clinical 
investigators. 

BEN VENUE LABORATORIES. INC. (N01-CM5-7595) 

The objectives of this contract are to provide facilities and capabilities 
for the development and production of parenteral investigational dosage 
forms for the Division of Cancer Treatment. The contractor is responsible 
for conformity to U.S. FDA Current Good Manufacturing Practices and for 
completing all required analytical testing on each product prepared. All 
products are packaged, labeled, and shipped to the National Cancer Institute 
for subsequent redistribution to clinical investigators. 

BOWMAN GRAY SCHOOL OF MEDICINE (N01-CM3-7603) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the 
prompt solicitation and award of projects to be conducted in this area from a 
pool of highly qualified contractors. 

BOWMAN GRAY SCHOOL OF MEDICINE (N01-CM4-7564) 

This is one of five contracts awarded in May 1984 (a) to develop criteria 
and guidelines for clinical use of nuclear magnetic resonance imaging (MR!) 
systems in the detection and diagnosis of pathology in human subjects and 
in the noninvasive characterization of tissues, and (b) to carry out 
performance and evaluation studies to compare the diagnostic accuracy 
of MRI with that from other modalities (e.g., CT and radioisotope imaging). 
This contractor, working as a member of the MRI Collaborative Working 
Group (CWG), has contributed to the development of clinical imaging protocols 
for evaluation of MRI in the diagnosis of 1) musculoskeletal tumors, 2) cervical 
myelopathies, 3) uterine neoplasms, and 4) liver metastases. They have also 
provided coordination for the CWG of the development of a lung cancer protocol. 
Patient imaging has begun in each of these studies. A breast disease protocol 
has also been prepared for adoption in year 02. 



32 



BRISTOL LABORATORIES, INC. (N01-CM3-7556) 

This fermentation contract is designed primarily to discover novel antitumor 
agents from microbial fermentations. This contract includes: (1) Isolation 
of microbes and screening these for unique biological activities. The isolations 
and screening are done mostly at the Bristol Myers Japanese facility, Bristol 
Myers Research Institute - Tokyo (BMRIT); (2) Preparation of fermentation 
beers of the selected microbes; (3) Development and use of in vitro pre-screens 
to evaluate the fermentations; (4) Development of in vitro assays to assist 
in quickly isolating the anticancer agents; (5) Dereplication of the materials 
to determine novelty; (6) Chemical isolation and characterization of the 
active components; and (7) Production of large quantities of new agents for 
thorough evaluation in DCT screens. This contract will terminate in FY 1986. 

CALIFORNIA, UNIVERSITY OF (NOl-CMO-7439) 

On this contract. Phase I/I I clinical evaluations of Wei 1 feron are being 
conducted in patients with advanced breast cancer and multiple myeloma. 
Although the above trials have now been completed, an ongoing trial of 
natural interferon in hairy cell leukemia is continuing. This contract 
will expired June, 1985. 

CALIFORNIA, UNIVERSITY OF (N01-CM3-7568) 

Project objectives are (1) to determine the extent to which in vitro clono- 
genic assays, using transplantable human tumor/athymic mouse xenografts as 
the source of tumor cells are capable of identifying drugs with activity 
against the same tumors in vivo and (2) to develop disease-oriented pre- 
clinical screens that permit expression of drug activity as response rate. 
This project ended August, 1985. 

CALIFORNIA, UNIVERSITY OF (N01-CM4-7672) 

Under this contract, a Phase I clinical trial is to be conducted with a 
monoclonal immunoconjugate. Pharmacokinetics, clinical toxicity, and biologic 
response will be monitored. This trial is scheduled to begin in late spring. 
The contract will expire December, 1985. 

CALIFORNIA, UNIVERSITY OF (N01-CM4-7684) 

This is one of five contracts awarded in May 1984 (a) to develop criteria 
and guidelines for clinical use of nuclear magnetic resonance imaging (MRI) 
systems in the detection and diagnosis of pathology in human subjects and 
in the noninvasive characterization of tissues, and (b) to carry out 
performance and evaluation studies to compare the diagnostic accuracy 
of MRI with that from other modalities (e.g., CT and radioisotope imaging). 
This contractor, working as a member of the MRI Collaborative Working Group 
(CWG), has contributed to the development of clinical imaging protocols 
for evaluation of MRI in the diagnosis of: 1) musculoskeletal tumors, 
2) cervical myelopathies, 3) uterine neoplasms, 4) liver metastases, and 
5) brain neoplasms. They have also provided coordination for the CWG 
of the development of the uterine neoplasms protocol. Patient imaging 
has begun in each of these studies. A cardiovascular disease protocol 
has also been prepared for adoption in year 02. 

33 



CALIFORNIA, UNIVERSITY OF (N01-CM5-7710) 

This is one of three contracts devoted to the application of a human tumor 
colony forming assay (HTCFA) to drug screening. Contract efforts have focused 
on screening compounds inactive in the standard in vivo pre-screen (murine P388 
leukemia) to establish the potential of the HTCFA to identify compounds not 
detected by the in vivo screen. Materials active against fresh human tumor 
specimens are also tested in a P388 cell line colony forming assay, developed 
under this project for direct comparison with the HTCFA. Screening has been 
initiated on more than 300 compounds and several have been identified as anti- 
tumor drug leads. Activity in the HTCFA, P388 colony forming assay, in vivo 
tumor panel , and degree of structural novelty, are used to determine which are 
recommended to the Decision Network Committee for development to clinical trial. 
To date three compounds have been accepted into the formal drug development 
program. 

CALIFORNIA, UNIVERSITY OF (N01-CM9-7315) 

This contract provides for a cyclotron-based neutron therapy system, a clinical 
facility in which to house the equipment and personnel to support clinical 
neutron therapy research at UCLA. The proposed facility will be constructed 
on the grounds of the Wadsworth Veterans Administration Medical Center near the 
UCLA campus. The cyclotron and associated components of the neutron therapy 
system are being fabricated in Berkeley, California. Construction of the 
facility began in November, 1982, and was scheduled for completion in April, 
1984. The Cyclotron Corporation, which is manufacturing the cyclotron and 
neutron therapy system, filed for bankruptcy under Chapter 11 of the Bankruptcy 
Code. The neutron therapy system, which was approximately 90% complete, has 
been moved to the VA Hospital facilities for final assent)ly and testing. 
Patient treatments are expected to begin in November, 1985. 

CHARLES RIVER BREEDING LABORATORIES (N01-CM3-7526) 

This contract provides for the rederivation of approximately 16 mouse and rat 
strains and two guinea pig strains on an annual basis. Rederived strains will 
be distributed to genetic centers for expansion and replacement of producing 
strains. 

CHARLES RIVER BREEDING LABORATORIES (N01-CM3-7626) 

This contract is a Primary Genetic Center. Rederived associated flora pedigreed 
starts are supplied to this contract for propagation into isolator maintained 
foundation colonies. These foundation colonies supply breeders on a weekly 
basis to pedigreed expansion colonies which support the production colonies. 
Both the pedigreed expansion and production colonies are maintained in a barrier 
room environment. Offspring from the production colonies are used for hybrid 
production and the many NCI research activities. 

CHARLES RIVER BREEDING LABORATORIES (N01-CM5-7653) 

This Rodent Production Center contract, located in North Carolina, produces 
hybrid mice and nude mice in a maximum barrier environment. 



34 



CHARLES RIVER BREEDING LABORATORIES (N01-CM8-7212) 

This contract provides for the continual monitoring of the associated isolators 
within the OCT animal program to determine the flora status of these foundation 
isolators and to check for specified isolator contaminants. In addition, this 
contract will provide the correct organisms for establishing flora in new 
isolators. 

CHARLES RIVER BREEDING LABORATORIES (N01-CM9-7229) 

This rodent production center contract supports a production effort designed 
to furnish animals as required by laboratory programs. Breeding animals are 
furnished by the government from primary genetic centers. This contract 
terminated January, 1985. 

CHEMICAL ABSTRACTS SERVICE (N01-CM4-7709) 



This Contractor operates the NCI's Chemical Information System, a large com- 
puterized system holding the structures and associated data of over 350,000 
chemical compounds tested by the NCI as anticancer agents. The system is used 
to determine which actual or potential acquisitions are identical or similar 
to previous accessions, to maintain inventory control, to monitor the movement 
of each sample from its receipt through shipment to screener, to allow for 
online and offline querying of the file, to run a model that predicts activities 
and toxicities and novelty of potential acquisitions, and to coordinate the 
actions of the several contractors involved in the pre-screen operations. 
This contract was phased out April 15, 1985; the system, which is now ten years 
old, being replaced by a newer one. 

CLEVELAND CLINIC (N01-CM4-7685) 

This is one of five contracts awarded in May, 1984 (a) to develop criteria and 
guidelines for clinical use of nuclear magnetic resonance imaging (MRI) systems 
in the detection and diagnosis of pathology in human subjects and in the non- 
invasive characterization of tissues, and (b) to carry out performance and 
evaluation studies to compare the diagnostic accuracy of MRI with that from 
other modalities (e.g., CT and radioisotope imaging). This contractor, working 
as a member of the MRI Collaborative Working Group (CWG), has contributed to the 
development of clinical imaging protocols for evaluation of MRI in the diagnosis 
of 1) musculoskeletal tumors, 2) cervical myelopathies, 3) uterine neoplasms, 
4) liver metastases, and 5) brain neoplasms. They have also provided coordina- 
tion for the CWG of the development of the cervical myelopathies protocol. 
Patient imaging has begun in each of these studies. 

DUKE UNIVERSITY (NOl-CMO-7436) 

This contract is conducting Phase I/II clinical trials of recombinant gamma 
interferon in the treatment of patients with disseminated malignant disease. 
Sixteen patients have been accrued to the initial step of this study, which 
is now completed. Fever has been the major toxicity. The contract has been 
extended without additional cost through June 30, 1985 to permit an additional 
study of the effects of repeated administration of gamma interferon at a single 
dose to be performed. 



35 



DUKE UNIVERSITY (N01-CM4-7686) 

This is one of five contracts awarded in May, 1984 (a) to develop criteria and 
guidelines for clinical use of nuclear magnetic resonance imaging (MRI) systems 
in the detection and diagnosis of pathology in human subjects and in the non- 
invasive characterization of tissues, and (b) to carry out performance and 
evaluation studies to compare the diagnostic accuracy of MRI with that from 
other modalities (e.g., CT and radioisotope imaging). This contractor, working 
as a member of the MRI Collaborative Working Group (CWG) , has contributed to 
the development of clinical imaging protocols for evaluation of MRI in the 
diagnosis of 1) musculoskeletal tumors, 2) cervical myelopathies, 3) uterine 
neoplasms, 4) liver metastases, 5) brain neoplasms, and 6) lung cancer. They 
have also provided coordination for the CWG of the development of the brain 
neoplasms protocol. Patient imaging has begun in each of these studies. 

DYNAMAC CORPORATION (N01-CM3-7563) 

The objective of this project is to develop and maintain a systematic litera- 
ture surveillance effort to identify published compounds which warrent acqui- 
sition based on their structural characteristics and biological properties. 
This contract is monitoring a broad base of chemical, biochemical, biological 
and patent literature to identify compounds for potential acquisition or task 
order synthesis. 

EG&G MASON RESEARCH INSTITUTE (N01-CM4-7647) 

As the result of budgetary restraints, this contract was reduced during the 
current contract period to a level of 21,500 L1210 equivalents (an annualized 
level of 19,000 equivalents). The contract provides for in vivo testing in 
the P388 leukemia pre-screen, for the evaluation of materials in specified 
tumor panel models, for detailed evaluations as requested by members of the 
NCI staff, and for evaluations of tumor models as directed by the Project 
Officer. In addition to the P388, other tumor systems currently being utilized 
include the murine L1210, M5076 sarcoma, and B16 (being phased out). Xenograft 
models include the colon, breast and lung sub-renal capsule models, the LOX 
amelanotic melanoma and the HL-60 which is used for special testing of 
differentiating agents. 

EMMES CORPORATION (NOl-CMl-7371) 



This contract provides operations office support for the Gastrointestinal 
Tumor Study Group and the Intergroup Testicular Cancer Studies. Functions 
include coordination of protocol development, editing and preparing final 
form of protocols, forms design, randomization, quality control of data, 
editing and preparing meeting agenda which include clinical trials reports, 
writing and preparing minutes of meetings, preparing correspondence, record- 
keeping, and files maintenance. During this period the Contractor has also 
provided research and information management assistance to the CTEP in support 
of its mission of coordination of ongoing and planned clinical trials. 



36 



EMMES CORPORATION (N01-CM8-7193) 

The EMMES Corporation provides the statistical support for the Gastrointestinal 
Tumor Study Group. They assist in design of protocols, perform statistical 
analyses of studies, and assist investigators in preparing manuscripts presenting 
the data. 

ENERGY. DEPARTMENT OF (Y01-CM2-0110) 

This contract is part of a collaborative effort to improve treatment planning 
with neutrons and with charged particles (protons, helium ions, pi-mesons and 
heavy ions). In addition to developing optimal treatment plans for the treat- 
ment of tumors in various anatomic sites with the particle beam at its own 
institution, the collaborative working group is conducting a comparison of 
treatment plans for selected tumors in each anatomic site. Whenever possible, 
measurements in patients or phantoms will be made to check the accuracy of the 
treatment planning. 

ENGLE LABORATORY ANIMALS (N01-CM3-0225) 

This contract furnishes approximately 104,000 hybrid mice for compound evalua- 
tion studies. Breeding animals are furnished from genetic centers and/or rodent 
production center colonies. This contract terminated January, 1985. 

FEIN-MARQUART ASSOCIATES, INC. (N01-CM4-7683) 

Under this contract, work has continued for the past year upon the development 
of the NCI Drug Information System (DIS). The major subsystems of the DIS, the 
Chemistry, Inventory and Shipping modules have all been installed, tested, and 
released for use. Trouble shooting in the first two of these is essentially 
complete. Some problems remain in the Shipping module and are currently being 
addressed. Meanwhile, the Biology module is being readied for installation. 
The previous computer system, which the DIS is replacing, has now been frozen 
and all new data have been flowing into the DIS since 3/85. The old contract 
has been terminated. The troublesome problem of documentation has been 
addressed addressed and a major documentation effort, with a high priority, 
has begun. This should be largely complete by 9/85 when the contract ends. 

FLOW LABORATORIES, INC. (NOl-CMl-7398) 

This resource contract provides the Division of Cancer Treatment with storage 
and distribution capabilities for the large volume of investigational and 
commercial drugs used in clinical trials. Approved orders for clinical drugs 
are packaged and shipped to destinations around the world. The contractor 
also provides a computerized inventory management system. This assures the 
proper rotation of stock, an adequate lead time to obtain new supplies of drugs, 
and the prompt removal of expired materials. 



37 



FLOW LABORATORIES, INC. (N01-CM2-7505) 

This contractor furnishes the National Cancer Institute with facilities and 
services for the storage and distribution of synthetic chemicals, bulk chemical 
drugs and crystalline natural products. Samples are weighed, packaged and 
shipped to contract screening laboratories and also to various domestic and 
foreign research institutions. The contract also provides for the maintenance 
of accurate inventory records. This is an on-going operation and supports many 
activities of the Developmental Therapeutics Program. 

FOX CHASE CANCER CENTER (NOl-CMO-7432) 

This contract is now being conducted by Jefferson Medical Center, Pennsylvania, 
under a subcontract to the Fox Chase Cancer Center. It was designed to study 
the Phase I effects of decreasing the number of circulating suppressor T cells 
in patients with disseminated malignancy by the infusion of anti-T suppressor 
cell antibody (anti-Leu 2a, Becton-Dickinson). Doses of 1, 5, 25, and lOOmg 
have been administered by infusions lasting from 2 hours to 24 hours. Sixteen 
patients have been accrued to the first step of this protocol. Pharmacokinetic 
data and immunologic data are being evaluated at this time. A second step of 
the study will evaluate the effects of repetitive administration of the optimal 
biologic dose. Toxicity has included fever and shaking chills. The contract 
was extended through June, 1985. 

FOX CHASE CANCER CENTER (N01-CM9-7314) 

This contract provides for the support of a clinical neutron therapy program 
at the University of Pennsylvania - Fox Chase Cancer Center using a DT generator 
developed under the direction of the University of Pennsylvania, in part through 
grant support from NCI. The addition to the Fox Chase Cancer Center to house 
the neutron therapy system was completed in October, 1981. After several 
delays, the first DT generator tube was shipped to Philadelphia in March, 1982. 
Unexpected problems with a series of tubes caused interruptions during 1982, 
1983, and 1984. Patient treatments began in January, 1984, continued for about 
two months and then were suspended for about a year, resuming in February, 1985. 

FRANKLIN RESEARCH INSTITUTE (N01-CM3-7632) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program 
for development. Unique compounds of interest emerging from the literature 
that cannot be obtained in sufficient quantities from the original investi- 
lators are synthesized by this mechanism. This mechanism is also used for 
the resynthesis of a limited number of panel compounds, and compounds of 
interest to intramural scientists. Potential radiosensitizers and radiopro- 
tectors are also synthesized in support of the Radiation Research Program. 

FRED HUTCHINSON CANCER RESEARCH CENTER (N01-CM4-7668) 

On this contract, a Phase I clinical trial is to be conducted with a monoclonal 
immunoconjugate directed against malignant melanoma. Pharmacokinetics, clinical 
toxicity, and biologic response will be monitored. This trial is scheduled to 
begin in late spring. The contract will expire December, 1985. 



38 



GEORGETOWN UNIVERSITY (NOl-CMO-7437) 

On this contract Phase II clinical trials of Wellferon in patients with meta- 
static melanoma. In the first step of this study, three different schedules 
were compared in a total of 32 patients. At present four patients have shown 
a complete or partial response. Responses were seen in both the alternate day 
and daily schedule, but not on the weekly schedule. The most significant 
toxicity was observed in one patient, who developed a lupus-like reaction 
while receiving the alpha interferon. This contract has received supplemental 
funding to conduct an additional clinical study in patients with malignant 
melanoma. The contract expiree August, 1985. 

GEORGIA, UNIVERSITY OF (N01-CM2-7401) 

This contract with the Department of Pharmaceutics of the University of Georgia 
has the responsibility of performing shelf life evaluation of clinical drugs. 
This stability data is supplied to the Food and Drug Administration in support 
of the NCI's IND filings. The contractor monitors the stability of dosage 
forms at several storage temperatures. The testing involves the use of multiple 
analytical methods. The method most frequently used for assay of the stability 
samples is high performance liquid chromatography (HPLC). This contractor also 
has the responsibility of conducting reserve sample inspections as required by 
the Current Good Manufacturing Practices (CGMPs). 

GEORGIA TECHNICAL RESEARCH CORPORATION (N01-CM2-751 7) 

This project is one of the three contracts whose objectives are to design 
and synthesize the following: (1) congeners of lead compounds to enhance 
the activity or broaden the antitumor spectrum; (2) "pro-drugs" that are 
chemically altered transport forms of compounds to modify both biological and 
pharmaceutical properties, such as (a) improve bio-availability by increasing 
aqueous solubility; (b) increase compound stability; and (3) compounds related 
to products of natural origin and other heterocycles with improved antitumor 
activity and decreased toxicity. These modifications include partial structures, 
structural analogs and novel heterocycles. 

HARLAN/SPRAGUE-DAWLEY, INC. (N01-CM2-3911) 

This contract operates the Animal Production Area at the Frederick Cancer 
Research Facility (FCRF). The contract operates as a Primary Genetic Center, 
Rederivation Center and Embryo Freezing Center. Strains are received from the 
NIH Repository and rederived for use at the FCRF and distribution to other NCI 
contract activities. The bulk of the production on this contract is for 
supplying the animal needs of the researchers located at the FCRF. Animals 
are also sent from FCRF to other NCI funded research activities. 

HARLAN/SPRAGUE-DAWLEY. INC. (N01-CM3-7623) 

This contract is a Primary Genetic Center. Rederived associated flora pedigreed 
starts are supplied to this contract for propagation into isolator maintained 
foundation colonies. These foundation colonies supply breeders on a weekly 
basis to pedigreed expansion colonies which support the production colonies. 
Both the pedigreed expansion and production colonies are maintained in a barrier 
room environment. Offspring from the production colonies are used for the many 
NCI research activities. 

39 



HARLAN/SPRAGUE-DAWLEY, INC. (N01-CM5-7728) 

This Rodent Production Center contract produces CD2F1 hybrid mice under 
maximum barrier conditions. 

HEALTH RESEARCH. INC. (N01-CM9-73n ) 

The objective of this program is to evaluate in a Phase II study, photo- 
radiation therapy as a means of local treatment of various malignancies in 
man. It is planned to determine its scope and limitations and especially 
to identify situations where it may offer a unique advantage over existing 
therapies as a treatment for patients who have failed other modalities. 
Photoradiation therapy involves irradiating hematoporphyrin derivative, 
which accumulates in malignant tissue, with appropriate laser light in 
the presence of oxygen. This process generates singlet oxygen, a highly 
toxic substance. A minimum of 25 patients per year will be studied. 

HOWARD UNIVERSITY (N01-CM2-7543) 

This contract is part of a collaborative effort among three institutions to 
investigate the role of intraoperative radiotherapy in the treatment of intra- 
abdominal malignancies and to develop guidelines for intraoperative radiotherapy 
techniques for use by other investigators and by clinical cooperative groups 
in Phase II and III clinical trials. An optional task is to investigate the 
use of radiation modifiers in conjunction with intraoperative radiotherapy. 
The collaborative effort will facilitate the evaluation of this new radiothera- 
peutic modality with respect to normal tissue tolerance, safety, and efficacy. 

HYBRITECH, INC. (N01-CM2-6010) 

This contract performs coupling of chemotherapeutic drugs, toxins and radio- 
isotopes to monoclonal antibodies directed against specific antigens found 
on human tumor cells. Appropriate tests are carried out on conjugates to 
demonstrate that the cytotoxic agent-antibody conjugates retain antigen-antibody 
specificity comparable to the unmodified antibody and cytotoxicity in excess 
of the nonderivatized cytotoxin. The contractor is required to scale up the 
appropriate conjugation procedure to provide sufficient quantities of a human 
use product for preclinical and preliminary clinical trials. Experiments have 
been ongoing to couple adriamycin, methotrexate, ricin A chain, Yttrium-90, 
Indium-Ul and Iodine-131 to three monoclonal antibodies: TlOl, an antibody 
directed against a human T-cel 1 differentiation antigen, 9.2.27, an antibody 
directed against a human melanoma cell antigen and D-3, an antibody directed 
against a tumor specific guinea pig antigen. The contractor has been supplying 
TlOl conjugated to indium and iodine for biodistribution and imaging studies 
and is scaling up to provide TlOl and 9.2.27 drug conjugates for clinical 
studies. This contract expired September, 1985, and is being recompeted. 

IIT RESEARCH INSTITUTE (N01-CM4-7567) 

This contract, awarded as a result of the recompetition of the in vivo screening 
program, has a current level of testing in mice of potential anticancer agents 
of 19,000 L1210 equivalent tests per year, having been reduced during FY-85 due 
to budgetary restrictions. The contract provides for in vivo testing in the 
P388 leukemia pre-screen, for evaluation of materials in specified tumor panel 



40 



models, for detailed evaluations requested by members of the NCI staff, and for 
characterizations and evaluations of tumor models as directed by the NCI Project 
Officer. All testing is carried out in accordance with the protocols of the NCI 
Developmental Therapeutics Program. Materials tested in the P388 leukemia pre- 
screen are new synthetic compounds and fractions of natural products provided by 
the NCI. Models of the conventional tumor panel now in use under this contract 
include the M5076 sarcoma and L1210 leukemia. Xenograft models include the 
breast sub-renal capsule model and the LOX amelanotic melanoma. 

ILLINOIS. UNIVERSITY OF (N01-CM3-7513) 

The objective of this contract is to do a worldwide survey of all the natural 
products literature, identifying new structures and reports of specific bio- 
logical activity of these compounds which may be related to cancer. Reports 
of biological activity of plant and animal extracts are also provided. This 
is a key contract for acquisition of new agents for the DTP screening program. 
This contract is currently being recompeted. 

ILLINOIS CANCER COUNCIL (N01-CM4-7667) 

This contract will evaluate the clinical effects of a murine anti-T-cell 
monoclonal immunoconjugate, TlOl-I^^l in a Phase I clinical trial. Pharmaco- 
kinetics, relevant biologic responses, and clinical toxicity will be monitored. 
In addition, localization in tumor and normal tissues will be compared. The 
trial began in mid-April of this year. 

This contract will also conduct a Phase I trial evaluating the clinical toxicity 
and effects of recombinant interleukin-2 and natural interleukin-2. Five 
patients will be initiated at each starting dose level and be escalated through 
four additional doses. This trial began in early May of this year. This 
contract will expire December, 1985. 

INFORMATION MANAGEMENT SERVICES, INC. (N01-CM2-7510) 

This contract supports the information needs of the Cancer Therapy Evaluation 
Program, DCT (CTEP, OCT) by providing comprehensive information management 
during the protocol review process, providing data on the objectives of both 
active and completed protocols, and providing data on the results of active and 
completed protocols. The system provides scientific and administrative infor- 
mation on: 1) treatment modalities (e.g. drugs, biological response modifiers, 
radiation, and surgery); 2) diseases; 3) protocols; and 4) investigator teams. 

In addition, a subcontracted effort to VSE Corporation provides for the main- 
tenance and operation of the Drug Distribution and Protocol Monitoring System 
(DDPMS). The DDPMS is an automated procedure used to verify the accuracy of 
investigator drug requests, thus fulfilling our legal (FDA) requirements in 
that regard. Verified data is retained and forms a drug distribution history 
which is used to monitor protocol activity as clinical trials progress. The 
system also provides management information for the program, the cooperative 
study group and private organizations. 

The contract will be extended noncompetitively during Fiscal Year 1985. The 
project will be recompeted in Fiscal Year 1986. 



41 



INFORMATION MANAGEMENT SERVICES. INC. (N01-CP3-1009) 

An interdi visional transfer of funds to DOE, Biometry Branch, to support 
statistical services for the Intergroup Testicular Studies. Monies support 
extramural ly contracted data management with IMS. 

INSTITUT JULES BORDET (N01-CM5-7645) (FORMERLY NOl-CMO-7350) 

Materials collected in Western Europe are screened in vivo against animal tumors 
in accordance with established NCI protocols. Materials that originated in the 
U.S. or other countries may be sent to this laboratory for testing. Testing is 
currently being conducted at a level of approximately 11,000 L1210 test equiva- 
lents per year. More detailed evaluation of drugs of interest to NCI is 
conducted upon request or agreement of the Project Officer. 

INSTITUTE OF CANCER RESEARCH (N01-CM4-3736) 

The objectives of this cost-sharing contract are to (1) study the biochemical 
and pharmacological bases for treatment failure or response; (2) acquire or 
synthesize potential anticancer agents designed to increase the therapeutic 
efficacy of known drugs; (3) evaluate new compounds synthesized by the con- 
tractor, or of interest to NCI, against human tumor xenografts and mouse tumors 
unique to the contractor; and (4) conduct toxicological studies to establish 
safe dosage levels and regimens for clinical evaluation of the drugs in the 
United Kingdom. This contract expired June, 1985. 

INSTITUTE OF MICROBIAL CHEMISTRY (N01-CM4-7593) 

The major objective of this contract is the isolation of new antitumor agents 
from fermentations of marine and terrestrial microorganisms. These fermenta- 
tions are screened against various enzymatic and other biochemical screens. 
Active products are isolated in sufficient quantities to be evaluated at the 
National Cancer Institute. In addition, various immunogen tests have been 
developed to evaluate the organisms and their metabolites as potential immune-^ 
logical stimulators specific for cancers. One chemotherapeutic agent from 
this contract, aclacinomyci n, is in Phase II clinical trials in the U.S.A. 
and another, deoxyspergualin, will enter Phase I trials shortly. 

ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUMORI (NOl-CMO-7338) 

A major effort in breast cancer has been through this contract. It has dealt 
primarily with adjuvant therapy of resectable disease, and its results have 
set a standard for therapeutic studies in this country and around the world. 
The Istituto has shown an improved overall survival for premenopausal patients 
treated with CMF, and that twelve months of CMF is no more effective than six 
months. More recent analyses indicate that survival of patients following 
relapse is unaffected by whether or not they received adjuvant chemotherapy, 
and that estrogen receptor status is an independent prognostic indicator for 
pre-menopausal women. In addition, they have developed an extensive data 
base on the utility of labelling index, which appears to be a more powerful 
prognosticator than hormone receptor status. 



42 



IOWA. UNIVERSITY OF (N01-CM3-7561) 

This contract provides services involving dosage form development and manu- 
facture of investigational drugs for subsequent clinical evaluation. Compounds 
to be formulated are selected and provided by the National Cancer Institute. 
The contractor has developed and produced primarily sterile freeze-dried 
injectable products under this contract. However, this contractor has the 
capability to produce a wide variety of pharmaceutical dosage forms. The 
contractor is also responsible for completing all required analytical and 
safety tests on each lot of drug. All products are packaged, labeled, and 
shipped to the National Cancer Institute for redistribution to clinical 
investigators. 

IOWA, UNIVERSITY OF (N01-CM3-7579) 

This contract provides capabilities to chemically characterize peptides, 
proteins and glycoproteins that may be used experimentally and/or clinically 
to modify tumor growth. Assay methods are developed to analyze the substance 
in bulk dosage form and in common pharmaceutical vehicles. Studies include 
determination of amino acid composition, molecular weight, isoelectric point, 
terminal sequence and development of suitable immunological measurement 
(radioimmunoassays, etc.) and suitable biological assays for qualitative and 
quantitative evaluations. In the past year this contractor has analyzed and 
characterized several lots of naturally occuring alpha, beta and gamma inter- 
ferons for activity and purity, analyzed several natural and recombinant IL-2 
preparations, natural B-cel 1 growth factor preparations, purified the tetrapep- 
tide tuftsin for preclinical screening, examined a murine tumor necrosis factor 
preparation for purity and provided near homogeneous material for monoclonal 
antibody production. 

IOWA, UNIVERSITY OF (N01-CH4-7594) 

This resource contract provides the Division of Cancer Treatment with facilities 
for development, formulation and production of oral dosage forms of investiga- 
tional drugs. The dosage forms are manufactured in conformity to FDA Current 
Good Manufacturing Practices and in a manner to protect personnel from exposure 
to potentially toxic agents. The contractor is also responsible for all required 
quality control tests on each product prepared. These dosage forms are packaged, 
labeled, and shipped to the National Cancer Institute for subsequent redistribu- 
tion to clinical investigators. 

JAPANESE FOUNDATION FOR CANCER RESEARCH (N01-CM3-6011) 

The objective of this contract is the maintenance of a chemotherapy liaison 
office at the Japanese Foundation for Cancer Research in Tokyo to provide 
up-to-date information services in support of our cancer treatment program, 
both preclinical and clincial. This is a cost-sharing Contract and is strongly 
supported by the Japanese Foundation for Cancer Research and the Japanese 
Society for the Promotion of Science. 



43 



JOHNS HOPKINS UNIVERSITY (NQ1-CM2-7509) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct 
Phase I clinical studies with new combinations or regimens mutually agreed 
upon. The Contractor is presently conducting Phase I studies with Spiro- 
mustine and Taxol and has just started a new trial with HMBA. This contract 
expi red July, 1985. 

JWK INTERNATIONAL CORPORATION (N01-CM2-5602) 

This project provides technical support services to the Office of the Director, 
OCT, as well as to the program areas of OCT in the performance of the planning 
and analytical tasks and general logistial support in the development of related 
or otherwise required documentation and conference support activities of the 
Division. This contract was awarded in December, 1981, for a three-year period, 
and expired March, 1985. This contract was recompeted during FY 1985 and was 
awarded to another firm. 

KANSAS, UNIVERSITY OF (N01-CM3-7562) 

This contract investigates approaches to resolve difficult dosage form develop- 
ment problems not amenable to usual solubilization or stabilization methods. 
This contractor has considerable expertise in the application of molecular 
complexes and reversible derivatives to Improve solubility. The contractor 
also is responsible for pilot scale preparation and chemical analysis of the 
formulations developed under this contract. 

KENTUCKY RESEARCH FOUNDATION, UNIVERSITY OF (N01-CM3-7620) 

Difficult dosage form development projects not amenable to the usual solu- 
bilization and/or stabilization approaches are assigned to this contractor. 
This contractor has particular expertise in the application of reversible 
derivatives (prodrugs) to improve drug solubility. Pilot batch preparation 
and chemical analysis of these novel formulations are carried out under this 
contract. 

KING ANIMAL LABORATORIES, INC. (N01-CM3-0228) 

This contract furnishes approximately 104,000 hybrid mice for compound evalua- 
tion studies. Breeding animals are furnished from genetic centers and/or rodent 
production center colonies. This contract terminated January, 1985. 

KOBA ASSOCIATES (N01-CM4-7661) 

This contract provides technical support for the collection, review and compi- 
lation of available information on biological response modifiers for possible 
development through preclinical and clinical trials. The contractor has respon- 
sibility for obtaining pertinent information from sources in Industry, institutes, 
universities as well as through literature review. Information and references 
obtained are submitted to the program in the form of review articles and one page 
synopses. Information gathered is stored in a computer data bank. Each year the 
program specifies about 100 compounds for the contractors to research. The 
contractor has provided about 50 one page synopses and 30 five page reviews. 

44 



LEO GOODWIN INSTITUTE FOR CANCER RESEARCH (N01-CM3-7625) 

This contract is a Primary Genetic Center. Rederived associated flora pedigreed 
starts are supplied to this contract for propagation into isolator maintained 
foundation colonies. These foundation colonies supply breeders on a weekly 
basis to pedigreed expansion colonies which support the production colonies. 
Both the pedigreed expansion and production colonies are maintained in a 
barrier room environment. Offspring from the production colonies are used 
for the many NCI research activities. 

LITTON BIONETICS. INC. (NOl-CMO-5724) 

This contract provides animal facilities to house small and large animals, 
including rats, cats, rabbits, goats and monkeys. The animals are used for 
the preparation of antibodies as well as for inoculation of tumor cells and 
virus preparations for tumorigenicity testing. 

LITTON BIONETICS, INC. (N01-CM2-5616) 

The major objectives of this contract are: (1) to purify and supply factors 
that promote growth and differentiation of myelogenous leukemic cells and T 
cells; (2) to purify the envelope and internal structural proteins of type C 
RNA tumor viruses; (3) to prepare monoclonal antibodies against the purified 
structural proteins; (4) to provide cultured T and B cells from human cord 
blood, peripheral blood and leukemic cells; and (5) to prepare and supply 
radiolabeled cDNA and RNA probes from type C retroviruses. 

LITTON BIONETICS, INC. (N01-CM3-7575) 

The major objectives of this contract are: (1) the use of immunofluoresence 
and radioimmunoassays to screen human T cells and T cell lines for viral 
structural protein expression; (2) the use of ELISA assays to detect anti- 
bodies against a putative human virus in serum from leukemic patients and 
normal donors; (3) to test culture fluids from short-term and long-term cul- 
tured cells for the presence of viral DNA polymerase activity; and (4) to test 
sera from patients with T cell leukemia for antibodies to human type C RNA 
tumor virus (HTLV). 

LITTON BIONETICS, INC. (N01-CM3-7582) 

This contract provides supportive services in the supply of small quantities 
of tissue culture cells, radiolabeled DNA and RNA probes. 

LITTON BIONETICS, INC. (N01-CM4-7651) 

The major objectives of this contract are to prepare and supply large quanti- 
ties of concentrated and purified human type C RNA tumor viruses. This con- 
tract is being recompeted. 



45 



LITTON BIONETICS, INC. (N01-CM4-7657) 

This contract supports Surgery Branch research by providing appropriate amounts 
of human and murine Interleukin-2 to conduct experiments. This research is 
directed toward developing new adoptive immunotherapies for the treatment of 
cancer using specifically sensitized lymphoid cells expanded in Interleukin-2 
or using Interleukin-2 directly as an immune adjuvant. This contract terminated 
April, 1985. 

MARYLAND. UNIVERSITY OF (N01-CM2-7541) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct 
Phase I clinical studies with new combinations or regimens mutually agreed 
upon. The Contractor is presently conducting Phase I studies with Spi ro- 
mustine and Menogaril and has started a new trial with HMBA. This contract 
expired July, 1985. 

MASSACHUSETTS GENERAL HOSPITAL (NOl-CMl-7481) 

This contract is part of a collaborative effort among three institutions to 
investigate the role of intraoperative radiotherapy in the treatment of intra- 
abdominal malignancies and to develop guidelines for intraoperative radiotherapy 
techniques for use by other investigators and by clinical cooperative groups in 
Phase II and III clinical trials. An optional task is to investigate the use 
of radiation modifiers in conjunction with intraoperative radiotherapy. The 
collaborative effort will facilitate the evaluation of this new radiotherapeutic 
modality with respect to normal tissue tolerance, safety, and efficacy. 

MASSACHUSETTS GENERAL HOSPITAL (N01-CM2-7532) 

This contract is part of a collaborative effort to improve treatment planning 
with neutrons and with charged particles (protons, helium ions, pi-mesons and 
heavy ions). In addition to developing optimal treatment plans for the treatment 
of tumors in various anatomic sites with the particle beam at its own institution, 
the collaborative working group is conducting a comparison of treatment plans 
for selected tumors in each anatomic site. Whenever possible, measurements in 
patients or phantoms will be made to check the accuracy of the treatment planning. 

MASSACHUSETTS GENERAL HOSPITAL (N01-CM4-7616) 



The objective of this collaborative effort is to develop criteria, guidelines, 
and methodology for the performance and evaluation of state-of-the-art high 
energy photon external beam treatment planning. This will be accomplished 
by extensive treatment planning for actual patients and by using state-of- 
the-art beam delivery, computerized treatment planning, and imaging systems. 
The results of this work will be described in a final report which will 
define state-of-the-art treatment planning and provide criteria, guidelines, 
and methodology for its application and evaluation. 



46 



MASSACHUSETTS GENERAL HOSPITAL (N01-CM4-7687) 

This is one of five contracts awarded in May, 1984 (a) to develop criteria 
and guidelines for clinical use of nuclear magnetic resonance imaging (MRI) 
systems in the detection and diagnosis of pathology in human subjects and in 
the noninvasive characterization of tissues, and (b) to carry out performance 
and evaluation studies to compare the diagnostic accuracy of MRI with that 
from other modalities (e.g., CT and radioisotope imaging). This contractor, 
working as a member of the MRI Collaborative Working Group (CWG), has contributed 
to the development of clinical imaging protocols for evaluation of MRI in the 
diagnosis of 1) musculoskeletal tumors, 2) cervical myelopathies, 3) uterine 
neoplasms, 4) liver metastases, and 5) brain neoplasms. They have also provided 
coordination for the CWG of the development of the musculoskeletal tumor and 
liver metastases protocols. Patient imaging has begun in each of these studies. 
This contractor has been influential in guiding the statistical design of several 
of the protocols. 

MASSACHUSETTS INSTITUTE OF TECHNOLOGY (N01-CM2-7525) 

This contract is for the Phase I evaluation of equipment for the hyperthermic 
treatment of cancer in conjunction with radiotherapy, chemotherapy, surgery, 
or immunotherapy. Five contractors are collaborating in a working group in 
conjunction with the National Center for Devices and Radiological Health to 
develop guidelines for the use of heat generating and thermometry equipment 
to facilitate the development of Phase II and III clinical studies for the 
adjuvant treatment of deeply seated tumors with heat. 

MAYO FOUNDATION (N01-CM2-7528) 

This contract is part of a collaborative effort among three institutions to 
investigate the role of intraoperative radiotherapy in the treatment of intra- 
abdominal malignancies and to develop guidelines for intraoperative radiotherapy 
techniques for use by other investigators and by clinical cooperative groups in 
Phase II and III clinical trials. An optional task is to investigate the use 
of radiation modifiers in conjunction with intraoperative radiotherapy. The 
collaborative effort will facilitate the evaluation of this new radiotherapeutic 
modality with respect to f;ormal tissue tolerance, safety, and efficacy. 

MAYO FOUNDATION (N01-CM2-7548) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct 
Phase I clinical studies with new combinations or regimens mutually agreed 
upon. The Contractor is presently conducting Phase I studies with Tiazofurin, 
Hexamethylmel amine IV, a combination trial with PALA/5-FU/TdR, and a new study 
will begin shortly with BIDA. This contract expired July, 1985. 

MAYO FOUNDATION (N01-CM3-7601) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism wil 1 enable the prompt 

47 



solicitation and award of projects to be conducted in this area from a pool 
of highly qualified contractors. The contractor is currently working on tasks 
to characterize the preclinical pharmacology of Pibenzimol, NSC 322921, 
Phyllanthoside, NSC 328426 and Teroxirone, NSC 296934. 

MAYO FOUNDATION (N01-CM5-7711) 

This is one of three contracts devoted to the application of a human tumor 
colony forming assay (HTCFA) to drug screening. Contract efforts have focused 
on screening compounds inactive in the standard in vivo pre-screen (murine P388 
leukemia) to establish the potential of the HTCFA to identify compounds not 
detected by the in vivo screen. Materials active against fresh human tumor 
specimens are also tested in a P388 cell line colony forming assay, developed 
under this project for direct comparison with the HTCFA. Screening has been 
initiated on more than 300 compounds and several have been identified as anti- 
tumor drug leads. Activity in the HTCFA, P388 colony forming assay, in vivo 
tumor panel, and degree of structural novelty, are used to determine which 
are recommended to the Decision Network Committee for development to clinical 
trial. To date three compounds have been accepted into the formal drug 
development program. 

MELOY LABORATORIES, INC. (N01-CM4-7650) 

The purpose of this contract is to provide effective inventory, distribution 
and quality assurance confirmation for biological response modifiers. The 
contractor is responsible for receipt, dispensing, storage, distribution and 
inventory control of biological agents. Quality assurance evaluation involves 
specific assays to confirm sterility and assays to determine pyrogenicity and 
endotoxin levels. The contractor performs general safety tests for biological 
agents in compliance with Government regulations intended for clinical use and 
helps in the development of master files and IND's for biologies. Currently, 
the contractor provides for storage and distribution of approximately 60 
different biologies. In the past year the contractor has performed general 
safety, pyrogenicity, purity and other relevant testing on several lots of 
monoclonal antibody preparations for use in clincial trials from within the 
program, as well as preparations submitted from other NCI and NIH scientists. 
The contract also provides for ascites production and purification of monoclonal 
antibody and has produced monoclonals specific for melanoma, colon, and breast 
cancers. 

MELOY LABORATORIES, INC. (N01-CM5-7688) 

This contract supports Surgery Branch research by providing appropriate amounts 
of human and murine Interleukin-2 to conduct experiments. In addition, murine 
and human lymphokine activated killer cells are generated for therapeutic 
experiments. This research is directed toward developing new adoptive immuno- 
therapies for the treatment of cancer using specifically sensitized lymphoid 
cells expanded in Interleukin-2 or using Interleukin-2 directly as an immune 
adjuvant. 



48 



MEMORIAL HOSPITAL FOR CANCER AND ALLIED DISEASES (NOl-CMO-7337) 

The Contractor conducts Phase I I/I 1 1 studies in patients with solid disseminated 
tumors. The tumors included are of the testicle, esophagus, lung (small cell), 
gastric, sarcoma, urothelial, and head and neck. A minimum of 200 patients is 
studied with no less than 25 patients in any tumor type. The patients are 
treated intensively with chemotherapy either alone or in combination with radio- 
therapy, immunotherapy, or surgery in protocols agreed upon by the NCI and the 
Principal Investigator. This contract expired June, 1985. 

MEMORIAL HOSPITAL FOR CANCER AND ALLIED DISEASES (N01-CM2-7546) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct 
Phase I clinical studies with new combinations or regimens mutually agreed 
upon. The Contractor is presently conducting Phase I studies with Fluoro- 
Ara-AMP in pediatric patients, a combination Phase I trial with PALA + MTX + 
5-FU, Tiazofurin, and Trimetrexate. This contract expired July, 1985. 

MEMORIAL HOSPITAL FOR CANCER AND ALLIED DISEASES (N01-CM4-7695) 

The objective of this collaborative effort is to develop criteria, guidelines, 
and methodology for the performance and evaluation of state-of-the-art high 
energy photon external beam treatment planning. This will be accomplished 
by extensive treatment planning for actual patients and by using state-of- 
the-art beam delivery, computerized treatment planning, and imaging systems. 
The results of this work will be described in a final report which will 
define state-of-the-art treatment planning and provide criteria, guidelines, 
and methodology for its application and evaluation. 

MICHIGAN, UNIVERSITY OF (NOl-CMO-7405) 

This contract conducts Phase II/III studies in patients with solid disseminated 
tumors. The tumors included are of the lung, breast, prostate, bladder, kidney, 
testicle, ovary, endometrium, cervix, head and neck, stomach, pancreas, and 
colon, as well as lymphomas, melanomas, and bone and soft tissue sarcomas. A 
minimum of 200 patients a year is studied, with no less than 25 patients in any 
tumor type. These patients are treated intensively with chemotherapy either 
alone or in combination with radiotherapy, immunotherapy, or surgery in pro- 
tocols agreed upon by the NCI and the Principal Investigator. Patient accrual 
continued through July, 1985, with follow-up ending in February, 1986. 

MICHIGAN TECHNOLOGICAL UNIVERSITY (N01-CM3-7633) 



This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program for 
development. Unique compounds of interest emerging from the literature that 
cannot be obtained in sufficient quantities from the original investigators are 
synthesized by this mechanism. This mechanism is also used for the resynthesis 
of a limited number of panel compounds, and compounds of interest to intramural 
scientists. Potential radiosensitizers and radioprotectors are also synthesized 
in support of the Radiation Research Program. 



49 



MICROBIOLOGICAL ASSOCIATES (N01-CM9-7287) 

This contract functions in four major areas: 1) To operate and maintain a 
virus serum diagnostic laboratory. Serum samples are submitted from contract 
animal suppliers and testing laboratories; 2) to test experimental tumors 
(animal and human) for viral contaminants; 3) to perform ELISA tests annually 
for the detection of mouse hepatitis virus (MHV); and 4) to produce vaccinia 
virus which is used for immunizing mice against infectious ectromelia. 

MIDWEST RESEARCH INSTITUTE (N01-CM3-7555) 

Midwest Research Institute is one of the three contractors responsible for the 
chemical analysis of bulk chemicals and clinical formulations which are to be 
investigated in the pharmacological, toxicological and clinical phases of the 
Developmental Therapeutics Program. The contractor determines the identity 
and purity of the compounds by appropriate methods. The contractor also 
determines solubility, stability and other physical -chemical properties to aid 
in formulation development and selection of storage conditions. Techniques 
commonly used include elemental analysis, chromatography (paper, thin layer, 
gas liquid and high pressure liquid), spectroscopy (ultraviolet, infrared and 
nuclear magnetic resonance), gas chromatography /mass spectrometry and other 
methods as needed. Reports of the work performed by the contractor provide 
data to be included in IND filings with the Food and Drug Administration and 
for quality assurance monitoring. 

MIDWEST RESEARCH INSTITUTE (N01-CM3-7604) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the 
prompt solicitation and award of projects to be conducted in this area from a 
pool of highly qualified contractors. The contractor is currently working on 
a task to characterize the preclinical pharmacology of Caracemide, NSC 253272 
and Melphalan, NSC 8806. 

MIDWEST RESEARCH INSTITUTE (N01-CM3-7634) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program for 
development. Unique compounds of interest emerging from the literature that 
cannot be obtained in sufficient quantities from the original investigators 
are synthesized by this mechanism. This mechanism is also used for the re- 
synthesis of a limited number of panel compounds, and compounds of interest 
to intramural scientists. Potential radiosensitizers and radioprotectors 
are also synthesized in support of the Radiation Research Program. 

MISSOURI, UNIVERSITY OF (N01-CM2-7534) 

This contract monitors the animal production and screening laboratory by 
testing for the presence of Salmonella and Pseudomonas. Samples are received 
on a scheduled basis from the animal producers and screening laboratories and 
approximately 9,000 fecal samples are tested per year. 

50 



MISSOURI, UNIVERSITY OF (N01-CM8-7157) 

This contract will provide for a complete pathological, parasitical, and 
microbiological work-up of breeding stock primarily sent from barrier room 
expansion colonies and the nude mouse production colonies. Special testing is 
provided on a priority basis when a suspected animal disease problem occurs at 
an animal supplier facility in a contract research facility. All testing is 
scheduled by the Project Officer. 

MONSANTO RESEARCH CORPORATION (N01-CM2-7516) 

This service preparative contract provides for the large-scale synthesis 
of compounds required for preclinical and clinical studies. The compounds 
prepared are not readily available on the open market or from the original 
supplier in the amounts and/or quality required. The effort of this contract 
is devoted to the preparation of large quantities of materials, in the multi- 
kilogram range, requiring pilot plant facilities. 

MOUNT SINAI MEDICAL CENTER (N01-AI2-2669) 

An inter-institutional transfer of funds to the NIAID supports the International 
Bone Marrow Transplant Registry. The specific aims are 1) to maintain a statis- 
tical center for the collection, organization and analysis of clinical data 
provided by bone marrow transplant teams throughout the world; 2) to disseminate 
the results of clinically relevant analyses of pooled Registry data to bone 
marrow transplant teams, and to the medical profession for the earliest possible 
benefit to patients who might be helped by bone marrow transplantation treatment; 
and 3) to aid in designing, organizing and providing statistical support for con- 
trolled, cooperative clinical trials utilizing bone marrow for transplantation. 
NCI support for this contract will terminate in Fiscal Year 1985. NIAID will 
continue to support this project. 

MURPHY BREEDING LABORATORIES (N01-CM3-0229) 



This contract furnishes approximately 104,000 hybrid mice for compound evalua- 
tion studies.' Breeding animals are furnished from genetic centers and/or rodent 
production center colonies. This contract terminated January, 1985. 

NATIONAL ACADEMY OF SCIENCES (N01-CM5-7644) 

This contract Task Order serves to develop standards for animal care and 
maintenance; shipping standards for the various species of laboratory animals, 
standards for nomenclature used to identify stocks and strains of laboratory 
animals; standards for animal maintenance in the research laboratory; and 
laboratory animal procurement standards. These standards are formulated by 
ad hoc commmittees whose membership represent commercial animal production 
colonies, governmental and academic institutions, and non-profit research 
Institutions. 



51 



NATIONAL CENTER FOR DEVICES AND RADIOLOGICAL HEALTH. FDA (Y01-CM2-0107) 

This Interagency Agreement provides technical support to NCI and to the five 
contractors participating in the collaborative Phase I evaluation of equipment 
for the hyperthermic treatment of cancer. The Division of Physical Sciences, 
National Center for Devices and Radiological Health, Food and Drug Administra- 
tion, has a number of highly recognized experts in electromagnetic radiation 
and in ultrasound who are available on a consultative basis to assist in the 
hyperthermia research program. 

NORTHERN CALIFORNIA CANCER PROGRAM (N01-CM3-7578) 

The capability for evaluating chemical compounds for radiation sensitizing 
and/or radiation protective properties is provided by this resource. Various 
physico-chemical parameters are determined on a large number of compounds 
representing a wide range of chemical structures. Compounds showing potential 
radiosensitizing or radioprotective characteristics will undergo in vitro 
testing to evaluate their cytotoxicity and degree of radiosensitization using 
mammalian cell cultures. Potential radiosensitizing compounds which appear 
to be superior to the standard - misonidazole - will be evaluated in vivo , 
using mice and/or rats. The effect of the compound in modifying the response 
to radiation will be measured in three different tumor systems (from the OCT 
panel of mouse tumor screens as stated in the Treatment Linear Array for 
Radiosensitizers) , each measured by a separate endpoint. The endpoints will 
include: the regrowth delay of tumors, tumor cell survival and the modification 
of the radiation dose required for curing 50% of the tumors. All radioprotective 
compounds tested will be compared with the standard - WR-2721. The contract 
also provides for the option of assessing potential long-term tissue injury 
by performing histopathol ogy on a limited number of tissues and compounds. 
This contract should provide new radiosensitizers and radioprotectors or leads 
in developing new types (classes) of radiation modifying compounds. 

NORTHWESTERN UNIVERSITY (N01-CM3-7538) 

This contract is designed to monitor and maintain genetic control of tumor 
strains and inbred mouse stocks. This service was established to assure 
continuous control of the biological materials used in program studies. 
The contractor carries out the service by performing skin grafts and anti- 
genic studies of mouse strains to assure their continuous genetic integrity 
and histocompatibility with other sublines maintained in counterpart genetic 
production centers. 

OHIO STATE UNIVERSITY (NOl-CMO-7442) 

This Phase I/I I clinical contract trial, evaluating both beta and gamma 
recombinant interferon in patients with renal cell carcinoma is continuing. 
Seven patients have been studied with gamma interferon. In four patients, 
the maximally tolerated dose (MTD) was 12 million units; however, in another 
three patients, the MTD ranged from 30-70 million units. Three additional 
patients have received beta interferon. This contract expired June, 1985. 



52 



OHIO STATE UNIVERSITY (N01-CM4-7622) 

The principal objective of this project is to collect pharmacokinetic data on 
new and established antitumor agents in patients undergoing treatment for 
malignant disease during Phase I studies to help establish the most effective 
dosage schedule. Two new or established cancer chemotherapeutic agents are 
evaluated annually. The studies involve approximately 30 patients per drug. 
During this period the Contractor evaluated Pibenzimol. 

OHIO STATE UNIVERSITY RESEARCH FOUNDATION (N01-CM2-7540) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct 
Phase I clinical studies with new combinations or regimens mutually agreed 
upon. The Contractor is presently conducting Phase I studies with Acodazole 
and 2-FAMP, and has started two new trials with Pibenzimol and BIDA. This 
contract will expired July, 1985. 

OHIO STATE UNIVERSITY RESEARCH FOUNDATION (N01-CM3-7598) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the 
prompt solicitation and award of projects to be conducted in this area from a 
pool of highly qualified contractors. The contractor is currently working on 
a task to characterize the preclinical pharmacology of Pibenzimol, NSC 322921, 
Aphidicolin Glycinate, NSC 303812 and Merbarone, NSC 336628. 

ORI, INC. (N01-CM3-7591) 

This contract is intended to provide quick response programming support, to 
enable the ITB to deal with the unanticipated problems or urgent requirements 
that are encountered in the operation of the ITB chemical and biological 
information system. Tasks have included, thus far, the transfer of the Hodes 
model to the DIS Pre-Registry subsystem, and Systems Analysis support for the 
DIS graphic subsystem (laser printer) and the DIS data entry module. 

ORKAND CORPORATION (N01-CM3-6010) 

This contract supports the Clinical Oncology Program of the Division of Cancer 
Treatment with computer programming expertise for the development of clinical 
information systems and with data technician services for the maintenance and 
utilization of these systems. A wide variety of systems have beep developed 
and are maintained for the Clinical Branches of the Clinical Oncology Program, 

PAN AMERICAN HEALTH ORGANIZATION (N01-CM2-7391) 

The Collaborative Cancer Treatment Research Program of paired U.S. - Latin 
American investigators is currently concentrating efforts towards Phase II 
studies in diseases such as gastric carcinoma, vulvar, penile and cervical 
squamous cell carcinomas very prevalent in Latin American Countries expected 
to generate important data in clinical oncology. Lesser numbers of Phase III 
studies are left at this point. 

53 



PAPANICOLAOU CANCER RESEARCH INSTITUTE (N01-CM8-7230) 

This contract will provide for a complete pathological, parasitical, and 
microbiological work-up of breeding stock primarily sent from barrier room 
expansion colonies and the nude mouse production colonies. Special testing 
is provided on a priority basis when a suspected animal disease problem occurs 
at an animal supplier facility or a contract research laboratory. All testing 
is scheduled by the Project Officer. 

PENNSYLVANIA, UNIVERSITY OF (N01-CM2-7529) 

This contract is part of a collaborative effort to improve treatment planning 
with neutrons and with charged particles (protons, helium ions, pi-mesons and 
heavy ions). In addition to developing optimal treatment plans for the treatment 
of tumors in various anatomic sites with the particle beam at its own institution, 
the collaborative working group is conducting a comparison of treatment plans 
for selected tumors in each anatomic site. Whenever possible, measurements in 
patients or phantoms will be made to check the accuracy of the treatment planning. 

PENNSYLVANIA, UNIVERSITY OF (N01-CM4-7697) 

The objective of this collaborative effort is to develop criteria, guidelines, 
and methodology for the performance and evaluation of state-of-the-art high 
energy photon external beam treatment planning. This will be accomplished 
by extensive treatment planning for actual patients and by using state-of- 
the-art beam delivery, computerized treatment planning, and imaging systems. 
The results of this work will be described in a final report which will define 
state-of-the-art treatment planning and provide criteria, guidelines, and 
methodology for its application and evaluation. 

PHARM-ECO LABORATORIES, INC. (N01-CM4-7587) 

This service preparative contract provides for the resynthesis of a variety 
of compounds required for clinical or preclinical evaluation. The compounds 
prepared are not readily available on the open market or from the original 
supplier in the amounts and/or quality required. About 50% of the effort 
of this contract is devoted to the preparation of large quantities of mate- 
rial, in the multikilogram range. 

PGLYSCIENCES, INC. (N01-CM3-7557) 

This service preparative contract provides for the large-scale extraction of 
various plants and isolation and purification of the active materials for pre- 
clinical development and clinical trials. Cost and yield data are obtained 
on these processes. In the case of those plant processes which will be used 
again, process development optimization studies are conducted. Materials 
worked on include pancratistatin, phyl lanthoside, and taxol . Process develop- 
ment on these plant products has been completed. This contract is currently 
being recompeted. 



54 



PROGRAM RESOURCES. INC. (N01-C02-3910) 

This contract is located at the Frederick Cancer Research Facility in Frederick, 
Maryland. The effort is divided into the following six segments: 

Administrative Support 

Provides for one Administrative Specialist and one Information Specialist in 
the Office of the Animal Genetics and Production Branch (AG&PB), Developmental 
Therapeutics Program, OCT. 

Genetic Monitoring 

Monitors all of AG&PB contract rat colonies for genetic purity. In addition 
this contract monitors the starts received from VRB-NIH both pre and post 
rederivation. 

Partial Support for Harlan/Sprague-Dawley 

Provides Work Orders and Shared Services type functions for the Harlan Sprague 
Dawley Animal Production contract. It serves as a method for paying the PRI 
for services performed at the Animal Production Area. 

Tumor Bank 

This segment has as its major goal the maintenance of approximately 20,000 
frozen tumor vials. This contractor furnishes needed tumors to the various 
DTP laboratories, as well as to other research institutions, both domestic 
and foreign. The tumors are supplied both in vivo and in vitro . 

In Vitro Cell Line Screening Project 

This new project (began in September, 1984) is devoted to the development and 
operation of in vitro disease-oriented antitumor drug screening models based 
on the use of established human tumor cell lines. This activity is planned 
to represent the primary DTP drug screening effort and will interface with 
other components of the drug development program. 

Metastatic Models Development 

This activity is closely related to the in vitro cell line screening project 
described above. Selected human tumor cell lines are being developed for use 
in metastatic models for in vivo drug evaluations. 

RAYLO CHEMICAL, LTD. (N01-CM3-7635) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program 
for development. Unique compounds of interest emerging from the literature 
that cannot be obtained in sufficient quantities from the original investi- 
gators are synthesized by this mechanism. This mechanism is also used for 
the resynthesis of a limited number of panel compounds, and compounds of 
interest to intramural scientists. Potential radiosensitizers and radio- 
protectors are also synthesized in support of the Radiation Research Program. 

55 



RESEARCH TRIANGLE INSTITUTE (N01-CM2-7515) 

This service preparative contraa provides for the synthesis of radioactive 
labeled chemicals and drugs for use in preclinical pharmacological and clinical 
studies. Many of the materials prepared are not available from commercial 
sources. All materials, whether prepared at the Institute or acquired from 
other sources, are analyzed for purity and identity by autography assay, etc 
This contract also provides storage facilities for labeled materials and 
distributes such as directed by the National Cancer Institute staff. 

RESEARCH TRIANGLE INSTITUTE (N01-CM3-7599) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the prompt 
solicitation and award of projects to be conducted in this area from a pool of 
highly qualified contractors. 

RESEARCH TRIANGLE INSTITUTE (N01-CM3-7619) 

Research Triangle Institute is one of the three contractors responsible for 
the chemical analysis of bulk chemicals and clinical formulations which are to 
be investigated in the pharmacological, toxicological and clinical phases of 
the Developmental Therapeutics Program. The contractor determines the identity 
and purity of the compounds by appropriate methods. The contractor also 
determines solubility, stability and other physical chemical properties to aid 
in formulation development and selection of storage conditions. Techniques 
commonly used include elemental analysis, chrcxnatography (paper, thin layer 
gas liquid and high pressure liquid), spectroscopy (ultraviolet, infrared and 
nuclear magnetic resonance), gas chromatography /mass spectroscopy and other 
methods as needed. Reports of the work performed by the contractor provide 
data to be included in IND filings with the Food and Drug Administration and 
for quality assurance monitoring. 

RESEARCH TRIANGLE INSTITUTE (N01-CM3-7636) 



This IS one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program 
for development. Unique compounds of interest emerging from the literature 
that cannot be obtained in sufficient quantities from the original investi- 
gators are synthesized by this mechanism. This mechanism is also used for 
the resynthesis of a limited number of panel compounds, and compounds of 
interest to intramural scientists. Potential radiosensitizers and radio- 
protectors are also synthesized in support of the Radiation Research Program. 

SIMONSEN LABORATORIES (N01-CM3-0230) 

This contract furnishes approximately 104,000 hybrid mice for compound evalua- 
tion studies. Breeding animals are furnished from genetic centers and/or rodent 
production center colonies. This contract terminated January, 1985. 



56 



SIMONSEN LABORATORIES (N01-CM3-7624) 

This contract is a Primary Genetic Center. Rederived associated flora pedigreed 
starts are supplied to this contract for propagation into isolator maintained 
foundation colonies. These foundation colonies supply breeders on a weekly 
basis to pedigreed expansion colonies which support the production colonies. 
Both the pedigreed expansion and production colonies are maintained in a barrier 
room environment. Offspring from the production colonies are used for hybrid 
production and the many NCI research activities. 

SIMONSEN LABORATORIES (N01-CM5-7729) 

This Rodent Production Center contract produces three hybrids (B6C3F1, B6D2F1, 
CD2F1) under barrier conditions. 

SIMONSEN LABORATORIES (N01-CM9-7247) 

This contract provides for the maintenance of a rodent production center. This 
produces stocks and strains of rodents for research and testing laboratory pro- 
grams. Production levels for individual colonies are correlated with requirement 
for special investigations. The breeding stock is received from the primary 
genetic centers. This contract terminated January, 1985. 

SISA. INC. (N01-CM3-7637) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program 
for development. Unique compounds of interest emerging from the literature 
that cannot be obtained in sufficient quantities from the original investi- 
gators are synthesized by this mechanism. This mechanism is also used for 
the resynthesis of a limited number of panel compounds, and compounds of 
interest to intramural scientists. Potential radiosensitizers and radio- 
protectors are also synthesized in support of the Radiation Research Program. 

SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH (N01-CM2-5600) 

This contract is for the production of murine monoclonal antibodies directed 
against human cytokines. The contractor develops appropriate immunizing 
protocols to confirm the immunogenicity of the human cytokine in mice, and 
produces and isolates individual hybridoma clones secreting monoclonal anti- 
body. Appropriate radioimmune assays are developed for screening individual 
hybridoma clones for antibody reactivity and ability of monoclonal antibody to 
specifically bind to and inhibit each cytokine. The contractor provides anti- 
cytokine secreting hybridomas and semi-purified immunoglobulin derived from 
the various hybridomas. The contractor has developed hybridomas secreting , 
monoclonal antibodies against human IL-2, and human gamma interferon and has 
undertaken development of monoclonal antibodies against human and murine tumor 
necrosis factor, human B-cel 1 growth factor, interleukin-1, and human alpha 
and beta interferons. This contract will expire September, 1985 and is being 
recompeted. 



57 



SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH (N01-CM3-75651 



The purpose of this contract is to provide methods for dose calculations for 
cancer therapy using radioactively labelled antibodies directed to tumor 
associated and/or tumor specific antigens. Dose calculations are to be 
developed for radiation doses to tumors and nonnal tissues resulting from 
the use of various isotopes (alpha, beta, or gamma emitters) tagged to 
antibodies for therapy purposes. Calculational methods are to be correlated 
with three-dimensional patient anatomy and the distribution and specific 
activity of radioactivity [as defined by scanners such as CT, single 
photon emission computed tomography (SPECT), positron emission tomography 
(PET) or gamma cameras] and should be compatable with conventional radio- 
therapy treatment planning systems insofar as possible. 

SMALL BUSINESS ADMINISTRATION (BIOTECH RESEARCH LABORATORIES) (N01-CM3-7558) 

This contract provides personnel to assist the staff of the Drug Evaluation 
Branch with the scheduling and evaluation of compounds for antitumor activity 
in the pre-screen and tumor panel test systems. They also assist in requesting 
required follow-up testing of compounds of interest and the scheduling of test- 
ing for those compounds recommended for further evaluation in additional anti- 
tumor test systems. The contractor provides for the coordination and data entry 
tor these compounds to both manual and computerized files which provide manage- 
ment reports for staff to monitor compounds in the linear array. 

SMALL BUSINESS ADMINISTRATION (BIOTECH RESEARCH LABORATORIES) (N01-CM3-7590) 

This contract supplies the Government with substantial quantities of well- 
characterized normal and neoplastic mammalian tissue culture cells and receives 
processes, and distributes fresh human leukemic cells and tissues. Complete ' 
records are maintained on all biological materials handled under the contract. 

SMALL BUSIN ESS ADMINISTRATION (BIOTECH RESEARCH LABORATORIES) (N01-CM3-7609) 

The purpose of this contract is to provide supportive services in small animal 
studies, immunology, and tissue culture. At the present time, these functions 
include: (1) detailed karyotypic analysis, including Giemsa banding, of a 
variety of monolayer and suspension cultured cells; (2) evaluation of tumori- 
genicity of various cultured cells by inoculation into nude mice; (3) testing 
the tumon genie potential of selected primate retroviruses; (4) preparing 
small quantities of selected cells and retroviruses; and (5) testing various 
tissue cultured cell specimens for mycoplasma contamination. 

SMALL BUSINESS ADMINISTRATION (MAXIMA CORPORATION) (N01-CM4-7628) 

The objective of this small business contract is to perform a variety of 
computer searches such as full structure searches, substructure searches 
and data item searches in support of the DTP program. The contractor uti- 
lizes several data bases such as NCI, Dare, Questel . NLM. and Dialog. 
Another newly added task under this contract is the development of chemical 
names for compounds of interest. 



58 



SOCIAL & SCIENTIFIC SYSTEMS. INC. (N01-CM2-5606) 

This contractor provides support services for conference management and 
associated general logistical activities for the Cancer Therapy Evaluation 
Program. Logistics support includes various technical and clerical tasks 
ranging from report design and preparation to routine typing. Conference 
support includes both pre- and post-conference management activities neces- 
sary to successfully conduct large as well as small meetings and provide the 
results thereof to the biomedical research community. 

SOCIAL & SCIENTIFIC SYSTEMS. INC. (N01-CM3-7576) 

Social & Scientific Systems, Inc. provides technical assistance and support 
services in the area of investigational drug regulations and clinical research. 
Information is gathered and assembled for the preparation of Investigational 
New Drug Applications (IND's). This includes screening information, animal 
toxicology, chemistry, bibliographic information, drug labeling and the clinical 
protocol. This information is submitted to the Food and Drug Administration, 
and an IND is awarded. This Contractor then maintains the files by providing 
in-depth tracking of drugs and amending IND information as necessary. The 
contractor also provides drug distribution monitoring assistance and is involved 
with the preparation of IND annual reports, the establishment of drug master 
files, the distribution of clinical brochures, and the dissemination of adverse 
drug reaction information. Additional effort was added to this contract to 
provide support for the receipt, organization, and disbursement of all clinical 
protocols and related materials involved in the Cancer Therapy Evaluation Program 
protocol review process. 

SOUTH FLORIDA, UNIVERSITY OF (N01-CM3-7639) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program for 
development. Unique compounds of interest emerging from the literature that 
cannot be obtained in sufficient quantities from the original investigators 
are synthesized by this mechanism. This mechanism is also used for the 
resynthesis of a limited number of panel compounds, and compounds of interest 
to intramural scientists. Potential radiosensitizers and radioprotectors are 
also synthesized in support of the Radiation Research Program. 

SOUTHERN ANIMAL FARMS (N01-CM3-0227) 

This contract furnishes approximately 104,000 hybrid mice for compound evalua- 
tion studies. Breeding animals are furnished from genetic centers and/or rodent 
production center colonies. This contract terminated January, 1985. 

SOUTHERN ANIMAL FARMS (N01-CM5-7727) 

This Rodent Production Center contract produces CD2F1 hybrid mice and the 
CD8F1 hybrid. In addition, the pure strains BALB/c-CMc and DBA/8 are 
maintained within this contract. 



59 



SOUTHERN ANIMAL FARMS (N01-CM9-7245) 

This contract provides for the maintenance of a Rodent Production Center. This 
center produces stocks and strains of rodents for research and testing labora- 
tory programs. Production levels for individual colonies are correlated with 
requirements for specific investigations. This contract terminated January, 1985. 

SOUTHERN CALIFORNIA. UNIVERSITY OF (NOl-CMO-7421) 

On this contract a Phase I/I I clinical trial was conducted, evaluating 
recombinant beta interferon in the treatment of patients with disseminated 
malignant disease. One more patient is required for completion. Toxicities 
have included high fevers up to 40°C lasting for 12 hours at doses ranging 
from 30-60 million units. Some patients have tolerated the dose escalation 
schema better than others. This contract expired April, 1985. 

SOUTHERN CALIFORNIA, UNIVERSITY OF (N01-CM2-7483) 

This contract is conducting Phase II studies of photoradiation therapy for 
local malignancies. Efforts will be primarily focused on lung lesions with a 
minimum of 25 patients per year being studied. It is planned to determine its 
scope and limitations and especially to identify situations where it may offer 
a unique advantage over existing cancer therapies as a treatment for patients 
who have failed other modalities. Photoradiation therapy involves irradiating 
hematoporphyrin derivative, which accumulates in malignant tissue, with 
appropriate laser light in the presence of oxygen. This process generates 
singlet oxygen, a highly toxic substance. A minimum of 25 patients per year 
will be studied. 

SOUTHERN CALIFORNIA, UNIVERSITY OF (N01-CM3-7600) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 28 in the OCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the 
prompt solicitation and award of projects to be conducted in this area from a 
pool of highly qualified contractors. The contractor has completed a task to 
characterize the preclinical pharmacology of 5-Azacytidine, NSC 102816. 

SOUTHERN RESEARCH INSTITUTE (N01-CM3-7552) 

This contract utilizes a variety of murine leukemia and solid tumor models 
to (1) evaluate the antitumor activity of congeners or prodrugs of new lead 
compounds; (2) conduct special non-protocol studies on promising compounds 
in order to identify the best candidates for further development; and (3) 
evaluate the activity of purified natural products. The special non-protocol 
studies may include experiments to determine whether the antitumor activity 
of an antimetabolite can be reversed by endogenous substances and whether 
congeners retain activity against tumors with acquired resistance to clinical 
agents. This contract is currently being recompeted. 



60 



SOUTHERN RESEARCH INSTITUTE (N01-CM3-7597) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the prompt 
solicitation and award of projects to be conducted in this area from a pool of 
highly qualified contractors. 

SOUTHERN RESEARCH INSTITUTE (N01-CM3-7638) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program for 
development. Unique compounds of interest emerging from the literature that 
cannot be obtained in sufficient quantities from the original investigators 
are synthesized by this mechanism. This mechanism is also used for the 
resynthesis of a limited number of panel compounds, and compounds of interest 
to intramural scientists. Potential radiosensitizers and radioprotectors are 
also synthesized in support of the Radiation Research Program. 

SOUTHERN RESEARCH INSTITUTE (N01-CM4-7580) 

This contract provides DCT with a resource to conduct detailed preclinical 
evaluations of anticancer agents identified as promising on primary screening 
contracts. Agents are subjected to rigorous testing to select those most 
deserving of development to clinical trial. In addition, an effort is made 
to develop treatment strategies to address common causes of treatment failure, 
such as the growth of tumor cell populations which are resistant to standard 
agents. As appropriate, studies are conducted in vitro or in vivo against 
transplantable animal tumors and human tumor xenografts. Principal tasks 
include the conduct of schedule dependency studies, activity evaluations of 
clinically formulated products, and evaluations of agents in tests which are 
different or more challenging than those used in primary screening (e.g., 
tests in animals bearing advanced tumors). 

SOUTHERN RESEARCH INSTITUTE (N01-CM4-7581) 

This contract provides assistance to the Drug Evaluation and Animal Genetics 
and Production Branches in protocol development and quality control of tumor 
lines and host animals. Tentative protocols are developed for new test systems 
or refinements suggested to enhance current systems. Animals and tumors used 
in the screening program are monitored for reproducibility and consistency of 
response. Cell kinetics are elucidated for in vivo systems utilized by the 
screening program. 

SOUTHERN RESEARCH INSTITUTE (N01-CM4-7615) 

The objective of the contract is to provide preliminary information on the 
cytotoxic and biochemical effects of new antitumor agents being considered 
for development to clinical trial. Experiments are conducted (1) to establish 
whether agents with novel chemical structures have biochemical activities 
similar to those of clinically evaluated drugs; (2) to answer specific bio- 
logical questions on new antitumor agents that are raised by the Decision 

61 



Network Committee; and (3) to determine the drug concentration and time of 
exposure required for significant tumor cell kill. The latter will be used 
to help treatment schedule selection and dose escalations for Phase I clinical 
trials. 

SOUTHERN RESEARCH INSTITUTE (N01-CM4-7646) 

This contract provides for the testing of materials in the P388 pre-screen and 
the mouse host antitumor models in the DCT panel of in vivo test systems at a 
level of 25,000 L1210 equivalents per year. The contract provides for special 
studies as requested by the Project Officer such as the detailed evaluation of 
compounds and the characterization and evaluation of tumor models. All testing 
is carried out according to the protocols of the Drug Evaluation Branch, DTP. 
Conventional mouse tumor panel models now in use at this laboratory are the B16 
melanoma, the CD8F1 mammary adenocarcinoma, the colon 38, the L1210 leukemia, 
and the M5076 sarcoma mouse tumor test systems. The panel of test systems is 
currently being modified, for example, the mouse tumor, B16 melanoma, is being 
phased out of use and testing with the human tumor, LOX amelanotic melanoma, 
is bei ng initiated. 

SOUTHWEST RESEARCH INSTITUTE (N01-CM3-7640) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program for 
development. Unique compounds of interest emerging from the literature that 
cannot be obtained in sufficient quantities from the original investigators 
are synthesized by this mechanism. This mechanism is also used for the 
resynthesis of a limited number of panel compounds, and compounds of interest 
to intramural scientists. Potential radiosensitizers and radioprotectors are 
also synthesized in support of the Radiation Research Program. 

SRI INTERNATIONAL (N01-CM2-7560) 

This service preparative contract provides for the synthesis of radiolabeled 
chemicals and drugs for use in preclinical pharmacologic and clinical studies. 
Many of the materials prepared are not available from commercial sources and 
are synthesized. All materials, whether prepared by the contract group or 
acquired from other sources, are analyzed for purity and identity by radio- 
autography assay, etc. This contract also provides storage facilities for 
labeled materials and distributes such as directed by the National Cancer 
Institute. 

SRI INTERNATIONAL (N01-CM3-7605) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism wil 1 enable the prompt 
solicitation and award of projects to be conducted in this area from a pool of 
highly qualified contractors. The contractor is currently working on a task 
to characterize the preclinical pharmacology of an L-cysteine Derivative, 
NSC 303861. 



62 



SRI INTERNATIONAL (N01-CM3-7618) 

SRI International is one of the three contractors responsible for the chemical 
analysis of bulk chemicals and clinical formulations which are to be investi- 
gated in the pharmacological, toxicological and clinical phases of the Develop- 
mental Therapeutics Program. The contractor determines the identity and purity 
of the compounds by appropriate methods. The contractor also determines 
solubility, stability and other physical-chemical properties to aid in 
formulation development and selection of storage conditions. Techniques 
commonly used include elemental analysis, chromatography (paper, thin layer, 
gas liquid and high pressure liquid), spectroscopy (ultraviolet, infrared and 
nuclear magnetic resonance), gas chromatography /mass spectrometry and other 
methods as needed. Reports of the work performed by the contractor provide 
data to be included in IND filings with the Food and Drug Administration and 
for quality assurance monitoring. 

SRI INTERNATIONAL (N01-CM3-7641) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program for 
development. Unique compounds of interest emerging from the literature that 
cannot be obtained in sufficient quantities from the original investigators 
are synthesized by this mechanism. This mechanism is also used for the 
resynthesis of a limited number of panel compounds, and compounds of interest 
to intramural scientists. Potential radiosensitizers and radioprotectors are 
also synthesized in support of the Radiation Research Program. 

SRI INTERNATIONAL (N01-CM4-7611) 

The objective of this contract between NCI, SRI International and Stanford 
University is the synthesis and biological evaluation of novel radiosensi- 
tizers. The primary focus of the work is the identification of leads other 
than 2-nitroimidazoles. Several new types of compounds have been investi- 
gated including quinoxaline, 1,4-di oxides, pyridine N-oxides, benzotriazoles 
and glutathione depleters ( di ethyl ma leate analogs). Benzamide 3-hydroxy- 
(NSC 379289) has confirmed in vitro and in vivo radiosensitization that is 
as good if not better than Misonidazole. Benzotri azine dioxides have also 
demonstrated in vitro radiosensitizer activity. These novel leads are being 
devel oped. 

STANFORD UNIVERSITY (NOl-CMl-7480) 

This contract is for the Phase I evaluation of equipment for the hyperthermic 
treatment of cancer in conjunction with radiotherapy, chemotherapy, surgery, 
or immunotherapy. Five contractors are collaborating in a working group in 
conjunction with the National Center for Devices and Radiological Health to 
develop guidelines for the use of heat generating and thermometry equipment 
to facilitate the development of Phase II and III clinical studies for the 
adjuvant treatment of deeply seated tumors with heat. 



63 



STARKS ASSOCIATES. INC. (N01-CM3-7642) 

This is one of the 12 master contractors for the synthesis of a variety of 
organic and/or inorganic compounds which have been identified by program for 
development. Unique compounds of interest emerging from the literature that 
cannot be obtained in sufficient quantities from the original investigators 
are synthesized by this mechanism. This mechanism is also used for the 
resynthesis of a limited number of panel compounds, and compounds of interest 
to intramural scientists. Potential radiosensitizers and radioprotectors are 
also synthesized in support of the Radiation Research Program. 

STARKS ASSOCIATES. INC. (N01-CM4-7588) 

This service preparative contract is for the resynthesis of bulk chemicals and 
drugs required for completion of drug evaluation studies, with approximately 
50% of the effort being devoted to the production of clinical materials. 
The materials assigned for resynthesis are not readily available in the 
quantities and/or quality needed from the original supplier or on the open 
market. Preparations vary in quantity from gram to multikilogram ?cale. 

STARKS C. P. (N01-CM8-7206) 

This contract is in support of the Drug Synthesis and Chemistry Branch's 
fundamental responsibility to acquire selected novel synthetic compounds for 
evaluation as potential anticancer agents - the initial step in the National 
Cancer Institute's Linear Array for drug development. The major focus of this 
contract is the active solicitation, acquisition documentation and management 
of the flow of approximately 10,000 compounds per year of diverse structural 
types. These compounds are selected by the Drug Synthesis and Chemistry 
Branch from a much larger pool of compounds provided through this contract in 
quantities adequate for the primary anticancer screen. This contract also 
acquires a significant proportion of the larger samples needed for secondary 
screening (Tumor Panel) of the many new leads that are identified. 

STATE UNIVERSITY OF NEW YORK (N01-CM2-7570) 

This project is one of the three contracts whose objectives are to design and 
synthesize the following: (1) congeners of lead compounds to enhance the 
activity or broaden the antitumor spectrum; (2) "pro-drugs" that are chemically 
altered transport forms of compounds to modify both biological and pharmaceutical 
properties, such as (a) improve bio-availability by increasing aqueous solubility; 
(b) increase compound stability; and (3) compounds related to products of natural 
origin and other heterocycles with improved antitumor activity and decreased 
toxicity. These modifications include partial structures, structural analogs and 
novel heterocycles. 

TACONIC FARMS (N01-CM3-0226) 

This contract furnishes approximately 104,000 hybrid mice for compound evalua- 
tion studies. Breeding animals are furnished from genetic centers and/or rodent 
production center colonies. This contract terminated January, 1985. 



64 



TACONIC FARMS (N01-CM5-7730) 

This Rodent Production Center contract will provide athymic nude mice under 
maximum barrier conditions. 

TECHNASSOCIATES. INC. (N01-CM3-7612) 

The purpose of this contract is to establish the need and requirements of the 
extramural community for an acquisition, quality assurance and distribution 
program for biological response modifiers. The contractor wil 1 conduct a 
survey to define potential suppliers and general availability of biologies as 
well as the amount, time intervals, levels of quality assurance, and standard- 
ization, toxicity testing, formulations and relevant in vitro and in vivo 
biological testing required by potential scientific investigators involved in 
testing and evaluating biologies. The survey is being conducted by phone, 
mail questionnaires and personal contact. Individuals are being contacted in 
private industry, academic institutions and government. The final document 
will be a written report summarizing all survey results and providing con- 
clusions and interpretation. This contract expired June, 1985. 

TECHNICAL RESOURCES. INC. (N01-CM5-7658) 

This project provides technical support services to the Office of the Director, 
OCT, as well as to the program areas of OCT in the performance of the planning 
and analytical tasks and general logistial support in the development of related 
or otherwise required documentation and conference support activities of the 
Division. This contract was awarded in April, 1985 for a five-year period. 

TENNESSEE, UNIVERSITY OF (N01-CM3-7607) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the prompt 
solicitation and award of projects to be conducted in this area from a pool of 
highly qualified contractors. 

TEXAS A&M RESEARCH FOUNDATION (N01-CM3-7536) 

This contract montitors the genetic purity of the strains produced at the 
Genetic Centers and Rodent Production Centers. The testing is done by checking 
biochemical markers, and animals are sent for monitoring on a weekly basis 
scheduled by the Project Officer. 

TEXAS. UNIVERSITY OF (NOl-CMl-7524) 

This contract is for the Phase I evaluation of equipment for the hyperthermic 
treatment of cancer in conjunction with radiotherapy, chemotherapy, surgery, 
or immunotherapy. Five contractors are collaborating in a working group in 
conjunction with the National Center for Devices and Radiological Health to 
develop guidelines for the use of heat generating and thermometry equipment 
to facilitate the development of Phase II and III clinical studies for the 
adjuvant treatment of deeply seated tumors with heat. 

65 



TEXAS. UNIVERSITY OF. HEALTH SCIENCE CENTER (N01-CM2-7542) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct 
Phase I clinical studies with new combinations or regimens mutually agreed 
upon. The Contractor is presently conducting Phase I studies with Tiazofurin 
and Menogaril and has started a new trial with Didemnin. This contract 
expi red July, 1985. 

TEXAS, UNIVERSITY OF (M.D. ANDERSON HOSPITAL & TUMOR INSTITUTE) (N01-CM2-7531) 

This contract is part of a collaborative effort to improve treatment 
planning with neutrons and with charged particles (protons, helium, ions, 
pi-mesons and heavy ions). In addition to developing optimal treatment 
plans for the treatment of tumors in various anatomic sites with the 
particle beam at its own institution, the collaborative working group is 
conducting a comparison of treatment plans for selected tumors in each 
anatomic site. Whenever possible, measurements in patients or phantoms 
will be made to check the accuracy of the treatment planning. 

TEXAS UNIVERSITY OF, SYSTEM CANCER CENTER (M.D. ANDERSON HOSPITAL AND TUMOR 
INSTITUTE) (N01-CM2-7550) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct Phase I 
clinical studies with new combinations or regimens mutually agreed upon. The 
Contractor is presently conducting Phase I studies with Trimetrexate and Taxol . 
This contract expired July, 1985. 

TEXAS, UNIVERSITY OF, SYSTEM CANCER CENTER (M.D. ANDERSON HOSPITAL & TUMOR 
INSTITUTE) (N01-CM3-7602y ~ ' 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the DCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the prompt 
solicitation and award of projects to be conducted in this area from a pool of 
highly qualified contractors. 

TEXAS, UNIVERSITY OF, SYSTEM CANCER CENTER (M.D. ANDERSON HOSPITAL AND TUMOR 
INSTITUTE) (N01-CM7-7T497 

This contract provided follow-up support for patients included in three 
adjuvant clinical trials for non-small cell lung cancer through the Lung 
Cancer Study Group. Patients were followed at regular intervals as deter- 
mined by the protocols. This contract expired December, 1984. 



66 



THERADEX SYSTEMS. INC. (N01-CM3-7553) 

The object of this contract is to provide a Clinical Trials Monitoring Service 
for the Phase I/I I Cancer Therapy Evaluation Program and Biological Response 
Modifiers Program investigators and all other investigators using NCI-sponsored 
investigational drugs. This service has four components: (1) a central data 
management resource for investigators conducting Phase I clinical trials; 
(2) a site visit monitoring resource for DCT to assure that Phase I/II 
contractors are in compliance with federal regulations; (3) co-site visiting 
cooperative groups as observers of peer audits; and (4) site visit monitoring 
of all other individual investigators conducting investigational drug trials. 

UTAH. UNIVERSITY OF (NOl-CMl-7523) 

This contract is for the Phase I evaluation of equipment for the hyperthermic 
treatment of cancer in conjunction with radiotherapy, chemotherapy, surgery, 
or immunotherapy. Five contractors are collaborating in a working group in 
conjunction with the National Center for Devices and Radiological Health to 
develop guidelines for the use of heat generating and thermometry equipment 
to facilitate the development of Phase II and III clinical studies for the 
adjuvant treatment of deeply seated tumors with heat. 

VERMONT, UNIVERSITY OF (N01-CM2-7547) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct Phase 
I clinical studies with new combinations of regimens mutually agreed upon. The 
Contractor is presently conducting a Phase I study with Spiromustine and has 
just started a a new trial with Didemnim. This contract expired July, 1985. 

VERMONT. UNIVERSITY OF (N01-CM3-7606) 

This Master Agreement for preclinical pharmacology task orders was established 
to conduct preclinical studies of the pharmacology of new or established anti- 
tumor agents. Most of the drugs to be studied will be under development and 
have passed Decision Point 2B in the OCT Decision Network, i.e. scheduled to 
begin preclinical toxicology. This task order mechanism will enable the prompt 
solicitation and award of projects to be conducted in this area from a pool of 
highly qualified contractors. 

VETERANS ADMINISTRATION MEDICAL CENTER (VAMC) (Y01-CM3-0256) 

This Interagency Agreement provides for the space and care of small animals 
required for the research purposes of the NCI-Navy Medical Oncology Branch. 
Space is provided to house conventional mice, nude mice, rats and rabbits. 
This Agreement also includes technical services on an occasional basis by 
VAMC animal room technicians. These services are the embedding, cutting 
and staining of tissue sections. This Agreement was terminated March, 1985, 
and the animal program is being transferred to the Frederick Cancer Research 
Center. 



67 



VSE CORPORATION (N01-CM5-6754) 

This contract is in the first year of a five (5) year period. It is the result 
of a competition held during 1984. Data processing services are provided to the 
Developmental Therapeutics Program by this contract. The scope of work includes; 

(1) reducing and disseminating data resulting from the screening program of the 
Drug Evaluation Branch to staff and suppliers of the compounds being tested; 

(2) writing new computer programs to process data from new methods of screening; 

(3) maintaining and modifying existing computer programs so that they are able 
to run at the Division of Computer Research and Technology; (4) improving data 
col lection methods; (5) documenting all computer programs and Contractor's 
procedures for data handling and running computer programs; compounds relating 
to collection and dissemination of data; (7) performing statistical analyses 
of test systems and test system parameters; (8) extensively collaborating with 
the Drug Information System Contractor; and (9) participating in scientific 
meetings. 

WARNER-LAMBERT COMPANY (N01-CM3-7285) 



This no-cost contract provides for developing and marketing AZQ as an antitumor 
agent. Its purpose is to facilitate development of the drug to the NDA stage. 
Warner-Lambert has conducted both pre-clinical and clinical studies with AZQ. 
All the additional toxicology studies required for FDA approval and foreign 
registration have been completed or will be initiated soon. Warner-Lambert 
defined a comparative trial of AZQ and BCNU in primary main tumors as the most 
important study to be conducted. The study is now activated and accruing 
patients. 

WARNER-LAMBERT COMPANY (N01-CM3-7614) 

This fermentation contract is designed primarily to obtain novel antitumor 
agents. This contract includes: (1) the preparation of fermentation beers 
from various microbes isolated from unique substrates from various parts of 
the world and fermented under a bevy of environmental and stress situations; 
(2) an in vitro tissue culture assay laboratory which assists in prescreening 
fermentation broths for cytotoxicity and is used to help assay chemical frac- 
tions, fermentation improvement samples and large pilot plant batches more 
quickly; (3) the isolation work required to obtain the active components from 
the confirmed active beers; and (4) the production of large quantities of anti- 
neoplastic agents selected for advanced development. One compound, a pyranone 
phosphate, is currently in toxicology study. Several other promising candidates 
are in early development. This contract will terminate in FY 1986. 

WARNER-LAMBERT COMPANY (N01-CM4-7589) 

This service preparative contract provides for the resynthesis of a variety 
of compounds required for clinical or preclinical evaluation. The compounds 
prepared are not readily available on the open market or from the original 
supplier in the amounts and/or quality required. About 30% of the effort 
on this contract is devoted to the preparation of large quantities of 
materials, in the multikilogram range, requiring pilot plant facilities. 



68 



WASHINGTON UNIVERSITY (N01-CM4-7696) 

The objective of this collaborative effort is to develop criteria, guidelines, 
and methodology for the performance and evaluation of state-of-the-art high 
energy photon external beam treatment planning. This will be accomplished 
by extensive treatment planning for actual patients and by using state-of- 
the-art beam delivery, computerized treatment planning, and imaging systems. 
The results of this work will be described in a final report which will 
define state-of-the-art treatment planning and provide criteria, guidelines, 
and methodology for its application and evaluation. 

WASHINGTON, UNIVERSITY OF (N01-CM9-7282) 

This contract provides a cyclotron -based neutron therapy system, a clinical 
facility in which to house the equipment and personnel to support a clinical 
neutron therapy research program at the University of Washington. The facility 
construction was completed in March 1984. The cyclotron was accepted in September 
1984 and patient treatment began in November 1984. 

WAYNE STATE UNIVERSITY (NOl-CMO-7404) 

The Contractor conducts Phase II/III studies in patients with advanced solid 
tumors. A minimum of 200 patients a year were studied, with no less than 
25 patients in any tumor type. These patients are treated intensively with 
chemotherapy either alone or in combination with radiotherapy, immunotherapy 
or surgery in protocols agreed upon by the NCI and the Principal Investigator. 
Accrual to new protocols ended in January, 1985; treatment and follow-up will 
continue through January. 1986. 

WAYNE STATE UNIVERSITY (N01-CM2-7551) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct Phase I 
clinical studies with new combinations or regimens mutually agreed upon. The 
Contractor is presently conducting Phase I studies with Acodazole, Caracemide, 
and Spi romustine. This contract expired July, 1985. 

WISCONSIN, UNIVERSITY OF (NOl-CMO-7434) 

This contract is conducting three Phase I/I I clinical trials of recombinant 
interferon in the treatment of patients with disseminated malignancies. 
Clinical trials with the gamma interferon are now reaching completion. Due 
to a delay in initiating the trial, this contract has received a no cost 
extension through June 30, 1985 in order to complete the final third step 
in the study to evaluate further the biologic effect of gamma interferon. 

WISCONSIN, UNIVERSITY OF (N01-CM2-7549) 

This contract is designed to conduct Phase I clinical studies with new anti- 
cancer drugs sponsored by the Division of Cancer Treatment or to conduct 
Phase I clinical studies with new combinations or regimens mutually agreed 
upon. The Contractor is presently conducting Phase I studies with Acivicin, 
Azodazole, and CBDCA-cisplatinum. This contract expired July, 1985. 



69 



WISCONSIN. UNIVERSITY OF (N01-CM4-7669) 

On this contract, a Phase I trial will investigate a single, rapid infusion 
versus a more prolonged infusion of recombinant interleuki n-2 in patients with 
advanced malignancies. Clinical toxicity, pharmacokinetics and immunologic 
monitoring will be conducted. This trial begin in early May of this year. 
This contract will expire October, 1985. 



70 



SUMMARY REPORT 

ASSOCIATE DIRECTOR FOR DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

NATIONAL CANCER INSTITUTE 

October 1, 1984 - September 30, 1985 



I. Introduction 

The Developmental Therapeutics Program (DTP) has primary operational 
responsibility for all aspects of the preclinical development of anti- 
tumor drugs for the Division of Cancer Treatment (DCT). The extramural 
component of the DTP is located in the Landow Building in Bethesda, 
Maryland, where directed drug development activities are contract 
supported and research in biochemistry and pharmacology is administered 
through the grant mechanism. The DTP intramural laboratory operation 
conducts anticancer drug and other pre-clinical cancer treatment related 
research in Building 37 on the NIH campus in Bethesda. 

The extramural program, which is devoted to the acquisition, antitumor 
evaluation, formulation, large-scale drug production and toxicology studies 
on new candidate anticancer drugs, is managed by seven Branches: Drug 
Synthesis and Chemistry, Natural Products, Drug Evaluation, Animal Genetics 
and Production, Pharmaceutical Resources, Toxicology, and Information 
Technology. An eighth Branch, Extramural Research and Resources, is 
responsible for the management of cancer related chemistry, biochemistry 
and pharmacology grants. 

The intramural program is conducted through five Laboratories: Biological 
Chemistry, Experimental Therapeutics and Metabolism, Pharmacology and 
Experimental Therapeutics, Tumor Cell Biology and Molecular Pharmacology. 
Intramural research is supportive of both new drug studies and basic 
investigations in cancer related biochemical processes and molecular 
biology. 

The Office of the Associate Director is responsible for the leadership 
and management of the DTP and the accomplishment of the goals and objec- 
tives of the DCT pre-clinical program. The progress of potential clinical 
candidates through the Decision Network process is summarized in Table 1. 

During this year a major reorientation of the drug development program has 
been initiated. A workshop, sponsored by DTP, was held in January entitled 
"Disease Oriented Antitumor Drug Discovery and Development." This led to a 
recommendation by a subcommittee of the DCT Board of Scientific Counselors 
that the essentials of the proposal presented at the workshop be adopted. 
The full Board approved the recommendation of the subcommittee and a phased 
implementation of the disease-oriented screen has begun. At the same time 
a major area of emphasis has been established in the natural products 
arena. The search for novel compounds has been broadened to encompass 

71 



sources such as fungi, bluegreen algae, marine organisms obtained at 
different ocean depths and plant collections from remote geographical 
locations such as Southeast Asia. The forthcoming year is expected to 
see the initiation of several projects in this area and the natural 
products so obtained will enter the disease-oriented screen of human 
tumor cell lines. 

II. Accomplishments 

A. Extramural Program 

1. Acquisition of new materials as potential anticancer drugs 

a. Drug Synthesis and Chemistry Branch (DS&CB) 

The fundamental responsibility of the DS&CB is the acquisition 
and management of the flow of unique synthetic compounds for 
evaluation as potential anticancer agents in the DTP. 

The DS&CB achieves its central mission by engaging in a variety 
of Program activities, namely, selective acquisitions, pre- 
selection model development, task order syntheses, contract 
syntheses, storage and distribution and computer assisted 
structure-activity analysis. The DS&CB works closely with the 
Information Technology Branch in restructuring the automated 
chemical data base system. In addition, DS&CB supports the 
Radiation Research Program, the European Organization for 
Research on Treatment of Cancer Program and the US Japan 
Cooperative Cancer Research Program. Recently, the DS&CB has 
assumed the responsibility for the radiolabel led synthesis. A 
collaborative program has been initiated with the Laboratory 
of Biological Chemistry for the design and synthesis of 
inhibitors of phospholipid metabolism. 

During the past year as a result of acquisition activities, 
360 new suppliers were added to the current list and seven 
synthetic compounds passed Decision Network (DN) 2A. The 
congeners and prodrug synthesis contracts have produced two 
improved drugs, a platinum (IV) complex, NSC 363812, which has 
advanced to DN 2B and a pyrazinediazohydroxide, NSC 361456, to 
the DN 2A level. 

The new screening approach will undoubtedly have some impact 
on the activities of DS&CB. In particular, the amounts of 
material needed for primary screening will be greatly reduced 
and this may make accessible to the program compounds which 
are only available in very limited quantities. 

b. Natural Products Branch (NPB) 

The objectives of the Natural Products Branch are: (a) to 
acquire a wide spectrum of unique chemical compounds of 
natural origin through donations and contracts for evaluation 
as potential antitumor agents; (b) to conduct a world-wide 

72 



program of literature surveillance to identify materials of 
interest for active acquisition; (c) to collaborate with 
suppliers of compounds to develop new derivatives and analogs 
of compounds of interest; (d) to procure or produce additional 
quantities of compounds under study to assure a sufficient 
supply of material for detailed biological evaluation and 
subsequent developmental studies including pharmaceutics and 
toxicology. 

During the past year fermentation contractors have evaluated 
24 762 new cultures. Of these 5,476 (22%) were active in one 
or'more of the prescreens. About 2.262 (49%) of these active 
cultures were regrown and retested and 1,619 (60%) were then 
selected for evaluation in vivo against P388; 346 (21%) were 
found to be active. 

In 1984 372 pure natural products were acquired, of which 344 
were donated and the remaining 28 were obtained from con- 
tractors. Also, through the literature surveillance program, 
165 compounds (44% of new acquisitions) were received in 
direct response to requests for specific compounds. The 
overall response rate to the literature requests is 28% and 
the project continues to be a very cost-effective way of 
acquiring compounds of interest from around the world. 

The commitment of the DTP to the compound-oriented screen will 
provide the opportunity for the NPB to examine a multiplicity 
of diverse materials of natural origin for their potential as 
selective antitumor agents. The future yield of active 
materials remains a question, but there is no doubt that 
natural products have an enormous potential for a variety of 
biological effects and there is every reason to anticipate 
that some of them will display useful antitumor activity. 

c. Drug Evaluation Branch (DEB) 

During the past year, 10,554 materials were examined in the 
in vivo P388 pre-screen. Of these 9,770 were synthetic 
chenncals and 784 were natural products. Three hundred and 
forty (3.2%) of the materials screened demonstrated confirmed 
activity in the pre-screen and entered the secondary phase of 
evaluation against a spectrum of transplantable mouse tumors. 

The human tumor colony forming assay (HTCFA) has continued to 
be used with emphasis being placed on those compounds which 
were inactive in the in vivo P388 pre-screen. Small numbers 
of the materials displayed possible evidence of differential 
or selective toxicity and will be examined further in other- 
tumor types. It is now very much apparent that the HTCFA is 
not viable as a primary screen for antitumor activity. Evalu- 
ation of its potential as a predicting system for human tumors 
in vivo will have to await the outcome of clinical trials or 
thosTagents identified by the assay. The first of these 
agents, dihydrolenperone (NSC 343513), will begin Phase I 
clinical trial in lung cancer patients in the summer. 

73 



Work has continued on the development of an in vitro disease- 
oriented antitumor screening model involving about 30 different 
human lung cancer cell lines. This Lung Cancer Drug Discovery 
Project (LCDDP) is the vanguard of the proposed new antitumor 
screen and it is expected that this LCDDP will become opera- 
tional in the fall of 1985. 

Development of metastatic human tumor models has progressed to 
the point that metastatic strains of several human tumors from 
lung, liver and lymph nodes have been selected. The human 
amelanotic melanoma cell line LOX appears to be particularly 
well suited for use in experimental metastatic chemotherapy and 
several lung tumors also appear promising in this regard. 

Treatment schedule and/or route dependency studies were 
completed for five clinical candidate drugs including flavone 
acetic acid, deoxysperguali n, tetracarcin and an anthra- 
pyrazole derivative. Biochemical studies suggest that 
deoxyspergualin may require activation by an amine oxidase to 
an aldehyde before tumor inhibition can occur. Congeners of 
several drugs are being evaluated for improved properties. 
Included in these evaluations are congeners of isopropylpyro- 
lizine, camptothecin melamines, triazenes and some cisplatin 
analogs, in particular, tetraplatin. 

There are now four National Cooperative Drug Discovery Groups 
(NCDDG) which will be in existence in FY 85. The four groups 
encompass 20 laboratory projects in 14 academic, research, and 
industrial organizations. The two NCDDGs awarded this fiscal 
year will investigate topoi some rases as therapeutic targets 
and the use of antireceptor monoclonal antibodies in cancer 
treatment. An ongoing collaboration has been established 
between DEB and the initial two NCDDGs and this relationship 
satisfactorily fulfills the requirement that the Government 
actively participate in work conducted under a Cooperative 
Agreement. 

2. Biological evaluation 

The primary function of the Animal Genetics and Production Branch 
(AG&PB) is the production of healthy laboratory animals with 
properly defined genetic characteristics. The AG&PB is also 
responsible for the proper functioning of the the Tumor Repository. 

During the past year, in anticipation of the forthcoming changes 
in the screening program, the AG&PB has begun to make the 
necessary changes in program emphasis which will be needed to 
accomodate the new activities. The branch will continue to 
provide, the quality control measures already in place for all 
DTP animals, during the transition period. 



74 



It has now been established that it is possible to freeze 
fertilized mouse embryos so that strains not in current use can 
be alternatively "stored" in this fashion until needed. 

3. Formulation and bulk chemical procurement 

The Pharmaceutical Resources Branch (PRB) has successfully 
accomplished its objectives in providing high quality bulk 
chemicals and pharmaceutical products to the various Programs in 
the Division of Cancer Treatment. During this reporting period, 
the prep lab contractors prepared over 181 compounds totaling more 
than 100 kilograms. Examples of bulk pharmaceutical substances 
which when delivered for fomiulationn included: Carboplatin 
(NSC 241240); 4-Ipomeanol (NSC 349438); Methotrexate (NSC 740); 
Ara-AC (NSC 281272); Fludarabine (NSC 312887); and L-Buthionine-S, 
R-sulfoximine (BSO) (NSC 326231). The Clinical Products Section 
directed the contract production of more than 450,000 injectable 
units and more than 880,000 oral dosages for clinical distribution. 
More than 20 BRMs have been received under this project and many 
more are expected in the next several months. The products 
received during this period included Interleukin-2; Thymosin; 
Alpha- Interferon; Lymphoblastoid Interferon; and Monoclonal 
Antibodi es. 

The pharmaceutical research and development activities were also 
quite successful in completing development of nine formulation 
assignments including parenteral products of Tetraplatin 
(NSC 349438); anthrapyrazole (NSC-349174); and others. 

4. Preclinical toxicology 

During this fiscal year the direction of the Toxicology Branch (TB ) 
has shifted from an unidirectional course to two highly integrated 
paths. The first path continues to be the elucidation of the 
potential adverse effects of new anticancer agents. The second 
path is the acquisition and use of pharmacokinetic information to 
reliably extrapolate toxic effects across species by relating 
plasma drug levels (peak and steady state) to the appearance and 
severity of toxicity. Integration of these two courses permits 
a more rational evaluation of the role of schedule dependence in 
efficacy of drug as well as in development of toxicity. 

Data on the following agents have been, or are anticipated to be, 
presented to the Decision Network Committee in FY'85. 

Flavone Acetic Acid NSC 347512 

Merbarone NSC 336628 

Deoxyspergualin NSC 356894 

Pibenzimol NSC 322921 

Triazine Antifol NSC 127755 



75 



Toxicology studies have been, or are anticipated to be, initiated 
in FY'85 on the following compounds. 

4-Ipomeanol NSC 349438 

Azacytosine Arabinoside NSC 281272 

Anthrapyrazole NSC 349174 

Tetraplatin NSC 363812 

Buthionine Sulfoximine NSC 326231 

Discreet Agent NSC 339004D 

Hexamethylene Bisacetamide (po) NSC 95580 

5. Information technology 

A fully operational version of the Drug Information system (DIS) 
was installed in the Information Technology Branch (ITS) in March 
1985. That allowed the NCI contract with the Chemical Abstracts 
Service (CAS) to be terminated the following month. The NCI-CAS 
contract had been in existence for a dozen years and its end, with 
the start-up of the DIS, marks a turning point in computer use in 
DTP. The DIS represents a consolidation of most of the disparate 
computer systems used by the DTP; it permits interactive control 
of acquisition, shipping, inventory and testing of chemicals. The 
major DIS databases are self-updating and the updating proceeds 
automatically at pre-determined dates and times. All output from 
the DIS is directed to a high-speed laser printer in the Landow 
Building. This printer is capable of graphical and non-graphical 
printing and represents a major advance over the earlier 
procedures which required the use of several different printers 
and which, as a result, could take hours or even days to complete. 
Since the DIS was released, it has logged tens of thousands of 
transactions. Numerous minor problems have been encountered and 
and resolved and many of the important DIS procedures have been 
greatly optimized. This process has been aided significantly 
by the installation at DCRT of a third KL-10 processor in the DEC 
System 10 computer. 

In a new excursion, the Branch is involved in the design of a 
computer system which is to be central to the new cell line project 
upon which the DTP is embarking. The new data system will run on 
a series of independent local microprocessors, controlled by and 
reporting to the DIS. In this project the throughput requirements 
are very high. Accordingly, a commitment has been made to 
robotics, which promises to deliver such a high volume of tests. 

6. Grants in pharmacology and biochemistry 

The Extramural Research and Resources Branch (ERRB) is responsible 
for the administration of preclinical grant-supported research 
leading to the development of effective anticancer drugs which act 
specifically or selectively against malignant growth with minimal 
toxicity to the host. The major areas of emphasis in the 
Biochemistry and Pharmacology Research Program are: drug design 
and synthesis, natural products development, experimental 
therapeutics, comparative pharmacology and toxicology, and 

76 



mechanisms of drug action. As new findings and significant 
developments occur, appropriate changes are made in programmatic 
emphasis and support in each of its major research categories. 
This Branch maintains liaison and coordinates its research 
activities with those of other Divisions of the National Cancer 
Institute. 

During FY 85, the Branch supported 283 research projects totalling 
$38.1 million dollars. 

B. Intramural Program 

1. Laboratory of Biological Chemistry (LBC) 

The Laboratory of Biological Chemistry was established in 1985 to 
identify as targets for drug design, cellular reactions that are 
critical to the control of tumor cell proliferation or differenti- 
ation. Recent advances in cell biology are evaluated for possible 
targets. Agents are designed to interact with these targets and 
are evaluated for biochemical and antitumor effectiveness. An 
important aspect of this mission is to develop appropriate in vivo 
systems to evaluate the chemotherapeutic effectiveness of agents 
shown to be active in simpler in vitro model systems. Accordingly, 
the Laboratory is involved in identifying endogenous factors 
present in vivo that modify drug action and influence differential 
toxicity with the aim of manipulating these factors to enhance 
antitumor activity. Ongoing research projects were reevaluated 
and approximately 50% of the resources was applied to non- 
traditional targets for antitumor drug design and study. These 
non-traditional targets include early key biochemical events 
signaling cell proliferation or differentiation. The other 50% of 
laboratory resources was applied to the study of either traditional 
targets or active compounds with traditional or unknown mechanisms 
of action. 

Reduced requirements for stimulation by growth factors may be the 
fundamental characteristic of transformed (neoplastic) cells. 
Projects have therefore been initiated to develop new chemo- 
therapeutic agents to block the action of growth factors. 
Non-traditional targets selected for drug design and study include: 
second messengers inositol triphosphate and di acyl glycerol ; 
myristoylation of cellular oncogene products; and protein kinase C. 

The availability of tissue culture cell lines has made it possible 
to study the regulation of proliferation and differentiation of 
specific hematopoieti-c cell types and the effects on these cells of 
known or suspected mediators and modulators. It was found 
previously that retinoic acid (RA) is a potent inducer of terminal 
differentiation of the human promyel ocytic cell line, HL-60, and the 
human monoblast- and monocyte-like cell lines, U-937 and THP-1. 
Studies are now in progress to apply the results obtained in vitro 
with HL-60 to a transplantable HL-60 tumor carried in athymic nude 
mice. 



77 



2. Laboratory of Experimental Therapeutics and Metabolism (LETM) 

The LETM encompasses an interdisciplinary research program in tumor 
biology, pathology, biochemistry, pharmacology and toxicology that 
include 1) utilization of the biochemical and pathobiol ogical 
characteristics of hepatic and extrahepatic tumor cell populations 
to define the histogenesis of specific cancers; 2) establishment 
of specific animal and human tumor cell lines to study new and 
existing anticancer drugs for their mechanism of toxicity; 3) 
investigation of biochemical and molecular mechanisms of drug 
metabolism and drug toxicity; 4) elucidation of the metabolic and 
physiological factors that may underlie target organ and target 
cell specific toxicity and prevention of toxicity by cellular 
defense mechanisms and 5) the continuation of the development of 
suitable cellular and animal models to elucidate mechanisms of 
toxicity of anticancer drugs and chemical modulators of anticancer 
drug effects. 

Although all of the current research is based on information 
generated in earlier animal experiments, the major emphasis during 
this report period has been on the characterization and establish- 
ment of human lung cancer cell lines (HLCCL) for research on cell 
type specific chemical/drug interactions. The experience generated 
through such studies has also enabled the initiation and design of 
a number of experiments primarily conducted by investigators of 
the Pharmacology and Toxicology Section, LETM. In summary, it was 
found that: 

1. Of seven investigated non-small cell derived (HLCCL), only two 
adenocarcinomas (NCI-H322 and NCI-H358) exhibited unequivocal 
well differentiated features of one pulmonary cell type (as 
assessed by electron microscopy) while all others demonstrated 
either dual differentiation (e.g., adenosquamous) or poor dif- 
ferentiation which made a diagnosis by cell type questionable. 
Evidence was also found that well differentiated lung tumors 
undergo pronounced morphological changes during prolonged 
maintenance in vitro . This exemplifies the need for careful 
electron microscopic assessment of HLCCL before and during 
their use for biochemical and pharmacological experiments. 

2. Similar to in vivo studi es in hamsters, the pulmonary 
carcinogen, diethylnitrosamine (DEN), is preferential ly metab- 
olized by HLCCL with features of pulmonary Clara cells in vitro 
and such metabolism is inhibi table by inhibitors of cytochrome 
P-450 enzymes. 

3. HLCCL of different cell types metabolize DEN via different 
enzyme systems, e.g., Clara cells via cytochrome P-450 and 
alveolar type II cells via prostaglandin endoperoxide synthe- 
tase. Cell type specific differences such as this in 
nitrosamine activation may well be responsible for the known 
organ and cell specific carcinogenicity of these compounds. 



78 



4. Similar to experiments in rats, the pulmonary toxin and 
candidate anticancer drug, 4-ipomeanol, is preferentially 
metabolized by an HLCCL with characteristics of Clara cells 
and such metabolism is mediated by cytochrome P-450 enzyme 
systems. The selective toxicity of the compound for this cell 
line suggests that its usefulness as an anticancer drug for 
pulmonary adenocarcinomas and other solid tumors of similar 
cell types should be pursued. 

5. The xenobiotic-metaboli sm enzymes, cytochrome P-450, 

a ryl hydrocarbon hydroxylase and ethoxycoumarin 0-deethylase 
activity are selectively present in non-small cell derived 
HLCCL. 

6. Normal alveolar type II cells of rat lung proliferate in vitro 
for 7 days in culture, retain their typical ultrastructure 
and phospholipid synthesis and can be identified by immuno- 
peroxidase using an antiserum specifically for rat surfactant 
apoprotein. 

3. Laboratory of Pharmacology and Experimental Therapeutics (LPET) 

LPET (formerly the Laboratory of Medicinal Chemistry and Biology) 
was established in 1975 in order to provide a facility capable of 
antitumor drug development from the stage of design and synthesis 
through biochemical and pharmacological characterization to Phase I 
clinical trial. In addition, the laboratory as a whole develops 
analytical methodology for the detection of antitumor drugs and 
their metabolites, and also participates actively in the clinical 
pharmacology/Phase I trials of new agents which either originated 
within the laboratory or which were derived from extramural sources 
but in whose preclinical development the laboratory has played a 
major role. Included among the many investigations which have been 
conducted during the reporting period are the following studies: 
Arabinosyl-5-azacytosine (ara-5-AC), a compound originally synthe- 
sized within LPET and presently under active development toward 
clinical trial, exhibits unexpectedly high intracerebral antitumor 
activity in the IC L1210 murine test system (i.e., activity greater 
than that of ara-C, and comparable to that of the prototype 
reference compound, BCNU). A rapid and sensitive HPLC assay for 
the quantitation of ara-5-AC in biological fluids has been 
developed. These findings will be of direct applicability in the 
projected Phase I/I I clinical trials of this compound shortly to 
be initiated by DCT. 

All the possible phosphonate analogs of the active metabolite of 
TAD (the active anabolite of tiazofurin) have now been prepared. 
One of these (beta-methylene TAD) was equi potent to TAD as an IMPD 
inhibitor and possesses the advantage of being resistant toward 
cleavage by TAD-inactivating enzymes. 



79 



In studies of the differences between metastatic cells and their 
non-metastatic counterparts, an L1210 system utilizing tumor 
cells isolated from murine liver has been utilized. Marked 
differences have been observed between liver metastatic L1210 
tumor cells and their ascitic counterparts, e.g., a 2-fold 
increase in glutathione concentration and a 5-fold greater level 
of surface SH-groups in the liver metastatic cells. The metastatic 
cells are more resistant to melphalan, but can be resensitized by 
blocking the surface sulfhydryls with 6,6-dithionicotinic acid. 

The new agent, merbarone (NSC 336629), has been extensively 
examined in terms of possible mechanism of action. The compound is 
an effective but atypical inhibitor of DNA synthesis; among a 
number of factors examined, this inhibition appears to correlate 
most closely with the ability of the compound to generate single- 
strand DNA breaks which do not, however, appear to be protein- 
associated. Investigation of the molecular pharmacology of this 
unusual wide-spectrum antitumor agent is continuing. 

4- Laboratory of Tumor Cell Biology (LTCB) 

While the overall objectives of the LTCB are to develop, implement 
and analyze data obtained from studies of cellular proliferation 
cell differentiation, and biochemical growth characteristics of ' 
normal and malignant mammalian cells both in vivo and in vitro 
the major activities during this reporting period haveTocused'on 
ut."w^?tt''^^^^^"^ ^° *^® proposed etiologic agent of AIDS namely, 
HILV-III. Among some of the many accomplishments by the laboratory 

1. More than 100 isolates of HTLV-III have been obtained from 
patients with acquired immune deficiency syndrome (AIDS) and 
pre-AIDS. ' 

2. Seroepidemiological studies with HTLV-III show that 95% of the 
AIDS and pre-AIDS patients and approximately 45% of healthy 
homosexuals carry HTLV-III antibodies. 

3. Development of an assay for detection of neutralizing 
antibodies to HTLV-III in sera of individuals exposed to the 
virus. 

4. Studies on the development of an animal model indicate 
chimpanzees to be a suitable animal. Seroconversion and 
HTLV-III isolation was demonstrated in chimps infected with 
HTLV-I II. 

5. Several drugs, including foscarnet, cis^-platinum and lapachone 
derivatives have been shown to inhibit HTLV-III replication. 

6. Determined the nucleotide sequence of the envelope gene of 
HTLV-III (RF). 



80 



5. Laboratory of Molecular Pharmacol goy (IMP) 

A major goal of the LMP is to develop strategies for the selective 
killing of particular human tumor cell types. Studies are being 
conducted in three major areas namely, 1) the mechanisms of action 
of alkylating agents; 2) topoisomerase II as a target for DNA 
intercalating agents and epipodophyllotoxins; 3) regulation of 
chromosomal protein biosynthesis in relation to the control of 
cell proliferation. 

The alkylating agent studies have shown the following: 1) there 
are marked differences in reactivity among guanines in a DNA 
sequence; 2) the differences are not markedly dependent on the 
solvent conditions of reactions; 3) the guanine selectivity 
pattern is different for different nitrogen mustards; 4) the 
potential inter-strand crosslinking sites -- e.g. 5'-GC-3' -- 
show relatively low reactivity for HN2, melphalan and spiro- 
hydantoin mustard; 5) these sites, however, show a markedly 
enhanced reaction with uracil mustard; 6) runs of G's (potential 
sites of intra-strand crosslinking) exhibited disproportionately 
high reactivity with most nitrogen mustards; 7) quinacrine mustard 
exhibited a uniquely different pattern of reactions. 

Topoisomerase II studies have shown that the enzyme's activity 
measured by the protein-associated strand break response is 
dependent on the proliferation state of the cells. In non- 
proliferating 3T3 cells, this activity was much less than in 
proliferating cells. When quiescent cells were stimulated to 
proliferate, the topoisomerase II response increased at about 
the time of the first wave of DNA synthesis, but did not then 
oscillate in phase with the cell cycle. Thus the observed 
alteration in topoisomerase II activity appears to be related to 
the proliferation state of the cells and not to phase of the cell 
cycle. 

Investigations of chromosomal protein synthesis and cell 
proliferation have resulted in further insight on the mechanism of 
the regulation of DNA and histone synthesis during S-phase. 
Studies were carried out of the effects of protein synthesis 
inhibitors applied to cells at relatively low doses such that 
protein synthesis was reduced by only 50%. Although DNA and 
histone syntheses were initially inhibited proportionately, these 
syntheses then recovered despite the continued inhibition of 
overall protein synthesis. This was found to be brought about by 
a compensatory elevation of histone m-RNA. A mechanism thus exists 
which tends to preserve the duration of S-phase when Gl is 
lengthened because of nutritional or metabolic deficiencies. The 
mechanism will be studied further by using other means to lengthen 
Gl phase. The relevance to cancer chemotherapy is that this 
metabolic regulatory mechanism may be defective in some neoplastic 
cell lines and could lead to a strategem by which such cells could 
be selectively killed. 



81 



COMPOUNDS THAT PASSED DECISION NETWORK (4/1/84 - 3/31/85) 



NSC 
Number 



Name 



Compound 
Type* 



Decision Network 2A 



201047D Discreet 

320486D Discreet 

321803 Estra-l,3,5(10)-trien-17-one, 3-methoxy-4-nitro-; 

Nitroestrone 

332598D Discreet 

338720D Discreet 

338947 Methanesulfonic acid, (methyl sul fonyl )-, 2- 

chloroethyl ester 

344007 Piperazine, l-(2-chloroethyl )-4-(3-ch1oropropyl )-, 

di hydrochloride 

348948 -1,5,2,4-Dioxadithiepane, 2,2,4,4-tetraoxide . 

349156 Pancratistatin 

349174 Anthra[l,9-cd]pyrazol-6(2H)-one, 5-[(3-aminopropyl ) 

amino]-7,10-di hydroxy-2-[2-[(2-hydroxyethyl )amino] 
ethyl]-, hydrochloride (10:17) 

355644 Anthra[l,9-cd]pyrazol-6(2H)-one, 7,10-dihydroxy-2- 

[2-[(2-hydroxyethyl)aminoJethyl]-5-[[2-(methylamino; 
ethyl ]ami no]-, acetate (salt) hydrobromide (10:5:21) 

36381 2D Discreet 

366241 7H-Pyrido[4,3-c]carbazolium, 2,2'-([4,4'- 

bipiperidine]-l,l'-diyldi-2,l-ethanediyl)bis[l- 
methoxy-, dimethanesulfonate, di sul fate; 
Pyridocarbazolium derivative 

368390D Di screet 



S 

s 
s 

NP 

S 

S 



S 

NP 

S 



82 



COMPOUNDS THAT PASSED DECISION NETWORK (4/1/84 - 3/31/85) 



NSC 
Number 



Name 



Compound 
Type* 



Decision Network 2B 



127755 Ethanesulfonic acid, cpd. with 3-chloro-4-[4-[2- 

chloro-4-(4,6-diamino-2,2-dimethyl-s^-triazin-l(2H)- 
yl ) phenyl ]-butyl]benzenesu1fonyl fluoride (1:1); 
Dihydrotriazine derivative 

281272 l,3,5-Triazin-2(l]i)-one, 4-amino-l-B-D- 

arabinofuranosyl-; Ara AC (5-Azacytosine 
Arabinoside) 

303812 G1ycine,[tetradecahdyro-3,9-dihydroxy-4 

( hydroxymethyl) -4, lib-dimethyl -8,1 la-met hano-llaH^- 
cycloheptal[£]naphtha1en-9-yl]methyl ester, 
hydrochloride, (3A,4A,4AA,6AB,8B,9B,11AB,11BB)-; 
Aphidicolin Glycinate 

303861 ethyl ester, methyl carbamate (ester), 

monohydrochloride; L-Cysteine analog 

308847 lH-Benz[de]isoqui noli ne-l,3(22[)-di one, 5-amino-2- 

[2-(dimethylamino)ethyl]-; Benzisoquinolinedione 

336628D Discreet 

339638 2H-Pyran-2-one, 5,6-dihydro-6-[3,6,13-trihydroxy- 

3-methyl-4(phosphonooxy)-l,7,9,ll-tridecatetraenyl]- 
monodsodium salt; Pyranone phosphate 

347512 4H-l-Benzopyran-8-acetic acid, 4-oxo-2-phenyl-; 

FTavone Acetic Acid 

356894 Heptanamide, 7-[(aminoiminomethyl )amino]-N-[2- 

[[4-[(3.aminopropyl)amino]butyl]amino]-l-hydroxy- 
2-oxoethyl]-, (+)-; Deoxyspergualin 



SS 

NP 
S 

S 

NP 

S 

NP 



83 



COMPOUNDS THAT PASSED DECISION NETWORK (4/1/84 - 3/31/85) 



NSC 
Number 



Name 



Compound 
Type* 



Decision Network 3 



322921 Phenol, 4-[5-(4-methyl-l-pipera2inly)][2,5'-bi-lH^- 

benzimidazol]-2'yl , tri hydrochloride; Pibenzimol 

325319 Didemnin B 

352122 D-Glucuronic acid, cpd. with 5-methyl-6-[[(3,4,5- 

trimethoxyphenyl )amino]methyl]-2,4- 
quinazoli nedi amine (1;1); Trimetrexate 



NP 
S 



Decision Network 4 



3051 N-Methylformamide 

264880 l,3,5-Triazin-2(lH)-one, 4-amino-5,6-di hydro-1-B-D- 

ribofuranosyl-, monohydrochloride; Dihydro-5- 
azacytidine 

267469 5,12-Naphthacenedione, 10-[(4-aminotetrahydro-2]^- 

pyran-2-yl)oxy]-7, 8,9, lO-tetrahydro-6, 8,11- 
trihydroxy-8-(hydroxyacetyl )-l-methoxy-, hydrochloride, 
(8S- cis )-; 4-Deoxydoxorubicin 

312887 9H^-Purin-6-amino, 2-fluoro-9-(5-0-phosphono-B-D- 

arabinofuranosyl )-; Fludarabine Phosphate 
(2-F-ARA AMP) 



S 
SS 

ss 



Decision Network Special 



324360 



Benzamide, N^, 3, 4- tri hydroxy-; Hydroxamic Acid 
derivative 



84 



COMPOUNDS THAT PASSED DECISION NETWORK (4/1/84 - 3/31/85) 



NSC 
Number 



Name 



Compound 
Type* 



Decision Network PROD 



45410 Pyrazole 

57155 N,N"-bis[p-(N'-methylamidino)phenyl]- 

terephthal ami dine; Tetrahydrochloride 

177248 l,2,4-Triazin-3(4H)-one, 4-B-D-ribofuranosyl-, 

1-oxide; Uricytin 

364989 l-Bis-(B-ch1oroethyl)amino-2-aminoethane 



S 
S 

NP 

S 



*S = synthetic 
NP = natural product 
SS = semi -synthetic (natural product modified synthetically) 



85 



ANNUAL REPORT OF THE DRUG SYNTHESIS & CHEMISTRY BRANCH 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 - September 30, 1985 

The fundamental responsibility of the Drug Synthesis and Chemistry Branch 
(DS&CB) is the acquisition and management of the flow of unique synthetic 
compounds for evaluation as potential anticancer agents in the Develop- 
mental Therapeutics Program (DTP). 

The DS&CB achieves its central mission by engaging in a variety of Program 
activities, namely, selective acquisitions, preselection model development, 
task order syntheses, contract syntheses, storage and distribution and com- 
puter assisted structure-activity analysis. The DS&CB works closely with 
the Information Technology Branch (ITB) in restructuring the automated 
chemical data base system. In addition, DS&CB supports the Radiation 
Research Program (RRP), the European Organization for Research on Treat- 
ment of Cancer (EORTC) Program and the U. S. Japan Cooperative Cancer 
Research Program. Recently, the DS&CB has assumed the responsibility for 
the radiolabelled synthesis. A collaborative program has been initiated 
with the Laboratory of Biological Chemistry for the design and synthesis 
of inhibitors of phospholipid metabolism. 

The DS&CB is gearing up in a variety of ways for the new opportunities 
for drug discovery through the emerging in vitro based, disease - oriented 
screens. 

The contracts managed by DS&CB are outlined in Table I. Presently, the 
DS&CB is staffed with six professionals and three clerical personnel. 

Acquisitions 

The function of the Acquisition Section is to ensure the continuous flow 
of approximately 10,000 compounds, selected from a large pool of available 
compounds for the primary screen. To fulfill this objective, the Section 
is engaged in the following activities: (1) developing and maintaining 
extensive contacts with industries and academic scientists for acquiring 
compounds with unique structural features and biological activity; (2) 
maintaining an effective selection ratio of inputs of compounds; (3) 
developing and implementing selection criteria; (4) developing computer 
models to facilitate selective acquisition; (5) acquiring compounds for 
the primary screen and Tumor Panel evaluations; (6) conducting structure- 
activity correlation studies, and (7) monitoring the discreet agreements 
with industrial suppliers. Specifically, the acquisition activities 
resulted in the following: (a) 360 new suppliers were added and (b) seven 
synthetic compounds passed DN2A; NSC's 95678, 291643, 326231, 343513, 
D-339004. D-339736 and D-361456. 

The Acquisition Section worked closely with ITB in restructuring the 
automated chemical data base system. The acquisition contractor performed 



87 



the difficult task of analyzing and reporting system problems for correction 
as each new computer subsystem was being introduced. 

The acquisition team has organized an intense effort to inform the 
scientific community of the new disease-oriented drug discovery 
approach. Compounds with biological rationale are being identified for 
evaluation in the new screens. 

Preselection Model 



Unless specially targeted, all potential acquisitions are rated for 
novelty and antitumor activity by computer programs based on molecular 
structure fragments. The programs are generated from compounds tested 
in P388, L1210, and B16. The novelty and activity ratings have been 
especially useful as P388 testing has been reduced. Refinements, 
modifications and continued development of the programs are conducted 
as an intramural research project. 

Structure-Activity Analysis 

Detailed structure-activity analyses based on our large chemical -biological 
data base are an essential part of our acquisition and synthesis activities. 
Such large-scale analyses of our data files have become feasible because of 
the chemistry- biology interlink. Examples of structure-activity analyses 
include: barbiturates, purines and pyrimidines. 

Resynthesis 

The Task Order mechanism is a cost-effective means of resynthesizing compounds 
of program interest that are not available in sufficient quantity from the 
original suppliers. Currently, we have 12 master contractors who have the 
expertise to synthesize a wide variety of organic and inorganic compounds. 
For this reporting period, we have awarded 20 individual projects consisting 
of 135 compounds. Compounds selected for synthesis include PS actives, toxics, 
LE actives, radiosensitizers/radioprotectors, special requests from intramural 
scientists, panel compounds, rationally designed compounds and bio-active 
compounds emerging from literature surveillance. During this reporting period, 
approximately 125 compounds synthesized through this mechanism were received. 

Synthesis of Congeners and Prodrugs 

The objective of this project is to advance promising leads that are flawed 
by limited solubility, stability or narrow spectrum of activity to DN2 level. 
A variely of approaches are brought into play to overcome these shortcomings: 
qualitative and in selected cases, quantitative structure-activity estimations, 
Craig plots, design of isosteres, prodrugs, and congeners. Examples of com- 
pounds under development are given below. NSC 363812, a platinum (IV) complex 
has advanced to DN2B and the pyrazinediazohydroxide, NSC 361456, to DN2A level. 
A third compound, NSC 373965, a water-soluble phenylalanyl derivative of 
mitindomide, is expected to advance to DN2 A/B level shortly. 

A collaborative program has been initiated with the Laboratory of Biological 
Chemistry for the design and synthesis of fradulent cyclitols as inhibitors of 
phospholipid metabolism. 



Literature Searches 

The chemical search component of the DS&CB plays an Integral part 1n 
supporting the search needs of the various Program elements of the 
Branch, namely, the acquisition of new novel synthetics, contract 
synthesis and grants. It also provides the scientific community with 
Information on the compounds screened In the Program. During this 
period, approximately 2,000 full structure and 400 substructure 
searches were performed. In addition, the Questel/Darc and Dialog 
data base systems were utilized to access synthesis methods for 
selected compounds. Systematic nomenclature was generated for 700 
compounds. 

The literature surveillance program reviews the massive 
number of compounds published each month, estimated at 30,000 per 
month, and prepares a list of approximately 1,000 of the most 
Interesting ones for review by DS&CB. These compounds are scored by 
the Nodes model and reviewed by chemists. The compounds that are 
finally selected are acquired either through mall requests or tasks 
order synthesis. 

The contract with the Japanese Foundation for Cancer Research assists 
DS4CB In identifying potential compounds for acquisition from Japan. 

Storage and Distribution 

This project provides the service needed by the Developmental Thera- 
peutics Program for the storage, inventory, distribution and docu- 
mentation of synthetic substances, crystalline natural products and 
bulk chemical drugs. Flow laboratories. Inc. has shipped more than 
15,000 compounds to contract screeners, formulation laboratories, NCI 
and NIH researchers and Investigators in 45 states of the U. S. and 
25 foreign countries. Flow has also processed (weighed, recorded 
and shelved) approximately 14,000 compounds that were returned by the 
screening laboratories. The Contractor interacts closely with members 
of DS&CB, DEB, ITB and PRB as well as the acquisitions contractor and 
DIS contractor. The Contractor is being equipped for weighing a large 
number of small samples for the forthcoming in-vitro screens. 

Radiation Research Program Support 

DS&CB works in close cooperation with the RRP and the Radiosensitizer/ 
Radioprotector (RS/RP) Working Group. The Branch monitors the radio- 
sensitizer synthesis contract; and also identifies and acquires novel 
compounds for the RS/RP screening contract. Three non-nitro compounds, a 
quinoxaline oxide and two benzamides have demonstrated radiosensitization 
as effective as misonidazole both in vivo and in vitro and are undergoing 
detailed evaluation. Phase I trials ornrsC-30TT67 (SRI2508) are nearly 
completed and protocols are being developed for Phase II trials. Both the 
synthesis and screening contracts are being recompeted. 



89 



TABLE I 





CONTRACTS - FY 85 


Contractor 


Investigator 


Alabama, University of 


Baker 


Dynamac Corporation 


Gray 


Flow Laboratories, Inc. 


Dorian 


Georgia Institute of 




Technology 


Zalkow 


Japanese Foundation for 




Cancer Research 


Sakurai 


Maxima Corporation 


Sobers 


Research Foundation of 




The State Univ. of N. Y. 




at Buffalo 


Anderson 


Research Triangle Institute 


Kepler 


SRI International 


Lee 


Starks C.P., Inc. 


Schultz 


12 Master Agreements (Task Order) Contracts: 


Alabama, University of 


Baker 


Eagle-Picher Industries, Inc. 


Roth 


Franklin Research Institute 


Kauffman 


Michigan Technological Univ. 


Swartz 


Raylo Chemicals, Ltd. 


Muhs 


Research Triangle Institute 


Seltzman 


SISA, Inc. 


Razdan 


Southern Research Institute 


Montgomery 


South Florida, Univ. of 


Owen 


Southwest Research Institute 


Lyle 


SRI International 


Smith 


Starks Associates 


Starks 



Contract No. 
NOl-CM-27571 
NOl-CM-37563 
N01-CM-27505 

NOl-CM-27517 

N01-CM-36011 
NOl-CM-47628 



NOl-CM-27570 
NOl-CM-27515 
NOl-CM-47611 
NOl-CM-47608 



NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 
NOl-CM- 



37631 
37634 
37632 
37633 
37635 
37636 
37637 
37638 
37639 
37640 
37641 
37642 



90 



PUBLICATIONS BY STAFF ; 

1. Paull, K. D., Nasr. M., and Narayanan. V. L. Computer Assisted Structure- 
Activity Correlations I: Evaluation of Benzo (de) Isoquinoline-l .3-diones 
and Related Compounds as Antitumor Agents. Arzneim.-Forsch./Drug Res. , 34 
Nr.lO, 1243-1246, 1984. 

2 Nasr, M.. Paull, K. D., and Narayanan, V. L. Computer Assisted Structure- 
Activity Correlations II: - Unsaturated Ketones, Lactones. Lactams 
and Related Michael Type Acceptors as Antitumor Agents. In Garattini . b. , 
Goldin, A., Hawking, F. , Schnitzer, R. J. (Eds.): Adv. Pharmacol. Chemo- 
ther . New York, Academic Press, Vol. 20, 1984, pp. 123-189. 

3. Johnston, T. P., Kussner, C. L. , Carter, R. L., Frye, J. L.. Lomax, N. R. , 
Plowman, J., and Narayanan, V. L.: Studies on Synthesis and Anticancer 
Activity of Selected N-(2-Fluoroethyl )-N-nitrosoureas. J. Med. Chem. . 27 
1422-1426, 1984. 

4 Nasr M., Paull, K. D., and Nar^anan, V. L.: Computer Assisted Structure- 
Anticancer Activity. Correlations of Carbamates and Thiocarbamates. 
J. Pharm. Sci . In Press 

5. Paull, K. D., Hodes, L., and Simon, R. M. Efficiency of Large-Seal e 

Antitumor Screening Relative to Activity Criteria. J. Natl. Cancer Inst.. 
In Press 

6 Brewer, A. D., Minatelli, J. A., Plowman, J., Paull, K. D., and Narayanan, 
V. L. 5-(N-Phenylcarboxamide)-2-Thiobarbituric and (NSC 336628). Biochem. 
Pharmacol . In Press 

PUBLICATIONS BY CONTRACTORS : 

During this year, contractors and subcontractors wrote four articles which 
involved NCI support. 

SEMINARS : 

1. "Synthesis of Mitomycin C Analogs". Professor D. N. Reinhoudt, Twente 

University of Technology, The Netherlands. 

2. "Second Generation Anthracyclines". Professor Federico Arcamone, Farmi- 

talia Carlo Erba, Milan, Italy. 

3. "Pyrimidines With New Biological Roles". Dr. Malcolm Stevens, Professor 

of Experimental Chemotherapy, Pharmacy Dept. , University of Aston in 
Birmingham, England. 

4. "Chemistry of 1 ,4,2-Dithiazines, Isomeric 1,3-Dithiolimines and Related 

Heterocycles With 1 ,2-Dithioethene Structure". Professor Egon Fanghanel, 
Technische Hochschule. Carl Schorlemmer. Leuna Merseburg. East Germany. 



91 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM-07101-10 |>SCB 



PERIOD COVERED 



O ctober 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must lit on one line tietween the borders.) 



Computer Methods for Prtig Preselection Based on Structure Activity 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute attlllatlon) 

Dr. Louis Hodes, Acquisition Section, DS&CB, DTP, DCT, NCI, NIH 



COOPERATING UNITS (If any) 

Chemical Abstracts Service 



LAB/BRANCH 



Drug Synthesis & Chemistry Branch 



Acquisition Section 



INSTITUTE AND LOCATION 

NCI, NIti, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



1.0 



PROFESSIONAL: 

1.0 







CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects D (b) Human tissues \^' (c) Neither 

n (a1) Minors 
D (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Molecular structure fragments and biological test results from those systems 
with sufficient data are fed to computer programs that provide estimates of 
antitumor activity and novelty. These estimates have been used to aid 
selection since 1980 and are especially useful as the number of compounds 
screened has been reduced repeatedly during the course of this project. 

The selection programs have been integrated into the pre-registry module 
of the Drug Information System. 

The programs have also provided a pioneering effort in automated literature 
surveillance by running all CAS registrations. This has been performed for 
all compounds registered in 1977, the year before DTP began its own literature 
searches. 

Also an innovative approach is being investigated combining physical parameters 
with structure features for our large diverse set of compounds. Early results 
with the octanol water partition coefficient are encouraging. 



92 



PHS 6040 (Rev. 1/84) 



ANNUAL REPORT OF THE NATURAL PRODUCTS BRANCH 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 - September 30, 1985 

One of the major objectives of the Division of Cancer Treament is to discover^ 
novel types of compounds with antineoplastic activity which can provide a basis 
for new chemotherapeutic agents. 

In this regard, natural product research has a long history of producing novel 
and unusual types of chemical structures which show many types of biological 
activity and indeed the basic structural types of various classes of natural 
oroducts have led to development of many major classes of chemotherapeutic com- 
pounds. The investigation of natural substances for anticancer activity provides 
new types of compounds for evaluation which possess unusual properties, and can 
lead to new drug classes for chemotherapeutic testing in cancer treatment. 

The Natural Products Branch has actively pursued acquisition, isolation, struc- 
ture determination, and testing of compounds from microbial, plant and animal 
sources in order to obtain new leads for further development in the NCI program. 

The major program areas of the Natural Products Branch are: (1) acquisition 
of crude biological materials of plant, marine, and microbial origin for the 
DTP screening program; {?.) contract research directed toward isolation of new_ 
agents from active extracts; (3) world-wide literature surveillance, and acqui- 
sition of natural products with demonstrated biological activity or novel struc- 
tural types for evaluation; (4) procurement and preparation of large quantities 
of active agents for drug formulation, tumor panel testing, toxicology and clin- 
ical studies. 

This year, with the commitment of the Developmental Therapeutics Program to a 
new direction in in vitro screening, a renaissance of interest in natural prod- 
ucts as potentially highly selective antitumor agents has begun with the ap- 
proval by the Board of Scientific Counselors of a major new program effort in 
collection of natural products from a wide variety of sources, including ter- 
restrial plants, marine invertebrates, algae and marine microorganisms. The 
Branch is currently very actively engaged in preparing the Requests for Pro- 
posals which will be issued for this work in FY '86. 

Objectives 

The objectives of the Natural Products Branch are: (a) to acquire a wide spec- 
trum of unique chemical compounds of natural origin through donations and con- 
tracts for evaluation as potential antitumor agents; (b) to conduct a world- 
wide program of literature surveillance to identify materials of interest for 
active acquisition; (c) to collaborate with suppliers of compounds to develop 
new derivatives and analogs of compounds of interest; (d) to procure or produce 
additional quantities of compounds under study to assure a sufficient supply of 
material for detailed biological evaluation and subsequent developmental studies 
including pharmaceutics and toxicology. 

93 



Organization and Staffing 

The Branch is organized into three segments, the Office of the Chief, the Fer- 
mentation Section, and the Plant and Animal Products Section. Many tasks re- 
quire interaction between these segments, and the Branch personnel are assigned 
duties in whichever of the areas requires their expertise depending on changing 
program needs. The present full time staff consists of four professionals and 
two secretaries. The contracts managed by the Branch are outlined in Table 1. 

Table 1. 



Natural Products Branch Contracts 



Contractor 

Bristol Myers 

Microbial Chemistry 
Research Foundation 

Polysciences, Inc. 

Warner-Lambert 

Univ. of Illinois 

( In Competition) 

Fermentation Section 



Investigator 

Claridge 

Umezawa 

Boettner 

French 

Farnsworth 



Contract No. 
N01-CM3-7556 

N01-CM4-7593 

N01-CM3-7557 
N01-CM3-7614 
N01-CM3-7553 
N01-CM5-7692 



Program Area 
Fermentation 
Fermentation 

Plant Products 

Fermentation 

Literature Surv, 

Fungal Fermenta- 
tion 



The objective is the discovery, isolation and development of novel antineoplas- 
tic drugs derived from microbial fermentations. The section maintains four 
contracts in support of these tasks. Three contracts are devoted to the isola- 
tion of unique organisms and systematic evaluation of the microbial world for 
its ability to produce novel anticancer agents. The work includes selection 
and screening of microbes, fermentation, fermentation development, genetic and 
culture research, chemical isolation, identification and scale-up production of 
active materials of interest for NCI evaluation. 

The program is using novel and/or improved techniques and unusual substrates to 
obtain unique organisms in an attempt to generate numerous new structural leads. 
In vitro prescreens are also constantly improved or changed with emphasis to- 
wards the use of mammalian cell lines. The objective is to increase the effi- 
ciency of the program in selecting potential leads and to decrease the in vivo 
testing load for primary fermentations. Currently used screens include enzyme 
inhibition, tubulin binding, microbial inhibition, phage induction, DMA binding, 
antimetabolite activity and cytotoxicity against various murine and human cell 
lines (e.g. human bronchioloalveolar carcinoma, L1210 murine leukemia and 
retrovirus transformed rat kidney cells). 

Active fermentations in the prescreens are tested in P388 in vivo and confirmed 
active leads are assigned to chemists for isolation, purification and identifi- 
cation of the active component for NCI evaluation. Further work on the compound 



94 



is based on its activity, novelty of structure, or superiority to the parent 
compound in the case of an analog. The antineoplastic agents are supplied by 
three contractors. This year the prescreens allowed the fermentation contrac- 
tors to evaluate 24,762 new cultures. Over 22% (5476) of these cultures were 
active in one or more of the prescreens. About 49% (2262) of these prescreen 
actives were regrown and retested for secondary screening, of which over 60% 
(1619) were selected for evaluation in vivo against P388, of which 21% (346) 
are active. 

The PRI-FCRF contract has been modified as a shared services contract and is 
being utilized primarily for pilot plant research and development, and for pro- 
duction of active agents in quantities sufficient for toxicology and clinical 
trials. About 1.77 million international units of Erwinia asparaginase have 
been produced, which is equivalent or better in purity than that currently used 
in the clinic. Six grams of 99% virenomycin have been produced to complete 
tumor panel evaluation. Attempts are continuing to produce prodrugs of Frederi- 
camycin. A new contract is being awarded in the area of fungal fermentation, 
which will emphasize the evaluation of fermentations from unusual organisms. 
New projects have been approved in the areas of isolation and fermentation of 
marine microorganisms and blue-green algae. 

Plant and Animal Products Section 

The major function of this program is the acquisition of new active antineoplas- 
tic agents from higher plants and animal products (mainly marine). This has 
largely been achieved by provision of in vivo screening services to NIH grantees 
and interested scientists worldwide, wTiereby high priority leads have been estab- 
lished, while subsequent fractionation and isolation procedures have been aided 
by in vitro bioassay services. Valuable guidance in the collection of new plants 
and marine organisms has been provided by reference to data in the extensive NCI 
files on natural products already tested. The scope of the acquisition program 
will be greatly expanded by the implementation of new plant and marine animal 
collection programs, and specifications for requests for proposals (RFPs) from 
prospective contractors in these areas have been drawn up. Organisms collected 
in these programs will be extracted by a facility under contract to NCI, and 
extracts tested in the new in vitro primary screens based on panels of human 
tumor cell lines. The increased sensitivity of the new screens will have a dra- 
matic impact on the role played by natural products in the drug discovery pro- 
gram in that the number of novel active naturally derived agents will be greatly 
increased. It is planned to establish an in-house isolation group to concen- 
trate on the isolation and purification of compounds exhibiting selective acti- 
vity, while other active leads will be investigated by external research groups 
in collaboration with NCI. As before, pure compounds will be evaluated in NCI 
screens, and any additional quantities required for further investigation will 
be procured by purchase from the investigator, or will be produced by the con- 
tract which is maintained for scale-up isolations. This contract continues to 
provide valuable support in the production of large quantities of plant-derived 
drugs required for preclinical and clinical investigations, including taxol , 
pancrati statin and camptothecin. 

Worldwide Surveillance of Natural Products 

This function is primarily carried out in the Office of the Chief. The objec- 
tive is to acquire new natural products with biological activities which may 

95 



relate to anticancer effects, and compounds of a wide variety of new or unusual 
structural types which are worthy of screening for antitumor activity. The ap- 
proach to identification of compounds for acquisition is multifaceted, and in- 
cludes a contract for literature surveillance which identifies new natural prod- 
uct structures and biological activities, literature review by Branch staff, 
personal contacts with scientists in universities, research institutes and chem- 
ical and pharmaceutical companies, attendance at scientific meetings where new 
compounds are reported, and review of progress reports of NIH grantees. During 
calendar year 1984 a total of 372 new pure natural products were acquired, of 
which 344 (92%) were donated and the remaining 28 compounds (8%) were from 
contractors. 

The literature surveillance program continues to be successful in bringing in 
new materials for screening. In 1984 165 compounds (44% of new acquisitions) 
were received in direct response to requests for those specific compounds, while 
a substantial number of unsolicited compounds received have come from suppliers 
who were contacted by our program previously in regard to other compounds. The 
response rate to our literature requests is 28% with 18% of responders supply- 
ing the requested compounds and another 10% sending regret letters or supplying 
other compounds or making inquiries about the program. 

Additional details are found in Table 2 which shows that foreign suppliers are 
currently the source of 56% of the input. The number of compounds acquired for 
screening is up 22% over last year. 



Table 2. 
Pure Compounds Acquired in Calendar Year 1984 



Plant 

Fermentation 

Animal 



Industry 



Compounds by Type and Source 
Univ. Res. Inst. Other Domestic Foreign 



9 ( 4%) 


167 ( 74%) 


39 (17%) 


9 (4%) 


88 (39%) 


136 (61%) 


55 (58%) 


26 ( 27%) 


13 (14%) 


1 (1%) 


41 (43%) 


54 (57%) 





53 (100%) 








46 (87%) 


7 (13%) 



Plant 

Fermentation 

Animal 



Compounds by Type and Mechanism 
Grant Contract Lit. Sur. Unsol . 



48 (21%) 


1 (<1%) 


113 (50%) 


62 (28%) 


2 ( 2%) 


27 (28%) 


46 (48%) 


20 (21%) 


43 (81%) 





5 ( 9%) 


5 ( 9%) 



96 



Accompi i shments 

A major new program in collection of natural products for screening, extraction 
and storage of extracts, and isolation of active constituents from these extracts 
has been developed and approved as a major new FY '86 initiative of the Division 
of Cancer Treatment. The collection projects include plant collection contracts 
in Africa, Asia and South America, marine organism collections in the Indo- 
Pacific region, a deepwater marine collection project and fermentation projects 
utilizing blue-green algae and marine microorganisms. The first of these con- 
tracts, in the area of fungal fermentation, will be awarded at the end of FY '85 
and is expected to be a valuable source of new bioactive compounds. 

Natural Products have continued to be an important source of compounds in pre- 
clinical development and early clinical studies as outlined in Table 3. 

Collaborative programs have been initiated with outside investigators to develop 
improved second generation drugs which are designed to obviate the problems with 
the parent molecules. One project is underway with Dr. Leon Zalkow of Georgia 
Technical University and Dr. Garth Powis of the Mayo Clinic on analogs of indi- 
cine N-oxide designed to increase antitumor activity and decrease hepatotoxicity. 
Several compounds have been produced which are both more active and more potent 
than the parent and these are now undergoing preliminary hepatotoxicity evalua- 
tion. A second project is being done in the area of camptothecin derivatives 
with Dr. Monroe Wall of the Research Triangle Institute which has resulted in 
discovery of several new compounds with increased activity over the parent 
compound. 

A program to develop more soluble analogs of taxol is a high priority effort 
and two approaches have been taken, one through a collaborative arrangement with 
Dr. David Kingston at Virginia Polytechnic Institute and the second jointly with 
the analog and congener development program of the Drug Synthesis and Chemistry 
Branch. Several compounds from the effort are showing preliminary promise. 

A total of 19 compounds were added to the tumor panel as a result of activity 
in the P388 prescreen or as bypass compounds based on other biological acti- 
vities. Data on several of these are encouraging and we anticipate having two 
or three new Decision Network 2A candidates in the next year for advanced pre- 
clinical development. 

Didemnin B, the first antitumor agent of marine origin, entered clinical trials 
this year as has taxol a potent plant derived antimitotic agent. An INDA fil- 
ing is expected early next year for deoxyspergualin, an interesting polyamine 
analog. 



97 







Table 3. 










Natural Products in Advanced 


Development 




NSC# 


Drug 


Origin 




Status 




163501 


Advicin 


Fermentation 


Phase 


II Clinical 


Trial 


218321 


Pentostatin 


Fermentation 


Phase 


II Clinical 


Trial 


526417 


Echinomycin 


Fermentation 


Phase 


II Clinical 


Trial 


141633 


Homoharrlngtonine 


Plant 


Phase 


II Clinical 


Trial 


125973 


Taxol 


Plant 


Phase 


II Clinical 


Trial 


269148 


Menogaril 


Fermentation 


Phase 


II Clinical 


Trial 


325319 


Didemnin B 


Marine Animal 


Phase 


I Clinical 


Trial 


328426 


Phyllanthoside 


Plant 


Pharmacology 




356894 


Deoxyspergualin 


Fermentation 


Toxicology 




303812 


Aphidicolin Glycinate 


Fermentation 


Pharmacology 




339638 


CI-920 


Fermentation 


Toxicology 




349156 


Pancrati statin 


Plant 


Formulation 




332598 


Rhizoxin 


Fermentation 


Formulation 




364372 


Elactodn* 


Fermentation 


Formulation 





* Passed Decision Network 2A FY '85 



98 



staff Publications 

1. Pettit, G.R., Cragg, G.M., Suffness, M., Gust, D., Boettner, F.E., 
Williams, M., Saenz-Renauld, J. A., Brown, P., Schmidt, J.M., and 
Ellis, P.D.: Isolation and structure of the Phyllanthus acuminatius 
Vahl (Euphorbiaceae) glycosides. J. Org. Chem. 49: 42F8-4266, 1984. 

2. Suffness, M. and Cordell, G.: Antitumor alkaloids. In Brossi, A. (Ed.): 
The Alkaloids , New York, Academic Press, 1985, in press. 

3. Marsh, J.C, Shoemaker, R.H., and Suffness, M.: Stability of in vivo 
P388 leukemia model in evaluation of antitumor activity of natural 
products. Cancer Treat. Rep. 69: 1985, in press. 

4. Foster, B.J., Clagett-Carr, K., Shoemaker, D.D., Suffness, M., Plowman, J., 
Trissel, L.A., Grieshaber, C.K., and Leyland-Jones, B.: Echinomycin: The 
first bifunctional intercalating agent in clinical trials. Invest. New 
Drugs, in press. 



99 



ANNUAL REPORT OF THE ORUG EVALUATION BRANCH 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

Drug Evaluation Branch (DEB) objectives are discovery of new anticancer agents, 
conduct of preclinical tasks essential or complementary to development of new 
drugs to clinical trial, and development of improved methods for drug discovery 
and evaluation. Principal products are recommendations of new drugs to the OCT 
Decision Network Committee, efficacy reports for inclusion in Clinical Bro- 
chures and Investigational New Drug Applications (INDA's), and communication 
of findings to clinical groups and the scientific community orally and via pub- 
lication. Program was implemented through 16 contracts funded at more than 
$6,000,000 (Table 1). This report includes findings through March 31, 1985. 
The contractors contributing to specific findings are cited in the text by 
numbers in parentheses as listed in Table 1. 

Yield of New Agents for Development to Clinical Trial 

Eight agents were referred to the OCT Decision Network (DN) Committee as candi- 
dates for development to clinical trial (Table 2). Reports on the preclinical 
therapeutic activities of Didemnin B (NSC-325319) , Nafidimide (NSC-30a847) , and 
the differentiating agent, HMBA (NSC-95580) were submitted to the Cancer Ther- 
apy Evaluation Program for inclusion in INDA's and Clinical Brochures. Preclin- 
ical findings for Dihydrolenperone (NSC-343513) were submitted to CTEP and to 
the NCI-Navy Medical Oncology Branch to aid in the design of Phase I clinical 
trials. 

In Vivo Screening 

In vivo screening (1-5) was initiated for 10,554 materials (9,770 synthetic and 
784 natural products). Transplantable mouse leukemia P388 remained the initial 
in vivo screen (pre-screen). Three hundred forty materials demonstrated con- 
firmed activity in the pre-screen. Agents active in the pre-screen and materials 
selected to "bypass" the pre-screen because of special interest were tested 
against a spectrum of transplantable mouse tumors. This spectrum of secondary 
in vivo models is being modified concomitant with a progressive decrease in 
the level of in vivo screening and development of a project for screening large 
scale in vitro against human tumor cell lines at the Frederick Cancer Research 
Facility (FCRF). At the beginning of this reporting period, the secondary in 
vivo mouse tumor models included B16 melanoma, L1210 leukemia, M5076 sarcoma, 
and the MX-1 human mammary carcinoma xenograft. Other murine mammary and colon 
tumors have been used selectively. The human amelanotic melanoma xenograft, 
LOX, is being phased into use in lieu of B16. Additional human tumor xenografts 
are being derived from the human tumor cell lines under development (9, 11). 
These will be used selectively for in vivo screening of in vitro active mate- 
rials. Studies related to tumor qulTTity control and est"a5"lishment of in vivo 
testing protocols were initiated for human large cell lung carcinoma H-460, lung 
adenocarcinoma A-549, glioma U-251 and medulloblastoma TE-671 , as well as LOX 



101 



(9, 11). An in vivo model of human promyelocytic leukemia HL-60 was developed 
to complement in vitro studies of differentiation inducers (2). A variant of 
mouse leukemia P388, developed resistant to Adriamycin, has demonstrated pleio- 
tropic resistance to other large molecular weight agents and contains an abnormal 
membrane glycoprotein associated with such resistance (13). 

In Vitro Screening Using Human Tumor Colony Forming Assays 

Emphasis was placed on HTCFA testing of P388 in vivo inactive compounds (6-8). 
Sixty were found active in initial single concentration HTCFA tests and 32 re- 
tained in vitro response rates of >^20% in subsequent dose-response tests. Of 
the latter, 23 were tested in a P388 colony forming assay. Eight were inactive 
suggesting the possibility of differential cytotoxicity to human tumor cells in 
primary culture. Two compounds demonstrated non-uniform (selective) activity 
among the six human tumor types used in the HTCFA. ni hydrolenperone (MSC-343513) 
was selectively active against lung tumors and will begin Phase I clinical trial 
by the NCI-Navy Medical Oncology Branch. NSC-340307 showed some selectivity 
against human breast cancer. Selected compounds, chosen for development on the 
basis of in vivo mouse tumor screens, were tested in the HTCFA to aid in iden- 
tifying those with potential human tumor type selectivity. 

Development of an In Vitro Disease-Oriented Antitumor Drug Screening Model 

Work was initiated in October, 1984 at the FCRF (9) to develop in vitro disease- 
oriented antitumor drug screening models. This project, begun as the DCT Lung 
Cancer Drug Discovery Project, was expanded to include other human and selected 
murine tumors. Essential to this effort is the collaboration of other DCT staff 
including the NCI-Navy Medical Oncology Branch (J. Minna). Over 30 different 
tumor lines have been acquired, expanded in cell culture, and cryopreserved in 
quantities adequate for characterization and initial experimentation. An auto- 
mated growth inhibition assay suitable for large-scale screening is being de- 
veloped. Actual drug screening is planned to begin in October, 1985. Of the 
compounds to be tested, 2,000 will be screened in vivo against P388 as well in 
order to provide comparative information on the two approaches to discovery of 
new antitumor drugs. 

Development of Metastatic Human Tumor Models 

This effort, conducted at FCRF, is closely integrated with the in vitro model 
development work described above. Metastatic strains of several human tumors 
have been selected from tissues (lung, liver, or lymph nodes) after implantation 
of human tumor cells by various routes. Cell strains from metastases of the ag- 
gressively growing LOX tumor appear particularly suited for experimental chemo- 
therapy. Several lung tumors also appear promising for development of metastatic 
models. 

Detailed Drug Evaluation and Congener Development 

Treatment schedule and/or route dependency studies were completed for five clin- 
ical candidate drugs (13). Flavone acetic acid (NSC-347512) activity against 
mouse colon tumor 38 was greatest when a single massive dose was given. Multi- 
ple treatment regimens were less effective, probably related to the slow plasma 
clearance of the drug in mice. IP activity was retained when NSC-347512 was 



102 



given IV. Deoxyspergualin (NSC-356894) activity against SC implanted mouse 
leukemia L1210 was greatest on an intensive, intermittent schedule, qShrs. q4 
days. Experimental formulations were evaluated for six drugs. Results to date 
indicate that the formulations of the Sulfonate derivative, NSC-329680; Tetra- 
carcin, NSC-333856; and the Anthrapyrazole derivative, NSC-349174 retained the 
activity of the bulk drug (13). 

Studies to provide basic information on the cytotoxic and biochemical effects of 
seven antitumor agents being considered for development to clinical trial were 
initiated (14). Major emphasis was placed on elucidating the mechanism of action 
of the polyamine, Deoxyspergualin (nSG). Results have indicated that the cyto- 
toxicity of nSG in vitro requires activation to the aldehyde by monoamine oxi- 
dase or enzyme(syin calf serum. The properties of DSG appear similar to those 
of the endogenous polyamines. Spermine and Spermidine, which also require "ac- 
tivation" to aldehyde by calf serum enzyme(s) to inhibit growth of L1210 cells. 
Labelled precursor incorporation studies showed that the initial effect of ac- 
tivated [)SG was inhibition of DNA synthesis. 

In order to maximize the potential of lead compounds, new congener series were 
evaluated under specialized controlled experimental conditions based on the char- 
acteristics of the parent compound (12). Under this project special studies were 
conducted to answer questions arising during the development of a drug. During 
the past year, the contractor (12) received 470 new compounds for evaluation, in- 
cluding 68 synthesized under contract to ns&CB. Through congener development of 
the Isopropylpyrrolizine derivative, NSC-278214, a highly active agent which was 
dropped from development because of severe solubility and stability problems, a 
congener with equivalent P388 activity and increased aqueous solubility was found. 
NSC-369395, a congener of Camptothecin, with greater activity against L12in has 
an amine substituent which should facilitate formulation. Special studies showed 
that Physostigmine, used in the clinic to alleviate the CNS toxicities of Spiro- 
mustine, did not reverse the anti-P388 activity of the latter. 

Studies of melamines and triazenes, aimed at developing analogs with increased 
therapeutic efficacy were continued (15). Phase I clinical trial of Trimelamol, 
MSC-283162, is nearly complete. Twenty-nine patients have been treated by single 
dose rapid IV infusion e^ery three weeks. Nausea and vomiting, lethargy and 
myel osuppression were observed toxicities. Nausea and vomiting were far less 
severe than previously observed with Pentamethylmel amine (PMH) and, unlike PMM, 
Trimelamol did not cause acute sedation. Evidence of antitumor activity was 
observed in five patients. Rased on further evaluations of the phenyltriazenes, 
the contractor (15) selected l-p-Carboxy-3,3-dimethylphenyltriazene (CB 10-277, 
the sodium salt of NSC-208107) for development to clinical trial. The compound 
was found to be metabolized in both rats and mice to the carboxylic acid glucuro- 
nide of the dimethyltriazene. No evidence was found for conversion to the mono- 
methyl derivative; the monomethyl compounds are believed to be the active species 
of the carboxamido triazenes including DTIC. Presumably NSC-208107 does not re- 
quire N-demethylation as it demonstrated antitumor activity against two human 
melanoma xenografts. 

The Platinum Analog Working Group continued to coordinate the development of Tet- 
raplatin, NSC-363812. This Cisplatin analog, discovered by DTP, retains antitumor 
activity against animal tumors with acquired resistance to Cisplatin, is suffi- 
ciently soluble and stable for parenteral use, and is less nephrotoxic than Cis- 
platin in rats (C. Litterst et al_, DTP intramural laboratories). 

103 



National Cooperative Drug Discovery Groups 

Eight applications were received in response to reissuance of the RFA for NCDDG 
Cooperative Agreements, (wo were recommended for first year funding in FY 1985. 
The new five year awards are aiiTied at investigation of (a) topoi somerases as new 
therapeutic targets (W. E. Ross, University of Florida, P. I.); and (b) antire- 
ceptor monoclonal antibodies in cancer treatment (J. Mendelsohn, Hemorial Sloan- 
Kettering Cancer Center, P. I.). These added to the two existing Groups (first 
year funding in FY 1984) will give us four NCOnG's comprising 20 laboratory 
projects in 14 academic, research, and industrial organizations. Anticipated 
FY 1985 funding for the four groups is over $2,000,000. 

A distinguishing feature of Cooperative Agreements is the requirement for active 
participation of NCI staff during performance. DEB collaboration with the Group 
devoted to the synthesis of specific inhibitors of viral oncogene-encoded tyro- 
sine specific protein kinases (V. Levin, University of California, San Francisco, 
P.I.) included identification of 284 candidate compounds which have been sub- 
mitted to our antitumor screening program which have the desired structural 
characteristics of potential inhibitors. OEB collaboration with the Group (C. 
Porter, Roswell Park Memorial Institute, P.I.) devoted to the discovery of in- 
hibitors of polyamine biosynthesis as potential antitumor agents included (a) 
preclinical antitumor evaluation of Difluoromethylornithine (OFMO), an irrever- 
sible inhibitor of ornithine decarboxylase, in combination with AraC based on 
reports that DFMO arrested LI 210 cells and transformed SV3T3 cells in S-phase 
while mouse bone marrow and normal 3T3 cells were arrested i n Gl ; and (b) pre- 
sentation of Deoxyspergualin, a homolog of Spermidine, to the Group as a poten- 
tial inhibitor of polyamine biosynthesis with demonstrated in vivo antitumor 
activity (see earlier discussion of nSG). 

Publications by Staff 

1. Geran, R. I.: Evaluation of potential antineoplastic agents. In Ottenbrite, 
R. M. and Butler, G. B. (Eds.): Anticancer and Interferon Agents: Synthesis 
and Properties . New York, Mercel Oekker, Inc., 1984, pp. 25-49. 

2. Shoemaker, R., Wolpert-DeFilippes, M., Melnick, N., Venditti, J., Simon, R., 
Kern, D., Lieber, M., Miller, W., Salmon, S., and Von Hoff, P.: Recent re- 
sults of new drug screening trials with a human tumor colony forming assay. 
In Salmon, S. and Trent, J. (Eds.): Human Tumor Cloning . Florida, Grune S 
Stratton, 1984, pp. 345-355. 

3. Shoemaker, R. H., Wolpert-DeFilippes, M. K., and Venditti, J. M.: Potentials 
and drawbacks of the human tumor stem cell assay. Behring Inst. Mitt. : 74, 
262-272, 1984. 

4. Venditti, J. M., Wesley, R. A., and Plowman, J.: Current NCI pre-clinical 
antitumor screening in vivo. Results of tumor panel screening, 1976-1982 
and future directions. In Garattini, S., Goldin, A., Hawking, F. (Eds.): 
Advances in Pharmacology and Chemotherapy 20. Florida, Academic Press, 
1984, pp. 1-20. 

5. Goldin, A., Venditti, J. M., and Geran, R.: The effectiveness of the anthra- 
cycline analog 4'-epidoxorubicin in the treatment of experimental tumors. 
Invest. New Drugs 3: 3-21, 1985. 

104 



6. Shoemaker, R. H., Wolpert-OeFilippes, M. K., Kern, D. H., Lieber, M. M., 
Makuch, R. W., Melnick, N. R., Miller, W. T. , Salmon, S. K., Simon, R. M. , 
Venditti, J, M,, and Von Hoff, D. D.: Application of a human tumor colony 
forming assay to new drug screening. Cancer Res . 45: 2145-2153, 1985. 

7. Abbott, R. J.: In vitro screening systems. Principles of Cancer Chemo- 
therapy (in press). 

8. Marsh, J. C, Shoemaker, R. H., and Suffness, M.: Stability of the in vivo 
P388 leukemia model in evaluation of antitumor activity of natural products. 
Cancer Treat. Rep, (in press). 

9. Weisenthal, L. M. , Shoemaker, R. H., Marsden, J. A., Dill, P. L,, Raker, 
J, A., and Moran, E. M.: In vitro chemosensiti vity assay based on the 
concept of total tumor cell kill. Recent Results in Cancer Research (in 
press). 

Publications by Contractors 

1. Corbett, T. H., Roberts, R. J., Leopold, W. R., Peckham, J. C. , Wilkoff, 

L. J., Griswold, D. P., and Schabel , F. M.: Induction and chemotherapeutic 
response of two transplantable ductal adenocarcinomas of the pancreas in 
C57R1/6 mice. Cancer Res. 44: 717-726,1984. 

2. Dumont, P, Van Der Esch, E., Jabri , M., Lejeune, F. , and Atassi , G.: 
Chemosensiti vity of human melanoma xenografts in immunocompetent mice 
and its histological confirmation. Int. J. Cancer 33: 447-451, 1984. 

3. Griswold, n. P., Laster, W. R., Trader, M. W., and Dykes, n. J.: The 
preclinical scientific basis for adjuvant chemotherapy in breast cancer. 
In Senn, H. J. (Ed.): Recent Results in Cancer Research 96. Rerlin, 
Springer-Verlag, 1984, pp. 1-7. 

4. Houchens, 0. P. and Ovejera, A. A.: Heterotransplantation Models for 
Screening - Current Status and Future Prospects. Principles of Cancer 
Chemotherapy (in press). 

5. Simpson-Herren, L.: Autographic Techniques for Measurement of the 
Labeling Index. Techniques for Analysis of Cell Proliferation (in 
press). 



105 



TABLE 1 

CONTRACTS MANAGED BY DEB AND FUNHING LEVELS, FY 1985 

In Vivo Screening $3.140.458 

1. Southern Research Institute (4-7646) 780,033 

2. Mason Research Institute (4-7647) 767,500 

3. Battel le-Columbus Laboratories (4-7648) 661,911 

4. IIT Research Institute (4-7567) 780,802 

5. Institut Jules Bordet, Belgium (5-7645) 150,212 

In Vitro Screening (HTCFA) $ 661 ,875 

6. UCLA (5-7710) 187,079 

7. Mayo Foundation (5-7711) 216,015 

8. University of Arizona (5-7662) 258,781 

New Model Development $ 680,000 

9. PRI-FCRF 

In Vitro Human Tumor Cell Lines 440,000 

Discovery of New Biological Models 90,000 

A&E Phase I Construction 150,000 

10. University of California, San Diego (3-7568) 0* 
Tumor Quality Control $ 301 ,725 

11. Southern Research Institute (4-7581) 301,725 
Detailed Drug Evaluation $1 .076,504 

12. Southern Research Institute (3-7552) 286,374 

13. Southern Research Institute (4-7580) 611,630 

14. Southern Research Institute (4-7615) 178,500 

15. Institute of Cancer Research, England (4-3736) O"*" 

Support Services $ 216.366 

16. S.B.A. Biotech (3-7558) 216,366 

TOTAL $6,076,928 

♦Terminated August 31. 1985. 
^Terminated June 30. 1985. 



106 



TABLE 2 

COMPOlJNnS SELECTEn FOR nEVELOPMENT 

APRIL 1 , 1984 - MARCH 31, 1985 

(DECISION NETWORK 2A) 



NSC No. 



Name 



Assignment 
Date 



Selection Basis* 



95678 


3-Hydroxy-2-formylpyridi ne 
thiosemicarbazone (3-HP) 


84-06-12 


291643 


Pyrimidineacetaldehyde 
derivative 


84-08-14 


326231 


L-Buthionine Sulfoximine 


84-06-12 


3390040 


(Discreet) 




84-06-12 


339736D 


(Discreet) 




84-06-12 


343513 


Dihydrolenperone 




84-04-10 


3614560 


(Discreet) 




84-12-05 



364372 



Elactocin 



84-08-14 



(Project to Review 
Old Drugs) 

IP LI 210 Leukemia 
IP M5076 Sarcoma 
SRC MX-1 Mammary 
Tumor Xenograft 

(Radiosensitizer 
Chemosensitizer) 

(Human Tumor Colony 
Forming Assay) 

(Human Tumor Colony 
Forming Assay) 

(Human Tumor Colony 
Forming Assay) 

IP B16 Melanoma 
IP LI 210 Leukemia 
IP M5076 Sarcoma 
SRC MX-1 Mammary 
Tumor Xenograft 

IP B16 Melanoma 
IP M5076 Sarcoma 



* Tumor panel systems in which a compound (non-formulated product) has demon- 
strated sufficient in vivo antitumor activity to warrant consideration for 
development to clinical trial (ON 2 activity). Information in parenthesis 
indicates reason for passing ON 2A other than in vivo activity in a tumor 
panel model . 



107 



ANNUAL REPORT OF THE ANIMAL GENETICS & PRODUCTION BRANCH 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 



The primary function of the Animal Genetics and Production Branch (AG&PB), 
Developmental Therapeutics Program (DTP), Division of Cancer Treatment (DCT), ^ 
is to provide healthy laboratory animals with properly defined genetic charac- 
teristics to various research investigators as follows: (1) DTP Screening 
programs, tumor panel, and Toxicology Program; (2) other NCI research contracts 
(DCCP, DCBD, etc.); (3) NIH and FCRF Intramural Programs; (4) Research Grants; 
(5) NIEHS, and (6) Veterans Administration Research facilities (surplus animals 
which are offered when available). 

The AG&PB is responsible for the proper functioning of the Tumor Repository 
which includes: 

1. Scheduled distribution of human/animal tumors to DTP contract screening 
and associated laboratories. 

2. Distribution of tumors to qualified cancer research investigators upon 
request. 

3. Monitoring tumor lines maintained by the Repository and contract screening 
laboratories for pathogenic contaminants. 

4. Performing pilot studies with potentially metastatic human/animal tumors to 
evaluate their potential for DTP Program usage. 

The AG&PB assists the Toxicology Branch by assuring the production and distribu- 
tion of adequate numbers of rodents which meet specified standards for their 
program. 

The AG&PB is responsible for staffing and maintaining the DCT intramural Barrier 
Facility in Building 37. 

The objectives of the AG&PB are to: 

1. Continue to provide laboratory animals of the quality (from both an animal 
health and genetic integrity viewpoint) and quantity to meet the needs of the 
various programs using these services. 

2. To upgrade experimental tumor monitoring services and provide necessary mani- 
pulations to assure that those tumors of importance to NCI Research Programs: 



A. Are free of pathogenic contamination 



109 



B. Meet individual tumor performance standards, e.g., growth rate, life 
span, metastatic potential, etc. 

C. Meet individual tumor characterization requirements including the 
usage of histology, karyotyping, isozyme electrophoretic studies, etc., 
as needed. 

3. To provide metastatic human/murine tumor models which offer potential for 
usage on DTP tumor panel and other DCT cancer research programs. 

4. To maintain the Building 37 barrier facility in a fashion that will: 

A. Meet investigator requirements 

B. Exclude pathogenic contaminants 

C. Meet all accreditation requirements 

5. To provide assurance that the FCRF Animal Production Facility continues to 
provide laboratory animals of superior quality and remains cost effective. 

6. To assist the DTP in upgrading extramural contractor usage laboratory fa- 
cilities and management to the extent that experimental animals can be main- 
tained in the same pathogen free status as received from the AG&PB. 

Accomplishments: 

1. The AG&PB has adjusted to changing needs of DTP programs (e.g., tumor panel 
changes) and to unexpected delays in completing the phasing-out of several 
tumor panel models to the extent that screening laboratories have accom- 
plished requested testing in a timely fashion. 

2. Changing needs of other users including new strain requirements (primarily 
FCRF intramural) have been handled expeditiously. 

3. A program has been instigated to study the feasibility/cost efficiency of 
upgrading conventional laboratory facilities to a pathogen exclusion status. 

4. Experimental tumor lines for DTP programs have been delivered as scheduled 
by DEB/AG&PB staff. 

5. A pilot study regarding the practicability of freezing fertilized mouse 
embryos has progressed to the extent that strains that are currently not in 
usage, e.g., RFM/Un, but where future demand is a probability, may be alter- 
natively "stored" in this fashion. 



no 



ANIMAL GENETICS & PRODUCTION BRANCH PROGRAM FUNDING 
FY 1985 



PRIMARY GENETIC CENTERS (4) $4,703.000 

Supply breeding nucleus for the animal 
program and athymic mice for drug 
evaluation. 

RODENT PRODUCTION CENTERS (5) 767,000 

Large-scale production of inbred mice and 
nude mice under both conventional and barrier 
controlled environment. 

HYBRID MOUSE PRODUCTION CENTERS (6) 4,000 

Supply hybrid mice for the screening program. 
These contracts terminated January 31, 1985 and 
were not renewed. 

DIAGNOSTIC & HISTOCOMPATIBILITY PROJECTS (8) 901,000 

To monitor animal health and genetic integrity. 

DEVELOPMENT OF STANDARDS & GUIDELINES (1) 34,000 

For animal care and breeding. 

MAINTENANCE OF FROZEN TUMOR BANK (1) 300,000 

FREDERICK CANCER RESEARCH FACILITY (2) 3,308,000 

CENTRALIZED REDERIVATION (1) 105,000 

Rederiving new starts from the NIH Repository 
into associated flora status. 

TOTAL $10,122,000 

Less Reimbursements (Including Grantee Collection) 3,192,000 

111 



ANNUAL REPORT OF THE PHARMACEUTICAL RESOURCES BRANCH 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

The Pharmaceutical Resources Branch (PRB) is structured to provide comprehensive 
pharmaceutical services to the various Programs of the Division of Cancer Treat- 
ment. The primary objectives of the Branch are to supply high quality chemical 
substances and formulated products for investigative program use. These objec- 
tives are accomplished essentially through contract support activities. The 
Branch supervises a total of 22 contracts with a combined annual budget of 
approximately 6.9 million dollars. 

The major contract areas include: chemical preparation and pilot plant produc- 
tion; analytical services; pharmaceutical research and development; and pharma- 
ceutical manufacturing. The synthesis and distribution of radiolabeled chemi- 
cals and drugs are also provided through PRB contract sources. Additionally, 
the Branch is responsible for shelf life surveillance, storage and distribution, 
and computerized inventory maintenance of all drug products used in the Clinical 
Programs of the Division of Cancer Treatment (DCT). 

A formulation research laboratory on the NIH campus is operated and staffed by 
the Pharmaceutical Resources Branch. This laboratory is assigned research 
projects of high Program interest which present difficult drug delivery prob- 
lems. Most of the chemical agents developed by the Laboratory of Pharmacology 
and Experimental Therapeutics are assigned to the formulation laboratory. This 
arrangement facilitates a scientific exchange between the developer and formu- 
lator and a team approach to the ultimate product design. 

Staff 

The Pharmaceutical Resources Branch is presently staffed with six senior profes- 
sionals, one technical and two secretarial personnel. In addition, two visiting 
visiting fellows are assigned to the formulation laboratory. The classification 
of the senior professionals is as follows: four PHS Commissioned Corps Pharma- 
cists; one Ph.D. analytical chemist; and one Ph.D. medicinal chemist. 

The Branch consists of three Sections: 

1 . Chemical Resources Sectio n 

The primary functions of the Chemical Resources Section are to provide for re- 
synthesis, large-scale production and procurement services for the acquisition 
of chemical substances. These services are accomplished by the management and 
supervision of a contract program for resynthesis and pilot plant production of 
various quantities of bulk substances intended for tumor screening panels, pre- 
clinical toxicology and pharmaceutical manufacture of investigational products 
for clinical evaluation. 



113 



Another major function of the Section involves the management of a contract 
program to prepare and distribute radiolabeled materials. These materials are 
distributed to authorized investigators for clinical pharmacology and other 
related studies. 

The chemical and radiolabel preparation laboratories, taken collectively, pro- 
vide the means of obtaining a broad variety of chemical compounds and the 
ability of providing large quantities of high purity drug substances. The 
Section supervises seven chemical prep lab contracts consisting of a combined 
annual effort of approximately 34 man years. Additionally, two radiosynthesis 
laboratories are supervised by the Chemical Resources Section with a combined 
annual effort of 5.5 man years. 

2. Analytical and Product Development Section 

This Section has two major functions: (1) the analytical assessment of chemi- 
cals and fonnulated products and (2) the development of investigational pharma- 
ceutical dosage forms for clinical trial. 

The analytical component of the Section supervises contractors engaged in the 
development of analytical methodology to determine the purity of chemicals, 
potency of active ingredients in pharmaceutical formulations, stability of' 
formulated products under accelerated and simulated use conditions, and identi- 
fication of impurities and/or degradation products. The Section's staff pre- 
pares bulk chemical specifications used for acceptance criteria of additional 
supplies either from commercial sources or chemical preparation contractors. 
The specifications and validated analytical methodology are prepared in a format 
suitable for, submission to the Food and Drug Administration as part of the NCI's 
Investigational New Drug Application. The Section is also responsible for chem- 
ical evaluation of new investigational dosage forms and for the shelf life sur- 
veillance of all formulated investigational products. 

Presently, the Section's analytical chemist supervises three analytical con- 
tracts representing a combined 18 staff year effort and a shelf life surveil- 
lance contract for clinical products involving an annual three and one-half 
staff year effort. These contractors have the expertise to chemically charac- 
terize a very structurally diverse group of chemicals. These contractors are 
also responsible for the development and application of stability-indicating 
methods for all new drug substances. 

The dosage form development component of the Section is responsible for conver- 
sion of bulk chemical into a pharmaceutical product suitable for clinical use. 
Essentially all products are developed for intravenous use. About one-half of 
the drugs do not exhibit adequate solubility or stability and some form of 
pharmaceutical intervention is required. Standard approaches (salts, solvents 
and surfactants) are initially evaluated. Emphasis is also given to evaluation 
of newer techniques to improve solubility or stability (emulsions, prodrugs and 
complexation). The developed dosage form is evaluated for chemical content, 
antitumor activity in the rodent screen and feasibility to be manufactured 
on production scale. 

Most of the product development effort is conducted under contract with the Sec- 
tion staff serving as project monitors. In addition, an intramural formulation 

114 



laboratory, supervised and maintained by this Section, is investigating 
methods to solve drug formulation problems. 

The Analytical and Product Development Section is responsible for the super- 
vision and management of eight contracts: three analytical contracts with a 
combined annual effort of 18 man years; three pharmaceuticdl R 4 D contracts 
with a combined annual effort of eight man years; one combined R&D (one and 
one-half man years) and pharmaceutical contract; and one shelf life contract 
involving an annual three and one-half man year effort. 

Analytical data developed on new investigational compounds is assembled and 
published in a book entitled " NCI Investigational Drugs - Chemical Information ." 
This text contains stability-indicating methods, spectral data, approximate 
solubility and stability data, and other appropriate information on a large 
number of agents. The publication is distributed to investigators throughout 
the country. 

3. Clinical Products Section 

The Clinical Products Section manages four pharmaceutical mission type contracts 
with capabilities to produce a broad variety of pharmaceutical products. The 
Section also manages a storage and distribution contract with computer capabil- 
ities for accurate accountability of the disposition of all investigational 
products. 

In addition, this Section manages a sizeable intramural budget for the direct 
purchase of chemicals and formulated products. During this report period, drug 
purchase expenditures were in excess of 2.0 million dollars. A significant 
amount of staff time is expended in this area in preparing purchase specifica- 
tions, award justifications and periodic budget projections throughout the year. 
Several types of NIH contract mechanisms are utilized for these procurement 
actions including bids from suppliers, blanket purchase orders, direct purchase 
contracts, etc. 

Investigational product literature in the form of Investigational Drug - Pharma- 
ceutical Data sheets is prepared by the Section. These information sheets are 
also supplied in bound book form (NIH Publication No. 85-2141) which is updated 
periodically. During this reporting period, over 4,000 issues were distributed. 

Goals and Accomplishments 

The Pharmaceutical Resources Branch has successfully accomplished its objectives 
in providing high quality bulk chemicals and pharmaceutical products to the 
various Programs in the Division of Cancer Treatment. During this reporting 
period, the prep lab contractors prepared over 181 compounds totaling more than 
100 kilograms. Examples of bulk pharmaceutical substances which when delivered 
for formulation Included: Carboplatin (NSC-241240); 4-Ipomeanol (NSC-349438); 
Methotrexate (NSC-740); Ara-AC (NSC-281272); Fludarabine (NSC-312887) ; and L- 
Buthionine-S, R-sulfoximine (BSO) (NSC-326231 ). The Clinical Products Section 
directed the contract production of more than 450,000 injectable units and more 
than 880,000 oral dosages for clinical distribution. 



115 



The pharmaceutical research and development activities were also quite success- 
ful in completing development of nine formulation assignments including paren- 
teral products of Tetraplatin {NSC-363812); Pyrazole (NSC-45410); 4-Ipomeanol 
(NSC-349438); Anthrapyrazole (NSC-349174) ; and others. 

Many of the new agents submitted for development present formulation problems 
due to poor aqueous solubility and/or stability. In view of these problems, 
the Pharmaceutical Resources Branch submitted a Request for Proposal to the 
Small Business Incentive Research (SBIR) Program for research proposals in the 
area of Novel Drug Formulation and Delivery Systems. Nine proposals were re- 
ceived and multiple awards are expected. An additional Request for Proposal 
was submitted under this same Program for a Computerized Pharmaceutical Tracking 
System which is expected to provide historical and control data on all products 
under investigation. Seven proposals were received for the project and multiple 
awards may be made. 

Also during this reporting period, the Clinical Products Section became increas- 
ingly involved with the management of acquisition and distribution of biological 
products for the Biological Response Modifiers Program (BRMP). More than 20 
BRMs have been received under this project and many more are expected in the 
next several months. The products received during this period included Inter- 
leukin-2; Thymosin; Alpha-Interferon; Lymphoblastoid Interferon; and Monoclonal 
Antibodies. The Pharmaceutical Resources Branch was also involved in providing 
pharmaceutical support for the Division of Cancer Prevention and Control (DCPC). 

During this reporting period, the Pharmaceutical Resources Branch participated 
in the newly formed NCI/European Collaborative Program which is an experimental 
scientist exchange program. Two scientists. Dr. John Slack from the University 
of Aston in Birmingham, England and Ms. Pamela Warrington, Senior Oncology 
Pharmacist from Edinburgh, Scotland, spent four weeks and three weeks, respect- 
ively, with PRB staff learning pharmaceutical aspects of investigational drugs. 

Publications by Staff 

1. Cheung, W. Y., Vishnuvajjala, B. R., and Flora, K. P.: Stability of 
cytarabine, methotrexate sodium, and hydrocortisone sodium succinate 
admixtures. Am. J. Hosp. Pharm . 41: 1802-1806, 1984. 

2. Cheung, W. Y., Vishnuvajjala, B. R., Morris, N. L., and Flora, K. P.: 
Stability of azacitidine in infusion fluids. Am. J. Hosp. Pharm. 41: 
1156-1159, 1984. 

3. Davignon, J. P. and Cradock, J. C: Pharmaceutical aspects of antitumor 
agents. Pharmaceutisch Weekblad 46: 1144-1150, 1984. 

4. McCarthy, L. E., Flora, K. P., and Vishnuvajjala, B. R.: Disparaties in 
the antiemetic and behavorial actions of delta-9-tetrahydrocannabinol and 
its 11-OH-metabolite in the cat. Res. Comm. Sub. Abuse 5: 103-114, 1984. 

5. McCarthy, L. E., Vishnuvajjala, B. R. , and Flora, K. P.: Antiemetic 
properties and plasma concentrations of delta-9-tetrahydrocannabinol 
against cisplatin vomiting in cats. In Agurell, S., Dewey, W. L., and 



116 



Willette, R. E. (Eds.): The Cannabinoids; Chemical, Pharmacologic, and 
Therapeutic Aspects . New York, Academic, 1984, 909 pp. 

6. Vishnuvajjala, B. R. and Cradock, J. C: Compatibility of plastic 
infusion devices with diluted N-methylformamide and N,N-dimethylacetamide. 
Am. J. Hosp. Pharm . 41: 1160-1163, 1984. 

7. O'Dwyer, P. J., Shoemaker, D. D., Plowman, J., Cradock, J. C, Grill o- 
Lopez, A., and Leyland-Jones, B.: Trimetrexate: a new antifol entering 
clinical trials. Investiga. New Drugs 3; 71-75, 1985. 

8. Perlin, E., Smith, C. G., Nichols, A. I., Almirez, R., Flora, K. P., 
Cradock, J. C, and Peck, C. C: Disposition and bioavailability of 
various formulations of tetrahydrocannabinol in the rhesus monkey. 
J. Pharm. Sci . 74: 171-174, 1985. 

9. Trissel, L. A., Davignon, J. P., Kleinman, L. M., Cradock, J. C, and 
Flora, K. P.: NCI Investigational Drugs - Pharmaceutical Data 1985 . 
NIH Publ. No. 85-2141, 1985, 192 pp. 



117 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl-CM-03584-13 PRB 



PERIOD COVERED 

October 1. 1984 to September 30. 1985 



TITLE OF PROJECT {80 characters or less. Title must lit on one line between the borders.) 

Research in the Development of Dosage Forms of New Antitumor Drugs 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: James C. Cradock 
Other: Karl P. Flora 

Babu R. Vishnuvajjala 

Yuen Cheung 



Head A&PDS 

Chemist A&PDS 
Visiting Assoc. A&PDS 
Visiting Fellow A&PDS 



PRB 


NCI 


PRB 


NCI 


PRB 


NCI 


PRB 


NCI 



COOPERATING UNITS (if any) 



Dr. Charles Litterst, DTP, DCT, NCI 



LAB/BRANCH 



Pharmaceutical Resources Branch 



Analytical and Product Development Section 



INSTITUTE AND LOCATION 



NCI. NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 



1.5 



PROFESSIONAL; 



1.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



n (b) Human tissues \3 (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project describes the activities of the formulation laboratory of 
the Pharmaceutical Resources Branch. These studies are directed toward 
resolving problems in the intravenous delivery of antitumor agents and 
involve methods to evaluate and then improve drug solubility and stability. 
Pharmaceutical aspects of the new platinum analogue, tetraplatin, were 
examined. Stability-indicating HPLC methods were developed and applied 
to an evaluation of this compound under a variety of conditions and a 
freeze dried dosage form was developed. 

The safety of certain antitumor agents has been a topic of considerable 
concern to personnel involved with the handling and administration of 
these compounds. Procedures to decompose (two-three log reduction) 
fourteen compounds have been developed using simple procedures: base, 
hydrogen peroxide, and/or heat. These compounds represent most of the 
chemical categories of clinically active antitumor agents. 

Methods to improve drug solubility have also been evaluated. Several 
water soluble prodrugs of camptothecin have been prepared and evaluated 
in suitable tumor models. The N,N-diethyl glycine derivative exhibited 
activity and potency similar to that demonstrated by camptothecin. 
Also the influence of the polymer, poly 1 -methionine sulfoxide on the 
solubility of several antitumor agents was studied. 



118 



PHS 6040 (Rev. 1/84) 



ZOl-CM-03584-13 PRB 

1. Stability, Formulation Development and Pharmacology of Tetraplatin 
(Cheung, Vishnuvajjala, Flora, Cradock and Litterst). 

Tetraplatin (NSC-363812) is a new antitumor platinum derivative under 
development in the Developmental Therapeutics Program. The rationale 
for pursuing the eventual clinical trials of this complex lies mainly 
in its activity in cisplatin resistant L1210 murine tumor systems. The 
predecessor to this compound was NSC-271674 a DACH Pt with a tricarboxy- 
benzene leaving group. This compound was not amenable to analysis by 
any of the usual analytical assay techniques. In addition, there was 
substantial evidence that the compound was extremely unstable in aqueous 
solution. Tetraplatin, however, can be assayed by HPLC and charac- 
terized by other standard analytical techniques. Although aqueous 
instability is apparent for tetraplatin, it is possible to significantly 
inhibit the decomposition of the intact drug by adjusting the chloride 
content of solutions. A stability-indicating HPLC method was developed 
to monitor the drug in various solutions and infusion fluids. The 
influence of pH on drug stability was studied in drug solutions 
(0.9 mg/ml) buffered at pH values between 3.0 and 7.0. The rate of 
decomposition of tetraplatin increases as the solution pH decreases. 
Solutions of tetraplatin in distilled water. Sterile Water for Injection 
and 5% Dextrose Injection show concentration-related differences in the 
rate of decomposition. The magnitude and time course of these differences 
has been somewhat erratic and irreproducible. However, simultaneous 
monitoring of chloride ion release using a chloride selective electrode 
in conjunction with the HPLC assay of the same solution indicate that the 
observed differences are real and are not an artifact of the chromato- 
graphic assay procedure. Studies on the stability of these solutions are 
ongoing. 

The stability of tetraplatin is greatly enhanced in chloride containing 
solutions. Drug concentration effects on stability are less marked. 
Drug concentrations of 0.1 - 5 mg/ml in 0.9% sodium chloride show less 
than 10% decomposition in 6 hours in all situations and are suitable 
for administration by either infusion or direct intravenous injection. 
Tetraplatin can be freeze dried from solutions containing sodium chloride 
and mannitol . 

Dr. Litterst and his colleagues are evaluating the pharmacology of tetra- 
platin in rodents. We are collaborating on a method to detect drug and 
metabolites in biological samples. This procedure will use HPLC separation 
and analysis of the eluates for platinum by atomic absorption. The ultra- 
violet absorption of tetraplatin is inadequate at concentrations found in 
biologic samples to use conventional HPLC separation and UV detection. 
Preliminary protein binding studies have been conducted and other plasma 
level measurements are being pursued. 

2. Methods for the Forced Decomposition and Decontamination of Residues of 
Formulated Antitumor Agents (Cheung, Vishnuvajjala, Cradock and Flora). 



119 



ZOl-CM-03584-13 PRB 

The Pharmaceutical Resources Branch, Developmental Therapeutics Program, 
Division of Cancer Treatment, National Cancer Institute is responsible for 
producing and supplying formulated antitumor agents for clinical studies. 
In recent years some medical personnel have expressed concern regarding 
the handling and disposal of these compounds which may be potential 
mutagens or carcinogens. 

We have recently evaluated a number of drugs in hopes of providing simple 
procedures to rapidly decompose the drug entity as it exists in the dosage 
form. It is hoped that these procedures will aid in the safe and efficient 
disposal of these agents. Also these procedures may be useful in the 
decontamination and clean-up of inadvertent spills in hospital and clinic 
settings. 

Results have been obtained on fourteen drugs. Decomposition is forced by 
the use of sodium hydroxide, hydrogen peroxide or heat or combinations of 
the above. The disappearance of drug was monitored by high performance 
liquid chromatography (HPLC) in all cases. The target for each drug was 
to force decomposition to the point that no HPLC peak for the intact drug 
was observed (2-3 log reduction). Using these procedures the following 
drugs have been decomposed: Adriamycin, Daunorubicin, Aclarubicin, 
Amsacrine, Menogaril, Mitomycin, Dactinomycin, Diaziquone, PCNU, BCNU, 
Azacitidine, Melphalan, and Mitoxantrone. Ames testing will be performed 
on each of the intact formulations and on the decomposed solutions to 
determine if the chemical treatments are associated with a comparable 
loss of biologic activity. 

3. Camptothecin (Drs. Vishnuvajjala, Cheung, Flora and Cradock). 

Camptothecin is a poorly water soluble natural product that is very active 
in experimental tumor models. The sodium salt of the lactone is active 
but at tenfold higher doses. Several prodrugs of camptothecin were pre- 
pared that have greater water solubility but retain the lactone ring. 

The new derivatives involve esterification with substituted amino acids 
of the hydroxyl group at position 20. Hydrochloride salts were then 
prepared from the bases. Five prodrugs were prepared, characterized 
chemically and found to be the expected structure with a purity by TLC 
and HPLC of > 95%. Solubilities ranged from 2 to 8 mg/ml and were 
adequate to deliver the projected doses. Antitumor data on the N,N 
diethyl glycine derivative indicates that the activity is comparable to 
camptothecin at similar doses. The other derivatives are substantially 
less potent than camptothecin. 

A HPLC method was developed to determine concentrations of the N,N- 
diethylglycine derivative in plasma. Studies to evaluate the rate of 
reversion of the ester to camptothecin were conducted in out-dated 
human plasma and in a solution containing a non-specific esterase. A 
paraben ester was used as a positive control in the non-specific 
esterase solution study. No significant ester hydrolysis was noted 
after incubation at 37° for 1 to 2 hours in either matrix. A small 
study in mice is planned to see if any significant conversion takes 
place in vivo. 



120 



ZOl-CM-03584-13 PRB 
Several small projects were also carried out during this period. Formu- 
lation work on the antitumor agent diaziquone was completed in this 
laboratory several years ago using 5% di methyl acetamide (DMA) to achieve 
the required solubility. Diaziquone has subsequently undergone extensive 
clinical trial mostly by the intravenous route but has also been tested 
intrathecal ly. Recently Dr. Poplack, COP, NCI, noted elevations of inyelin 
basic protein in humans receiving diaziquone to treat CNS leukemia and in 
monkeys injected with the DMA containing vehicle. The product was 
reevaluated in an attempt to reduce or eliminate this solvent. The same 
drug solubility could be achieved by reducing the DMA content by one-half 
or substituting DMSO at one-fifth the amount. However, adequate solubility 
could not be achieved in the absence of an organic solvent. The DMSO 
formulation will likely be evaluated intraventricularly in monkeys. 

Poly 1 -methionine sulfoxide was reported by others to improve the water 
solubility of several compounds. The influence of this polymer on the 
solubility of several poorly water soluble antitumor agents was evaluated. 
Pharmaceutical ly significant solubility increases have not been observed. 

Publications 

1. Cheung, Y.W., Vishnuvajjala, B.R., Morris, N.L. and Flora, K.P.. The 
Stability of Azacitidine in Infusion Fluids. Am. J. Hosp. Pharm. 

41: 1156-1159 (1984). 

2. Cheung, Y.W. , Vishnuvajjala, B.R. and Flora, K.P.. Stability of an 
Admixture of Cytarabine, Methotrexate and Hydrocortisone Sodium 
Succinate in Infusion Fluids. Am. J. Hosp. Pharm. 41: 1802-1806 (1984). 

3. Tabibi, S.E. and Cradock, J.C.. Stability of Melphalan in Infusion 
Fluids. Am. J. Hosp. Pharm. 41: 1380-1382 (1984). 

4. Vishnuvajjala, B.R. and Cradock, J.C. Compatability of Plastic Infusion 
Devices with N-Methylformamide and Dimethyl acetamide Containing Solutions. 
Am. J. Hosp. Pharm. 41: 1160-1163 (1984). 

5. McCarthy, L.E., Flora, K.P., Vishnuvajjala, B.R.. Disparities in the 
Antiemetic and Behavioral Actions of Delta-9-Tetrahydrocannabinol and 

its 11-OH Metabolite in the Cat. Res. Comm. Sub. Abuse. ^:103-114 (1984). 

6. Ray, G., Crook, M., West, N. , Kwoka, M., Rehagen, G., Cox, J., Murrill, 
E. and Flora, K.. Comparison of the Analysis of Delta-9-Tetrahydro- 
cannabinol Capsules by High-Performance Liquid Chromatography and 
Capillary Gas Chromatography. J. Chromatogr. 317:455-462 (1984). 

7. Perlin, E., Smith, C.G., Nichols, A. I., Almirez, R., Flora, K.P., 
Cradock, J.C. and Peck, C.C. Disposition and Bioavailability of Various 
Formulations of Tetrahydrocannabinol in the Rhesus Monkey. J. Pharm. 
Sci. 74:171-174 (1985). 



121 



ANNUAL REPORT OF THE TOXICOLOGY BRANCH 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

The mission of the Developmental Therapeutics Program centers on the discovery 
and preclinical development of agents with clinical anticancer potential. 
Investigations focusing on the hazards of antineoplastic compounds to healthy 
organs in intact experimental animals are the final steps in the preclinical 
stages of new drug development. Such investigations comprise the primary respon- 
sibility of the Toxicology Branch. Toxicology studies designed to meet this 
responsibility involve three major objectives: 

1. Determination and safety assessment of an initial dose for 
clinical trial. 

2. Determination of primary organ systems adversely affected by drug 
administration. 

3. Determination of the reversibility of the adverse effects. 

Data generated from studies on each new drug and evaluated in light of potential 
human toxicity comprise the major portion of the evaluable information required 
by the Food and Drug Administration for an Investigational New Drug Application. 

During this fiscal year the direction of the Toxicology Branch has shifted from 
an unidirectional course to two highly integrated paths. The first path contin- 
ues to be the elucidation of the potential adverse effects of new anticancer 
agents. The second path is the acquisition and use of pharmacokinetic informa- 
tion to reliably extrapolate toxic effects across species by relating plasma 
drug levels (peak and steady state) to the appearance and severity of toxicity. 
Integration of these two courses permits a more rational evaluation of the 
role of schedule dependence in efficacy of drug as well as in development of 
toxicity. 

In the main, the Branch achieves its primary responsibility through the operation 
and management of a prime contract in which the qualitative and quantitative 
toxicological profiles of antitumor drugs and modalities are determined in 
experimental animals. For management, the prime contract is divided into four 
definitive tasks. Task I is devoted to the complete preclinical toxicologic 
evaluation of cytotoxic agents, radiosensitizers, radioprotectors, etc. 
Standardized guidelines using mice, rats and dogs are followed to determine the 
initial dose for Phase I clinical trials, to verify safety of the initial clin- 
ical dose and to elucidate specific target organ toxicity and its reversibility. 
Task II studies are concerned with limited evaluations of drugs. These studies 
are performed to complete the toxicity profile on compounds for which some 
toxicology data is available. Task III involves the development and implemen- 
tation of in vivo and in vitro tests to evaluate organ specific toxicity. The 



123 



"special studies" carried out under this task yield important information 
leading to development of new, more meaningful toxicity testing studies. Task 
IV of the Prime Contract deals with the administrative aspects of toxicity 
testing such as data handling, subcontractor monitoring as required by Good 
Laboratory Practice Regulations, and financial and program management. 

An extramural contract effort in pharmacology and pharmacokinetics, a Master 
Agreement arrangement, has been functional for two years. The majority of the 
preclinical pharmacology data used in our analysis of kinetics and continuous 
delivery is developed under this project. Protocols are divided into three 
phases, with phases 2 and 3 usually performed in mice. 

1. Analytical Methods Development. 

2. Plasma elimination following a bolus dose. 

3. Plasma pharmacokinetics during continuous drug delivery. 

Information from these studies form the basis for similar studies in dogs. All 
data are then integrated with the toxicity studies to provide the cornerstone 
for drug characterization which more closely meets clinical needs. 

The Division of Cancer Treatment maintains a master file with the FDA which 
contains toxicity testing protocols for antitumor agents. These protocols set 
forth jointly agreed to procedures for animal toxicity testing of antineoplastic 
drugs. Data from studies conducted under the protocols are accepted for 
regulatory purposes in INDA approval. The Toxicology Branch has amended the 
protocols to accurately reflect newer methods and techniques. Additionally, 
individualized protocols (drug specific) are routinely developed to account for 
agent specific chemical and/or physical properties. During the past year, the 
Branch has collected and evaluated pharmacokinetic data in addition to toxicity 
data in an effort to understand the in vivo behavior of new agents related to 
toxic findings. New guidelines, dependent on drug characteristics, for 
pharmacokinetic studies and continuous delivery procedures in mice and dogs have 
been prepared and submitted to the Food and Drug Administration for inclusion in 
the Master File. 

Data on the following agents have been, or are anticipated to be, presented to 
the Decision Network Committee in FY'85. 

Flavone Acetic Acid NSC-347512 

Merberone NSC-336628 

Deoxyspergualin NSC-356894 

Pibenzimol NSC-322921 

Triazine Antifol NSC-127755 

Flavone Acetic Acid should be an exciting and important addition to the anti- 
cancer drug arsenal. The agent appears to be exceptionally active against 
solid tumors of endothelial origin, notably colon cancers in mice. The drug is 
relatively non toxic to dogs at doses, and concomitant blood levels, which are 
very effective in murine tumor systems. Sufficient preclinical data are avail- 
able to suggest a clinical dose escalation scheme based on pharmacokinetic 



124 



parameters. Merberone and Deoxyspergualin are active in experimental leukemias 
and lymphomas. Pharmacokinetic data suggest that efficacious plasma levels of 
these two drugs can be achieved and maintained in the absence of life-threatening 
toxicity. 

Toxicology studies have been, or are anticipated to be, initiated in FY '85 on 
the following compounds: 

4-Ipomeanol NSC-349438 

Azacytosine Arabinoside NSC-281272 

Anthrapyrazole NSC-349174 

Tetraplatin NSC-363812 

Buthionine Sulfoxamine NSC-326231 

Discreet Agent NSC-339004D 
Hexamethylene Bisacetamide (po) NSC-95580 

Studies on these agents likely will be completed during FY'86 and will provide 
a complete picture on their toxic effects and pharmacokinetic behavior using the 
integrated approach developed over the past two years. 

Publications and Presentations 

Staff 

1. Grieshaber, C.K.: Preclinical cardiotoxicity of two mitoxantrone analogues. 
Cancer Treat. Symp : 3: 19-23, 1984. 

2. Ginsburg, E., Gram, T.E. and Trush, M.A.: Comparison of the pulmonary 
toxicity and chemotherapeutic activity of bleomycin-BAPP to bleomycin and 
pepleomycin. Cancer Chemother. Pharmacol . 12: 111-115, 1984. 

3. Krijgsheld, K.R. and Gram, T.E.: Selective induction of renal microsomal 
cytochrome P-450-linked monooxygenases by 1 ,1-dichloroethylene in mice. 
Biochem. Pharmacol . 33: 1951-1956, 1984. 

4. Krijgsheld, K.R., Lowe, M.C., Mimnaugh, E.G., Trush, M.A., Ginsburg, E. and 
Gram, T.E.: Selective damage to nonciliated bronchiolar epithelial cells 
in relation to impariment of pulmonary monooxygenase activities by 1,1- 
dichloroethylene in mice. Toxicol. Appl . Pharmacol . 74: 201-213, 1984. 

5. Tong, S., Hirokata, Y., Litterst, C.L. and Gram, T.E.: Interaction of the 
oncolytic drug, l-(2-chloroethyl )-3-cyclohexyl-l-nitrosourea (CCNU) with 
the mixed function oxidase system in rats. Chem. Biol. Interact . 49: 
105-119, 1984. 

6. Mimnaugh, E.G., Trush, M.A., Bhatnagan, M., and Gram, T.E.: Enhancement 
of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by 
the anticancer drug, Adriamycin. Biochem. Pharmacol . 34: 847-856, 1985. 

7. Gram, T.E.: The pulmonary mixed function oxidase system. In Witschi, 
H.P. and Brain, J.D. (Eds.): The Toxicology of Inhaled Materials . 
Springer-Verlag, Berlin, 421-4/0, 198b. 



125 



8. Foster, B.J., Clagett-Carr, K., Shoemaker, D.D., Suffness, M., Plowman, 
J., Trissel, L.A., Grieshaber, C.K. and Leyland-Jones, B.: Echinomycin: 
The first bifunctional intercalating agent in clinical trials, J. Clin . 
Oncol . in press. 

9. Grieshaber, C.K. and Marsoni , S. The relation of preclinical toxicology 
to findings in early clinical trials. Cancer Treat. Rep , in press. 

10. Okine, L.K., Goochee, J.M,. and Gram, T.E.: Studies on the distribution 
and covalent binding of [^^C] 1 ,1-dichloroethylene in the mouse: effects 
of various pretreatments on covalent binding in vivo . Biochem. Pharmacol , 
in press. 

11. Okine, L.K. and Gram, T.E.: Protection by methyl prednisolone against 
butylated hydroxy-toluene-induced pulmonary damage and impairment of micro- 
somal monooxygenase activity in the mouse: lack of effect on fibrosis. 
Exp. Lung Res , in press. 

12. Gram, T.E.: Metabolism of drugs. In Craig, C.R. and Stitzel , R.E. (Eds.): 
Modern Pharmacology . Boston, Little, Brown & Co., in press. 

13. Okine, L.K.N, and Gram, T.E.: Drug absorption and distribution. 

In Craig, C.R. and Stitzel, R.E. (Eds.): Modern Pharmacology . Boston, 
Little, Brown & Co., in press. 

Contractors 

During this year contractors and subcontractors to the Toxicology Branch 

presented 6 papers or abstracts which involved NCI support. 



126 



ANNUAL REPORT OF THE INFORMATION TECHNOLOGY BRANCH 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

A fully operational version of the Drug Information System (DIS) was installed 
in March 1985. That allowed the NCI contract with the Chemical Abstracts 
Service (CAS) to be terminated the following month. The NCI-CAS contract had 
been in existence for a dozen years and its end, with the start-up of the DIS, 
marks a turning point in computer use in DTP. The DIS represents a consoli- 
dation of most of the disparate computer systems used by the DTP; it permits 
interactive control of acquisition, shipping, inventory and testing of chemi- 
cals. The major DIS databases are self-updating and the updating proceeds 
automatically at pre-determined dates and times. All output from the DIS is 
directed to a high-speed laser printer in the Landow Building. This printer is 
capable of graphical and non-graphical printing and represents a major advance 
over the earlier procedures which required the use of several different printers 
and which, as a result, could take hours or even days to complete. Since the 
DIS was released, it has logged tens of thousands of transactions. Numerous 
minor problems have been encountered and resolved and many of the important 
DIS procedures have been greatly optimized - a process which has been aided 
significantly by the installation at DCRT of a third KL-10 processor in the 
DEC System 10 computer. 

Meanwhile, the ITB supports the information needs of the DTP by providing data 
to the program in many ways. With DTP's access to data supported by the DIS, 
the requirements for reports which have to be generated by ITB staff are actu- 
ally decreasing. The ITB staff is, therefore, able for the first time to 
devote its attention to collecting and organizing much data that is ancillary 
to the screening work, e.g. flow of mice and data pertaining to plant collec- 
tion. During this year, furthermore, there has been increasing emphasis on the 
use of graphics to represent the complex data used by DTP. In this connection, 
several distinct programs have been written for the display of screening data 
and some of these are now being used regularly. 

Finally, in an exciting new excursion, the Branch is involved in the design of 
a computer system which is to be central to the new cell line project which the 
DTP is embarking upon. The new data system will run on a series of independent 
local microprocessors, controlled by and reporting to the DIS. In this project 
the throughput requirements are very high. Accordingly, a commitment has been 
made to robotics, which promises to deliver such a high volume of tests. 

Office of the Chief, ITB 

This Office supervises and coordinates the activities of the two Sections of 
this Branch, directs the progress of the DIS and initiates and pursues appro- 
priate information activities with other units within NCI and NIH, other 
Agencies of the Government, and private sector organizations. 



127 



1. Drug Information System. 

After the first release of the DIS on March 1, 1985, careful monitoring of the 
system for accuracy and performance began. Very few inaccuracies (data errors) 
have been found; those that were discovered have been corrected and the DIS is 
thought currently to be essentially error-free. The initial slow response 
encountered was traced to two problems; computer overloading and inadequate 
optimization in some parts of the programs. The first of these problems was 
essentially eliminated at mid-April with the expansion of the DEC-10 computer 
by a factor of over 50%. Work on optimization still continues, but the major 
difficulties were resolved during M^, and since then, response time has not 
been cited as a serious problem. 

The interfacing of the high-speed laser printer to the computer system has been 
completed, but handling of graphics by this printer is not yet trouble-free. 
In the online environment, the electronic balances are working well and the 
bar code technology is proving to be a major strength of DIS. An extensive 
effort to provide systems level documentation of the DIS has begun and should 
be completed before the end of 1985. 

Support for users of the DIS is provided by ITB. Five different User's Manuals 
have been written and published and two more are in preparation. In addition, 
a total of over 400 online "HELP" messages have been written and made available 
to online users. Finally, during May and June, a series of 2-hour seminars on 
use of the DIS was presented to DTP staff. 

Future work on the DIS will be directed at remaining DTP requirements such as 
animal and plant resource files, the toxicology file and the file of bulk 
chemicals prepared under Good Manufacturing Practices. 

2. Computer System for the Cell Line project. 

The DTP has begun a major new initiative which involves the testing of chemi- 
cals jn vitro for selective cytotoxicity towards specific cell lines. It is 
hoped ultimately to be able to complete as many as a million such tests per 
year. Such a level of testing mandates strong computer support. Accordingly, 
a system, centered upon a group of microprocessors in the laboratory has been 
designed. These processors will accept strategic commands from the DIS and 
return semi -processed data to the DIS. The microprocessors will achieve much 
of the necessary work by issuing commands to robots, which will do a great deal 
of the actual sample handling, and can readily do so under sterile conditions. 
A prototype version of this system should be running by the end of 1985. 

3. Automation of Literature Surveillance. 

Literature surveillance, a manpower-intensive task, is currently carried out by 
two contractors to the DTP. During this year, the ITB monitored the entire 
literature for two months, comparing the results of a manual review against one 
performed by a computer, using a sophisticated model. The results demonstrated 
that the computer is considerably cheaper and more effective than a manual 
reviewer. It reviews many more published compounds and flags active compounds 
at a much higher rate (18% versus 3%). On the other hand, the computer cannot 
easily deal with non-algorithmic constraints. 



128 



4. Natural Products File. 

The "Berdy File" of antibiotics is a useful resource whose value has always 
been compromised by the absence in the file of chemical structures. During 
this reporting period ITB, taking advantage of the programs developed in the 
Branch, has begun systematically to add structural data to the Berdy File. 
Some 6,000 structures of compounds in this file are known; over half of these 
have now been entered and completion of this file is scheduled for late 1985. 
It will then become searchable by structure and substructure as well as by 
other information types (micro-organism, author and so on) and should prove 
to be a valuable resource to DTP. 

5. Automation of EL ISA Assays. 

In connection with the AIDS program, LTCB conducts a large number of EL ISA 
and EL ISA-like assays each year. The large amounts of data so produced 
proving to be a problem, ITB designed, developed and installed a data system 
on the Apple II computers in LTCB. This system works very well and has greatly 
assisted the LTCB in maintaining the flow of samples through their analytical 
laboratories. 

6. ITB Reports. 

This year, ITB has prepared, written and distributed 6 Reports dealing with 
subjects ranging from computer coding of tautomeric structures to new 
Investigational New Drug Applications filed by DCT during the year. 

Biological Information Section, BIS 

1. Screening Database. 

Maintenance of this database has proceeded very smoothly during the year. 
Files resulting from the annual update in March were passed to the DIS and, for 
the first time, were made searchable online by the DIS. Data from the in vivo 
screening as well as from the HCTFA flow uneventfully into the Biology database. 
Retrieval of these data in a variety of forms is provided for and thousands of 
such retrievals are carried out each year. Many of the retrievals are com- 
pleted by ITB staff themselves, using systems provided by ITB. 

2. Data Retrieval and Report Generation. 

The standard support for DTP data retrieval has been provided. A total of 324 
Screening Data Summary reports have been run during the year - the number of 
NSC Numbers in such a report can range from one to several hundred. In addi- 
tion to such routine support, a detailed correlation of preclinical and clinical 
activities for about 100 drugs was completed for the January 1985 review of the 
screening program. Another study which was completed, compared the data from 
in vivo testing to that from in vitro testing for some standard agents. This 
was done as a preliminary to the Cell Line project. A report containing 
detailed chemistry and summary biology data for 1300 active, open compounds was 
produced in October 1984. These compounds, no longer under active investigation 
by NCI, are of possible value to other organizations interested in development 
of antitumor agents. 



129 



3. Microprocessors. 

Among the many strengths of the microprocessor is its ability to serve as a 
very powerful communications node. The IBM PC which the BIS acquired this 
year is used very heavily in this manner. It is frequently necessary to move 
several thousand NSC Numbers and records from one computer to another - for 
instance from the DCRT IBM system to a private sector computer for literature 
searching or to the IBM PC in the Brussels liaison office. Such transfers are 
now done with the PC. It can be connected first to one, then to the other 
computer, or alternatively, it can be used to collect data and write it to a 
floppy disk which can be mailed to Brussels. 

4. Animal Utilization. 

A computer system has been established which monitors the flow of mice from the 
suppliers to the screeners. Each screening laboratory now reports regularly 
(along with screening data) the numbers of mice ordered, received, used, and so 
on. These data are collected by programs which process them and automatically 
produce, upon a monthly basis, curves of these data for each strain/screener 
combination. This system is now working well and has permitted DTP staff to be 
alerted to and to resolve some major problems in the utilization of mice by the 
DTP. A parallel program handles the cost informationn of the mouse supply 
program; this is provided to the Animal Genetics and Production Branch as a 
management tool . 

5. Plant Collection Database. 

A system has been developed to manage the needs of the increasing effort in 
plant collection of the Natural Products Branch. Collectors in the field will 
enter data concerning their collection activities into a locally operated IBM 
PC. These data will be copied to a disk which will be mailed to DTP. Programs 
have been written to. read such a disk and to use the data to update the "Plant 
Header File". 

Chemical Information Section, CIS 

1. Input of Chemical Structures. 

The structure-input program that runs on a light-pen driven PC has been opti- 
mized and enhanced. It now can accept a large structure in about one minute 
and, as such, is no longer the rate-limiting step in data entry. Accordingly, 
this program is now in routine production use by the acquisitions contractor. 
A newer, radically different program which runs on an IBM PC has been com- 
pleted. It is based upon a markedly different philosophy and, as hoped, it 
allows one to enter structures much more rapidly - as little as a few seconds 
suffices in many cases. The new program has been patented in behalf of the 
U.S. Government; enquiries as to licenses have already been received, and the 
massive data input to which it seems to lend itself is currently being 
investigated. A good deal of time has been devoted to the implementation of a 
number of graphical approaches to the presentation of screening data. This has 
led to "flowers", bar-graphs, and survival curves. All three of these 
techniques have been made available to the general user via interactive 
programs and the latter two are finding some measure of acceptance. 



130 



2. Output of Chemical Structures - Computer Graphics. 

The procediires laid down within the DIS for the input, storage, and presenta- 
tion of chemical structures are now working, apparently successfully. The last 
link in this chain is the printing of the structure on the laser printer and 
this step proved to be quite difficult, because of the number of useful vari- 
ables such as the size of the structure, its orientation (and that of the 
accompanying text) on the page and the desirability of access to different type 
fonts (italics, greeks, bolds etc). Once these questions had all been resolved 
and structure drawing had become more routine, the more general problem of 
handling by the printer of generalized graphics was addressed. Work in this 
area, which is still going on, is aimed at printing a wide variety of graphics 
such as plots, bar-graphs, flow-charts or logos and signatures. 

Publication : 

Kerekes, P., Sharma, P. N., Brossi, A., Chignell, C. F. , and Quinn, F. R.: 
Synthesis and Biological Effects of Novel Thiocolchicines. 3. Evaluation of 
N-Acyl Deacetylthiocolchicines, N-Alkoxycarbonyl-Deacetylthiocolchicines and 
0-Ethyl Demethylthiocol chicines. New Synthesis of Thiodemecolcine and Anti- 
leukemic effects of 2-Demethyl- and 3-Demethylthiocolchicine. J. Med. Chem. , 
in press. 



131 



ANNUAL REPORT OF THE EXTRAMURAL RESEARCH AND RESOURCES BRANCH 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 



Description 

The Extramural Research and Resources Branch is responsible for the 
administration of preclinical grant-supported research leading to the develop- 
ment of effective anticancer drugs which act specifically or selectively 
against malignant growth with minimal toxicity to the host. The major areas 
of emphasis in the Biochemistry and Pharmacology Research Program are: Drug 
design and synthesis, natural products development, experimental therapeutics, 
comparative pharmacology and toxicology, and mechanism of drug action. As new 
findings and significant developments occur, appropriate changes are made in 
programmatic emphasis and support in each of its major research categories. 
This Branch maintains liaison and coordinates its research activities with 
those of other Divisions of the National Cancer Institute, other Federal 
agencies, and academic institutions at the national and international levels. 
Projects of high programmatic interest are funded through the special ex- 
ceptions procedure provided by the NCI Executive Committee, while proposals 
with objectives not of immediate relevance to drug development are trans- 
ferred to other institutes or to more appropriate programs within the National 
Cancer Institute. 

During Fiscal Year 1985, the Branch supported 283 research projects totalling 
$38.1 million dollars. The distribution of projects among the major categories 
shown in the following table is in accordance with the need and current 
priorities for research in the respective drug development area. 



1 

1 BIOCHEMISTRY AND PHARMACOLOGY 


PROGRAM 1 


1 BY SUB 


-CATEGORY 




1 FY 


1985 






Number of 


Total Amount 1 




Grants 


(Thousands) 1 


1 SYNTHESIS & CHEMISTRY 


83 


$ 9,464 1 


1 NATURAL PRODUCTS 


31 


3,538 1 


1 SCREENING & EXPERIMENTAL 






1 THERAPEUTICS 


32 


3,507 1 


1 COMPARATIVE PHARMACOLOGY 


19 


1,824 1 


1 OTHER PRECLINICAL ASPECTS 


8 


753 1 


1 MECHANISM OF ACTION 


103 


11,708 1 


1 PROGRAM PROJECTS 


7 


7,272 1 


1 TOTAL 


283 


$ 38,076 1 



133 



Significant Recent Results 

Chemistry of Platinum Anticancer Drugs 

The clinical effectiveness of cis- diamminedichloroplatinum (II), cis -DDP, 
against testicular, ovarian and bladder tumors has encouraged research into 
its mechanisms of antitumor activity, and into its synergistic reactions 
with other drugs in combination chemotherapy protocols. Pretreatment of DNA 
restriction fragments with cis- DDP modulated and activated the sequence 
specific cleavage by bleomycin. Cis -DDP may modify DNA structure in a way 
that facilitates action by another agent. By chemically linking a cis-PtCl2 
fragment to another dye or drug it should be possible to produce a compound 
having unique antitumor properties, or utility as probes of antitumor action. 
Recently, a compound having the covalent DNA-binding properties of an analog 
and the intercalating features of acridine orange has been synthesized. Upon 
binding to DNA, high concentrations of the dye increase the accessibility of 
bases in DNA to chemical reactions. The same synthetic scheme permits attach- 
ment of fluorescent rhodamine dyes to the "PtCl2" moiety. Follow-up research 
relates to conversion of a modified bleomycin with a spermidine tail into a 
platinum derivative which could significantly affect the binding of cis-DDP 
to DNA jji vivo. 

These studies lead to better understanding of the mechanism of action 

of cis- DDP and contribute to development of more effective cancer therapeutic 

agents. (CA 34992, Lippard). 

Forskolin as a Potential Antimetastatic Agent 

Continued emphasis is placed on the discovery and development of new natural 
products for eventual clinical application. Studies have revealed that shortly 
after adhesion to blood vessel endothelium, metastatic cells become surrounded 
by thrombotic material, apparently induced by the transient tumor cells. The 
search for a compound which would act as a potent platelet aggregation inhibitor 
while causing minimal host toxicity has centered on forskolin, a diterpene iso- 
lated from the roots of the Indian plant, Coleus forskohlii . Forskolin induces 
significant reductions (over 70%) in numbers of pulmonary tumor foci. No overt 
toxicity was observed. 

Forskolin is a powerful stimulator of platelet adenylate cyclase. It causes a 
several -fold increase in intracellular concentrations of c-AMP. It is likely 
that forskolin may act synergistically with these natural compounds in vivo 
to block tumor cell-platelet interactions. The low observed toxicity in mice 
and its effective inhibition of in vitro tumor induced aggregation of human 
platelets may make it a potentiaT~drug for treating rapidly metastasizing human 
tumors. (CA 07340, Parks) 

Cell Kinetics-Directed Treatment Schedules 

Solid tumors contain relatively low growth fractions. Therefore, agents which 
induce cell cycle phase synchrony or those that direct non-dividing cells into 
the cell cycle for therapeutic attack are valuable adjuncts in combination 
chemotherapy. Considerable research effort is directed at means of manipulating 
tumor cell cycle kinetics to achieve maximum cell kill by phase-specific agents. 



134 



Compound l,2:5,6-di-anhydro-galactitol (GAL) was tested in Chinese hamster 
ovary cells (CHO), human adenocarcinoma of the stomach and an Ehrlich ascites 
tumor system in vivo . In each case, transient, reversible blocking in the S 
phase was folTowed by several -fold increases of cell fractions in the G2-M 
phases. Subsequent to GAL treatment in CHO cells, bleomycin, an agent most 
effective in the G2-M phases, was administered. At 30 hour post-GAL treatment, 
cell survival had decreased 300% from that observed when bleomycin was used 
as a single agent. 

Further studies on enhancement of therapeutic response are planned using agents 
which induce kinetic changes combined with cycle specific cytotoxins. They 
provide the basis for development of clinical protocols involving kinetics-based 
treatment schedules for more effective treatment. (CA 15397, Barranco). 

Gene Amplification and Methotrexate Resistance 

The role of gene amplification in the acquisition of cellular resistance to 
methotrexate (MTX) has been studied in the human, mouse, rat and hamster cell 
lines. In each case, the gene coding for dihydrofolate reductase (DHFR), the 
target enzyme for MTX, undergoes repeated amplification with a concurrent 
increase in gene product. Recombinant DNA studies and DNA sequencing in each 
of the cell lines shows remarkable similarity in nucleotide base composition. 
Recently, amplification of the DHFR gene has been demonstrated in blood cell 
samples drawn in vivo from a human patient with chronic myeloid leukemia. 
Post-treatment levels of DHFR, following administration of MTX, were two to 
threefold higher than pretreatment levels. Amplification occurred primarily 
if not exclusively, in neoplastic cells. Repeated samples drawn from the 
same patient were employed to avoid problems associated with the development of 
increasing resistance in cultured tumor lines. Although the mechanism of ampli- 
fication is not known as yet, studies with mouse cell lines indicate that 
brief inhibition of DNA replication or damage to DNA itself, increases the 
occurrence of MTX resistance due to gene amplification. Development of multi- 
drug protocols utilizing an inhibitory agent that does not damage DNA initially 
may help circumvent this problem. (CA 16318, Schimke) 

Multi drug-Resistant Human Tumor Cells 

Following the significant finding by Schimke that amplified genes in tumor cells 
which code for dihydrofolate reductase play an important role in methotrexate 
resistance, other researchers are examining the molecular basis for this action 
as it relates to acquired drug resistance. In particular, evidence is being 
sought that the mechanism may initiate pleiotropic or cross resistance to agents 
that share similar chemical structures. 

The experimental plans include the isolation of the human homologue of the 
Chinese hamster gene associated with multidrug resistance and analysis of its 
amplification and expression in human tumors resistant to chemotherapy. Studies 
include correlation of expression of specific genes with the tumor drug re- 
sistance profile and the tumor type; isolation of full-length cDNA clones 
corresponding to the cloned genes and determination of their DNA sequence; 
analysis by gene transfer of the functions of the cloned genes in the develop- 
ment of multidrug resistance. These studies will elucidate mechanisms of 
pleiotropic drug resistance to provide information critical to successful 
clinical treatment of resistant leukemias in humans. (CA 40333, Roninson). 



135 



Areas for Future Emphasis 

Tumor Heterogeneity 

Research emphasis continues to be placed on studies designed to characterize 
genetic, biochemical and pharmacological differences among cell populations 
in a given tumor type. Other support is directed at understanding hetero- 
geneous cell population response to single and multi-agent treatment, and 
to the mechanisms of development of cross-resistance to commonly used anti- 
cancer agents. The results provide a rationale for preparing drugs designed 
to exploit cell differences by novel or improved target specific delivery 
systems, and for the biochemical manipulation of mechanisms unique to tumor 
cells. 

In Vitro Clinical Tumor Model Systems 

This program strongly emphasizes research leading to the development of in 
vitro assays which will effectively predict patient tumor response before 
chemotherapy commences. In the coming year support will be directed pri- 
marily to rapid and inexpensive screening assays designed to predict and 
quantitate drug sensitive and drug resistant tumor responses. Two advan- 
tages of this strategy are foreseen: clinicians will be able to administer 
more efficacious disease specific drug regimens than at present, and 
continued dependence on more costly in vivo animal model systems will be 
diminished. 

Counteractive Measures Against Clinical Toxicity 

There is increasing awareness that the use of known and established clinical 
drugs can be manipulated to induce more effective and longer responses in 
cancer patients. Toxicity of drugs such as methotrexate and adriamycin in 
higher doses can be modified by manipulation of dosage regimens or by employ- 
ment of rescue factors with ultimate protection to patients from severe 
toxic side effects. Use of high-dose therapy with appropriate counteractive 
rescue agents appears to be especially promising for patients with metastatic 
solid tumors. 

Other Program Activities 

This program provided support for the following conferences in FY 1985. 

1) Workshop on Folyl and Antifolyl Polyglutamates , held in Airlie, Virginia, 
November 14-17, 1984, organized by Dr. I. David Goldman, Medical College of 
Virginia, Richmond, VA. 2) Cancer Drug Resistance Workshop, XIV International 
Chemotherapy Congress , held in Kyoto, Japan, June Z3-Z8, 1985, organized by 
Dr. Thomas C. Hall, University of Hawaii, Honolulu, HI. 3) Gordon Conference 
on Polyamines and Cancer , held in Meriden, New Hampshire, July 1-5, 1985, 
organized by Dr. David R. Morris, University of Washington, Seattle, WA. 
4) Gordon Research Conference on Chemotherapy of Cancer , held in New London, 
New Hampshire, July 22-26, 1985, organized by Dr. Franco Muggia, New York 
University Medical School, New York, NY. 

During the last year, 887 research papers have been published with partial 
or total support provided by this program. 

136 



ANNUAL REPORT OF THE LABORATORY OF BIOLOGICAL CHEMISTRY 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

The Laboratory of Biological Chemistry was established in 1985 to identify 
as targets for drug design, cellular reactions that are critical to the control 
of tumor cell proliferation or differentiation. Recent advances in cell 
biology are evaluated for possible targets. Agents are designed to interact 
with these targets and are evaluated for biochemical and antitumor effective- 
ness. An important aspect of this mission is to develop appropriate in vivo 
systems to evaluate the chemotherapeutic effectiveness of agents shown to be 
active in simpler in vitro model systems. Accordingly, the Laboratory is 
involved in identifying endogenous factors present in vivo that modify drug 
action and influence differential toxicity with the aim of manipulating these 
factors to enhance antitumor activity. To accomplish this mission, several 
existing research units were combined through an extensive reorganization of 
the DTP intramural research program to form the new Laboratory of Biological 
Chemistry. These units were the former Drug Metabolism Section, LCP, the 
Applied Pharmacology Section, LMCB, the Cellular Pharmacology Section, LMCB, 
and Dr. Breitman's research unit from LMCB. Ongoing research projects were 
re-evaluated and approximately 50% of the resources was applied to non-tradi- 
tional targets for antitumor drug design and study. These non-traditional 
targets include early key biochemical events signaling cell proliferation or 
differentiation. The other 50% of Laboratory resources was applied to the 
study of either traditional targets or active compounds with traditional or 
unknown mechanisms of action. 

Reduced requirements for stimulation by growth factors may be the fundamental 
characteristic of transformed (neoplastic) cells. Research in molecular 
biology has identified several specific biochemical changes produced by intro- 
duction or overexpression of oncogenes which may reduce the levels of exogenous 
growth factors needed to trigger cell replication. These findings suggest 
that a more selective approach to chemotherapy may focus on the interaction of 
growth factors with cells rather than on basic metabolic reactions such as 
those involved in nucleic acid synthesis. We have therefore initiated projects 
to develop new chemotherapeutic agents to block the action of growth factors. 
Non-traditional targets selected for drug design and study include: 
second messengers inositol triphosphate and diacyl glycerol ; myristoylation of 
cellular oncogene products; and protein kinase C. 

Recent findings indicate that inositol -phosphates formed from phosphatidyl - 
inositides may be the second-messengers which mediate the action of many growth 
factors. In addition, the product of two distinct oncogenes (src and ros) 
phosphorylate phosphatidyl inositol and increase the levels of phosphatidyl - 
inositol polyphosphates in the membranes of cells transformed by these onco- 
genes. The synthesis of phosphatidylinositol polyphosphates and their sub- 
sequent hydrolysis therefore provide attractive well-defined targets for the 
design of inhibitors for use in chemotherapy. A project was initiated that 

137 



has two related goals. First, to determine the relative importance of the 
enzymatic reactions involved in inositol phosphate production as a signal 
initiating cell replication. Secondly, to design, synthesize, and evaluate 
drugs to inhibit this process, specifically inhibitors of the reactions of 
phosphatidylinositol polyphosphate synthesis and of phospholipase C, the enzyme 
which produces the inositol phosphates. Test systems have been developed to 
identify drugs that inhibit phosphatidylinositol turnover or synthesis. 
Several compounds are currently being synthesized for evaluation in these 
systems. These include: 2-deoxy-myo-inositol and 5-deoxy-myo-inositol which 
lack critical hydroxyls; and phosphonate analogues of phosphatidylinositol as 
potential non-hydrolizable antimetabolites. 

It may be possible to alter the activity of an oncogene product by interfering 
with its localization in the plasma membrane. The early events signaling cell 
proliferation occur in the plasma membrane of the cell, the location of most of 
the known cellular oncogene products. Myristoylation has been shown to be 
critical for the membrane localization and cellular transforming activity of 
p60src and has been implicated for other transforming proteins. Three mechan- 
isms for blocking myristoylati on-dependent transformation are under investiga- 
tion: (1) direct chemical inhibition of transforming protein myristoylation; 
(2) inhibition of translocation of the myristoylated transforming protein to 
the plasma membrane; and (3) displacement of the myristoylated transforming 
protein away from the membrane, into the cytoplasm, and thus away from its 
substrate. Methods developed in the p60src system will be applied to malignant 
cells where membrane-associated oncogene products are suspected. 

Inducers of protein kinase C activity such as diacyl glycerols and phorbol 
esters, have shown the importance of this enzyme in the regulation of growth 
factor activity, lymphoblastogenesis, normal cell proliferation, cellular 
differentiation and oncogene expression. Studies in this Laboratory of protein 
kinase C activity during differentiation of human promyelocytic leukemia cell 
line HL-60 have indicated that not only is this enzyme activity increased in 
cells induced to differentiate along the myeloid or monocytic pathways, but 
that there is the rapid appearance of differentiation specific peptides which 
are phosphorylated in a calcium- and phospholipid-dependent manner. Natural 
metabolites that produce differentiating activity and activation of protein 
kinase C and phosphatidylinositol turnover are the diacylglycerols. Related 
dialkyl glycerols and dialkylphospholipid analogues that are more metabolically 
stable are under investigation for their potential as selective anticancer 
drugs via their ability to induce differentiation of HL-60 cells. Currently 
1-0-hexadecyl -2-0-methyl -3-phosphochol i ne and 1-0-hexadecyl -2-0-methyl -3-phos- 
phate are being evaluated. Other analogues are being synthesized. 

The availability of tissue culture cell lines has made it possible to study the 
regulation of proliferation and differentiation of specific hematopoietic cell 
types and the effects on these cells of known or suspected mediators and modu- 
lators. It was found previously in this Laboratory that retinoic acid (RA) 
is a potent inducer of terminal differentiation of the human promyelocytic cell 
line, HL-60, and the human monoblast- and monocyte-like cell lines, U-937 and 
THP-1. In addition RA was found to induce differentiation of fresh cells in 
primary culture of patients with acute promyelocytic leukemia. While retinoic 
acid alone is capable of inducing terminal differentiation, combinations of a 
physiological concentration of RA (10 nM) and either cAMP inducing agents (e.g., 



138 



cAMP, prostaglandin E, or cholera toxin) or the conditioned medium from either 
activated T-cells or human leukemic T-cell lines synergistically induced differ- 
entiation of HL-60. An activity called "differentiation inducing activity" or 
DIA has been purified from these conditioned media and attempts are now being 
made to produce monoclonal antibodies against this protein. Studies are now 
in progress to apply the results obtained in vitro with HL-60 to a transplan- 
table HL-60 tumor carried in athymic nude mice. 

The technique of photoaffinity labeling is being used to identify intracellular 
drug binding proteins associated with the cytotoxicity or resistance of anti- 
tumor agents. A radioactive photoactive analogue of vinblastine was prepared 
and found to interact with alkaloid binding proteins that have not been des- 
cribed previously. The relationship of drug-binding proteins to cellular 
drug-sensitivity and drug-resistance is being investigated. In a similar study, 
a radioactive photoactive analogue of daunorubicin was found to bind to speci- 
fic proteins in a heart homogenate. Identification of these intracellular 
drug-binding proteins could lead to a better understanding of the cytotoxic 
effects of these agents. 

The methods developed to study the non-traditional targets described above are 
also being used to study the biochemical effects of new or established anti- 
tumor agents. Investigators in this Laboratory have extensive experience in 
nucleotide metabolism. Cyclopentenyl cytidine (cCyd) was shown to have in 
vivo antitumor activity against L1210 and P388 leukemias. cCyd produced a 
rapid decrease in CTP synthesis in vitro and in vivo , concomitant with an 
inhibition of rRNA transcription, and to a lesser extent, tRNA, without changes 
in the processing of nucleolar RNA. In HL-60 cells, inhibition of CTP synthe- 
tase leads to rapid inhibition of DNA and c -myc mRNA synthesis which precedes 
the appearance of differentiated cells. The cyclopentenyl adenosine analogue, 
neplanocin A, was found to be an effective inhibitor of RNA and DNA methylation 
resulting from its conversion to an analogue of AdoMet, and that these effects 
appear to be responsible for reduced c-myc RNA expression and the induction 
of myeloid differentiation in this cell line. The cyclopentenyl analogues of 
other nucleosides (including modified nucleosides such as 5-fluorouridine) 
will be studied in these systems. 

A project involving traditional targets for drug design and study is a continu- 
ing project to determine the relative dependency of host and tumorous tissues 
on de novo vs salvage pathways for the synthesis of pyrimidine and purine 
nucleotides in vivo . This project is divided into the following specific 
aims: to determine the physiologic importance of circulating pyrimidines and 
purines and their role in modulating the antitumor activity of antipyrimidine 
and antipurine chemotherapeutic agents; to study the liver as a modulator of 
circulating nucleosides and as a possible target for chemical manipulation; 
to develop agents to interfere with nucleoside salvage to be used in combina- 
tion with inhibitors of de novo synthesis; to develop methodology for monitor- 
ing and quantitating theTlux through the de novo pathways of host and tumorous 
tissues in vivo . Studies from our Laboratory demonstrated that low levels of 
pyrimidines and purines, within the physiological range, are capable of 
reversing the toxicity of inhibitors of purine and pyrimidine de novo synthesis. 
Also, at these low concentrations, cultured cells turn off their de novo path- 
ways and use exclusively their salvage mechanisms. Thus, circulating nucleo- 
sides may be an important factor in antipyrimidine and antipurine chemotherapy. 



139 



To evaluate to what extent tumors in vivo depend on their salvage pathways to 
fulfil their nucleotide requirements, we have adapted the tumor model system 
developed by Gullino and coworkers, where a tumor (in our case Walker 256) is 
implanted into the ovarian fat pad of female Sprague-Dawley rats and isolated 
in a parafilm pouch in the subcutaneous space. This system will permit 
direct sampling of the interstitial fluid and a measurement of the blood flow 
and trans-tumor differential of nucleoside concentrations. With this informa- 
tion it will be possible to calculate for the first time the actual net hourly 
contribution of circulating nucleosides and bases to tumor nucleotide pools. 

Research from this laboratory has shown that the isolated rat liver can act as 
a donor organ of nucleosides that can be salvaged in vivo by other organs or 
tumors. Manipulation of this donor organ to inhibit or increase biosynthesis 
of salvageable nucleosides could be useful in cancer chemotherapy. A recently 
completed study from our laboratory indicated that the hepatic production of 
pyrimidines utilizes intermediates of the urea cycle. This study utilized 
■^\-bi carbonate. We are currently using -^^NH^Cl and ■'•^N-glutamine to determine 
directly the contribution of CPS-I and CPS-II to the N^ and N3 positions of 
the uracil nucleus. 

The data so far discussed in this section indicate that inhibition of nucleo- 
side salvage could enhance the effectiveness of inhibitors of de^ novo nucleo- 
tide synthesis. An in vivo model system developed to study the metabolism of 
pyrimidine nucleosides present in the plasma of the mouse is currently being 
used to assess the effects on salvage, in vivo , of agents designed to block 
pyrimidine salvage. 3-Deazauridine (500 mg/kg) produced a marked (>70%) inhi- 
bition of uracil nucleotide formation from uridine in liver, kidney, and 
L1210 tumor cells. Dipyridamole (100 mg/kg) did not significantly reduce 
salvage of uridine by liver or kidney and produced only small, transient reduc- 
tions in salvage by L1210 tumors. Dipyridamole pretreatment did not alter the 
rate of clearance of uridine from the plasma. We examined a series of 29 
analogs of uridine, most with modifications at the 5'-position, as inhibitors 
of uridine kinase in vitro and of uridine salvage by intact L1210 cells. 
CyclopentenyluraciT~is promising as an inhibitor of uridine salvage since 
it selectively blocks the salvage of uridine by cells, is resistant to phos- 
phorolysis, and is not cytotoxic at biochemically active concentrations. 
Further development and assessment of the in vivo effectiveness of this 
analogue is underway. 

Uridine Phosphorylase is under evaluation as a chemotherapeutic agent. Twenty- 
five units/mouse of uridine phosphorylase, purified by affinity chromatography, 
produced marked (>90%) inhibition of uracil nucleotide formation by L1210 
tumor cells, with a recovery to 40% of control seen 12 h after injection of 
enzyme. Uridine phosphorylase alone produced a modest (20%) decrease in uracil 
nucleotides of L1210 ascites cells, but greatly depleted (>95%) pools in com- 
bination with PALA, an inhibitor of pyrimidine synthesis de novo . Studies in 
progress will establish the therapeutic effect of combinations of PALA and 
uridine phosphorylase. 

We have recently developed a method, which is the only method available, to 
monitor the flux through the de novo pyrimidine pathway in vivo . ■^ CO2 is 
administered to mice and the incorporation of ^^C into pyrimidine nucleotides 
is quantitated by GC/MS. Using this method we studied the effect of PALA, a 

140 



potent inhibitor of de novo pyrimidine synthesis, on pathway flux in tumors 
sensitive and resistant to PALA. During the past year, this method was used 
to study the effect of acivicin, a glutamine analogue undergoing clinical 
evaluation, on de novo pyrimidine synthesis in L1210 (sensitive to acivicin) 
and B16 melanoma (resistant to acivicin) in vivo . GC/MS techniques were devel- 
oped to quantify isotopic abundances in cytosine, cytidine, adenine, adenosine, 
quanine, guanosine, inosine, and hypoxanthine. These techniques will allow us 
to quantitate the flux through the de novo purine pathway in vivo simultaneous 
with measurements of the de novo pyrimidine pathway. We developed a method to 
quantify de novo pyrimidine biosynthesis based on the isotopic enrichment of 
Nl and N3 atoms of the uracil nucleus when hepatocytes are exposed to -^^N-NH^Cl 
or 15[^„gTijtamine. From these values we are able to calculate, utilizing 
probability statistics, the number of uracil molecules produced by the de novo 
pathway that are labeled and those that are not labeled. By determining tlie 
enrichment of specific atoms of the product (uridine) formed in a pathway we 
know the isotopic enrichment of the immediate precursor pool for that atom. 
Comparison of this value with the precursor enrichment value obtained for the 
cell homogenate will indicate if compartmentation of precursors is occurring. 
We have developed a general mathematical framework using linear algebra that 
is implemented in a computer program that will calculate the results of 
isotope dilution experiments for an arbitrary number of components. The 
generality of the framework has allowed us to develop a detailed mathematical 
treatment of biosynthetic studies which measure incorporation of a stable 
labeled precursor into a metabolic product. 

The experience of investigators of this Laboratory in the pharmacology of 
antitumor agents was applied to several active antitumor agents under develop- 
ment by DTP for clinical trial. The time course and dose response curve for 
inhibition of DNA synthesis in mouse spleen and jejunum was determined for 
aphidicolin glycinate, a water soluble ester of aphidicolin currently under- 
going preclinical toxicological evaluation. Studies of the biochemical and 
pharmacologic effects of tiazofurin were continued. The combination of tiazo- 
furin and 5-FU was synergistic in toxicity to L1210 cells; tiazofurin enhanced 
the toxicity of 5-FU in non-tumored mice. These effects may be related to a 
tiazofurin-induced increase in PRPP pools. The perfluorocarbon emulsion 
fluosol-DA was evaluated for its ability to increase the perfusion of solid 
tumors in vivo . Blood flow in a s.c. implanted solid Walker 256 tumor and in 
normal rat tissues was determined by 14C-iodoantipyrine and autoradiographic 
techniques. Fluosol-DA did not increase flow to the whole tumor and the pat- 
tern of regional flow within the tumor was not substantially altered; whereas 
cerebral flow was increased two-fold. A gas chromatographic method was devel- 
oped using nitrogen-phosphorous detectors to quantify thiotepa and its metabo- 
lite, TEPA, in extracts of blood and CSF. This technique was used to study 
the pharmacokinetics of these two compounds in monkey and man following i.v. 
and i.t. administration. A method was developed to quantify intracellular 
thioinosine monophosphate and thioguanosinfe monophosphate to be used in a 
clinical study of 6-mercaptopurine. A GC/MS technique was developed for dihy- 
drolemperone, an agent scheduled for clinical evaluation against lung cancer. 

The preceding summary outlines the objectives of the Laboratory of Biological 
Chemistry and describes some of the research carried out within the Laboratory 
during the past year. The individual Project Reports, which follow, describe 
this research in greater detail. 

141 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED 

October 1. 1984 to September 30, 1985 



PROJECT NUMBER 



ZOl CM 06162-01 LBC 



TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) 

Pharmacol oq.y of Antitumor Agents 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: 
Other; 



R.L. Cysyk 

S.D. Averbuch 

P. A. Monks 

J.D. Moyer 

P.J. O'Dwyer 

R.L. Felsted 

C.J. Glover 

J.D. Strong 



Chief LBC, NCI 

Med. Staff Fellow LBC, NCI 
Visiting Associate LBC, NCI 



C.A. Chisena Biologist LBC, NCI 
N.R. Bachur, Med. Research 

Officer LBC, NCI 



Staff Fellow 

Research Chemist 
Microbiologist 
Sr. Investigator 



LBC, NCI 

CTEP, NCI 

LBC, NCI 

LBC, NCI 

LBC, NCI 



COOPERATING UNITS (if any) 

George Washington University (P. Klubes) 

Pediatric Oncology Branch, NCI (S. Zimm and D. Poplack) 

Clinical Pharmacology Branch, NCI (J. Collins) 



Laboratory of Biological Chemistry 

SECTION 



INSTITUTE AND LOCATION 



NCK NIH. Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 
4.0 



PROFESSIONAL: 

1.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
n (a2) Interviews 



2.5 



K (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The time course and dose response curve for inhibition of DNA synthesis in mouse 
spleen and jejunum was determined for aphidicolin glycinate, a water soluble ester 
of aphidicolin currently undergoing preclinical toxicological evaluation. Studies 
of the biochemical and pharmacologic effects of tiazofurin were continued. The 
combination of tiazofurin and 5-FU was synergistic in toxicity to L1210 cells.; 
tiazofurin enhanced the toxicity of 5-FU in non-tumored mice. These effects may be 
related to a tiazofurin-induced increase in PRPP pools. Cyclopentenyl cytosine was 
found to have reproducible antitumor activity against L1210 and P388 leukemia in 
the mouse. A sharp drop in CTP pools of L1210 cells in vivo was observed after 
treatment, indicating an effect on CTP synthetase in vivo . The perfluorocarbon 
emulsion fluosol-DA was evaluated for its ability to increase the perfusion of 
solid tumors in vivo . Blood flow in a s.c. implanted solid Walker 256 tumor and ir 
normal rat tissues was determined by 14C-iodoantipyrine and autoradiographic 
techniques. Fluosol-DA did not increase flow to the whole tumor and the pattern of 
regional flow within the tumor was not substantially altered; whereas cerebral flovi 
was increased two-fold. A gas chromatographic method was developed using nitrogen-' 
phosphorous detectors to quantify thiotepa and its metabolite, TEPA, in extracts o1 
blood and CSF. This technique was used to study the pharmacokinetics of these two 
compounds in monkey and man following i.v. and i.t. administration. A method was 
developed to quantify intracellular thioinosine monophosphate and thioguanosine 
monophosphate to be used in a clinical study of 6-mercaptopurine. A radioactive 
photoactive anthracycline analog was used to photoaffinity label anthracycline 
binding proteins in P388 cells sensitive and resistant to anthracyclines, and in 
rat heart homogenates. Specific polypeptides were isolated which may function as 
mediators of anthracycline activity. 

142 



PHS 6040 (Rev. 1/84) 



GPO SI 4-918 



ZOl CM 06162-01 LBC 

Aphidicolin glycinate: Antitumor activity and biochemical effects in vivo . 
Aphidicolin, a specific inhibitor of DNA polymerase is active against experi- 
mental murine tumor models. Its poor solubility has led us to investigate 
aphidicolin glycinate (AG, NSC-303812), a water-soluble ester currently under- 
going preclinical toxicological evaluation. AG has demonstrated good activity 
against the i.p. implanted B16 melanoma producing maximum increased life spans 
of 75% following i.p. administration q3h t.i.d. on days 1-9. Treatment sche- 
dules involving both single injections per day on days 1-9 and multiple injec- 
tions per day on days 1,5, and 9 were less effective. Similarly, greater 
activity was observed against the i.p. M5076 sarcoma when 3 daily injections 
were given on days 1-9 (57% ILS) than with a single injection on either days 
1-9 (36% ILS) or on days 1,5, and 9 (inactive). On the assumption that DNA 
polymerase inhibition is the basis for this activity, inhibition of DNA syn- 
thesis in CDFl mice was evaluated by measurements of incorporation of ^H-thymi- 
dine (20 liCi iv) into DNA of spleen and jejunum. At 2h after administration 
of AG, inhibition was dose dependent (ID5Q 60 mg/kg in both tissues) and was 
>99% at 300 mg/kg. The inhibition was rapid in onset; AG 100 mg/kg i.p. pro- 
duced maximal (>98%) inhibition in both tissues at 30 min. Recovery occurred 
in intestine within 16h; in spleen recovery was delayed to 24h, and was fol- 
lowed by a rebound incorporation at 48h (203%). We conclude that AG produces 
marked inhibition of DNA synthesis at doses below the LDjo in mice. Similar 
experiments with sensitive and resistant tumors may reveal selectivity in 
the inhibition of DNA synthesis, and guide optimal scheduling. 

Tiazofurin: Biochemical and pharmacologic effects . We have shown that Tiazo- 
furin (TR) enters human erythrocytes by utilizing their facilitated nucleoside 
transport system. TR competes with endogenous nucleosides for this transport 
mechanism, thereby reducing nucleoside uptake into the cells. The Km and Vmax 
(± SE) for radio-labeled TR transport into erythrocytes are 170 ± 26 pM and 
55 ± 13 nmol/h/10^ cells, respectively, which is similar to the kinetic con- 
stants measured for uridine transport into erythrocytes (Km = 168 ± 37 uM and 
Vmax =61 ±16 nmol/h/lO^ cells). The Ki (± SE) of TR for uridine transport is 
178 ± 11 vM and for thymidine transport is 102 ± 59 ]M. The clinical signifi- 
cance of TR sharing the same transport protein as endogenous nucleosides is 
unclear. TR could potentially reduce the entry of circulating nucleosides 
into cells and might thereby alter the effects of antipyrimidine antitumor 
agents, or it may directly alter the cellular transport of antitumor agents 
which utilize the nucleoside transport system. 

Preliminary results from cell survival studies (using a cell cloning assay) 
show that a combination of 100 yM TR and 100 \M 5FU increases the killing 
of cultured L1210 cells by 1 log as compared to each of these agents alone. 
Furthermore, there is a significantly greater cell killing effect when these 
2 drugs are combined if TR and 5FU are administered simultaneously or when TR 
incubation precedes TR incubation. This indicates that TR may be enhancing 
the cytotoxicity of 5FU. To test this hypothesis we studied biochemical 
changes in L1210 cells in vitro following TR treatment and showed that incuba- 
tion of L1210 cells for 4 h with 0.5 or 2 mM TR resulted in a 7-9 fold increase 
in their intracellular concentration of PRPP. In addition, further incubation 
of these cells with 14c_5fu for 1 h resulted in a 4-7 fold increase in the 
formation of phosphorylated anabolites of 5FU. These results indicate that 
the increased toxicity resulting from the combination of TR and 5FU may be 



143 



ZOl CM 06162-01 LBC 

due to Increased PRPP concentrations that enhance the anabolism of 5FU to 
toxic species. 

When non-toxic doses (250-500 pM) of TR precede administration of 5FU to CDFi 
mice, there is an increase in host toxicity in response to this combination 
as compared to the administration of 5FU as a single agent. Administration of 
200 mg/kg 5FU (i.p.) alone, resulted in a 1% mortality rate, which was increased 
to 27 and 63% with prior administration of 250 or 500 mg/kg TR. When mice 
bearing L1210 tumors were pretreated with 250 mg/kg TR, there was an improvement 
in the antitumor effect of a low dose of 5FU (50 mg/kg) (42% ILS 5FU alone to 
85% ILS 5FU/TR combination), but this combination was not superior to the 
antitumor properties of an optimal dose (100 mg/kg) of 5FU alone (85% ILS). 
Thus, it appears that the dose response curve for 5FU toxicity is shifted to 
lower concentrations by TR. 

Pharmacology of cyclopentenyl cytosine . Cyclopentenyl cytosine is currently 
under evaluation as an antitumor agent: reproducible activity has been seen 
against both L1210 and P388 leukemia in the mouse. In L1210 a >100% ILS was 
achieved when mice were treated with 1 mg/kg qdx9. Biochemical studies in vivo 
and in cell cultures suggest inhibition of cytidine nucleotide formation as the 
mechanism of action. In vivo treatment of mice bearing L1210 leukemia produces 
a sharp drop in CTP pooTs within 4h. In L1210 cell cultures, cyclopentenyl 
cytosine inhibits cell replication at concentrations as low as 100 nM, but 
provision of 20 pM cytidine permits cell growth at normal rates even in the 
presence of 1 jjM cyclopentenyl cytosine. 

Attempts to increase blood flow in the rat solid Walker 256 tumor by the use 
of perfluorochemical emulsion fluosol-DA (F-DA). Solid tumors may be refrac- 
tory to antineoplastic agents because of poorly perfused regions within the 
tumor. The small particle size and decreased viscosity of the F-DA emulsion 
as compared to blood may allow for increased perfusion throughout the tumor. 
We have examined the effect of F-DA on blood flow in male Wistar rats bearing 
a Day-7 solid Walker 256 tumor implanted s.c. in the neck. F-DA-treated rats 
maintained under 100% O2 underwent an isovolumic F-DA-blood exchange which 
reduced their hematocrits to less than 3%. Blood flow in tumor and normal 
tissues was determined using l^c.iodoantipyrine and autoradiographic techniques. 
The results indicate that F-DA blood exchange did not increase flow to the 
whole tumor. Similarly the pattern of regional flow within the tumor was not 
substantially altered. In contrast, flow to cerebral tissue was increased two- 
fold. The findings do not support the use of F-DA to enhance blood flow to the 
s.c. implanted solid Walker 256 tumor. 

Anthracycline photoaffinity labeling of an 18 Kdalt protein in P388 sensitive 
and resistant cell lines . N-(p-Azidobenzoyl )-daunorubicin, ([^HJNABD), a 
radioactive photoactive anthracycline analog was used for photoaffinity label- 
ing of anthracycline binding polypeptides (ABPs) in P388 murine leukemic cells. 
Anthracycline sensitive (P388/S) and resistant (P388/DRN) cell homogenates 
were mixed with 50 nM [3h]NABD, exposed to UV-light, and analyzed by SDS-PAGE 
for 3h incorporation. Fluorography showed incorporation of radioactivity 
into a Mp 18 kdalt component independent of prominently stained polypeptides. 
Radioactive incorporation into this component was 3966 ± 355 dpm and 6487 ± 533 
dpm per 50 ifl cellular protein for P388/S and P388/DRN cells, respectively 
(p<.005). Photolabeling of P388/S subcellular fractions showed predominant 

144 



ZOl CM 06162-01 LBC 

mitochondrial localization of the Mp 18 kdalt radiolabel. This was confirmed 
by a relative quantitative distribution of radiolabel corresponding to that of 
the mitochondrial marker, cytochrome oxidase. ABP photolabeling was unaffected 
by DNAse or RNAse, and radioactivity was not extracted into chloroform :methanol 
(2:1). The protein composition of the photolabeled constituent was confirmed 
by the reduction of radioactivity to ambient background following treatment 
with proteinase K. The quantitative photolabeling of Mp 18 kdalt was maximum 
after 10 min of UV-light exposure, and was linear with respect to protein. 
Saturation of photolabeling was observed for higher concentrations (>100 nM) 
of [3h]NABD. The possible role of this polypeptide as a mediator of anthracy- 
cline activity remains to be determined. 

Rat heart anthracycline binding polypeptides (ABPS) identified by photo-affinity 
labeling" The same technique described above was used to photo-affinity label 
ABPs in rat heart homogenate. The most prominently labeled species were Mp 18.3 
and 31.2 Kd polypeptides located in the 100,000 x g sedimented fraction. Mp 
18.3 exhibited a saturable photolabeling of 2.42 ± 0.47 pmoles/mg homogenate 
protein above 0.15 uM ^HNAB-daunorubicin while Mp 31.2 exhibited an apparent 
linear increase in photolabeling up to 0.62 i/l 3H_NAB-daunorubicin. A 16-fold 
molar excess of NAB-daunorubicin over 0.16 \t\ ^H-NAB-daunorubicin reduced 
the photolabeling of Mp 18.3 by 90% confirming a high degree of analog labeling 
specificity. Mp 31.2 was reduced 25% suggesting both specific and nonspecific 
labeling. Mp 18.3 and 31.2 were not labeled by photoactive model compounds 
lacking the amino sugar and/or the anthracyclinone ring. Also, photolabeling 
was inhibited by the addition of daunorubicin and N-substituted anthracycline 
analogs. 

Clinical pharmacologic studies with 6-mercaptopurine . A method was developed 
to quantify intracellular thioinosine monophosphate (TIMP) and thioguanosine 
monophosphate (TGMP) in patients' leukemic cells which were treated with 6-mer- 
captopurine (6MP). The feasability of the method was demonstrated by quanti- 
fying TIMP and TGMP in leukemic cells obtained 24 h and 48 h after start of 
infusion with 6MP. These studies will be used to correlate TIMP and TGMP 
intracellular levels with plasma levels of 6MP and the patient's response. 

Blood-CSF studies with thiotepa . The intent of this study was to evaluate the 
therapeutic advantage of intrathecal administration of thiotepa using a monkey 
model. A gas chromatographic method was developed using nitrogen-phosphorous 
detectors to quantify thiotepa and its metabolite, TEPA, in extracts of blood 
and CSF. The results of these studies showed that after iv administration of 
thiotepa, the drug was rapidly equilibrated with CSF (<10 min.), and that 
thiotepa was rapidly cleared from CSF and plasma (t 1/2 of approx. 20-30 min). 
However, after iv administration of thiotepa, considerable TEPA was formed and 
found to have a much longer t 1/2 (approx. 5 h). A comparison of total tissue 
exposure (AUC) of thiotepa and TEPA to the CSF after iv and i.t. administration 
of Thiotepa indicated a 20:1 therapeutic advantage when the drug was given iv 
(assuming that the potency of thiotepa and TEPA are equivalent). Subsequent 
data obtained from a patient who received an i.t. dose and another patient who 
received an iv dose of Thiotepa indicated species differences in man and monkey 
in that the amount of TEPA formed in man was considerably less than that obser- 
ved in the monkey. These results suggest no therapeutic advantage of iv admin- 
istration of Thiotepa and demonstrate that the monkey model is a poor predictor 
for man in this instance. 

145 



ZOl CM 06162-01 LBC 
Publications : 

1. Arnold, S.T., Jayaram, H.N., Harper, G.R., Litterst, C.L., Malspeis, L., 
Desouza, J.J.V., Staubus, A.E., Ahluwalia, G.S., Wilson, Y.A. , Cooney, D.A. 
and Johns, D.G.: The disposition and metabolism of tiazofurin in rodents, 
rabbits and dogs. Drug Metab. Disp . 12: 165-173, 1984. 

2. Blasberg, R., Horowitz, M. , Strong, J., Molnar, P.. Patlak, C, Owens, E., 
and Fenstermacher, J.: Regional measurements of [^'^Clmisonidazole distri- 
bution and blood flow in subcutaneout RT-9 experimental tumors. Cancer 
Res. 45: 1692-1701, 1985. 

3. Clendeninn, N.J., Jolivet, J., Curt, G.A., Cysyk, R.L., Myers, C.E. and 
Chabner, B.A.: Cytotoxic drugs active in leukemia. In Goldman, J.M. and 
Preisler, H.D. (Eds.): Leukemia , Boston, MA, Butterworth, 1984, pp. 
35-103. 

4. Dodion, P., Egorin, M.J., Tamburini, J.M., Riggs, C.G., Jr. and Bachur, 
N.R.: The murine metabolism and disposition of marcel lomycin. Drug Metab. 
Disp . 2: 209-216, 1984. 

5. Dodion, P., Riggs, C.E., Jr., Akman, S.R., Tamburini, J.M., Colvin, O.M., 
and Bachur, N.R.: Interactions between cyclophosphamide and adriamycin 
metabolism in rats. J. Pharmacol, and Exp. Ther . 229: 66-69, 1984. 

6. Dodion, P., Rozencweig, M. , Nicaise, C, Walthien, M. , Tamburini, J.M., 
Riggs, C.E., Jr. and Bachur, N.R.: Human pharmacokinetics of marcello- 
mycin. Cancer Chemother. Pharmacol . 14: 42-48, 1985. 

7. Egorin, M.J., Fox, B.M., Spiegel, J.F., Gutierrez, P.L., Friedman, R.D. 
and Bachur, N.R.: Cellular pharmacology in murine and human leukemic cell 
lines of diaziquone (NSC 182986). Cancer Res . 45: 992-999, 1985. 

8. Gormley, P., Riccardi, R., O'Neill, D. and Poplack, D. : Intrathecally 
administered m-AMSA in the Rhesus monkey. Cancer Drug Delivery 1: 
101-107, 1984. 

9. Horowitz, M., Blasberg, R., Molnar, P., Strong, J., Kornblith, P., 
Pleasants, R. and Fenstermacher, J.: Regional l^c-misonidazole distri- 
bution in the RT-9 brain tumors. Cancer Res . 43: 3800-3807, 1983. 

10. Kalyanaraman, B., Sealy, R.C., and Sinha, B.K.: An electron spin reso- 
nance study of the reduction of peroxides by anthracycline semiquinones. 
Biochem. Biophys. Acta , 799: 270-275, 1984. 

11. Kurlansik, L., Williams, T.J., Strong, J.M., Anderson, L.W., Campana, J.E.: 
Desorption ionization mass spectrometry of synthetic porphyrins. Biomed . 
Mass Spectrum 11: 475-481, 1984. 

12. Lucignani, G., Nehlig, A., Blasberg, R., Patlak, C.S., Anderson, L., 
Fieschi, C., Fazio, F., Sokoloff, L.: Metabolic and kinetic considera- 
tions in the use of [125j]hipdm for quantitative measurement of regional 
cerebral blood flow. J. Cereb. Blood Flow Metabol . 5: 86-96, 1985. 

146 



ZOl CM 06162-01 LBC 

13. Nakazawa, H., Andrews, P. A., Gallery, P.S. and Bachur, N.R.: Superoxide 
radical reactions with anthracycline antibiotics. Biochem Phamacol . Vol. 
34 4: 481-490, 1985. 

14. Pan, S., Andrews, P. A., Glover, C.J., and Bachur, N.R.: Reductive activa- 
tion of mitomycin C and mitomycin C metabolites catalyzed by NADPH-cyto- 
chrome P-450 reductase and xanthine oxidase. J. Biol. Chem . 259: 959-966, 
1984. 

15. Shoemaker, D.D., Cysyk, R.L., Gormley, P.E., DeSouza, J.J.V. and Malspeis, 
L. : The metabolism of m-AMSA by rat liver microsomes. Cancer Res . 44: 
1939-1949, 1984. 

16. Sinha, B.K.: Metabolic activation of procarbazine: Evidence for carbon- 
center free radical intermediates. Biochem. Pharmacol . 33: 2777-2781, 1984. 

17. Sinha, B.K., Trush, M.A., Kennedy, K.A. and Mimnaugh, E.G.: Enzymatic 
activation and binding of adriamycin to nuclear DNA. Cancer Res . 44: 
2892-2896, 1984. 

18. Akman, S., Dodion, P., Tamburini, J.M., Riggs, C.G., Jr., Colvin, D.M. and 
Bachur, N.R.: Interactions between cyclophosphamide and adriamycin 
metabolism in rats. J. Pharmacol. Exp. Ther ., 1985, in press. 

19. Dodion, P., Chang, B.K., Egorin, M.J., Olman, E.A., English, K.A. and 
Bachur, N.R.: The metabolism and disposition of the new anthracycline 
antibiotic menogarol (7-OMEN) in mice. Drug. Metab. Disp ., 1985, in 
press. 

20. Dodion, P., Riggs, C.E., Jr., Akman, S.R. and Bachur, N.R.: The effect of 
hyperthermia on the in vitro metabolism of adriamycin. Cancer Treat. Rep . 
1985, in press. 

21 Felsted, R.L., Gupta, S.K., Glover, C.J. and Gallagher, R.E.: Surface 
membrane glycoproteins of wild type and differentiation-inducer-resistant 
HL-60 cells. Blood , 1985, in press. 

22. Kalyanaraman, B. , Sealy, R.C. and Sinha, B.K. : An electron spin resonance 
study of the reduction of peroxides by anthracycline semiquinones. 
Biochim. Biophys. Acta , 1985, in press. 

23. Klecker, R.W., Jr., Jenkins, J.F., Kinsella, T.J., Fine, R.L., Strong, J.M. 
and Collins, J.M. : Clinical pharmacology of 5-iodo-2'-deoxyuridine 

and 5-iodo-uracil and endogenous pyrimidine modulation. Clin. Pharma. Ther . 
1985, in press. 

24. Mimnaugh, E., Kennedy, K., Trush, M. and Sinha, B.K. Adriamycin-enhanced 
membrane lipid peroxidation in isolated nuclei. Cancer Res . 1985, in press. 

25. Monks, A., Marquez, V.E., Mao, D.T. , and Cysyk, R.L.: Inhibition of 
nucleoside transport by 2-B-D-ribofuranosylthiazole-4-carboxamide 
(Tiazofurin) and related analogues. Cancer Lett. , 1985, in press. 



147 



ZOl CM 06162-01 LBC 

26. Nakazawa, H., Riggs, C.E., Jr., Egorin, M.J., Redwood, S.M., Bachur, N.R., 
Bhatnagara, R. and Ito, Y.: Continuous extraction of urinary anthracycline 
antitumor antibiotics with the horizontal flow-through coil planet centri- 
fuge. J. Chromatogr ., 1985, in press. 

27. Sinha, B.K., Strong, J., Gibson, N. and Kalyanaraman, B., Mechanism of DNA 
strand breaks by mitonafide, an imide derivative of 3-nitro-l,8-naphthalic 
acid. Biochem. Pharm . 1985, in press. 

28. Sinha, B.K., Strong, J., Gibson, N.W., and Kalyanaraman, B.: Metabolism 
and mechanism of action of imide derivative of 3-nitro-l,8-naphthalic acid; 
mode of action. Biochem. Pharm . 1985, in press. 

29. Sinha, B.K., Trush, M.A. and Kalyanaraman, B.: Microsomal interactions and 
inhibition of lipid peroxidation by etoposide (UP-16, 213); implication for 
(mitonafide). Chem. Biol. Interaction, 1985, in press. 



148 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 06163-01 LBC 



PERIOD COVERED 

October 1, 1984 to September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line tjetween the borders.) 

Pharmacologic Aspects of Nucleotide Metabolism 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 



PI: 
Other: 



R. Cysyk 

L. Anderson 

S. Arnold 

0. Ayers 

C. Chisena 

N. Malinowski 

P. Monks 

J. Moyer 



Chief LBC, 

Chemist LBC, 

Chemist LBC, 

Lab. Technician LBC, 

Biologist LBC, 

Chemist LBC, 

Visiting Associate LBC, 

Sr. Staff Fellow LBC. 



NCI J. Strong Pharmacologist LBC, NCI 

NCI D. Zaharevitz Staff Fellow LBC, NCI 

NCI 

NCI 

NCI 

NCI 

NCI 

NCI 



COOPERATING UNITS (if any) 

Medicinal Chemistry Section, LPET, NCI (V. Marquez) 
Ohio State University (L. Malspeis) 



LAB/BRANCH 

Laboratory of Biological Chemistry 



INSTITUTE AND LOCATION 



NCI. NIH. Bethesda. Maryland 20205 



TOTAL MAN- YEARS: 

4.5 



PROFESSIONAL: 

2 



2.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



n (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) , 

The isolated perfused rat liver exports the purines adenine and hypoxantnine at a 
rate such that 0.5-1.0 \i^ of each compound is maintained in the perfusate. It 
was found that this concentration of either purine completely reversed the growth 
inhibition of L1210 and HL-60 cells caused by DON, an inhibitor of de novo purine 
biosynthesis. Also, 1 ]i^ adenine (in the absence of DON) completely inhibited de 
novo purine synthesis. Thus, physiologic concentrations of these purines can sup- 
port cell growth in the absence of de novo synthesis. An in vivo tumor model was 
established to study the actual utiTTzatTon of circulating purines and pyrimi dines 
by a solid tumor. Studies with 15NH4CL and 15N-glutamine demonstrated extensive 
involvement of the urea cycle in the formation of pyrimidines, de novo , in isolated 
rat hepatocytes. 3-Deazauridine and dipyridamole were evaluated as inhibitors of 
uridine salvage in vivo . 3-Deazauridine produced a marked, but short-lived, inhi- 
bition of uridine~salvage in liver, kidney, and L1210 tumor cells. Dipyridamole 
did not significantly reduce salvage. A series of 29 analogues of uridine was 
examined as inhibitors of uridine kinase in vitro and of uridine salvage by intact 
L1210 cells. Of these compounds, cyclopentenyl uracil is most promising since it 
selectively blocks the salvage of uridine by L1210 cells, is resistant to phosphor 
olysis, and is non-cytotoxic at biochemically active concentrations. Uridine phos- 
phorylase was purified to homogeneity and tested in vivo for its ability to reduce 
the concentration of circulating uridine that wouTcT be available for salvage. 
Uridine phosphorylase alone produced a modest (20%) decrease in uracil nucleotides 
of L1210 ascites cells, but greatly depleted (>95%) pools in combination with PALA, 
an inhibitor of pyrimidine synthesis de novo . A series of uridine analogues modi- 
fied in the 5'-position of the ribosyTmoiety was evaluated as inhibitors and 
substrates of uridine phosphorylase. Replacement or esterification of the 5!- 
hydroxyl group led to nearly complete loss of susceptibility to phosphorolysis. 

149 



PHS 6040 (Rev. 1/84) 



GPO 9l4>«ie 



ZOl CM 06163-01 LBC 
Objective : 

The overall objective of this project is to determine the relative dependency 
of host and tumorous tissues on de novo vs salvage pathways for the synthesis 
of pyrimidine and purine nucleotTJes in vivo . The de novo biosynthetic pathways 
supply pyrimidines and purines for nucleic acid synthesis and are therefore 
considered to be important pathways for cell proliferation. Therefore, 
there has been a considerable effort throughout the past several decades to 
develop specific inhibitors of enzymes of these pathways. Although a number 
of potent inhibitors (e.g. PALA, pyrazofurin, 6-azauridine) exhibit excellent 
in vitro activity against isolated enzymes and cultured cells and in vivo 
activity against certain murine tumors, only marginal clinical success has 
been achieved with these agents. It would appear that factors other than 
potency of inhibition are important because very potent enzyme inhibitors 
(PALA, pyrazofurin) are only marginally effective. Accordingly, lack of clin- 
ical success with the pathway inhibitors developed to date might be an indica- 
tion that the importance of the de novo pathway to cell survival in vivo might 
be over-estimated. If so, then the development of additional inhibitors of 
the de novo pathway would be a futile effort unless there is a coordinate 
development of agents that either interfere with the salvage pathway or with 
the synthesis and export of preformed nucleosides by donor organ(s). 

This Project is divided into the following specific aims: to determine the 
physiologic importance of circulating pyrimidines and purines and their role 
in modulating the antitumor activity of antipyrimidine and antipurine chemo- 
therapeutic agents; to study the liver as a modulator of circulating nucleo- 
sides and as a possible target for chemical manipulation; to develop agents to 
interfere with nucleoside salvage to be used in combination with inhibitors of 
de novo synthesis; to develop methodology for monitoring and quantitating the 
flux through the de novo pathways of host and tumorous tissues in vivo . 

Major Findings : 

Physiologic and pharmacologic importance of circulating nucleosides . Previous 
studies from our Laboratory (Karle, Anderson, and Cysyk, J. Biol. Chem. 259: 
67-72, 1984) demonstrated that uridine, at concentrations found in plasma, 
reversed the growth inhibition by PALA of cultured L1210 cells. Also, in the 
presence of plasma concentrations of uridine, cultured L1210 cells turned off 
de novo pyrimidine synthesis and utilized their salvage pathway nearly exclu- 
sively. Thus, circulating uridine may be an important factor in antipyrimidine 
chemotherapy. Purine nucleosides and bases were studied in a similar manner. 
It was determined that the isolated perfused rat liver exports the purines 
adenine and hypoxanthine at a rate to maintain the perfusate at 0.5-1.0 iiM 
in each of these compounds. HL-60 cells were treated with 3 pM DON and 
L1210 cells were treated with 0.3 liM DON. Concurrently, the cells were 
treated with 0.5 to 10 ;jM adenine or hypoxanthine. Treatment with DON, 
only, resulted in inhibition of cell growth to 30% of control growth. Co- 
incubation with low levels of either adenine or hypoxanthine reversed this 
toxicity such that the cells grew 80-95% of control growth. Thus, low levels 
of purines, within the physiological range, are capable of reversing the 
toxicity of an inhibitor of purine de novo synthesis. It was found that 
adenine concentrations as low as 1.0 vM completely inhibit purine de novo 
synthesis. At this concentration, PRPP levels are unaffected as is undine 

150 



ZOl CM 06163-01 LBC 

synthesis. Thus, if maintained at a constant concentration, 1 pM adenine is 
able to provide sufficient purines to maintain the purine nucleotide pool via 
the salvage pathway alone. In addition, the regulation of the de novo synthe- 
tic pathway is accomplished by a means other than lowering of the PRPP pool, 
since the PRPP concentration was unaffected by an adenine concentration capable 
of completely inhibiting purine de novo synthesis. 

Development of an in vivo tumor model to study the utilization of circulating 
preformed purines and pyrimidineT I To evaluate to what extent tumors in vivo~ 
depend on their salvage pathways to fulfil their nucleotide requirements, we 
have adapted the tumor model system developed by Gull i no and coworkers, where 
a tumor (in our case Walker 256) is implanted into the ovarian fat pad of female 
Sprague-Dawley rats and isolated in a parafilm pouch in the subcutaneous space. 
This "tissue-isolated" tumor is connected to the host by a single artery and 
vein from which blood can be withdrawn. Also, a chamber can be inserted into 
the growing neoplastic mass from which to sample the interstitial fluid. 
Guillino and coworkers used this system to analyze for amino acids, proteins, 
and a variety of small molecules (other than nucleosides). This system 
permits direct measurement of the blood flow and trans-tumor differential 
of nucleoside concentration. With this information it will be possible to 
calculate for the first time the actual net hourly contribution of circulating 
nucleosides and bases to tumor nucleotide pools. 

Hepatic regulation of circulating nucleosides . Research from this laboratory 
(Monks and Cysyk, Am. J. Physiol. 242, 465, 1982) has shown that the isolated 
rat liver can act as a donor organ of the nucleoside uridine which could be 
salvaged in vivo by other organs or tumors. Manipulation of this donor organ 
to inhibit or increase biosynthesis of salvageable uridine could be useful in 
cancer chemotherapy. A recently completed study from our laboratory (Monks, 
Chisena, and Cysyk, Arch. Biochem. Biophys. 236: 1, 1985) indicated that the 
hepatic production of pyrimi dines utilizes intermediates of the urea cycle^ 
This study utilized ■'■^C-bi carbonate. We are currently using -^^NH^Cl and ^^N- 
glutamine to determine directly the contribution of CPS-I and CPS-II to the Nj 
and N3 positions of the uracil nucleus. The extent of incorporation is deter- 
mined by GC/MS using our recently developed method (Strong et al . J. Biol. 
Chem. 260: p. 4276, 1985). 

Development of inhibitors of nucleoside salvage . The results discussed in the 
earlier sections of this project indicate that inhibition of nucleoside salvage 
could enhance the effectiveness of inhibitors of de novo nucleotide synthesis. 
An in vivo model system (Moyer, Malinowski, and Ayers, J. Biol. Chem. 260 : 2812- 
2818, 1985) developed to study the metabolism of pyrimidine nucleosides present 
in the plasma of the mouse is currently being used to assess the effects on 
salvage, in vivo , of agents designed to block pyrimidine salvage. A series 
of studies were performed to evaluate the effects of 3-deazauridine and the 
nucleoside transport inhibitor dipyridamole (Persantin) on uridine salvage in 
vivo . 3-Deazauridine (500 mg/kg) produces a marked (>70%) inhibition of 
uracil nucleotide formation from uridine in liver, kidney, and L1210 tumor 
cells. The inhibition was only short-lived however, even at this high dose. 
Dipyridamole (100 mg/kg) did not significantly reduce salvage of uridine by 
liver or kidney and produced only small, transient reductions in salvage by 
L1210 tumors. Dipyridamole pretreatment did not alter the rate of clearance 
of uridine from the plasma. 

151 



ZOl-CM-06163-01 LBC 

We examined a series of 29 analogs of uridine, most with modifications at the 
5'-position, as inhibitors of uridine kinase in vitro and of uridine salvage 
by intact L1210 cells. Substitution at the 5"^position resulted in decreased 
efficacy as inhibitors of uridine kinase, particularly if the substituent was 
large. None of the analogs with 5'-position modifications effectively inhibited 
salvage of uridine by intact L1210 cells. A series of four carbocyclic pyrimi- 
dine nucleoside analogs were all effective competitive inhibitors of uridine 
kinase and of uridine salvage by intact L1210 cells. Cyclopentenyl uracil is 
promising as an inhibitor of uridine salvage since it selectively blocks the 
salvage of uridine by cells, is resistant to phosphorolysis, and is not cyto- 
toxic at biochemically active concentrations. Although not a particularly 
potent inhibitor of uridine kinase it may nonetheless be effective in vivo , 
because physiological levels of uridine phosphorylase should prevent accumula- 
tion of uridine behind the enzymic block. Further development and assessment 
of the in vivo effectiveness of these analogues is underway. 

Evaluation of Uridine Phosphorylase as a chemotherapeutic agent . Results pub- 
lished this year (Moyer and Henderson, Biochem. Pharm. 34: lOlTlOS, 1985) 
indicated that administration of uridine phosphorylase could reduce the utiliz- 
ation of endogenous uridine by conversion to uracil which is very poorly sal- 
vaged. We have subsequently found that 25 units/mouse of uridine phosphory- 
lase, purified by affinity chromatography, produced marked (>90%) inhibition 
of uracil nucleotide formation by L1210 tumor cells, with a recovery to 40% of 
control seen 12 h after injection of enzyme. Uridine phosphorylase alone 
produced a modest (20%) decrease in uracil nucleotides of L1210 ascites cells, 
but greatly depleted (>95%) pools in combination with PALA, an inhibitor of 
pyrimidine synthesis de novo . Studies in progress will establish the therapeu- 
tic effect of combinations of PALA and uridine phosphorylase, but initial 
results indicate that the combination is more toxic than PALA alone, and this 
increase in toxicity may preclude successful use of this drug combination. 

Development of inhibitors of nucleoside catabolism . The enzyme uridine phos- 
phorylase (EC 2.4.2.3) catalyzes the first step of uridine catabolism and also 
cleaves the chemotherapeutic agents 5-fluorouridine, 5-bromo-2'-deoxyuridine, 
and 5-iodo-2'-deoxyuridine. In vivo this enzyme is extremely active and pro- 
duces a very rapid cleavage of uridine in rats or mice. Susceptibility to 
cleavage by this enzyme is therefore a major consideration for the design of 
pyrimidines as chemotherapeutic agents. We examined a series of uridine ana- 
logs modified in the 5'-position of the ribosyl moiety as inhibitors and sub- 
strates of uridine phosphorylase f rom £. Coli and from rat liver. Replacement 
or esterification of the 5'-hydroxyl group led to nearly complete loss of 
susceptibility to phosphorolysis by either enzyme. 5'-Chloro-deoxyuridine 
and 5'-benzoyl-uridine, although not substrates, were competitive inhibitors 
of uridine phosphorylase f rom JE. Coli with Ki's of 370 yM and 49 yM, 
respectively. 5'-Ainino-5'deoxyuridine activated uridine phosphorylase from 
E. Col i with both a decrease in Km for uridine and an increased V^ax observed. 
These results are important to the design of uridine analogs and for the 
mechanism of uridine phosphorylase. 



152 



ZOl-CM-06163-01 LBC 
Publications ; 

1. Karle, J.M., Anderson, L.W. and Cysyk, R.L.: Effect of plasma concentration 
of uridine on pyrimidine biosynthesis in cultured L1210 cells. J. Biol . 
Chem . : 259, 67-72, 1984. 

2. Karle, J.M. and Cysyk, R.L.: Regulation of pyrimidine biosynthesis in 
cultured L1210 cells by 3-deazauridine. Biochem. Pharmacol . 33: 
3739-3742, 1984. 

3. Karle, J.M., Monks, A., Wolfe, R.M. and Cysyk, R.L.: Effect of 2-0-D- 
Ribofuranosylthiazole-4-carboxamide on salvage of nucleosides by cultured 
L1210 cells. Cancer Lett . 24: 4276-4281, 1985. 

4. Lim, M. , Moyer, J.D., Cysyk, R.L. and Marquez, V.E.: Cyclopentenyl 
uridine and cytidine analogs as inhibitors of uridine-cytdine kinase. 
J. Med. Chem . 27: 1536-1538, 1984. 

5. Monks, A., Chisena, C.A. and Cysyk, R.L.: Influence of ammonium ions on 
hepatic de novo pyrimidine biosynthesis. Arch. Biochem. Biophys . 236: 
1-10, 19'55. 

6. Moyer, J.D. and Paterson, A.R.P. and Henderson, J.F.: Effect of a 
nucleoside transport inhibitor on the disposition of uridine in mice. 
Biochem. Pharmacol . 33: 2327-2328, 1984. 

7. Moyer, J.D. and Henderson, J.F.: Salvage of uridine in the mouse: The 
effect of uridine phosphorylase pretreatment. Biochem. Pharmacol . 34: 
101-105, 1985. 

8. Moyer, J.D., Malinowski, N., and Ayers, 0.: Salvage of circulating 
pyrimidine nucleosides by tissues of the mouse. J. Biol. Chem . 260: 
2812-2818, 1985. 

9. Strong, J.M., Upton, O.K., Anderson, L.W. , Monks, A., Chisena, C.A. and 
Cysyk, R.L.: A novel approach to the analysis of mass spectrally assayed 
stable isotope labeling experiments. J. Biol. Chem . 260: 4276-4281, 1985. 

10. Zimm, S., Grygiel, J.J., Strong, J.M., Monks, T. , Poplack, D.G. : Indentifi- 
cation of 6-mercaptopurine riboside in patients receiving 6-mercaptopurine 
as a prolonged intravenous infusion. Biochem. Pharmacol . 33: 4089-4092, 
1984. 

11. Karle, J.M., Cowan, K.H. and Cysyk, R.L.: Uracil nucleotide synthesis 

is a human breast cancer cell line (MC-7) and in two drug resistant sublines 
that contain increased levels of enzymes of the de novo pyrimidine pathway. 
J, Biol. Chem., 1985, in press. 

12. Moyer, J.D. and Henderson, J.F.: Compartmentation of intracellular 
nucleotides in mammalian cells. Crit. Rev. Biochem., 1985, in press. 



153 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06164-01 LBC 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Inhibitors of Phospholipid Metabolism as Potential Chemotherapeutic Agents 



PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: J. Moyer Staff Fellow LBC, NCI 



Other: 



N. Dean 
0. Ayers 



Visiting Fellow 
Lab. Technician 



LBC, NCI 
LBC, NCI 



COOPERATING UNITS (if any) 

Medicinal Chemistry Section, LPET, OCT, NCI 

Drug Synthesis and Chemistry Branch, DCT, DTP, NCI, 



LAB/BRANCH 

Laboratory of Biological Chemistry 



INSTITUTE AND LOCATION 

NCI. NIH. Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

2.0 



PROFESSIONAL: 

1.5 



0.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



D (b) Human tissues ^ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Agents which block the formation of second messengers which mediate growth factor 
action may be of value in cancer chemotherapy. We have developed test systems to 
identify drugs which interfere with phosphatidylinositol formation or hydrolysis, 
because the formation of inositol phosphates has been implicated in the action 
of a number of growth factors including platelet derived growth factor and 
bombesin. A series of rationally-designed inhibitors of enzymes involved in 
phosphatidylinositol metabolism are currently being synthesized for evaluation. 



154 



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ZOl CM 06164-01 LBC 

Objectives -. Reduced requirements for stimulation by growth factors may be the 
fundamental characteristic of transformed (neoplastic) cells. Research in 
molecular biology has identified several specific biochemical changes produced 
by introduction or overexpression of oncogenes which may reduce the levels of 
exogenous growth factors needed to trigger cell replication. These findings 
suggest that a more selective approach to chemotherapy may focus on the inter- 
action of growth factors with cells rather than on basic metabolic reactions 
such as those involved in nucleic acid synthesis. We have therefore initiated 
a project to develop new chemotherapeutic agents to block the action of growth 
factors. 

Recent findings indicate that inositol -phosphates formed from phosphatidyl - 
inositides may be the second-messengers which mediate the action of many growth 
factors. In addition, the product of two distinct oncogenes (src and ros) 
phosphorylate phosphatidyl inositol and increase the levels of phosphatidyl- 
inositol polyphosphates in the membranes of cells transformed by these onco- 
genes. The synthesis of phosphatidyl inositol polyphosphates and their sub- 
sequent hydrolysis therefore provide attractive well-defined targets for the 
design of inhibitors for use in chemotherapy. This project has two related 
goals. First, to determine the relative importance of the enzymatic reactions 
involved in inositol phosphate production as a signal initiating cell repli- 
cation. Secondly, to design, synthesize, and evaluate drugs to inhibit this 
process, specifically inhibitors of the reactions of phosphatidylinositol 
polyphosphate synthesis and of phospholipase C, the enzyme which produces the 
inositol phosphates. 

Major Findings : A series of test systems have been developed to identify drugs 
that inhibit phosphatidylinositol turnover or synthesis. These test systems 
fall into three categories: Functional tests of mitogen action, biochemical 
assay of inositol phosphate production, and assays of the enzymes involved in 
phosphatidylinositol metabolism. 

Functional Assays of Mitogen Action ; Two assays have been developed based on 
the response of quiescent Swiss 3T3 cells to serum or purified factors such as 
phorbol esters or platelet derived growth factor (PDGF). The first is [^H]- 
thymidine incorporation as a measure of DNA synthesis, the other is [3H]uridine 
uptake. Uridine uptake is reproducibly enhanced 3 to 5-fold within 1 h of 
stimulation of 3T3 cells by serum, phorbol esters, or PDGF. This assay should 
identify agents which prevent the action of growth factors, and is rapid and 
simple enough to permit testing of about 20 compounds per day. 

Biochemical Assays of Inositol Phosphate Production: We have developed an 
assay for measuring L^HJ-inositol phosphates based on separation with high 
performance liquid chromatography. Use of an automatic sample injector and a 
microprocessor-controlled fraction collector has permitted automation of this 
assay. Initial studies demonstrate a 3-5 fold increase in [3H]-inositol 
phosphate concentrations in 3T3 cells prelabeled with [3H]-inositol and stimu- 
lated with serum. This assay is being further characterized and should iden- 
tify agents which block the formation of inositol phosphates in 3T3 cells. 

A second assay employs GH3 cells, a rat pituitary tumor cell line. These cells 
were found to produce particularly large amounts of inositol phosphates when 
stimulated by TRH in the presence of lithium. The large production of inositol 



155 



ZOl CM 06164-01 LBC 

phosphates (50-fold controls) permits an estimate of inositol phosphate forma- 
tion without chromatography and makes these cells an excellent test system. 
We anticipate about 30 compounds per day could be evaluated. 

Phosphatidyl inositol synthesis by intact GH3 cells can also be measured in a 
short term assay of [3H]-inositol incorporation. Incorporation is directly 
proportional to time for at least 4 h and >90% of the label present in the 
lipid fraction is phosphatidyl inositol as determined by thin layer chroma- 
tography. 

Enzyme Assays ; A soluble preparation of phosphatidyl inositol synthetase 
permits enzyme activity measurements and the identification of inhibitors and 
substrates for this enzyme. Initial results show that simple di -substituted 
cyclohexanes are not inhibitors nor is epi-quercitol (4-deoxy-myo-inositol). 
The enzyme is inactivated by a, 3 unsaturated cyclohexone. The Km for 
Inositol was measured at 1.7 mM. 

Proposed Course ; Several compounds are currently being synthesized for evalua- 
tion in the assays described above. In particular, the 2-deoxy-myo-inositol 
and 5-deoxy-myo-inositol , which lack critical hydroxyls, will be evaluated. 
Mechanistic considerations suggest that these compounds should be unable to 
serve as a source of second messengers if they can be Incorporated into phos- 
pholipids. Phosphonate analogs of phosphatidyl inositol are potential non- 
hydrolizable analogs with possible inhibitory action. We will also attempt to 
alter cellular content of phosphatidyl inositides to determine the relative 
importance of phosphatidyl inositide availability versus phospholipase activa- 
tion in the production of inositol phosphates after stimulation by growth 
factors. These studies may provide useful inhibitors of phospholipid meta- 
bolism and should further clarify the potential of phospholipid metabolism as 
a target for cancer chemotherapy. 



156 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06165-01 LBC 



PERIOD COVERED 

October 1, 1984 to September 30, 



1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Stable Isotope Studies of de Novo Pyrimidine and Purine Pathways 



PRINCIPAL INVESTIGATOR (List Other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: J. Strong Pharmacologist LBC, NCI 



Other: 



L.W. Anderson 
D.W. Zaharevitz 
R.L. Cysyk 



Chemist 
Staff Fellow 
Pharmacologist 



LBC, NCI 
LBC, NCI 
LBC, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Biological Chemistry 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



1.5 



PROFESSIONAL: 
1 



0.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues 1^ (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Existing stable isotope tracer methodology was used to monitor the flux through 
the de novo pyrimidine pathway in vivo . Recovery of pathway activity after 
acivicin treatment in acivicin-sensiti ve and acivicin-resistant tumor lines was 
determined. In conjunction with these studies a new tumor model using invaded 
spleens was evaluated. Stable isotope tracer methodology was developed for 
studying the de novo pyrimidine and purine pathways. Methodology was developed 
using GC/MS techniques in combination with HPLC separations for quantifying iso- 
topic abundances in both purine and pyrimidine bases and nucleosides. In addition, 
a fully automated GC/MS technique was developed to simultaneously quantify the 
amount and isotopic ^^H enrichment in amino acids in sample extracts of various 
tissues. A computer model using linear algebra techniques was developed which 
allows us to interpret data obtained from multiple stable label experiments. The 
predictive nature of this technique will allow us to study the pharmacologic 
effects of compartmentation as it relates to antitumor activity of agents which 
act to inhibit pyrimidine and purine biosynthesis. 



157 



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ZOl CM 06165-01 LBC 

Radiolabeled tracer methodology has played a major role in the elucidation of 
metabolic pathways. Such information, coupled with specific activity determin- 
ations of enzymes in cell-free extracts, is a valuable indicator of the impor- 
tance of a particular pathway in the intact cell. Studies designed to measure 
pathway activity need to be carried out in intact cells because of the impro- 
bability of duplicating, in vitro , the intracellular microenvironment in which 
the enzyme functions. Moreover, the concept of intracellular compartmentation 
of pathway activities has now replaced the simplistic view of the cell as a 
bag of uniformly dispersed enzymes acting on a homogeneous supply of metabo- 
lites. Intracellular and intercellular compartmentation of enzymes and small 
molecules necessitates experimentation with intact cells and animals, however 
at the same time, complicates the interpretation of results obtained using 
tracer methodology. The use of stable labeled compounds as tracers combined 
with mass spectrometric techniques in some cases are more advantageous for 
these types of studies. Precursors can be labeled with the appropriate stable 
isotope to essentially 100% enrichment without posing a biohazard. These high 
enrichments minimize the dilution effects which occur especially in vivo . 
Stable isotopes such as nitrogen and oxygen which have no radiola¥el isotopes 
easily available can be used as tracers and most importantly, these techniques 
are easily adapted to studies where several atoms in the product molecule are 
labeled. The latter advantage will allow us to address some of the problems 
with tracer studies such as the fact that isotope enrichment determinations in 
pathway products following administration of labeled precursors represent an 
average enrichment value which includes (a) isotopically enriched product 
molecules that were formed from the labeled pathway under study and (b) non- 
isotopically enriched product molecules that were either present prior to the 
labeling period or were produced by an alternate pathway during the course of 
the labeling experiment. A method which would distinguish a-type molecules 
from b-type molecules would be a desirable technique to study pathway activity 
and regulation. Furthermore, such a method would be valuable in compartmenta- 
tion studies since the enrichment of the newly synthesized product molecules 
would be a measure of the isotopic enrichment of the precursor molecules in 
the cellular compartment from which the product is derived. 

Objective : 

The objective of this project is two-fold: (1) to develop stable-label method- 
ology to quantitate the flux through the de novo pyrimidine and purine pathways 
in vivo and in intact cells in vitro , ar\(f~(2) to develop stable-label methodo- 
logy and computer models to study compartmentation of reaction pathways in 
vivo and in intact cells in vitro . 

Measurement of Flux through the de novo pyrimidine and purine pathways . We 
have recently developed a method (Strong et. a1.. Anal. Biochem. 132~ 243-253, 
1983), which is the only method available, to monitor the flux through the de 
novo pyrimidine pathway in vivo . •'•^002 is administered to mice and the 
incorporation of ^\ into pyrimidine nucleotides is quantitated by GC/MS. 
Using this method we studied the effect of PALA, a potent inhibitor of de novo 
pyrimidine synthesis, on pathway flux in tumors sensitive and resistant to PALA 
(Monks et. al., J. Biol. Chem. 258, 13564-13569, 1983). During the past year, 
this method was used to study the effect of acivicin, a glutamine analogue 
undergoing clinical evaluation, on de novo pyrimidine synthesis in L1210 
(sensitive to acivicin) and B16 melanoma (resistant to acivicin) in vivo . 

158 



ZOl CM 06165-01 LBC 

In conjunction with these studies, a new tumor model (tumor invaded spleens 
contained in L1210 sc tumored mice), was investigated. Flux through the de 
novo pathway as measured by incorporation of -^002 into the Z uracil pools 
of both tumors and L1210-invaded spleens was maximally suppressed 1 h after 
acivicin treatment. The inhibitory effect of acivicin was reversed by 4 h in 
L1210 sc and spleens; however, the inhibitory action of acivicin persisted for 
48 hrs in the B16 melanoma model. Investigations are continuing using stable 
isotope labeling experiments to determine the effect of acivicin on the conver- 
sion of UTP to CTP in vivo . 

Gas chromatographic/mass spectrometric (gc/ms) techniques were developed to 
quantify isotopic abundances in cytosine, cytidine, adenine, adenosine, quani- 
nine, guanosine, inosine, and hypoxanthine. These gc/ms techniques in combin- 
ation with HPLC separations and enzyme and chemical degradations, will allow 
us to quantify the isotopic abundances in the purine and pyrimidine bases 
arising from base, nucleoside, and individual nucleotide pools contained in 
various tissues. Investigations are presently underway to establish the 
appropriate precursors, i.e., NH^, -^^N-glutamine, ^ N-glycine, etc., which 
will produce sufficient labeling in the purine nucleotide pools; thus allowing 
quantification of flux through the de novo purine pathway in vivo simultaneous 
with measurements of the de novo pyrimidine pathway. Other drugs and combina- 
tions of drugs will be studied in this system. Also, the dilabel technique 
(described below) will be used to quantitate pathway flux. 

Pharmacologic considerations of pathway compartmentation . Recently we reported 
a method to quantify de novo pyrimidine biosynthesis based on the isotopic 
enrichment of Nl and N3 atoms of the uracil nucleus when hepatocytes are exposed 
to 1^N-NH4C1 or ^^N-glutamine, (Strong et. al., J. Biol. Chem. 260: 4276-4281, 
1985). From these values we are able to calculate, utilizing probabil ity 
statistics, the number of uracil molecules produced by the de novo pathway 
that are labeled and those that are not labeled. By determining the enrichment 
of specific atoms of the product (uridine) formed in a pathway we know the 
isotopic enrichment of the immediate precursor pool for that atom. Comparison 
of this value with the precursor enrichment value obtained for the cell homo- 
genate will indicate if compartmentation of precursors is occurring. We have 
developed a general mathematical framework using linear algebra that is imple- 
mented in a computer program that will calculate the results of isotope dilu- 
tion experiments for an arbitrary number of components. The generality of the 
framework has allowed us to develop a detailed mathematical treatment of bio- 
synthetic studies which measure incorporation of a stable labeled precursor 
into a metabolic product. 

In order to implement these studies, it was necessary to develop methods to 
quantify the isotopic enrichments in the amino acids which are intermediates 
in the pyrimidine de novo synthetic pathway in vivo , especially glutamine and 
aspartate. This past year we developed a fuTTy automated system using a 
Hewlett Packard gc/ms equipped with an automatic sampler to quantify both the 
amount of amino acid in the sample and the isotopic abundance of stable labeled 
atoms in the amino acids simultaneously, for aspartate, glutamine, glutamate, 
and the non-amino acid urea in a single gs/ms run. Preliminary studies in 
tumored mice which were infused i.p. with -^^NH^CI indicate the usefulness 
of predicting compartmentation of reaction pathways in vivo . Firstly, analysis 
of the uracil nucleotide pool formed during the course of the experiment showed 



159 



1.0 


77.3 


68.2 


11.2 


11.4 


55.8 


82.0 


8.3 


16.0 


59.8 


80.5 


9.8 



ZOl CM 06165-01 LBC 

different patterns of enrichments in the N3 (78%) and Nl (53%) contained in 
the liver. A comparison of this data with extracts of the tumor uracil nucleo- 
tide pool demonstrated inter-organ differences i.e. N3 (48%) and Nl (7%). 
Secondly, in a similar study enrichments in the Nl and N3 atoms contained in 
the fraction of the newly formed uracil nucleotide pool of liver extracts were 
compared to the ^^N enrichment in aspartate contained in the same liver 
extracts. The results of this study are shown in the table: 

TIME OF INFUSION (min) %F (1) % ^^Hl % 15^3 % I^n-ASP 

10 
45 
75 

(1) fraction of uracil nucelotide pool formed during the experiment 

These results imply intra-organ compartmentation since the isotopic enrichment 
found in Nl of the newly formed uracil nucleotide pool was much higher than the 
enrichment quantified for the ^^H contained in aspartate in the same cell. 
The aim of this research is to verify the predictive capability of this 
technique by isolating an individual compartment e.g. mitochondria and to show 
the actual compartmentation of reaction pathway intermediates, and then to 
exploit the predictive nature of this technique to study the pharmacologic 
effects of compartmentation as it relates to antitumor activity of agents which 
act to inhibit pyrimidine and purine biosynthesis. 

Publications : 

1. Strong, J.M., Upton, O.K., Anderson, L.W., Monks, A., Chisena, C.A., and 
Cysyk, R.L.: A novel approach to the analysis of mass spectrally assayed 
stable isotope-labeling experiments. J. Biol. Chem. 260: 4276-4281, 1985. 



160 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06166-01 LBC 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Macromolecular Interactions of Vinca Alkaloids 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute affiliation) 

PI: Ronald L. Felsted Research Chemist LBC, NCI 



Others; 



Ahmad R. Safa 
Constance Glover 



Visiting Fellow 
Microbiologist 



LBC, NCI 
LBC, NCI 



COOPERATING UNITS (if any) 

Ernest Hammel 



Sr. Investigator 



LMCB, NCI 



LAB/BRANCH 

Laboratory of Biological Chemistry 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

1.2 



PROFESSIONAL; 

1.1 



0.1 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n {a1) Minors 
D (a2) Interviews 



D (b) Hunnan tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This project involves the identification of Vinca alkaloid macromolecular inter- 
actions in cells and the characterization of the relationship of these associations 
to overall drug anti-neoplastic activity and drug-resistance mechanisms. Radio- 
active photoactive analogues of vinblastine have been synthesized. The pharmaco- 
logical activity of one of these analogues is compared to the parent drug. 
Macromolecular interactions are identified by photoaffinity labeling and by SDS- 
polyacryl amide gel electrophoresis. The labeled polypeptides are characterized 
and identified by immunoprecipitation. The mechanism of analogue binding to 
tubulin and inhibition of microtubule polymerization is being studied. New Vinca 
alkaloid binding proteins have been identified and their role in these processes 
is being examined. The relationship of drug-binding proteins to cellular drug- 
sensitivity and drug-resistance is being investigated. 



161 



PHS 6040 (Rev. 1/84) 



GPO SI 4-918 



ZOl CM 06166-01 LBC 

The Vinca alkaloids, vinblastine and vincristine, isolated from the plant 
Vinca rosea L. are important chemotherapeutic agents with clinical activity 
against a spectrum of human cancers. It is generally assumed that the mechan- 
ism for their cytotoxic, anti-mitotic and anti-neoplastic activity is related 
to their binding to the tubulin dimer of microtubules with the subsequent 
depolymerization and disruption of the cellular microtubular network including 
the mitotic spindle. However, evidence from several studies suggests that 
binding to mitotic spindle tubulin dimers cannot account for their pronounced 
cytotoxic effects on slowly proliferating sensitive cells and cells in interphase 
since in these cells cytotoxicity is evident long before mitotic arrest becomes 
manifested. The Vinca alkaloids have been shown to inhibit the incorporation 
of ^H-uridine into RNA and ^H-thymidine into DNA. These effects may be 
due to the specific inhibition of nucleic acid synthesis as well as the 
inhibition of nucleotide uptake into cells. Finally, our knowledge of the 
mechanism involved in the expression of -Vinca alkaloid resistance in cancer 
cells is limited. While oversynthesis of particular proteins in Vinca alkaloid 
resistant cells has been noted, these proteins have not been identified and 
their possible interactions with Vinca alkaloids have not been described. 

Affinity labeling of proteins with photoactive ligands is a powerful tool in 
probing biological targets. The photoaffinity labeling technique allows ident- 
ification of specific- acceptor molecules in a complex mixture of biological 
components. Under photolabeling conditions a reversible complex occurs between 
the photoactive analogue and unique acceptor sites of specific polypeptides 
which recognize the characteristic drug structure. Upon irradiation with UV 
light, the analogue is converted into a highly reactive nitrene intermediate 
which then covalently labels the acceptor site. 

Objectives ; 

This project involves the identification of specific Vinca alkaloid interac- 
tions with macromolecules in cell lines and mammalian tissues. The unique 
biological associations are identified by in vitro and in situ covalent label- 
ing with radioactive photoactive vinblastine (VB) analogLTesT Specific radio- 
labeled macromolecules will be identified by immunoprecipitation. The biolo- 
gical and pharmacological activities of the parent VB and photoactive VB ana- 
logues will be compared. The relationship of specific Vinca alkaloid binding 
macromolecules to VB cytotoxic, anti-mitotic and drug-resistance mechanisms 
will be examined. 

Major Findings ; 

Two photoactive VB analogues have been synthesized: (1) N-(p-azidobenzoyl )- 
N'-g-aminoethylvindesine (NABAV) and (2) N-(p-azidosalicilyl )-N'-3- 
aminoethylvindesine (NASAV). The NABAV has been prepared radioactive: 
N-(p-azido-3,5-[3H]-benzoyl )-N'-B-aminoethyl vindesine (^H-NABAV). The 
3H-NABAV has been used to probe Vinca alkaloid macromolecular interactions 
by photoaffinity labeling homogenates of calf brain and tumor cell lines. 
After photolabeling, Vinca alkaloid binding components were resolved by 
SDS-PAGE and identified by comparing the radioactivity in 1 mm gel slices 
to polypeptide MW standards. Prominently labeled components of Mp 54.3 and 
21.5 Kd corresponded to unique Coomassie blue stained polypeptides, and were 
superimposed upon a background of nonspecific radioactivity. The Mp 54.3 

162 



ZOl CM 06166-ni LBC 

polypeptide was found in both the 100,000 x g pellet and supernatant fraction 
whereas the Mp 21.5 polypeptide was exclusively located in the 100,000 x g 
pellet. Photolabeling was maximal after 10 min UV light exposure and linear 
to 150 jjg of protein. Photolabeling of Mp 54.3 and 21.5 with increasing 
concentrations of ^h-NABAV (0-1.32 gM) was biphasic, consistent with mixed 
specific and nonspecific labeling. After correcting for nonspecific labeling, 
Mp 54.3 and 21.5 exhibited a saturation above 0.33 viM ^H-NABAV. Increasing 
concentrations of non-radioactive NABAV or VB added to 0.44 uM 3h-NABAV 
resulted in a limited reversal of photolabeling of Mp 54.3 and 21.5 correspond- 
ing to 3.2 and 0.62 pmol/mg calf brain homogenate protein, respectively, 
confirming the specific photoaffinity labeling of these Vinca alkaloid 
acceptors. Ka(VB)/Ka(NABAV) for Mp 54.3 was 0.86. The Mp 54.3 component 
was also prominent in human KGl myeloid, MCF-7 breast cancer cells and P388 
mouse leukemia cells. The Mp 54.3 polypeptide was identified as a tubulin 
subunit by immunoprecipitation in calf brain and P388 supernatant fractions. 

The NABAV and VB blocked tubulin polymerization in vitro at similar concen- 
trations. The cytotoxic concentrations of NABAV and VB toward P388 cells were 
identical. Thin-section electron microscopy of NABAV or VB treated P388 and 
MCF-7 cells revealed similar multinucleated and mitotic arrested cells and 
induction of tubulin crystals, annulated lamellae and vaculolized rough endo- 
plasmic reticulum. 

Proposed Course: 

The above data confirm the similarities of NABAV to its parent VB and indicate 
its usefulness for identifying Vinca alkaloid binding proteins. These proteins 
may play significant roles in the mechanism of drug action. The NABAV will be 
used to probe for specific VB binding sites in pleiotropic drug resistant cell 
lines. Initially, wild-type and drug-resistant cells will be photoaffinity 
labeled and the radioactive labeled polypeptides will be compared in order to 
identify differences which might represent cellular components which play 
direct roles in drug resistance mechanisms. Specifically, we will examine 
cell lines previously shown to exhibit unique polypeptide changes proportional 
to the level of acquired drug resistance (i.e. CEM/VLB/100 human lymphoid 
cells obtained from W. Beck and the colchicine resistant Chinese hampster 
cells obtained from J. Biedler). We will also compare the following apparent 
pleiotropic cell lines: HL-60 (N. Ahmad), KB (I. Pastan) and MCF-7 (K. 
Cowen). Unique differences will be examined with 3h_nABAV. The ^^^l-H^S^\l 
analogue also will be used to increase the detection sensitivity. 



163 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06167-01 LBC 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Inhibition of Myristoylati on-Dependent Oncogene Mediated Cellular Transformation 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: R.L. Felsted Research Chemist LBC, NCI 



Other; 



C. Glover 



Microbiologist 



LBC, NCI 



COOPERATING UNITS (if any) 

Laboratory of Medicinal Chemistry and Pharmacology, DTP, DCT, NCI (V. Marquez); 
Frederick Cancer Research Facility (A. Shultz, S. Oroszlan). 



LAB/BRANCH 

Laboratory of Biological Chemistry 



SECTION 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 
1.9 



PROFESSIONAL: 

0.9 



1.0 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The modification of one-proteins with the fatty acid myristate is an early step 
associated with the transformation of normal to neoplastic cells. The exact signi- 
ficance of myristoylati on in transformation has not been established. However, it 
is thought to be part of the mechanism by which cytoplasmic oncogene kinases are 
localized to the inner plasma membrane surface. Since the transforming activity 
of onc-kinases is dependent upon this membrane binding, this project will investi- 
gate the role of myristoylati on as it relates to the mechanism of this subcellular 
localization. A specific myristoylati on assay is being developed using synthetic 
NH2-terminal polypeptide homologues of cellular and viral myristoylated proteins. 
The myristyl transferase(s) substrate specificity for polypeptide and fatty acid 
will be examined in order to define the myristoylati on mechanism. The role of 
covalently bound myristic acid in translocation of myristoylated proteins from 
their site of cytoplasmic synthesis to the plasma membrane will be examined using 
mutants of myristoyl -proteins which lack the fatty acid and with myristoyl trans- 
ferase inhibitors. The involvement of myristic acid in membrane binding will be 
examined by looking for specific membrane receptors of the myristoyl -proteins. 
Information on the enzymology of myristoylati on and the role of myristic acid in 
translocation and membrane binding will be used to design and synthesize specific 
inhibitors of myristoylati on in these related phenomena with the goal of developinc 
chemotherapeutic agents specific for a critical early step of malignant trans- 
formation. 



164 



PHS 6040 (Rev. 1/84) 



GPO 814-818 



ZOl CM 06167-01 LBC 

Tyrosine-specific protein kinase activity is associated with several known 
oncogenes and is an appealing target for the chemical manipulation of kinase 
associated cellular transformation. Selective inhibition of onc-kinases 
appears to be a formidable task, but may become possible when the cellular 
substrate(s) for the onc-kinases are identified. Recently it has been observed 
that direct inhibition of the onc-kinases may not be necessary in order to 
block (or reverse) transformation. The transforming activity of onc-kinases 
depends upon their association with the inner plasma membrane surface. It has 
been proposed that the mechanism by which viral encoded onc-kinases such as 
p60src become membrane bound is through a post- or co-translational addition 
of myristic acid to their NH2-terminal glycine via an amide linkage. In 
recently published experiments. Cross et aj_. constructed p60src NH2-terminal 
deletion mutants that failed to incorporate myristic acid. These mutant pro- 
teins were still functional kinases in vitro and in vivo , but instead of being 
localized to the membrane they were Tound in the cytoplasm. Most pertinent to 
this proposal, these mutant proteins no longer transformed cells. Presumably, 
myristoylation is critical to p60src subcellular localization and cellular 
transforming activity. 

Myristoylation is also necessary for the membrane localization of the cellular 
p60src tyrosine kinase encoded by the src proto-oncogene. In addition, several 
normal and neoplastic mammalian cells Tiave elevated levels of a similar size 
tyrosine kinase, p58. However, this p58 kinase is distinct from the cellular 
and viral p60src as well as from five other proto-oncogene cellular tyrosine 
kinases and it does not contain gag linked viral proteins. Most interestingly, 
one of these p58 kinases contains myristic acid. These results indicate that 
myristoylation may be an important regulator of the subcellular distribution 
and function of normal cellular tyrosine kinases. Thus, transformation 
resulting from the expression of structurally modified tyrosine kinases or 
elevated amounts of cellular kinases also may depend on myristoylation-directed 
subcellular distribution. 

Objectives i 

In this project we will examine the role of myristoylation of onc-tyrosine 
kinases as targets for the chemotherapeutic inhibition of cellular transform- 
ation. Specifically, we will study three mechanisms for blocking myristoyla- 
tion dependent transformation by p60src: these include, (i) inhibition of the 
myristoyl transferase(s) , (ii) the blocking of translocation of soluble 
myristoyl -proteins to their membrane binding sites, and (iii) inhibition of 
binding of myristoyl -proteins to potential membrane receptors. We will 
examine, also, the relationship of myristoylation to normal tyrosine kinases 
and the role of myristoyl ated-tyrosine kinases in normal cellular growth and 
differentiation. 

Proposed Course : 

i. Myristoyl Transferase(s) . Fatty acid amides are well known in nature and 
their syntheses by eucaryotic cell free systems have been described. Myristoyl 
amidation takes place during or immediately after protein synthesis so fatty 
acid addition may occur by a co-translational process as in the case for NH2- 
terminal acetylation. The myristoyl transferase(s) also are characterized by 
an absolute specificity for myristic acid. The question of substrate specifi- 

165 



ZOl CM 06167-01 LBC 

city will be examined in order to develop a specific enzyme assay for myristoy- 
lation. It is likely that nascent polypeptide chains are the actual substrates 
of the myristoyl transferase(s) . Studies with p29gag-ras and deletion con- 
struct mutants of pSOsrc indicate that the first 13-15 terminal amino acids 
may present a conformation-dependent myristoylation acceptor site as the 
substrate for myristoyl transferase(s) . In collaboration with Drs. Alan Shultz 
and Stephen Oroszlan of the Frederick Cancer Research Facility, we will synthe- 
size a 14-15 amino acid polypeptide corresponding to the NH2-terminal sequence 
of the p60src protein. In addition, a tyrosine residue will be added to the 
COOH-tepminal end of the synthetic polypeptide to allow for radiolabel ing 
with ^'^^I. This NHo-terminal glycine peptide will be tested as a substrate 
in a specific myristoyl transferase assay and used as a probe for myristoyl- 
protein acceptors (see ii and iii, below). 

Synthetic polypeptides corresponding to several known myristoyl acceptor amino 
acid sequences will be tested to assess relative myristoyl transferase sub- 
strate specificity. Polypeptide analogues of varying lengths and with selected 
amino acid substitutions will be tested as competitive inhibitors. In colla- 
boration with Dr. Victor Marquez of the Laboratory of Medicinal Chemistry and 
Pharmacology, DTP, OCT, NCI, we will design and synthesize polypeptide and/or 
fatty acid derivitives that will serve as specific irreversible (suicide) 
inhibitors of the myristoyl transferases(s) . Also, once the fatty acid myris- 
toyl donor has been identified, it might be possible to block myristoylation 
by inhibiting the de novo synthesis of the fatty acid donor. 

ii. Myristoyl -protein translocation . Shortly after synthesis on free ri bo- 
somes, the myristoyl -p60src kinase forms a soluble complex with two cellular 
proteins of 50 and 90 kilodaltons. It has been suggested that this complex 
may act as a device for transporting newly synthesized p60src kinase to its 
site of transforming action at the plasma membrane. Similar complexes have 
been reported for onc-kinases encoded by other retroviruses so "complex medi- 
ated" translocation might be another important step in onc-kinase cellular 
transformation. Although the p50 and p90 proteins are not thought to bind to 
the NH2-terminal region of p60src, the role of myristoylation in this complex 
is not understood. It may be possible to establish the function of myristic 
acid in the carrier mediated translocation by testing to see if mutants of 
p60src kinases which lack myristic acid are still able to form comparable 
complexes with p50 and p90. Ultimately, it may be necessary to use specific 
inhibitors of myristoyl transferases(s) (see i, above) or myristoyl -protein 
binding (see iii, below) in order to clarify the role of myristic acid in 
translocation. 

iii. Myristoyl -protein membrane binding . Myristic acid may play a direct 
role in the binding of one-proteins to the plasma membrane. This could occur 
by one of two basic mechanisms. In one mechanism, the hydrophobic fatty acid 
could serve as an anchor for the protein by embedding into the membrane lipid 
bi layer. In fact, this has been shown for some integral membrane proteins 
where the palmitic acid esterification of membrane bound nascent polypeptides 
is part of a maturing process which results in these proteins being more firmly 
anchored in the membrane. This anchor mechanism also is compatible with the 
carrier mediated translocation process described above (see ii.). Since myris- 
toylation occurs in the cytoplasm, premature anchoring to the nearest intra- 
cellar membrane bilayer could be prevented by complexing the newly modified 

166 



Zni CM 06167-01 LBC 

one-protein to specific carrier proteins. In turn the carrier proteins could 
provide the recognition signal that determines the ultimate membrane destina- 
tion. However, once the complex arrives at the membrane and the carrier pro- 
teins release the transported one-protein, membrane binding must be determined 
by intrinsic structural features of the myristoyl -one-protein; in this case, 
myristate insertion into the membrane bilayer. The problem with this hypothesis 
is that in contrast to the irreversible binding of palmitoyl ester modified 
integral membrane proteins, myristoyl -proteins are reversibly membrane bound. 

A second mechanism which could provide for reversibility would be the recog- 
nition of and binding to myristate by specific membrane receptors. If, in 
addition, the receptor recognizes the adjoining NH2-terminal polypeptide 
sequence, then an added degree of specificity would result (i.e. the binding 
of p60src specifically to membrane adhesion plaques). We propose to examine 
the role of myristoyl -protein binding by classical receptor techniques. Using 
the radiolabeled myristoylated synthetic polypeptides described above, we will 
look for saturable binding to membrane vesicles as well as to soluble binding 
proteins such as the p50 and p90 (see ii, above). In addition, specificity 
will be confirmed by competitive displacement of membrane bound radioactive 
myristoyl -one-proteins with nonradioactive synthetic peptides. This displace- 
ment assay also will be used to survey for potential inhibitors of myristoyl- 
protein receptor binding and/or myristoyl -protein carrier complex binding. 
Simple amide combinations of fatty acid and synthetic polypeptides will 
be examined as potential inhibitors of the translocation and membrane binding 
of myristoylated one-proteins. 



167 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06168-01 LBC 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must ti: on vne line betweer\ the borders.) 

Cellular Pharmacology of InLerferon and Double-stranded RNA 



PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Robert I. Glazer Senior Investigator LBC, NCI 



Others: Mrunal Chapekar 

Victor E. Marquez 



Visiting Associate 
Visiting Scientist 



LBC, NCI 
LPET, NCI 



COOPERATING UNITS (if any) 

Medicinal Chemistry Section, LPET, DTP, DCT, NCI (V. Marquez) 



LAB/BRANCH 

Laboratory of Biological Chemistry 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



1.5 



PROFESSIONAL: 



1.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



K (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This study will entail: 1) investigations of the cyclopentenyl and 5'-phosphonate 
analogs of the interferon-induced 2' ,5' -oligoadenylate as specific inhibitors of 
2' ,5'-oligo(A) phosphodiesterase, and 2) determination of the mechanism of the 
potentiation produced by poly(I)'poly(C) and its derivatives on the antitumor 
activity of immune interferon (IFN-y). This will include investigating the role 
of transcription and cellular oncogene expression by the synergistic combination 
of poly(I)'poly(C) and IFN-y in human colon carcinoma cell lines HT-29 and BE. 



l68 



PHS 6040 (Rev. 1/84) 



GPo si4-eta 



ZOl CM 06168-01 LBC 

This area of study involves two projects. The first will examine the activity 
of carbocyclic and phosphonate analogs of 2' ,5'oligo(A), the interferon-induced 
oligonucleotide believed to mediate the antiviral or possibly the antiprolifer- 
ative activity of interferons (Fig. 1). Dr. Chapekar's previous studies of 
2',5'-oligo(A) metabolism in response to interferon and/or double-stranded 
RNA (poly(I)'poly(C)), showed that 2' ,5'-oligo(A) phosphodiesterase (PDE) is 
rate-limiting in degrading 2' ,5'oligo(A) in the cell, and in fact, none could 
be detected by a sensitive radioligand binding assay. Thus, in collaboration 
with Dr. Victor E. Marquez, Medicinal Chemistry Section, LPET, we plan to 
investigate whether 5 '-phosphonate (i) or neplanocin A (£) analogs of 
2',5'-oligo(A) are specific, and possibly irreversible inhibitors of 2',5'- 
oligo(A) phosphodiesterase (Fig. 1), and thus lead to potentiation of the 
activity of interferon. 



HOI— PI f — I 



r^'-edgoM •ynUMUM 



per . AtrtnA 






FIG. 1 



'^. 



At tq 







Neplanocin A derivative 2 



Studies over the past three years in this Section have involved an examination 
of the mechanism of action of human leukocyte, fibroblast and immune interferons- 
a, 3 and y (IFN-a, g and y). These investigations showed that IFN-a 
and -e interferons were not highly cytocidal to HT-29 cells in vitro , and 
were in fact, inactive against several human leukemias. Coa^im strati on of 
poly(I)»poly(C) moderately increased cytotoxicity but it was only an additive 
effect. Further studies with natural and recombinant IFN-y have established 
that this lymphokine is a potent inhibitor of cell proliferation as well as 
being highly cytocidal. Three day treatment of HT-29 cells with 200 units/ml 
of IFN-Y resulted in 90% growth inhibition, and 99% reduction in colony 
format ioa. 



169 



Zni CM 06168-01 LBC 

The antitumor effects of IFN-y were further explored in combination with 
poly(I)«poly(C). Unlike combinations of IFN-a and IFN-gwith this double- 
stranded RNA, IFN-Y and poly(I)'poly{C) showed a syntergistic effect on 
cell viability in colon carcinoma cell lines HT-29 and BE. Three day treat- 
ment with 10-25 units/ml of IFN-y resulted in 30-40% reduction in cell 
viability in HT-29 cells and a 10% reduction in BE cells. Coaddition of 
poly(I)«poly(C) with IFN-y resulted in a 70-90% reduction in colony formation 
in HT-29 cells and a 50-60% reduction in BE cells. Most notable was the appear- 
ance of this effect in cell line BE which is resistant to either drug alone. 
The appearance of the synergistic effect on cell viability was accompanied by 
a corresponding reduction in transcription, but not by rRNA degradation, 
changes in 2', 5'-oligo(A) levels or alterations in protein phosphorylation in 
vitro . These results establish a clear association between RNA transcription 
and cell lethality. Thus, the second area of study related to the scheme in 
Fig. 1 is the mechanism of the potentiation of interferon activity by double- 
stranded RNA (dsRNA). The dsRNAs being evaluated are poly(I) 'polyCC), 
poly(I)'poly(C)«polylysine«-carboxymethylcellulose (ICLC) and mismatched 
poly(I)'poly{C) where there is a 13% substitution of uridylic acid in the 
poly(C) (also called "Ampligen", Hem Research Inc.). Since IFN-y, ICLC 
and "Ampligen" are undergoing phase I clinical trials, it will be important to 
assess the efficacy of these dsRNAs for potentiating the cytocidal effect of 
IFN-Y. 

Our data imply that there is a very sensitive dsRNA-regulated component to 
transcription and we plan to investigate this phenomenon by measuring the 
transcription of cellular oncogenes believed to be involved in cell cycle 
transit. Studies of this nature in human colon carcinoma cell lines HT-29 
(IFN-Y sensitive) and BE (IFN-y refractory) are currently in progress. 

Major Findings ; 

1. The effect of polyinosinicpolycytidylic acid (poly(I)«poly(C)) on the 
cell lethality produced by 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) 
was examined in human colon carcinoma cell line HT-29. Pretreatment of cells 
with poly(I)«poly(C), as well as during exposure to FUra or FUrd resulted in 
antagonism of the cell lethality generated by the fluoropyrimidines. Antagon- 
ism of FUra cytotoxicty was also produced by the 2'-0-methylated analog, 
poly(I)*poly(C^), but not by the individual single-stranded polynucleotides 

or by the component mononucleotides, CMP and IMP. In contrast, CMP blocked 
the toxicity of FUrd.- The antagonism by poly(I)«poly(C) of FUra and FUrd 
cytotoxicity was related to the inhibition of their metabolism to FUTP and 
their incorporation into RNA and not to inhibition of the synthesis of RNA. 
Antibodies to leukocyte and fibroblast interferons did not reverse the antagon- 
istic activity of poly(I)«poly(C). These results indicate that poly(I)'poly(C) 
may be interfering with the transport and/or initial metabolism of FUra and 
FUrd to FUMP which is independent of the ability of the double-stranded RNA to 
induce interferon. (ligo and Glazer, Cancer Res. 4^: in press). 

2. The effect of polyriboinosinic • polyribocytidylic acid (poly(I)'poly(C)) 
on the antitumor activity of 5-fluorouracil (FUra) and 5-fluorouridine (FUrd) 
was evaluated in mice bearing L1210 leukemia. Coadministration intravenously 
of poly(I)«poly(C) and either FUra or FUrd on days 1, 5 and 9 to mice-bearing 
L1210 leukemia implanted subcutaneously resulted in a 40% greater increase 

170 



ZOl CM 06168-01 LBC 

in lifespan at the optimal antitumor dose vs^. FUra or FUrd alone. This effect 
appeared to result from greater host tolerance of a dose of FUra or FUrd which 
would otherwise be cytotoxic. The protective effect of poly(I)'poly(C) was 
also evident in non-tumor-bearing mice as well as following administration of 
drug intraperitoneally to mice bearing the tumor implanted intraperitoneally. 
FUrd incorporation into RNA in the spleen, bone marrow and small intestine 
revealed little or no changes after coadministration of poly(I)'poly(C). 
{2',5')01igo(A) synthetase activity, an indication of interferon activity, 
was markedly depressed in the spleen and bone marrow following treatment with 
FUrd; however, poly(I)'poly(C) administered together with FUrd returned 
(2* ,5')oligo(A) synthetase activity to normal levels. These data indicate 
that poly(I)-poly(C) ameliorates the host toxicity of fluoropyrimidines, 
possibly via an interferon-mediated effect, and thereby resultsMn enhanced 
therapeutic efficacy of the antimetabolite as an antitumor agent. (ligo and 
Glazer, Cancer Res. 45: in press, 1985). 

3. The cytocidal activity of human immune interferon (IFN-y) in combina- 
tion with the synthetic double-stranded RNA, poly(I)-poly(C) , was investigated 
in human colon carcinoma cell line HT-29. Three day treatment with 10 to 25 
units/ml of IFN-y resulted in 30 to 40 % reduction in colony formation, 
whereas 25 to 100 pg/ml of poly(I)-poly(C) reduced cell viability by 10 to 
20% of control. The lethal effect of the combination of IFN-y and poly(I*- 
poly(C) was synergistic wherein 70 to 90% reduction in colony formation was 
observed. Measurements of DNA, RNA and protein synthesis after IFN-y and 
poly(I)'poly(C) treatment showed a dose-dependent reduction in all three 
parameters. Recombinant IFN-y in combination with poly(I)-poly(C) exhibited 
a similar effect. Studies evaluating the molecular mechanism of IFN-y and 
poly(I)'poly(C) toxicity indicate a lack of involvement of the double-stranded 
RNA-dependent (2' ,5' )oligo(A)-RNAse L and protein kinase pathways; however, 
the effect appears to be related to the inhibition of ribosomal RNA transcrip- 
tion in this cell line. (Chapekar and Glazer, Cancer Res. 45: in press). 

Publications : 

1. ligo, M. and Glazer, R.I.: Antagonistic effect of polyinosinic-polycyti- 
dylic acid on cell lethality produced by 5-fluorouracil in human colon 
carcinoma cells in vitro . Cancer Res . 45: 1953-1957, 1985. 

2. ligo, M., Chapekar, M.S. and Glazer, R.I.: Synergistic antitumor effect of 
fluoropyrimidines and polyinosinic-polycytidylic acid against L1210 
leukemia. Cancer Res . , in press. 

3. Chapekar, M.S. and Glazer, R.I.: Synergistic effect of human immune inter- 
feron and double-stranded RNA against human colon carcinoma cells. Cancer 
Res. , in press. 



171 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06169-01 LBC 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) 

Cellular and Molecular Pharmacology of Phospholipid and Diacyl glycerol Analogs 



PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atflllaVon) 

PI: Robert I. Glazer Senior Investigator LBC, NCI 



Others: Kathleen D. Hartman 
Marion C. Knode 
Ester Zylber-Katz 



Chemist LBC, NCI 

Biologist LBC, NCI 

Visiting Scientist LBC, NCI 
(DTP Exchange Program) 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Biological Chemistry 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



1.75 



PROFESSIONAL 



0.75 



1.0 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Dialkylphospholipid and diaUyl glycerol analogs will be investigated in human 
promyelocytic leukemia cell line HL-60. These studies will include examining the 
role of phosphatidylinositol metabolism and the cellular oncogenes c -myc , c-H -ras 
and c- fos in the differentiative and antiproliferative effects of these analogs. 
The compounds l-0-hexadecyl-2-0-methyl-3-phosphocholine and l-O-hexadecyl-2-0- 
methyl-3-phosphate are currently being evaluated. 



172 



PHS 6040 (Rev. 1/84) 



OPO •I4<«1t 



ZOl CM 06169-01 LBC 

One of the most intensively investigated areas of cell biology in the past few 
years is the process of calcium- and phospholipid-dependent protein phosphory- 
lation (Fig. 1). Inducers of the protein kinase activity (protein kinase C) 
believed to be responsible for this process, such as diacyl glycerol s(i) and 
phorbol esters(2), have shown the importance of this enzyme in the regula- 
tion of growth factor activity, lymphoblastogenesis, normal cell proliferation, 
cellular differentiation and oncogene expression. Studies in this laboratory 
of protein kinase C activity during differentiation of human promyelocytic 
leukemia cell line HL-60 have indicated that not only is this enzyme activity 
increased in cells induced to differentiate along the myeloid or monocytic 
pathways, but that there is the rapid appearance of differentiation specific 
peptides which are phosphorylated in a calcium- and phospholipid-dependent 
manner. 

In association with the activation of protein kinase C activity is the turn- 
over of phospholipids such as phosphatidylinositol-4,5-bisphosphate(£) to 
yield diacyl glycerol and inositol phosphates, which in turn activate phosphory- 
lation (Fig. 1). In addition, inositol phosphates are involved in the mobili- 
zation of calcium within the cell and thus set in motion calcium-dependent 
protein phosphorylation as well. One series of natural metabolites known to 







/^ ArachldonI 


-y 




Prostaglandins )<* 


VT 


P5)-»(lPi)-»^IP) 








V 


*| Ca'-'MoblliMtion | 










Activation of 








Protein Kinase C 








FIG. 1 




po-5-. 




pOC„H, 






-oJ-,. 

-OH 

acYlglvcerol 



1 


<h:h, 4 

Lo-P-0-CH^,-N, -CHj 
i CH, 


i-c-n.-c-if- 


(■nvaddil 






po-la 




oXo- 3 


OCICHiliiCH, 
LoH 


phox 


ilwliclyliioilmM.&OliphiJiphaw 




l2T«lr»<)0C 


«fW¥lphofbol-13acei8ie 
ITPAI 



173 



ZOl CM 06169-01 LBC 
produce differentiating activity and activation of protein kinase C and phos- ■ 
phatidylinositol turnover are the diacylglycerols. Related dialkylglycerols 1 
are more metabolically stable and more effective inducers of differentiation 
in HL-60 cells. Moreover, phospholipid analogs such as dialkylphosphatidyl- 
choline(ii) are also selective cytotoxic drugs against HL-60 cells vs. normal 
neutrophils and in some instances possess differentiating activity as well. 

Thus, it is proposed that dialkylglycerol and dialkylphospholipid analogs be 
investigated for their potential as selective anticancer drugs via their 
capacity to mobilize calcium- and phospholipid-dependent processes related to 
cellular differentiation. We are evaluating l-O-hexedecyl-2-O-methyl phospha- 
tidyl choline (ii) and its corresponding phosphatidic acid derivative (C-I6-OCH3PA). 
The dialkyl PC analog was originally synthesized as a derivative of platelet 
activating factor but was inactive as such due to the C-2 ether linkage. However, 
the drug was found to have preferential toxicity against HL-60 leukemia cells 
vs. normal neutrophils where the IC50 for HL-60 cells was about 50-fold less 
tFan for neutrophils. The other analog, C-I6-OCH3PA, is a soluble form of 
dialkylglycerol and is less cytotoxic to HL-60 cells. However, it induces mono- 
cytic differentiation in this cell line to a greater extent than C-I6-OCH3PC 
and may resemble more closely the action of diacyl glycerol and phorbol esters 
in inducing differentiation of HL-60 cells along the monocyte-macrophage 
pathway. 

Thus, we plan to examine perturbation of phosphatidylinositol metabolism and 
cellular oncogene expression in HL-60 cells in response to C-I6-OCH3PC and 
C-I6-OCH3PA. Other analogs will be synthesized in collaboration with Dr. 
Joseph Hajdu, California State University, and include l-O-hexadecyl-2-aceta- 
mide-phosphatidyl choline and l-O-hexadecyl-2-acetamido glycerol. We also 
propose to have synthesized dialkyl analogs of phosphatidylinositol, and 
possibly analogs modified in the inositol moiety. The latter compounds in 
particular, may serve as novel compounds for the delivery of inositol analogs 
into the cell since inositol itself is poorly transported. 

Major Findings: 

The effects of differentiating agents on the activity and phosphorylation 
pattern produced by phospholipid- and Ca++-dependent protein kinase (PL-Ca-PK) 
was examined in human promyelocytic leukemia cell line HL-60. Dimethyl - 
sulfoxide (DMSO), retinoic acid (RA) and 1,25-dihydroxyvitamin D3 {1,25(0H)2D3) 
increased the appearance of mature myelocytic (DMSO and RA) or monocytic 
(1,25(0H)2D3) cells. The tumor promoter, 12-^-tetradecanoyl phorbol -13-acetate 
(TPA) increased the appearance of adherent macrophage-like cells. Coincident 
with the appearance of differentiated cells induced by DMSO, RA and 1,25(0H)2D3 
was an increase in PL-Ca-PK activity. In contrast, TPA treatment resulted in 
the rapid disappearance of PL-Ca-PK and the induction of PL-Ca-independent 
protein kinase activity. The phosphorylation pattern resulting from endogenous 
PL-Ca-PK in extracts from cells treated with DMSO, RA or 1,25(0H)2D3 showed a 
prominent phosphorylated protein of 37 kilodaltons (pp37) and 38 kilodaltons 
(pp38) which was related to the appearance of the myelocyte/monocyte phenotype. 
pp37 and pp38 were also present in TPA-treated cells but their phosphorylation 
was no longer dependent on the presence of PL and Ca. Cells treated with DMSO 
and RA also exhibited a PL-Ca-dependent pp21 which was barely evident in 
l,25(0H)D3-treated cells, and thus, represented a myeloid cell marker. Also 

174 



ZOl CM 06169-01 LBC 

present was a prominent PL-Ca-dependent ppl9 which remained unchanged following 
treatment with DMSO, RA and l,25(0H)2n3, but markedly diminished in TPA-treated 
cells. On the other hand, TPA-treated cells exhibited a characteristic ppl30 
which was antigenically related to the actin binding protein, vinculin. These 
results indicate that there are characteristic PL-Ca-dependent phosphorylated 
proteins indicative of mature myelocytic and monocytic cells, as well as 
PL-Ca-independent phosphorylated proteins characteristic of the macrophage-like 
phenotype. (Zylber-Katz & Glazer, Cancer Res. in press.) 

Publications : 

1. Zylber-Katz, E. and Glazer, R.I.: Phospholipid- and calcium-dependent 
protein kinase activity and protein phosphorylation in human promyelo- 
cytic leukemia cell line HL-60. Cancer Res ., in press. 

2. Zylber-Katz, E. and Glazer, R.I.: Cooperative effects of retinoic acid 
and dimethyl sulfoxide on phorbol ester-mediated differentiation and phos- 
pholipid- and calcium-dependent protein phosphorylation in human promyelo- 
cytic leukemia cell line HL-60. F.E.B.S. Letters , in press. 

3. Glazer, R.I.: Differentiation of malignant cells as a new mode of 
chemotherapy. InrElsebai I. (Ed.): Current Treatment of Cancer . 
Heidelberg, VICC-Springer-Verlag, in press. 



175 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07109-09 LBC 



PERIOD COVERED 

October 1, 1984 to September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) 

Molecular and Cellular Pharmacology of Nucleoside Analogs 



PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Robert I. Glazer Senior Investigator LBC, NCI 



Others: Marvin B. Cohen 

Kathleen D. Hartman 
Marion C. Knode 
Kasturi Sri ram 
Victor E. Marquez 



Staff Fellow 
Chemist 
Biologist 
Visiting Fellow 
Visiting Scientist 



LBC, NCI 

LBC, NCI 

LBC, NCI 

LBC, NCI 

LPET, NCI 



COOPERATING UNITS (if any) 

Medicinal Chemistry Section, LPET, DTP, OCT, NCI (V. Marquez) 



LAB/BRANCH 

Laboratory of Biological Chemistry 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



2.5 



PROFESSIONAL: 



1.5 



1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



K (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The mechanism of action of nucleoside antimetabolites will be assessed in human 
tumor cells in tissue culture, and will include studies of ribosomal RNA proces- 
sing, transcription, methylation of RNA and DNA and cellular oncogene expression. 
These studies will deal with the cyclopentenyl analogs of adenosine (neplanocin-A), 
cytidine, uridine, 3-deazaadenosine 5-fluorouridine and 5-azacytidine. The 
activity of these drugs will be assessed by the clonogenic survival of human 
colon carcinoma cell line HT-29 and the differentiation response of human pro- 
myelocytic leukemia cell line HL-60. 



176 



PHS 6040 (Rev. 1/84) 



GPO S14-9ia 



ZOl CM 07109-09 LBC 

Molecular and Cellular Pharmacology of Anticancer Drugs . The area of research 
most intensively pursued over the past seven years by this Section has been the 
mechanism of action of adenosine analogs and pyrimidine antimetabolites. The 
newest compounds related to these anticancer drugs are the cyclopentenyl series 
typified by the adenosine analog, neplanocin A (i). This drug is unique 
that it is the only nucleoside analog which appears to act as a specific 
inhibitor of RNA methylation via its anabolism to a metabolite of S-adenosyl-L- 
methonine (Fig. 1) without itself being significantly incorporated~into RNA. 
It is proposed that other purine analogs in this series will yield equally 
interesting biological activities. For example, it is expected that the 
cyclopentenyl analog of 3-deazaristeromycin (3-deazacarbocyclic adenosine) will 
act as an irreversible inhibitor of ^-adenosyl homocysteine hydrolase (Fig. 1). 
Moreover, cyclopentenyl analogs of pyrimidine nucleosides have already yielded 
a nontoxic inhibitor of cytidine-uridine kinase (cyclopentenyluridine) and a 
highly potent and irreversible inhibitor of cytidine triphosphate synthetase 
(cyclopentenylcytidine) (£). 



NH, 



a> 



CvckHMnlenylcvtidifw 2 neplanocin A 1 

'^ (NPC) -^ 



MEimUTION rATHWAr 

ATT + i-M«| . ►■y.AdoMil 



t 



NPC 



-R-CHj, 

S-AdoHcy 



B—^-i^l liydtcy 



3-deazaNPC » I i>yiitoi.i. 

Ado, ♦ Hey 



FIG» 1 



It is expected that the corresponding analogs of 5-fluorouridine and 5-azacyti- 
dine will also yield unique and perhaps more selective cytotoxic and pharmaco- 
logical activities. Therefore, this laboratory in collaboration with the 
Medicinal Chemistry Section, LPET, will continue to investigate the mechanism 
of action as well as the cytocidal activity of these drugs in human tumor cell 
lines such as colon adenocarcinoma HT-29, the tissue culture counterpart of 
the colon tumor xenograft used for drug screening. In addition to this tissue 
culture line, we will also assess the effect of these agents on the human 
promyelocytic leukemia cell line HL-60. This cell line has the ability to 

177 



ZOl CM 07109-09 LBC 

differentiate along the myelo- or monocytic pathways in response to a diverse 
group of compounds. Thus far, our studies with the cyclopentenyl analogs have 
demonstrated that cyclopentenyl cytidine is a potent and rapid inducer of 
myeloid differentiation in HL-60 cells with activity superior to that of 
retinoic acid, and that neplanocin A produces a partial myelocytic response. 
Having drugs with precise mechanisms of action may help to elucidate the key 
cycle phase specific events required to induce differentiation. This approach 
is being utilized by measuring the expression of various cellular (proto) 
oncogenes believed to be involved in cellular proliferation and differentia- 
tion (Fig. 2). 




Phospholipase C 



DG -* I Protein Kinase C 



^IP, » Ca** 



PIP 



myc 
myb 



C 



Differentiation 




FIG. 2 



10"* 3.3x10-* W' 10"* 3.3 xlO"* 

DRUG CONCENTRATION (Ml 



FIG. 3 



It appears that for c -myc mRNA, there is a striking correlation between the 
degree of differentiation on day 5 or 6 and the reduction in c -myc mRNA levels 
1 hour after drug treatment (Fig. 3). This suggests that suppression of tran- 
scription of c- myc mRNA is a prerequisite for differentiation to occur. 

Since cyclopentenyl cytidine is the most potent inhibitor of DNA synthesis and 
c-myc RNA synthesis, as well as the most effective and rapid inducer of 



178 



ZOl CM 07109-09 LBC 

differentiation among the carbocyclic analogs thus far, these data suggest 
that c-myc RNA synthesis occurs in S phase and that accumulation of cells in 
the Gl pHase is a prerequisite for differentiation to proceed. Other cellular 
oncogenes such as c-fos and c-H-ras are also believed to be involved in cell 
cycle progression. THe H-ras oncogene codes for a GTP-binding protein believed 
to be involved in the reguTition of phosphatidyl inositol metabolism, whereas 
the c-myc and c-fos genes code for nuclear DNA-binding proteins. Thus, we 
plan tFTxamine tTie role of the mRNAs of these cellular oncogenes in differen- 
tiation in response to not only nucleoside analogs but other agents thought to 
perturb phosphatidyl inositol metabolism such as analogs of phospholipids and 
diacyl glycerol (see project "Cellular and Molecular Pharmacology of Phospho- 
lipid and Diacyl glycerol Analogs"). 

Major Findings ; 

I. Nucleoside Analogs and Cellular Oncogene Expression : 

1. The mechanism of action of the adenosine analog, neplanocin A (NPC) was 
investigated in human colon carcinoma cell line HT-29. Cell viability was 
reduced to 38 and 17% of control by 24 hr exposure to 10-^ and lO-'^ M NPC, 
respectively. Cytocidal activity was not affected by inhibition of adenosine 
deaminase with 2'-deoxycoformycin. Concomitant with decreased cell viability 
was the reduced incorporation of [14c]dThd and [3H]Leu, and to a lesser 
extent [3H]Urd, into acid-precipitable material. Labeling of rRNA and tRNA 
during drug treatment for 24 hr with [3H -methyl ]Met and [l^CjUrd revealed 
that NPC primarily inhibited RNA methylation, and to a lesser extent, RNA 
synthesis. RNase T2 digests of total RNA indicated that base and 2'-0-methy- 
lation were inhibited to approximately the same degree. Metabolites of NPC 
were measured by reverse-phase HPLC and it was found that the major drug meta- 
bolite was the drug analog of S^-AdoMet with little formation of the respective, 
S-AdoHcy metabolite. NPC was utilized to a very small degree for RNA synthesis 
where only 2 and 30 pmoles of NPC per A26p were incorporated into rRNA and 
tRNA after 24 hr exposure to 10-5 and 10"^ M NPC, respectively. These results 
indicate that NPC is metabolized to a metabolite of ^-AdoMet which is a poor 
methyl donor for RNA methyltransferases, and that the accompanying decrease 

in RNA methylation and protein synthesis appears to be related to its cytocidal 
activity. (Glazer & Knode, J. Biol. Chem. 259: 12964, 1985). 

2. The mechanism of action of the cyclopentenyl analogue of cytidine, cCyd, 
was investigated in human colon carcinoma cell line HT-29. Upon exposure of 
cells to 10-6 M cCyd, cell viability was reduced to 20% of control, whereas 
cytocidal activity was not present after 2 hr of drug exposure. Cell lethality 
was partially reversible by Urd, Cyd or dCyd at 10-° M cCyd, and fully rever- 
sible by these nucleosides at 2.5 x 10-7 m cCyd. The incorporation of 
[l^C]dThd and [^HlUrd into DNA and RNA was inhibited by 50% by exposure for 

2 hr to 2.5 X 10-/ and 1.5 x 10-6 m cCyd, respectively. Upon 24 hr of 
drug exposure, the IC50 for RNA synthesis was reduced 2.5-fold whereas DNA 
synthesis was almost totally inhibited. cCyd produced a rapid and preferential 
reduction of CTP synthesis with a half life of 1 hr at lO-o M drug. The 
ICko 0^ cCyd for reducing CTP concentrations after 2 hr of drug exposure was 
4 X 10-7m. Concomitant with the reduction of CTP levels was the inhibition 
of transcription of rRNA, and to a lesser extent, tRNA, without changes in 



179 



ZOl CM 07109-09 LBC 

the processing of nucleolar RNA. No changes in the size of DNA were produced 
following treatment with cCyd. These results indicate that cCyd is a potent 
and rapid inhibitor of CTP synthesis and that this effect correlates with its 
cytocidal activity. (Glazer, Cohen, Hartman, Knode, Lim and Marquez, Biochem. 
Pharmacol . , in press). 

3. The effect of the cyclopentenyl adenosine analogue, neplanocin A (NPC), on 
cell growth and differentiation was examined in the human promyelocytic leu- 
kemia cell line HL-60. Continuous exposure of HL-60 cells to 0.1 - 3.3 uM 
NPC resulted in a progressive reduction in cell growth which was accompanied 
by an increase in differentiation to cells with a myelocyte and neutrophil 
morphology. The latter effect was expressed as an increase in the capacity 

of cells to reduce nitroblue tetrazolium and reached a level of 40% of the 
total cell population. Preceding the phenotypic changes was the inhibition of 
RNA and DNA methylation in preference to inhibition of their synthesis which 
coincided with the formation of a metabolite of NPC with the chromatographic 
characteristics of S^-adenosyl-L-methionine (AdoMet). In addition, c- myc mRNA 
expression, which is amplified in HL-60 cells, was markedly reduced following 
NPC treatment. These results indicate that NPC is an effective inhibitor of 
RNA and DNA methylation resulting from its conversion to an analogue of AdoMet, 
and that these effects appear to be responsible for reduced c-myc RNA expres- 
sion and the induction of myeloid differentiation in this cell line. (Linevsky, 
Cohen, Hartman, Knode and Glazer, Mol . Pharmacol., in press). 

4. The effects of the cyclopentenyl (cCyd) and cyclopentyl (carbodine) ana- 
logues of cytidine on differentiation, and nucleic acid and nucleotide biosyn- 
thesis was examined in human promyelocytic leukemia cell line HL-60. Continu- 
ous exposure for 5 days to 10"^ to 10"^ M cCyd or 10"^ to 10-^ M carbodine 
produced progressive inhibition of cell growth. During this exposure interval, 
pronounced differentiation to mature myeloid cells occurred wherein 95% of the 
cell population reduced nitroblue tetrazolium four days after exposure to 

10"7 M cCyd or 10-5 carbodine. Preceding differentiation was the inhibition 
of DNA synthesis which reached 10% of control levels 24 hr after exposure to 
10"^ M cCyd or lO'^ M carbodine, while RNA synthesis was inhibited to a 
lesser extent. The appearance of mature myeloid cells by cCyd was preceded by 
the inhibition of c- myc mRNA synthesis which was more pronounced than the 
reduction in total cellular RNA synthesis. These biochemical effects were 
accompanied by a rapid and pronounced inhibition in the synthesis of CTP, but 
not of UTP, ATP or GTP, where the half-life for the disappearance of CTP was 
1.5 to 2 hr. Following drug removal, cells treated with cCyd showed sustained 
inhibition of CTP synthesis, whereas cells treated with carbodine showed almost 
complete recovery of CTP levels within 48 hr. These results indicate that 
inhibition of CTP synthetase leads to rapid inhibition of DNA and c -myc mRNA 
synthesis which precedes the appearance of differentiated cells, and that the 
cyclopentenyl analog, in contrast to the cyclopentyl derivative of cytidine 
leads to irreversible inhibition of CTP synthesis. (Glazer, Cohen, Hartman, 
Knode, Lim and Marquez, Biochem. Pharmacol., in press). 

5. The effects of the pyrrolopyrimidine antibiotics, sangivamycin and toyocamy- 
cin, on the synthesis of RNA and protein, ribosomal RNA processing, and cell 
vialDility were examined in colon carcinoma cell line HT-29. Exposure for 24 

hr to toyocamycin caused an exponential type of cell lethality resulting in a 
4 log reduction of cell viability, while sangivamycin produced a gradual and 

180 



ZOl CM 07109-09 LBC 

self-limiting type of cell lethality resulting in a 1 log reduction of cell 
viability. Toyocamycin at a concentration of 1 ;W produced total cessation 
of precursor rRNA processing, while 10 uM sangivamycin produced little or no 
effect on processing. On the contrary, sangivamycin caused a significant 
decrease in protein synthesis after 6 hr, while toyocamycin had less effect. 
The inhibition of protein synthesis by sangivamycin results from an inhibition 
of the formation of complexes essential to the initiation of protein synthesis. 
The results suggest that the mechanism of action of these closely related 
agents are quite distinct. The marked loss of cell viability caused by toyoca- 
mycin correlates with its effect on rRNA processing while the slow inhibition 
of protein synthesis appears to be secondary to the loss of nbosome synthesis. 
On the other hand, the lesser cytotoxicity produced by sangivamycin results 
from a more direct effect on protein synthesis. Importantly, cells are much 
less capable of resuming normal proliferative activity after 24 hr of impaired 
rRNA processing than after a similar interval of reduced protein synthesis. 
(Cohen and Glazer, Mol . Pharmacol. 27: 349, 1985). 

6. The effects of six nucleoside and base analogs, 5-fluorouracil , 5-azacy- 
tidine, sangivamycin, toyocamycin, 8-azaguanine, and tubercidin, on nbosomal 
RNA processing and cell viability were examined in the colon carcinoma cell 
line HT-29. Exposure of HT-29 cells to various concentrations of each of 
these compounds for 24 hr produced two distinct types of results. Toyocamycin, 
5-fluorouracil, and tubercidin caused an exponential type of cell lethality 
resulting in 3-4 log reduction of cell viability, while sangivamycin, 8-aza- 
guanine and 5-azacytidine produced a gradual and self-limiting type of cell 
lethality resulting in no greater than a 1 log reduction of cell viability. 
Likewise, the effects of these drugs on rRNA processing resulted in their 
classification into two groups: toyocamycin, 5-fluorouracil , and tubercidin 
caused an abnormal accumulation of the 45S precursor to rRNA, while sangiva- 
mycin, 8-azaguanine, and 5-azacytidine did not cause an accumulation of 45S 
RNA. Sangivamycin, 8-azaguanine and 5-azacytidine all produced an inhibitory 
effect on protein synthesis, while tubercidin inhibited protein synthesis at a 
concentration similar to that which caused the accumulation of 45S RNA, and 
toyocamycin and 5-flurouracil had no effect on protein synthesis at concentra- 
tions at which 45S RNA accumulated. These results show that cells are much 
less capable of resuming normal proliferative activity after exposure to drugs 
which caused the accumulation of 45S rRNA precursor, in comparison to nucleo- 
side or base analogs which act by other mechanisms. (Cohen and Glazer, Mol. 
Pharmacol. 27: 308, 1985). 

Publications : 

1. Glazer, R.I. and Knode, M.C.: Neplanocin A: a cyclopentenyl analog of 
adenosine with specificity for inhibiting RNA methylation. 0. Biol. 
Chem. 259: 12964-12969, 1984. 

2. Zwelling, L.A., Minford, J., Nichols, M., Glazer, R.I. and Shackney, S.: 
Enhancement of intercalator-induced deoxyribonucleic acid scission and 
cytotoxicity in murine leukemia cells treated with 5-azacytldine. 
Biochem. Pharmacol. 33: 3903-3906, 1984. 



181 



ZOl CM 07109-09 LBC 

3. Cohen, M.B. and Glazer, R.I.: Cytotoxicity and inhibition of ribosomal 

RNA processing in human colon carcinoma cells. Mol . Pharmacol . 27: 308-313, 
1985. 

4. Chapekar, M.S., Bustin, M. and Glazer, R.I.: Evidence that high mobility 
group protein 17 is not phosphorylated in human colon carcinoma cells. 
Biochim. Biophys. Acta 838: 351-354, 1985. 

5. Cohen, M.B. and Glazer, R.I.: Comparison of the cellular and RNA-dependent 
effects of sangivamycin and toyocamycin in human colon carcinoma cells. 
Mol. Pharmacol . 27: 349-355, 1985. 

6. Cohen, M.B. and Glazer, R.I.: Inhibition of interleukin-2 messenger RNA 
in mouse lymphocytes by 2'-deoxycoformycin and adenosine metabolites. 
Annals N.Y. Acad. Sci ., in press, 1985. 

7. Glazer, R.I., Knode, M.C., Lim, M.-I. and Marquez, V.E.: Cyclopentenyl 
cytidine analogue: an inhibitor of cytidine triphosphate synthesis in human 
colon carcinoma cells. Biochem. Pharmacol ., in press. 

8. Linevsky, J., Cohen, M.B., Hartman, K.D., Knode, M.C. and Glazer, R.I.: 
Effect of neplanocin A on differentiation, nucleic acid methylation and 
c-myc mRNA expression in human promyelocytic leukemia cells. Mol . 
Pharmacol . , in press. 

9. Glazer, R.I., Cohen, M.B., Hartman, K.D., Knode, M.C, Lim, M.-I. and 
Marquez, V.E.: Induction of differentiation in human promyelocytic 
leukemia cell line HL-60 by the CTP synthetase inhibitor, cyclopentenyl 
cytidine analogue. Biochem. Pharmacol., in press. 



182 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07156-02 LBC 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) 

Differentiation of Human Leukemia Cells 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Theodore R. Breitman Chemist LBC, NCI 



Other: 



Masue Imaizumi 
Jiro Uozumi 
Sharon Orndorf 



Visiting Fellow 
Visiting Fellow 
Microbiologist 



LBC, NCI 
LBC, NCI 
LBC, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 



Laboratory of Biological Chemistry 



INSTITUTE AND LOCATION 

NCI. NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS; 
4.0 



PROFESSIONAL: 

3.0 



1.0 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



K (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The availability of tissue culture cell lines has made it possible to study the 
regulation of proliferation and differentiation of specific hematopoietic cell 
types and the effects on these cells of l<nown or suspected mediators and modu- 
lators. It was found previously in this laboratory that retinoic acid (RA) 
is a potent inducer of terminal differentiation of the human promyelocytic cell 
line, HL-60, and the human monoblast- and monocyte-like cell lines, U-937 and 
THP-1. In addition RA was found to induce differentiation of fresh cells in 
primary culture of patients with acute promyelocytic leukemia. While retinoic 
acid alone is capable of inducing terminal differentiation, combinations of a 
physiological concentration of RA (10 nM) and either cAMP inducing agents (e.g., 
cAMP, prostaglandin E, or cholera toxin) or the conditioned medium from either 
activated T-cells or human leukemic T-cell lines synergistically induced differ- 
entiation of HL-60. An activity called "differentiation inducing activity" or DIA 
has been purified from these conditioned media and attempts are now being made to 
produce monoclonal antibodies against this protein. In addition to DIA, gamma- 
interferon also has differentiation inducing activity. However, polyclonal 
antibody against gamma-interferon does not neutralize DIA. Studies are now in 
progress to apply the results obtained in vitro with HL-60 to a transplantable HL- 
60 tumor carried in athymic nude mice. "These studies should have applicability in 
the treatment of patients with some leukemias. 



183 



PHS 6040 (Rev. 1/84) 



GPO 914-918 



ZOl CM 07156-02 LBC 



Objectives; 



This project seeks an understanding of the process of terminal differentiation 
of myeloid cells. It is now generally agreed that some leukemias, as well as 
other malignancies, are diseases resulting from a block in terminal differen- 
tiation. This view suggests that viable treatment may be possible with agents 
that induce differentiation. To aid in this search, studies are conducted to: 
a) better understand the mechanism{s) of terminal differentiation; b) study 
the metabolism of known inducers of differentiation, e.g., RA, to aid in the 
development of more potent inducers; 3) screen known and newly synthesized 
compounds for their differentiation inducing activity; d) employ an animal 
model system (transplantable HL-60 carried in athymic nude mice) to determine 
if inducers of differentiation in vitro are active by a similar mechanism in 
vivo ; e) establish as transplantable tumors in athymic nude mice other human 
myeloid leukemia cell lines to be used for testing protocols developed for 
treating the HL-60 transplantable tumor line. 

Methods Employed : 

The principal methods employed involve measurement of differentiation of human 
leukemia cell lines in cell culture. Most studies are conducted with the HL-60 
promyelocytic cell line. Differentiation is assessed primarily by morphology 
and the ability of cells to reduce nitroblue tetrazolium to a formazan after 
incubation of HL-60 cells for 4-5 days in the presence of an inducer. 

Major Findings ; 

1. Combinations of DIA and a physiological concentration of RA (10 nM) induce 
HL-60 to differentiate to cells that have monocytic markers and that can carry 
out several important functions of phagocytes including immunophagocytosis, 
development of receptors for the chemotactic peptide FMLP, and chemotaxis. In 
contrast, HL-60 induced with RA alone do not develop chemotactic peptide 
receptors and do not exhibit chemotaxis. These results suggest that therapy in 
vivo with RA as a sole agent may not be as effective as a combination therapy 
with DIA (or possibly other lymphokines such as gamma-interferon) and RA. 

2. A polyclonal antibody against gamma-interferon did not neutralize the 
activity of a more purified DIA. This result indicates that DIA is distinct 
from gamma-interferon even though gamma-interferon is a DIA. 

Proposed Course ; 

1. A large effort is underway to produce a monoclonal antibody to DIA. This 
antibody will be used to purify large quantities of DIA by affinity chroma- 
tography so that amino acid sequence data can be obtained. This information 
will allow us to construct the necessary probes for the isolation of the gene 
and the cloning of this potentially important product. 

2. Purified DIA will be labeled with 125i and studies conducted on its 
probable binding to specific receptors on responsive cells. 

3. The transplantable HL-60 tumor line will be used to determine if substances 
that induce differentiation in vitro also induce differentiation in vivo. In 



184 



ZOl CM 07156-02 LBC 

addition, attempts will be made to establish as transplantable tumors other 
leukemia cell lines such as KG-1, THP-1, and K-562. 

4. Studies will be conducted on the mechanism of RA-induction of HL-60. 
Initially, efforts will be directed to determine if a stable covalent bond is 
formed between the retinoyl moiety of RA and a protein(s) in a manner similar 
to fatty acid-acylation of proteins. 

Publications ; 

1. Hemmi, H. and Breitman, T.R. Induction of functional differentiation of a 
human monocytic leukemia cell line (THP-1) by retinoic acid and cholera 
toxin. JPN. J. Cancer Res. (Gann) 76: 345-351 (1985). 

2. Breitman, T.R., Keene, B.R. and Hemmi, H. : Growth and differentiation of 
human myelomonocytic leukemia cell lines in serum free medium. In Mather, 
J. (Ed.): Mammalian Cell Culture. The Use of Serum-Free and Hormone 
Supplemented Media . New York, Plenum Press, 1984, in press. 

3. Breitman, T.R., Keene, B.R. and Hemmi, H.: Studies of growth and differ- 
entiation of human myelomonocytic leukemia cell lines in serum-free 
medium. In Barnes, D. (Ed.): Methods in Molecular and Cell Biology . 
New York, A. Liss, 1984, in press. 

4. Hemmi, H. and Breitman, T.R.: Induction of differentiation of the human 
promyelocytic cell line HL-60 and primary cultures of human leukemia cells. 
A model for clinical treatment. In Glazer, R.I. (ED.) Developments in 
Cancer Chemotherapy . Boca Raton, FL, CRC Press, 1984, in press. 

5. Imaizumi, M. and Breitman, T.R. Retinoic acid-induced differentiation of 
the human promyelocytic leukemia cell line, HL-60, and fresh human leukemia 
cells in primary culture: a model for differentiation inducing therapy of 
leukemia. Leukemia Reviews Int., in press. 



185 



ANNUAL REPORT OF THE LABORATORY OF EXPERIMENTAL THERAPEUTICS AND METABOLISM 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

The Laboratory of Experimental Therapeutics and Metabolism (LETM) personnel 
conduct experiments and generate basic information that contributes to the 
improvement of cancer treatment. This interdisciplinary research program in 
tumor biology, pathology, biochemistry, pharmacology and toxicology has overall 
aims that include 1) utilization of the biochemical and pathobiological 
characteristics of hepatic and extrahepatic tumor cell populations to define 
the histogenesis of specific cancers; 2) establishment of specific animal and 
human tumor cell lines to study new and existing anticancer drugs for their 
mechanism of toxicity; 3) investigation of biochemical and molecular mechanisms 
of drug metabolism and drug toxicity; 4) elucidation of the metabolic and 
physiological factors that may underlie target organ and target cell specific 
toxicity and prevention of toxicity by cellular defense mechanisms and 5) the 
continuation of the development of suitable cellular and animal models to 
elucidate mechanisms of toxicity of anticancer drugs and chemical modulators of 
anticancer drug effects. 

The LETM is presently organized into two sections. 

Pharmacology and Toxicology Section. 

As part of the LETM, the overall goal of the Pharmacology and Toxicology section 
is to provide basic information pertaining to the biochemistry, pharmacology, 
and toxicology of chemicals that are useful in our understanding and improvement 
of cancer therapy. Personnel in the Pharmacology and Toxicology section utilize 
an interdisciplinary and collaborative approach to the project areas summarized 
below. 

a) In Vivo Metabolism and Toxicity; Effects of Thiol Modulation 

Two classes of chemical agents, 2-chloroethyl nitrosoureas and alkylfurans, have 
organ specific toxicity in vivo that relates to thiol modulation. In vivo 
administration of buthionine sulfoximine (BSO) depleted rat tissue thiols with 
the greatest depletion in kidney tissue and bone marrow thiols being the most 
resistant to thiol depletion. BSO pretreatment increased the renal and hepatic 
toxicity of MeCCNU in rats and increased the lethality of L-PAM in BDF mice. 
Liver GSH levels decreased after administration of 2-methylfuran which displayed 
enhanced toxicity after phenobarbital treatment of rats. Di ethyl maleate treat- 
ment enhanced toxicity and covalent binding of 2-methylfuran presumably due to 
GSH depletion. However, GSH depletion by BSO protected against 2-methylfuran 
toxicity and covalent binding. 

DNA alkylation and nephrotoxicity of MeCCNU indicate that it may not be possible 
to dissociate the antitumor activity of the nitrosoureas from their potential 
for nephrotoxicity. However, nephrotoxicity appears independent of carbamylat- 
ing activity of MeCCNU. Hepatic metabolism of MeCCNU by cytochrome P-450 

187 



dependent reactions to reactive intermediate(s) of the 2-chloroethyl side chain 
may contribute to the DMA alkylation and renal toxicity. Metabolic studies 
with A- (2-chloroethyl) conjugates of cysteine and glutathione indicate a pos- 
sible role of these metabolic intermediates in nephrotoxicity of chloroethyl- 
nitrosoureas. 

An animal model that utilizes multidosing has been utilized to examine the 
mechanism of BCNU-induced pulmonary toxicity. Electron microscopic studies 
have demonstrated that BCNU causes selective toxic changes in pulmonary alveolar 
type II cells suggestive of disturbed phospholipid synthesis at doses of BCNU 
that no other cell type examined was affected. A marked decrease in pulmonary, 
but not renal, fatty acid synthesis rates was noted after BCNU treatment. 

A novel protocol was utilized to evaluate the nephrotoxic potential of tetra- 
platin, a new platinum analog. The nephrotoxicity of tetraplatin was compared 
with cisplatin at equimolar doses and found to be less toxic and markedly 
different. The first indication of neurotoxicity was a severe diuresis and 
certain kidney function parameters were found to be inappropriate for assessing 
the onset of nephrotoxicity. Urinary excretion of proteins, glucose and enzymes 
was the most sensitive indicator for the onset of neurotoxicity. Differences 
between the toxicity of tetraplatin and cisplatin include hematologic disorders 
and gastrointestinal toxicity. 

b) In Vitro Nephrotoxicity Model with Renal Slices 

An in vitro model using renal cortical slices has been developed to evaluate 
acutely-acting proximal tubular nephrotoxicants. Fischer 344 rat kidney slices 
provided a useful model for mechanistic evaluations of several classes of 
nephrotoxicants. Data obtained with in vitro slice exposure appear to correlate 
well with in vivo nephrotoxicity; quantitative comparisons between different 
nephrotoxicants do not appear justified and the model does not appear to be 
useful for routine screening of unknown compounds. Mechanistic evaluations for 
certain classes of nephrotoxicants appear to be a very useful application of 
this UL vitro model . 

c) Human Lung Carcinoma Cell Line Model Systems 

Biochemical characterization of human lung carcinoma cell lines (HLCCL) has 
continued to provide a basis for target cell -oriented lung cancer therapy. The 
cell lines NCI-H332 and NCI-H358, derived from pulmonary adenocarcinomas metab- 
olized arachidonic acid (AA) to PGE2 which was characterized by negative ion 
chemical ionization GC-MS analysis. The prostaglandin endoperoxide synthetase 
(PES) activity was also measured by the amount of immunoreactive PGE2 formed 
under a variety of incubation conditions. No detectable PGE2 was formed by the 
small cell cancer cell lines NCI-H69 and NCI-H128 when incubated in the presence 
or absence of calcium ionophore A23187. NCI-H358 cells failed to metabolize 
polyunsaturated fatty acids other than AA. These findings suggest that powerful 
biochemical tools can be developed to understand important pathophysiological 
aspects of human lung cancer that may have useful diagnostic applications during 
cancer therapy. Other studies include the observation that a homogenous human 
pulmonary large cell carcinoma cell line NCI-H460 is unique from other HLCCL in 
that P6E2 synthesis activity is much higher in early passage as compared to that 
in late passage. These changes are being correlated with ultrastructural 
changes which may provide additional diagnostic application of clinical value. 

188 



d) Cell -Free In Vitro Formation of Reactive Intermediates 

Prostaglandin endoperoxide synthetase (PES) catalyzed cooxidation was found to 
occur with mitoxantrone, bromophenol and 2-bromohydroquinone. The human pul- 
monary adenocarcinoma cell lines, NCI-H322 and NCI-H358, which contain high PES 
activity are susceptible to mitoxantrone-induced cytotoxicity. 

Adriamycin stimulated NADPH-dependent lipid peroxidation several-fold in 
microsomes but not mitochondria isolated from EMT6 mammary carcinoma cells and 
B16 solid tumors. In separate experiments, it was found that in the presence 
of either NADPH or NADH, GSH increased endogenous and adriamycin-enhanced mouse 
kidney microsomal lipid peroxidation, in contrast to the known inhibitory effect 
of GSH in other peroxidation systems. These findings indicate much more needs 
to be understood about lipid peroxidation processes as related to both normal 
and tumor tissues during anthracycline chemotherapy. 

Pathology and Ultrastructural Oncology Section 

This Section's research focusses on mechanisms of development and therapy of 
lung cancer. Studies on the pathogenesis of chemically induced lung cancer in 
rodents as well as reports on human lung cancers have provided ample evidence 
that the histopathologic typing of lung cancers and their preneoplastic lesions 
is insufficient for research in that they lack information on cell type and 
degree of differentiation of subcellular organelles. Projects of this Section 
and collaborative efforts with the Pharmacology and Toxicology Section, LETM, 
as well as other laboratories are therefore based on the concept that response 
of cells to chemicals including drugs are dictated by specific biochemical 
reactions of cells in a manner which can be dependent of cell types and dif- 
ferentiation. This approach necessitates the application of electron microscopy 
for diagnosis of cell type and degree of differentiation at the organelle level 
to all experiments. Moreover, studies on the effects of chemicals and drugs 
are generally addressed with the same methodology to provide initial information 
on target cell type(s) and/or organelles that could then be utilized for the 
design of more focussed experiments on the underlying biochemical mechanisms. 

Although all of our current research is based on information generated in 
earlier animal experiments, our major emphasis during this report period has 
been on the characterization and establishment of human lung cancer cell lines 
(HLCCL) for research on cell type specific chemical /drug interactions. The 
experience generated through such studies has also enabled us to initiate and 
design an number of experiments primarily conducted by investigators of the 
Pharmacology and Toxicology Section, LETM. In summary, we found that: 

1. Of seven investigated non-small cell derived HCCL, only two adenocarcinomas 
NCI-H322 and NCI-H358) exhibited unequivocal well differentiated features 
of one pulmonary cell type (as assessed by electron microscopy) while all 
others demonstrated either dual differentiation (e.g., adenosquamous) or 
poor differentiation which made a diagnosis by cell type questionable. We 
also found evidence that well differentiated lung tumors undergo pronounced 
morphological changes during prolonged maintenance in vitro . This exempli- 
fies the need for careful electron microscopic assessment of HCCL before 
and during their use for biochemical and pharmacological experiments. 



2. Similar to in vivo studies in hamsters, the pulmonary carcinogen, di ethyl - 
nitrosamine'TDEN), is preferentially metabolized by HLCCL with features of 
pulmonary Clara cells in vitro and that such metabolism is inhibi table by 
inhibitors of cytochrome P-450 enzymes. 

3. HLCCL of different cell types metabolize DEN via different enzyme systems, 
e.g., Clara cells via cytochrome P-450 and alveolar type II cells via 
prostaglandin endoperoxide synthetase. Cell type specific differences such 
as this in nitrosamine activation may well be responsible for the known 
organ and cell specific carcinogenicity of these compounds. 

4. Similar to experiments in rats, the pulmonary toxin and candidate anti- 
cancer drug, 4-ipomeanol, is preferentially metabolized by an HCCL with 
characteristics of Clara cells and that such metabolism is mediated by 
cytochrome P-450 enzyme systems. The selective toxicity of the compound 
for this cell line suggests to pursue its usefulness as an anticancer drug 
for pulmonary adenocarcinomas and other solid tumors of similar cell types. 

5. The xenobi otic-metabolism enzymes, cytochrome P-450, aryl hydrocarbon hydrox- 
ylase and ethoxycoumarin 0-di ethyl ase activity are selectively present in 
non-small cell derived HCCL while they are lacking in small cell derived 
HCCL. 

6. Normal alveolar type II cells of rat lung proliferate in vitro until 7 days 
in culture, retain their typical ultrastructure and phospholipid synthesis 
and can be identified by immunoperoxidase using an antiserum specifically 
for rat surfactant apoprotein. 

Publications : 

1. Boyd, M.R., and Reznik-SchCTller, H.M.: Metabolic basis for the 
pulmonary Clara cell as a target for pulmonary carcinogenesis. Toxicologic 
Pathology 12: 56-61, 1984. 

2. McMahon, J.B., Reznik-Schaller, H.M., Gazdar, A.F., Becker, K.L.: 
Influence of priming with 5-hydroxytryptophan on APUD characteristics in 
human small cell lung cancer cell lines. Lung 162: 265-269, 1984. 

3. Ravindranath, V., Burka, L.T., and Boyd, M.R.: Synthesis of 2-([l*C]- 
methyDfuran and 4-oxo[5-l*C]-2-pentenal . J. Labelled Compds. Radiopharm. , 
21: 713-718, 1984. 

4. Ravindranath, V., Burka, L.T., and Boyd, M.R.: Reactive metabolites of 
toxic methylfurans. Science 224: 884-886, 1984. 

5. Schuller, H.M. and Ward, J.M.: Quantitative electron microscopic analysis 
of changes in peroxisomes and endoplasmic reticulum induced in mice during 
hepatocarcinogenesis by diethyl nitrosamine promoted by di (2-ethylhexyl )- 
phthalate or phenobarbital. J. Exp. Pathol. 1: 287-294, 1984. 

6. Sinha, B.K., Trush, M.A., Kennedy, K.A. and Mimnaugh, E.G.: Enzymatic 
activation and binding of adriamycin to nuclear DMA. Cancer Res. 44: 2892- 
2986, 1984. 



190 



7. Smith, A.C. and Boyd, M.R.: Preferential effects of 1 ,3-bis(2-chloroethyl )- 
1 -nitrosourea on pulmonary glutathione reductase and 6SH/GSS6 ratio: 
Possible implications for lung toxicity. J. Pharmacol. Exp. Ther. 229: 
658-663, 1984. 

8. Mimnaugh, E.G., Trush, M.A., Bhatnagar, M. and Gram, T.E.: Enhancement of 
reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the 
anticancer drug, adriamycin. Biochem. Pharmacol. 34: 847-856, 1985. 

9. Pour, P. and Reznik-Schaller, H.M.: Squamous cell carcinomas of the 
bronchi in Syrian golden hamsters. In Jones, T.C. and Mohr, U. (Eds.): 
ILSI Monographs on Pathology of Laboratory Animals, Vol. 2, Respiratory 
System . West Berlin, Springer Verlag, 1985, pp. 47-53. 

10. Stinson, S.F., Reznik-SchUller, H.M.: Neoplasms of the mucosa of the 
ethmoid turbinates in the rat. In Jones, T.C. and Mohr, U. (Eds.): ILSI 
Monographs on Pathology of Laboratory Animals, Vol. 2, Respiratory System . 
West Berlin, Springer Verlag, 1985, pp. 47-53. 

11. Boyd, M.R.., Ravindranath, V., Burka, L.T., Dutcher, J.S., Franklin, R.B., 
Statham, C.N., Hashek, W.M., Hakkinen, P.J., Morse, C.C. and Witshi, H.P.: 
Drug metabolizing enzyme systems and their relationship to toxic mechanisms. 
Proceedings of New Approaches in Toxicity Testing and their Application 

to Human Risk Assessment , in press. 

12. Falzon, M., McMahon, J.B., Gazdar, A.F., and Schuller, H.M.: Preferential 
metabolism of N-nitrosodi ethyl amine by two cell lines derived from human 
pulmonary adenocarcinomas. Carcinogenesis , in press. 

13. Falzon, M., McMahon, J.B., Schuller, H.M.: Xenobiotic-metabolizing enzyme 
activity in human non-small cell derived lung cancer cell lines. Biochem. 
Pharmacol . , in press. 

14. Falzon, McMahon, J.B., Schuller, H.M., Boyd, M.R.: Differential activation 
of 4-ipomeanol by human lung cancer cell lines (HLCCL). Cancer Res. , in 
press. 

15. Kensler, T.W., Schuller, H.M., Jayaram, H.N. and Cooney, D.A.: Characteri- 
zation of eleven important transplantable murine tumors from the standpoint 
of morphology, pyrimidine biosynthesis and responsiveness to pyrimidine 
antimetabolites. In Kaiser, H.E. (Ed.): Progressive Stages of Neoplastic 
Growth . New York, Pergamon Press, in press. 

16. Kramer, R.A., Schuller, H.M., Smith, A.C, Boyd, M.R.: Effects of buthio 
nine sulfoximine on the nephrotoxicity of l-(2-chloroethyl )-3-(trans-4- 
methylcyclohexyl )-l -nitrosourea (MeCCNU). J. Pharmacol. Exp. Ther. , in 
press. 

17. Litterst, C, Smith, J.H., Smith, M.A., Uozumi , J. and Copley, M.: 
Sensitivity of urinary enzymes as indicators of renal toxicity of the 
anticancer drug cisplatin. Proceedings from Newer Diagnostic Methods in 
Nephrology and Urology, in press. 



191 



18. Kramer, R.A., McMenamin, M.G. and Boyd, M.R.: Mechanism of chloroethyl- 
nitrosourea nephrotoxicity: Studies with MeCCNU. In Bach, P.H. and Lock, 
E.A. (Eds.): Renal Heterogeneity and Target Cell Toxicity . John Wiley & 
Sons, in press. 

19. Litterst, C, Smith, J.H., Smith, M.A., Uozumi , J. and Copley, M.: 
Sensitivity of urinary enzymes as indicators of renal toxicity of the 
anticancer drug cisplatin. Proceedings from Newer Diagnostic Methods in 
Nephrology and Urology , in press. 

20. McMahon, J.B., Smith, A.C., del Campo, A., Singh, G., Katyal , S., Schuller, 
H.M.: Characterization of proliferating rat alveolar type II cells in 
vitro by immunological, biochemical and morphological criteria. Exp. Lung 
Res. , in press. 

21. Mimnaugh, E.G., Kennedy, K.A., Trush, M.A. and Sinha, B.K.: Adriamycin- 
enhanced membrane lipid peroxidation in isolated nuclei. Cancer Res. , 
In press. 

22. Minchin, R.F., Ho, P. and Boyd, M.R.: Effects of oxygen on the disposition 
of nitrofurantoin in intact rat lung. Drug Metab. Dispos. , in press. 

23. Minchin, R.F., Johnston, M.R., Reznik-Schuller, H.M., Aiken, M.A. 

and Boyd, M.R.: Toxicity of doxorubicin in isolated dog lung perfused in 
situ . J. Pharmacol. Exp. Ther. , in press. 

24. Reznik-SchOller, H.M.: Experimental carcinogenesis of bronchiolo- 
alveolar neoplasms. In McDowell, E.M. (Ed.): Current Problems in Tumor 
Pathology . Vol. III. Lung Tumor Pathobiology. Edinburgh, Churchill- 
Livingstone, in press. 

25. Schuller, H.M., Smith, A.C., Gregg, M. and Boyd, M.R.: Sequential 
pathological changes induced in rats with the anticancer drug l,3-bis(2- 
chloroethyl)-l-nitrosourea (BCNU). Exp. Lung Res. , in press. 

26. Schuller, H.M., Stinson, S.F., Ward, J.M., McMahon, J.B., Singh, G., 
Katyal, S.L.: Loss of Clara cell antigens from neoplastic Clara cell 
derived lesions induced in the hamster lung by N-nitrosodiethylamine. 
Am. J. Pathol. , in press. 

27. Schuller, H.M. and McMahon, J.B.: Inhibition of N-nitrosodiethylamine 
induced respiratory tract carcinogenesis by piperonyl butoxide in hamsters. 
Cancer Res. , in press. 

28. Ward, J.M., Diwan, B.A., Oshima, M., Hu, H., Schuller, H.M., Rice, J.M.: 
Tumor initiating and promoting activities of Di(2-ethylhexyl )phtalate in 
vivo and in vitro . Proc. Intern. Conf. on Phtalic Acid Esters, in press. 



192 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07140-03 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

BCNU - Induced Pulmonary Fibrosis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: A. C. Smith Staff Fellow LETM, NCI 



Others: 



H. M. Schuller 

M. R. Boyd 

J. B. McMahon 

D. J. Reed 

R. A. Gram 



Vet. Med. Officer 

Associate Director 

Cancer Expert 

IPA 

Bio. Lab. Aid 



LETM, NCI 
DTP, NCI 
LETM, NCI 
LETM, NCI 
LETM, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI. NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 
1.0 



PROFESSIONAL: 
1.0 



_QJL 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues S (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

BCNU, a potent alkylating antitumor agent, causes a severe pulmonary injury in 
patients treated with high doses of the drug. We have described a multi-dosing 
animal model to study the mechanism of BCNU-induced pulmonary toxicity. After 2 
doses of BCNU (10 mg/kg cumulative dose), electron microscopic studies have shown 
changes in pulmonary Type II cells which are associated with cell toxicity and 
disturbed surfactant synthesis. Alveolar Type II cells are responsible for sur- 
factant production and have a very active lipid biosynthesis system. Therefore, 
fatty acid synthesis was measured as a marker for pulmonary Type II cell injury 
after BCNU administration. Single doses of BCNU caused a dose- and time-dependent 
decrease in fatty acid synthesis rates measured in lung tissue. A maximum of 48% 
of ^'*C-acetate incorporation into fatty acids occurred 24 hours after a dose of 
120 mg BCNU/kg. This depression in fatty acid synthesis lasted up to 48 hours 
after a single dose of BCNU. The multi-dosing model for BCNU-induced pulmonary 
fibrosis also resulted in a marked alteration of pulmonary fatty acid synthesis 
rates. After 6 doses of BCNU (cumulative dose 30 mg/kg) fatty acid synthesis was 
depressed by 64%. This inhibition was organ-specific, renal rates of ^^C-acetate 
incorporation into fatty acids did not change after 6 doses of BCNU when maximal 
changes in lung tissue occurred. This correlated with an absence of kidney damage 
measured by organic ion accumulation or lustology. Rates of fatty acid synthesis 
appear to be a good biochemical marker for pulmonary alveolar Type II cell injury 
produced by BCNU. Freshly isolated rat pulmonary Type II cells are sensitive 
to BCNU in vitro . Incubation of confluent cultures of Type II cells with BCNU 
resulted~Tn a depression of cellular glutathione. These alterations of the Type 
II cells antioxidant defense mechanism occurred with evidence of cell injury, but 
no cell death. This indicates, perhaps an additional oxidant challenge is neces- 
sary to produce overt cell death. 

193 



PHS 6040 (Rev. 1/84) 



GPO SI4-91B 



ZOl CM 07140-03 LETM 

Objectives : 

BCNU (l,3-bis(2-chloroethyl )-l-m"trosourea, Carmustine) is a very potent anti- 
tumor xenobiotic used in the treatment of a wide variety of human neoplasms. 
BCNU therapy is associated with numerous drug-related side effects, the one 
most recently described is the development of a life- threatening pulmonary 
fibrosis after BCNU treatment. Our laboratory has developed an animal model to 
study BCNU-induced pulmonary injury in F344 rats. Animals treated with 5 mg 
BCNU/kg once a week for 6 weeks develop a progressive pulmonary fibrosis 
which is histologically similar to the lung disease which develops in patients 
treated with high doses of BCNU. Electron microscopic studies aimed at follow- 
ing the progression of lung injury caused by BCNU treatment have demonstrated 
that relatively low doses of BCNU cause selective toxic changes in pulmonary 
alveolar Type II cells suggestive of disturbed surfactant synthesis. No other 
cell type examined (endothelial, Clara, alveolar Type I cells) was effected by 
small doses of BCNU. This led us to postulate that alveolar Type II cells may 
be the target cell population for BCNU-induced lung injury. 

Pulmonary Type II cells are located in the alveolar wall and are believed to be 
stem cells for replacement of alveolar Type I cells which line the alveolus. 
Type II cells are also thought to be the major site for the production of 
pulmonary surfactant. Surfactant, a phospholipid- rich material, which acts to 
coat the alveolar cell surface thus lowering the surface tension and preventing 
the collapse of the alveolus during respiration. Therefore, Type II cells have 
a very active lipid biosynthesis system and Type II cell toxins have a marked 
effect on total lung lipid biosynthesis. Studies were initiated to explore the 
effect of BCNU on Type II cell function in vivo by measuring pulmonary fatty 
acid synthesis rates. 

Other studies in our laboratory have been concerned with the mechanism of BCNU- 
induced lung damage. Recent work suggested that the effect of BCNU on the 
pulmonary glutathione redox system, an important cellular antioxidant defense 
mechanism, may result in the onset of pulmonary injury. We have demonstrated 
in vivo that there is a temporal relationship between the alteration of the 
"pulmonary glutathione system and the onset of severe lung damage. Since Type 
II cells appear to be the target cell for BCNU-induced lung injury, studies 
were initiated to explore the role of alterations in the antioxidant defense 
mechanism in the toxicity of BCNU on freshly isolated rat pulmonary Type II 
cells. 

Methods Employed : 

A. Effect of BCNU on fatty acid synthesis 

Male Fisher 344 rats (8 weeks old) were treated with BCNU intraperitoneally. 
For multi -dosing studies, rats were injected with 5 mg BCNU/kg dissolved in 10% 
ethanol. Higher concentrations of BCNU were needed for single dose studies and 
therefore BCNU was dissolved in sesame oil. 

After BCNU administration, rats were sacrificed by exsangui nation. Lungs were 
removed, trimmed, lobes were separated and rinsed in buffer. Slices were made 
by cutting the lobe with a series of razor blades separated by washers. Slices 

194 



ZIO CM 07140-03 LETM 

were very uniform and approximately 0.4 - 0.5 mm thick. Each incubation con- 
tained 100 mg lung tissue (about 6-8 slices) in 3 ml of PFMR-4 media. Slices 
were preincubated for 15 minutes at 37 °C under 95% 02:5% CO2. Acetate (5yCi 
sodium Cp--'-^C] acetate in 15 ymoles of cold acetate) was added and samples 
were incubated for an additional 60 minutes. The reaction was terminated when 
the lung slices were removed and homogenized in chloroform: methanol (2:1) to 
extract lung lipids. The organic layer was washed 2 times with 1.0 ml of 0.1 M 
phosphate buffer (pH 7.4) saturated with chloroformimethanol (2:1). The solvent 
was removed under nitrogen and lipids were resuspended in 1.5 ml chloroform. 
Radioactivity was determined in duplicate aliquots. 

B. Effects of BCNU on isolated rat pulmonary Type II cells in vitro 

F344 rat alveolar Type II cells were isolated by the method described in this 
annual report. Confluent 4 day Type II cell cultures containing approximately 
1.8 X 10° cells were suspended in F12 media containing no serum and exposed to 
50 p M BCNU for 4 hours at 37 °C under 5% CO2. Total glutathione levels were 
determined by high pressure liquid chromatography according to the method 
described by Reed and coworkers (Anal. Biochem. (1980) 106:55). Reduced 
glutathione was used to establish a standard curve and y-glutamylglutamate 
was used as an internal standard. 

Results : 

A. In vivo effect of BCNU on pulmonary fatty acid synthesis. 

Single doses of BCNU had a profound effect on pulmonary rates of fatty acid 
synthesis measured in lung slices. There was a rapid 25% decrease in fatty 
acid synthesis in lung tissue 4 hours after BCNU administration. This inhibi- 
tion of fatty acid synthesis remained up to 48 hours after a single dose of 
BCNU. Increased doses of BCNU resulted in a larger amount of fatty acid syn- 
thesis inhibition. A single dose of 90 mq BCNU/kg or 120 mg BCNU/kg inhibited, 
respectively, 36% and 48% of total lung ■'•^C-acetate incorporation into fatty 
acids 24 hours after BCNU. The multi -dosing model for BCNU-induced pulmonary 
fibrosis also resulted in a marked depression of pulmonary rates of fatty acid 
synthesis. Three doses of BCNU (cumulative dose 15 mg/kg) resulted in a 40% 
depression of ^*C-acetate incorporation into lung phospholipids. Six doses of 
BCNU (cumulative dose 30 mg/kg) caused fatty acid synthesis rates to fall to 
52% of control levels. Again, this inhibition of ^'^C-acetate incorporation in 
lung lipids by BCNU was persistant, lasting up to 48 hours after a dose of 
BCNU. 

The effects of BCNU on fatty acid synthesis rates were specific for lung tissue. 
After 6 doses of BCNU (cumulative dose 30 mg/kg) which resulted in a 50% inhibi- 
tion of ^^C-acetate incorporation into lung lipids did not result in any altera- 
tion of fatty acid synthesis in kidney tissue. This was also no evidence of 
kidney damage after BCNU administration when measured biochemically by organic 
ion accumulation or morphologically. This information demonstrates that changes 
in fatty acid synthesis rates after BCNU are specific for the target organ, the 
lung, and are presumably due to Type II cell injury caused by BCNU administra- 
ti on . 



195 



ZIO CM 07140-03 LETM 

B. The effect of BCNU on isolated pulmonary Type II cells in vitro . 

Preliminary experiments have been conducted to establish the sensitivity of 
freshly isolated rat pulmonary Type II cells to BCNU treatment in vitro . Of 
particular interest is to establish the role of the alteration of the antiox- 
idant defense mechanism in BCNU-induced Type II cell injury. BCNU causes a 
time- and dose-dependent decrease in cellular glutathione levels. Four hours 
after exposure of Type II cells to BCNU, approximately 58% of total glutathione 
was depleted due to a marked inhibition of cellular glutathione reductase by 
BCNU. With this marked alteration of the cellular antioxidant defense mecha- 
nism, there was an increase in cell injury measured by lactate dehydrogenase 
leakage, but no indication of cell death measured morphologically. These 
studies indicate that pulmonary Type II cells are sensitive to BCNU toxicity 
in vitro as wel 1 as j_n vivo . 

Significance : 

BCNU therapy often causes a fatal pulmonary fibrosis in patients treated with 
high doses of the drug. This research project is aimed at elucidating the 
mechanism of BCNU-induced pulmonary injury. We have shown by electron micros- 
copy that pulmonary Type II cells are the only cell type affected by low doses 
of BCNU and that this morphological evidence of injury correlates with bio- 
chemical changes (i.e., fatty acid synthesis rates) associated with pulmonary 
Type II cells. Fatty acid synthesis rates is a sensitive biochemical marker 
for pulmonary Type II cell injury caused by BCNU can be used to monitor and 
quantitate lung damage in vivo . We have also shown that freshly isolated rat 
pulmonary Type II cells are sensitive to BCNU-induced damage in vitro . Pre- 
liminary results indicate that after exposure to low concentrations of BCNU 
there are marked alterations in the glutathione redox systems and these changes 
occur with apparent cell toxicity but no cell death. These studies suggest 
that perhaps an additional oxidant challenge is necessary for BCNU to cause 
overt cell death. 

Proposed Course : 

Current studies are aimed at specifically addressing the effect of BCNU on the 
glutathione redox system in freshly isolated rat pulmonary Type II cells. 
There are several questions to be answered pertaining to the role BCNU-induced 
alterations of the glutathione redox status in Type II cells with oxidant 
damage. These studies will indicate perhaps the type and extent of oxidant 
challenge necessary for cell death and also may help to elucidate the mechanism 
of BCNUinduced pulmonary fibrosis in vivo . 

Publications : 

1. A.C. Smith and M.R. Boyd (1984) Preferential effects of l,3-bis(2-chloro- 
ethyl )-l-nitrosourea on pulmonary glutathione reductase and GSH/GSSG ratio: 
Possible implications for lung toxicity. J. Pharmacol. Exp. Ther. 229, 
658-663. 

2. H.M. Schuller, A.C. Smith, M. Gregg and M.R. Boyd (1985) Pathogenesis of 
pulmonary fibrosis induced in rats with the anticancer drug 1,3-bis 
(2-chloroethyl )-l-nitrosourea (BCNU). Exp. Lung Res, (in press). 

196 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07160-02 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

In vitro Model for Evaluating Nephrotoxicity 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

P. I.: J. H. Smith PRAT Staff Fellow NIGMS 



Others: S. J. Lovett 



Bio. Lab. Aid 



LETM, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI. NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 

0.6 



PROFESSIONAL: 

0.5 



0.1 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



D (b) Human tissues E (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

An in vitro model using renal cortical slices has been developed to evaluate 
acutely-acting proximal tubular nephrotoxi cants. The toxicity of several 
classical nephrotoxicants (including HgCl?, K2Cr207, CCI4, hexachlorobutadiene, 
cephaloridine, gentamicin and 4-ipomeanol) has been evaluated in this sytem 
using Fischer 344 rat kidney. Conditions have been established where several 
physiologic parameters can be monitored after a single incubation of 100 mg of 
renal cortical slices. Although the data for in vitro slice exposure appear to 
correlate well with in vivo nephrotoxicity, quantitative comparisons between 
different nephrotoxicants are probably inappropriate. Thus, this model will not 
be useful for routine screening of unknown compounds. Nevertheless, there are 
similarities in the characteristics of nephrotoxicity observed in vitro and those 
reported in vivo , suggesting the model may be useful for mechanistic evaluations 
of certain classes of nephrotoxicants. Preliminary data suggest this model may 
be of particular use in evaluating nephrotoxicity of platinum anticancer drugs. 



197 



PHS 6040 (Rev. 1/84) 



GPO 914-918 



ZOl CM 07160-02 LETM 

Objectives : 

Although the kidneys are susceptible to toxicity by a variety of xenobiotics, 
and especially anticancer drugs, relatively little is known regarding mecha- 
nisms of nephrotoxicity. Renal cortical slices perpared from laboratory 
animals have been used to evaluate renal toxicity, in combination with urinary, 
serologic and histologic evaluations. In acute toxicity evaluation, renal 
cortical slices usually are prepared 24 hr or longer after nephrotoxicant 
administration and several physiologic parameters are determined. Recently, 
the mechanism of chloroform nephrotoxicity was determined by monitoring organic 
ion accumulation after in vitro exposure of renal cortical slices from mice, 
rabbits and rats to chloroform. This in vitro model allowed an evaluation of 
the relationship between in situ renal metabolism of a compound and the subse- 
quent nephrotoxicity. The purpose of this investigation was to determine 
whether renal cortical slices could be used to evaluate other acutely-acting 
proximal tubular nephrotoxicants. Because decreases in organic ion accumulation 
do not necessarily reflect impaired proximal tubular function due to toxicity, 
particularly after in vitro exposure to a chemical, other measures of proximal 
tubular function and cell integrity were incorporated into the procedure. 

Methods Employed : 

Approximately 100 mg of thin renal cortical slices are prepared from freshly 
isolated Fischer 344 rat kidneys. Slices are placed in 2 ml of a phosphate- 
buffered medium on ice until all slices are prepared for the days' incubations, 
usually a period of 1 to 1 1/2 hr. This brief holding period serves to rinse 
the freshly sliced tissue. The slices are transferred to 25 ml Erlenmeyer 
flasks containing 4 ml of the preincubation media and various concentrations of 
the nephrotoxicant to be evaluated. Flasks are gassed with 100% O2 for 5 min, 
stoppered, and preincubated at 37°C for 2 hr in an oscillating water bath. 
After this preincubation, slices are rinsed in fresh media lacking the nephro- 
toxicant, blotted gently, and transferred to fresh media for a subsequent 
incubation at 25°C. This incubation is performed to assess proximal tubular 
cell function by monitoring gluconeogenesis, organic ion accumulation and 
tissue glutathione concentrations. The preincubation and incubation media are 
monitored for renal marker enzymes, which have been assessed in the urine 
clinically as a non-invasive monitor of nephrotoxicity. Enzyme activities 
originating from brush border [alkaline phosphatase (AP), y-glutamyl trans- 
peptidase ( yGT), maltase], cytosol [lactate dehydrogenase (LDH)] and lysosomes 
[N-acetyl- gglucosaminidase (NAG)] are determined. Additionally, preincubation 
media is monitored for the presence of malondi aldehyde (MDA) as an indication 
of lipid peroxidation. 

Major Findings : 

Conditions have been established where several physiologic parameters can be 
monitored after a single incubation of renal cortical slices {~100 mg); pre- 
vious investigations have assessed each of these individually. These param- 
eters are relevant to tests used for assessment of nephrotoxicity after in 
vivo administration of a nephrotoxicant. 



198 



ZOl CM 07160-02 LETM 

Compounds representing several classes of nephrotoxi cants have been tested in 
this in vitro slice model. In vitro concentrations were chosen which were 
equivTTent to or less than t"^ total number of moles administered in vivo to 
produce nephrotoxicity. The compounds evaluated include HgCl2, K2Cr207, CCI4, 
hexachlorobutadiene (HCBD), cephaloridine, gentamicin, and 4-ipomeanol. 4- 
Ipomeanol is not nephrotoxic to rats and was, therefore, used as a negative 
control. The concentration of 4-ipomeanol was based on the LD50 of this 
compound. 

Measurements of proximal tubular cell function appeared to be most sensitive in 
detecting cell toxicity. The concentration-response curves for organic ion 
accumulation and for gluconeogenesis were similar. On initial observation, the 
data from in vitro slice exposure appeared to correlate well with in vivo 
nephrotoxicity of these compounds. Upon more critical analyses of these data, 
however, it appeared that quantitative comparisons between different nephro- 
toxicants are probably inappropriate. Consistent with in vivo nephrotoxicity, 
the metals HgCl2 and K2Cr207 were the most nephrotoxic. The solvents CCI4 and 
HCBD followed in toxicity. At the higher concentrations, neither solvent was 
completely soluble in the aqueous buffer. Therefore, it was not clear whether 
the "toxicity" observed in this in vitro model represented a toxic mechanism 
similar to that occurring in vivo , or whether this " in vitro " toxicity repre- 
sented nonspecific solvent effects on membrane integrity. Relatively higher 
concentrations of cephaloridine and gentamicin were required to produce toxici- 
ty in this model This was consistent with these drugs where nephrotoxicity is 
delayed or requires multiple doses, and where toxicity appears to be correlated 
directly with renal accumulation of the drug. 4-Ipomeanol gave no indication 
of nephrotoxicity on any of the parameters measured in this model in the 
concentration range approximating the LD50 (1 mM in this system). 

Release of the cytosolic enzyme, LDH, is used routinely for assessing damage in 
cell cultures and freshly isolated cells. The relative responses of LDH release 
for CCI4, HCBD, cephaloridine and gentamicin were similar to slice function 
data. LDH release was not a good indicator of cell toxicity after exposure to 
HgCl2 and K2Cr207. Release of MAG was similar to LDH except HgCl2 dramatically 
increased NAG release as well. In general, release of brush border enzymes was 
a very poor indicator of cell damage in this model. The largest increase of 
AP, yGT and maltase release was observed only after exposure to the solvents 
CCl4 and HCBD. Thus, this elevation may be related to the physical condition 
of the cells and membrane integrity as a result of the solvent, detergent-like, 
effects. In all cases, it was preferable to monitor for enzyme activity in the 
incubation medium rather than the preincubation medium. This was because some 
compounds, such as HgCl2, directly inhibited the activity of several enzymes. 

There was evidence of lipid peroxidation, monitored by MDA formation, in slices 

preincubated with HgCl2 and cephaloridine. These data are consistent with_ 

other investigations reporting lipid peroxidation by these compounds both in 
vivo and vn^ vitro . 

Significance to Biomedical Research and the Program of the Institute : 

Nephrotoxicity is an undesirable side-effect of several commonly used anti- 
cancer agents. Traditionally, nephrotoxicity has been monitored and alterna- 

199 



ZOl CM 07160-02 LETM 

tive dosage regimens have been employed to minimize the potential for toxicity. 
Unfortunately, relatively little is known regarding the actual mechanism of 
nephrotoxicity of these- compounds. Such knowledge may lead to the develop- 
ment of second generation drugs with chemotherapeutic efficacy which lack the 
nephrotoxicity, or, alternatively, to the development of methods to avoid the 
nephrotoxic effects of currently used drugs. An in vitro model model offers 
the opportunity to assess directly the renal metabolism and toxicity of drugs 
and chemicals independent of variables such as pharmacokinetics or toxicity to 
other target organs. In addition, the development of an in vitro model for 
nephrotoxicity can provide a rapid, inexpensive in vitro target organ screen 
for a variety of chemicals and drugs. Thus far, this model appears to cor- 
relate with characteristics of nephrotoxicity observed in vivo . Since the 
tissue is isolated rapidly from the animal, this should^ecrease the potential 
for dramatic metabolic alterations and other similar artifacts that may be 
encountered when using isolated cells or cell culture techniques. These advan- 
tages may facilitate investigations of species, strain and sex differences in 
nephrotoxicity and, ultimately, may provide information on more appropriate 
animal models for toxicological evaluations. Specifically, the model offers 
the opportunity to utilize human renal tissue for comparative purposes. 

Proposed Course : 

Evaluation of a variety of classical nephrotoxi cants has indicated the benefits 
and limitations of this in vitro slice model. Currently, this in vitro slice 
model is being used to evaluate the relative nephrotoxicity of a group of 
platinum anticancer agents including cisplatin, CBDCA, CHIP and tetraplatin 
(NSC 119875, 241240, 256927 and 363812, respectively). These data obtained 
using Fischer 344 rat renal cortical slices will be compared to the in vivo 
nephrotoxicity of these drugs (see Annual Report ZOl CM 07169-01 LETMT. 
Attempts are being made to obtain human renal tissue within 24 hr after death 
for similar in vitro nephrotoxicity assessments. The goal of this investiga- 
tion will then be to use this model for mechanistic evaluation of the nephro- 
toxicity and of those agents which may reduce nephrotoxicity of platinum 
anticancer agents. Additionally, collaborative efforts are being made to 
evaluate histologically the condition of the renal cortical slices subsequent 
to the preincubation period. This evaluation will address the question of 
whether cell specific regions of the nephron are damaged in vitro similar to 
damage occurring in vivo. 



200 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07161-02 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. We must tit on one line t>etween the t)orders.) 

In Vivo Studies on the Toxicity of Alkylfurans 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute affiliation) 

P. I.: V. Ravindranath Visiting Fellow LETM, NCI 



Others: M. R. Boyd 



Associate Director 



DTP, NCI 



COOPERATING UNITS (If any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

0.6 



PROFESSIONAL: 
0.5 



0.1 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues S (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The mechanisms involved in the metabolic activation and toxicity of 2-methylfuran 
(2-MF), a naturally occurring cytotoxic furan found in cigarette smoke and coffee 
are being investigated. 

Following administration of 2-[(l^C)methyl]furan to rats, extensive covalent bind- 
ing of label to macromolecules in liver, lungs and kidney was observed. Smaller 
amounts were bound to tissues with little or no known mixed function oxidase 
activity. Maximal covalent binding to both protein and DNA was observed in the 
liver, the target organ where toxicity is manifested. Liver GSH levels decreased 
by a third, half an hour after administration of 2-MF indicating the formation of 
electrophilic metabolites. Pretreatment with various inhibitors and inducers of 
metabolism showed that phenobarbital potentiated toxicity of 2-MF and was followed 
by increased urinary excretion of label, while 3-methylcholanthrene or piperonyl- 
butoxide did not markedly alter the toxicity of 2-MF. Pretreatment with buthi- 
onine sulfoximine (BSO), a GSH depletor and a chemosensitizing agent, while de- 
creasing covalent binding, also decreased toxicity, whereas di ethyl maleate, also 
a GSH depletor, increased both covalent binding and toxicity of 2-MF. BSO, which 
depletes GSH levels by inhibiting cysteine synthetase is known to enhance cysteine 
levels in tissues. Thus, BSO probably decreases covalent binding by trapping the 
reactive intermediate as the stable cysteine conjugate. Pretreatment with L-2-oxo 
thiazoline 4-carboxylate (OTZ), promoter of GSH biosyntheisi potentiated hepato- 
toxicity and increased covalent binding to liver proteins, although coadministra- 
tion of OTZ and BSO prior to an i.p. dose of 2-MF, decreased the covalent binding 
to liver proteins and the serum GTP levels compared to that of rats that received 
2-MF alone. The GSH conjugate of the reactive metabolite of 2-MF is unstable, 
which ma.y explain the potentiation of hepatoxicity of 2-MF by OTZ. 

201 



PHS 6040 (Rev. 1/84) 



GPO 9l4>»le 



ZOl CM 07161-02 LETM 

Objectives : 

The toxic alkylfurans, namely, 3-niethylfuran (3-MF), a major constituent of 
atmospheric smog, and 2-methylfuran (2-MF), a natural product present in many 
foods, cigarette smoke and coffee, are activated by microsomal mono-oxygenase to 
reactive metabolites that bind covalently to microsomal proteins. Using 
semicarbazide as a trapping agent, acetyl acrolein (AA) and metylbutenedial 
(MBD) were isolated as the products of microsomal oxidation of 2-MF and 3-MF, 
respectively. A comparison of the covalent binding of •^H-3-MF and the amounts 
of methyl butenedial disemicarbazone produced in microsomal incubation in the 
presence and absence of NADPH and semicarbazide revealed an inverse relation- 
ship. Semicarbazide strongly inhibited the NADPH dependent covalent binding of 
the alkylfurans presumably by trapping the reactive aldehyde before it could 
react with tissue macromolecules ( Science , in press). 

Studies were designed to examine the metabolism and toxicity of 2-MF in vivo 
in rats and to correlate the toxicity with the observed covalent bindTng using 
time-course and dose-response relationship. The effect of various inducers 
and inhibitors of cytochrome P-450 metabolism on the toxicity and covalent 
binding of 2-MF was also to be examined. In view of the instability in vitro 
of the glutathione conjugate of the reactive metabolite of 2-MF, nameTy acetyl - 
acrolein, the effects of various inducers and depletors of glutathione on the 
toxicity of 2-MF was undertaken. 

Methods Employed : 

An original synthesis of 2-([l*C]methyl )furan was first developed using 2-furyl 
lithium and ([^^Clmethyl ) iodide. Male Sprague Dawley rats were injected with 
varying doses of ^^C-2-MF intraperitonially and were sacrificed 0.5 to 24 hr 
after dosage. Liver, lung, kidney, blood and muscle were monitored for total 
radioactivity, radioactivity extractable with methanol and radioactivity cova- 
lently bound to both protein and DNA. Glutathione levels were determined in 
liver, lungs and kidneys. Serum glutamic pyruvic transaminase levels were 
determined. Samples of liver, lung and kidney were fixed in formalin, sec- 
tioned and stained with hematoxylin and eosin and examined by light microscopy. 
Urine was collected after dosage with l^C-2-MF for isolation of mercapturic 
acid conjugates. 

Major Findings : 

When l*C-2-MF was administered in vivo to rats, large amounts of radioactivity 
were covalently bound to macromoTecules in the liver, lungs and kidney and 
smaller amounts were bound to tissues with little or no known amounts of mixed 
function oxidase activity. Maximal covalent binding to both protein and DNA 
was observed in the liver. Covalent binding to liver proteins was five times 
greater than kidney per mg protein. Damage to liver was observable biochem- 
ically by a rise in serum glutamic pyruvic transaminase levels and histopatho- 
logically by centri lobular necrosis of liver. Liver GSH levels decreased by 
33% half an hour after a dose of 2-MF indicating the formation of reactive 
electrophilic metabolites and the role of endogenous GSH as a protecting agent. 



202 



ZOl CM 07161-02 LETM 

Pretreatment with phenobarbital enhanced the toxicity of 2-MF. Over a 12 hr 
period there was an increased urinary excretion of 2-MF metabolites in rats 
pretreated with phenobarbital. Pretreatment of rats with phenobarbital also 
greatly enhanced the level of covalent binding of the label to proteins in 
liver, lungs and kidney. Covalent binding to liver proteins and DNA increased 
two-fold with phenobarbital pretreatment. Pretreatment with N-octyl imidazole 
(Wilkinson et al., 1976), a potent inhibitor of cytochrome P-450 markedly 
decreased the level of covalent binding of the label from l^C-2-MF to proteins 
and DNA in liver, lungs and kidney. N-octyl imidazole pretreatment completely 
ameliorated any increase in SGPT levels caused by 2-MF, pretreatment with 
N-octyl imidazole completely prevented the hepatotoxicity caused by 2-MF treat- 
ment. SGPT levels of rats treated with 3-methylcholanthrene or piperonyl 
butoxide prior to 2-MF administration were not different from rats that received 
2-MF alone. 

Prior dosing with diethylmaleate increased covalent binding of 2-MF metabolites 
in liver, lungs and kidney. However, pretreatment with buthionine sulfoximine 
(BSO), which depletes GSH by inhibiting GSH synthesis, decreased both toxicity 
and covalent binding of 2-MF metabolites in anamoly to the effects observed 
with diethylmaleate pretreatment. BSO is known to inhibit cysteine synthetase 
and increase the concentration of cysteine. In vitro , cysteine is a better 
inhibitor of covalent binding of 2-MF metabolites than GSH. Cysteine can form 
a cyclic conjugate with acetyacrolein which is more stable than the GSH-acetyl- 
acrolein conjugate. Thus, it seems that BSO inhibits covalent binding by 
trapping the reactive metabolites as cysteine conjugates. The effects of BSO 
pretreatment on 2-MF covalent binding is marked in the liver where there is a 
rapid turnover of GSH. Pretreatment with oxythiazolidine, an inducer of GSH, 
increased both toxicity and covalent binding of 2-MF metabolites in liver. But 
a combination of oxythiazolidine and BSO, which is known to nullify the effects 
of oxythiazolidine, decreased both toxicity and covalent binding significantly. 
The possible reason for the increased covalent binding observed with oxythi- 
azolidine could be that the increased GSH levels prevent the suicidal destruc- 
tion of cytochrome P-450 by the reactive metabolite of 2-MF by forming the 
GSH-conjugate. But, the GSH-acetyl acrolein conjugate is known to be unstable, 
hence, the acetyl acrolein released from the GSH conjugate could react with 
other proteins. Further, the acetyl acrolein-GSH conjugate can react with 
proteins by the virtue of its free aldehyde moiety. Thus, there is increased 
metabolism of 2-MF in the presence of increased levels of GSH, but due to the 
instability of the GSH conjugate it cannot effectively prevent the covalent 
binding of the reactive metabolite. 

Significance to Biomedical Research and the Program of the Institute : 

Identification of a - 6 unsaturated aldehydes as the major reactive metabolites 
of alkylfurans leads to a new class of reactive metabolites which can react 
specifically with tissue macromolecules in a number of ways. Unsaturated 
aldehydes can react with both proteins and DNA either via Michael addition 
across the activated double bond or nucleophilic addition to the aldehyde. In 
view of the bifunctional reactive sites interstrand crosslinks and protein and 
DNA crosslinks are possible, which may be important in view of the potential 
carcinogenicity and/or cytotoxicity effects of the toxic furans. The decreased 



203 



ZOl CM 07161-02 LETM 

covalent binding of 2-MF metabolites seen after pretreatment with BSO is also 
of importance in view of the role of BSO as a radiosensitizing agent in cancer 
chemotherapy particularly with alkylating agents. 

Proposed Course : 

The major objectives of this project are nearly complete and the manuscripts 
are in preparation. *^ 

Publications : 

1. Boyd M.R. Ravindranath. V., Burka, L.T., Dutcher, J.S., Franklin, R.B. 
Statham, C.N., Haschek, W.M.. Hakkinen. P.J., Morse, C.C. and Witschi 
H.P.: Drug metabolizing enzyme systems and their relationship to toxic 
mechanisms. In: Proceedings of New Approaches in T oxicity Testing and 
their Application to^Human Risk Assessment , in press. 



204 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07162-02 LETM 



PERIOD COVERED 



October 1, 1984 to September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Role of Arachidom'c Acid Metabolism in Human Lung Cancer 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



PI: 


W. 


C. 


Hubbard 


Others: 


E. 


G. 


Mimnaugh 




J. 


B. 


McMahon 




H. 


M. 


Schuller 




S. 


S. 


Lau 




M. 


R. 


Boyd 




M. 


G. 


McMenamin 




K. 


E. 


Greene 



Cancer Expert 

Chemist 

Cancer Expert 

Vet. Med. Officer 

Sr. Staff Fellow 

Associate Director 

Chemist 

Bio. Lab. Tech. 



LETM, NCI 

LETM, NCI 

LETM, NCI 

LETM, NCI 

LETM, NCI 
DTP, NCI 

LETM, NCI 

LETM. NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI. NIH. Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

1.0 



PROFESSIONAL: 



0.7 



0.3 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
n (a2) Interviews 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The metabolism of arachidonic acid (AA) has been determined in four lines. The 
cell lines NCI-H69 and NCI-H128 are derived from small cell carcinomas of the lung 
(SCCL), and the cell lines NCI-H322 and NCI-H358 are derived from pulmonary 
adenocarcinomas. AA did not undergo metabolism when incubated with suspensions 
of NCI-H69 and NCI-H128 cells. Significant metabolism did occur when AA was 
incubated with monolayers of unstimulated NCI-H322 and NCI-H358 cells. A single 
metabolite of AA isolated from these cells was identified as prostaglandin E2 
(PGE2) via combined gas chromatography-mass spectrometry (GC-MS). Additional 
details of arachi donate metabolism in monolayers of these cells are contained 
in a separate report (see No. 07173-01). The preferential synthesis of prosta- 
glandins by certain types of human lung carcinomas may have direct clinical 
implications. Elevated levels of production of selected prostanoids in patients 
could be indicative of the presence of certain cell types of lung carcinomas. 
The selective synthesis of prostanoids by certain human lung carcinomas may also 
confer significant capabilities for P-450 monooxygenase-independent metabolism 
of antitumor agents and other xenobiotics. The possibility that prostaglandin 
synthesis may be a unique feature of certain types of human lung carcinomas and 
the role of the prostaglandin endoperoxide synthase (PES) system in the metab- 
olism of antitumor agents and other xenobiotics are under investigation. 



205 



PHS 6040 (Rev. 1/84) 



GPO 9I4-9I8 



ZOl CM 07162-02 LETM 
Methods Employed : 

GC/MS analysis: PGE2 was identified as the major metabolite of arachidonic 
acid synthesized by the NCI-H322 and NCI-H358 cells via negative ion chemical 
ionization (NICI) GC-MS analysis. Aliquots of 200 ul of the HBSS removed after 
incubation with the NCI-H358 and NCI-H322 cells were placed in a vial containing 
ten nanograms of 3,3,4,4-tetradeutero (d4) prostaglandin E^. The pH of the 
HBSS was titrated to pH 3.2 followed by extraction with diethyl ether. The 
ether extract was transferred to a 1.0 ml conical vial and derivatized to the 
methyl oxime-pentafluorobenzyl-bistrimethyl si lyl ether (MO-PFBE-TMS) derivative. 
PGE2 was identified via monitoring the characteristic fragment ions m/z 524 
iroJ^x^J^^ ^^^^^ ^""^ ""/^ ^2^ (d4-PGE2), eluting at the retention time of the MO- 
PFBE-TMS derivative of PGE2 and the distribution of the ion current profiles 
and total ion current into syn- and anti-isomers characteristic of PGEo. 
Salient features of employing NICI GC-MS analysis are presented below (see 
Proposed Course of Studies). 

Characterization of the PES system: Characterization of the PES system requires 
frequent sampling of the incubation medium for determination of the quantities 
of PES products formed. In order to eliminate perturbations in temperature 
and gas equilibrium as a variable in these studies, we are presently adapting 
our procedures for the proliferation of different human lung carcinoma cells 
attached to microcarriers. 

In addition to adaptations of our culture methods for the proliferation of 
human lung carcinoma cells, we have investigated the fatty acid cyclooxygenase 
metabolism of four polyunsaturated fatty acids in monolayers of NCI-H358 cells 
These four fatty acids (linoleic, eicosatrienoic, eicosapentaenoic and docosa- 
hexaenoic acids) are significant lipid constituents of the culture medium in 
which the lung carcinoma cells are maintained in vitro and could contribute to 
the PES activity of human lung carcinoma cells. 

Major Findings : 

Studies of the metabolism of linoleic, eicosapentaenoic and docosahexaenoic 
acids in monolayers of NCI-H358 cells indicate that these polyunsaturated 
fatty acids are not metabolized by the PES system in these cells. Eicosatri- 
enoic acid undergoes oxygenation in the presence of the NCI-H358 cells, presum- 
ably to PGEi. The quantiative relationship between PGEi and PGE2 synthesis 
from endogenous fatty acid precursors is under investigation. The adaptation 
of our cell culture procedures have proven successful in the proliferation of 
the NCI-H358 cell line attached to microcarriers. 

Proposed Course : 

The future course of these studies can be subdivided into two major areas. 
First, the kinetics of PES metabolism of arachidonic acid and other polyunsat- 
urated fatty acids will be determined in cell lines NCI-H322 and NCI-H358. 
These studies will allow us to determine total PES activity of these human 
lung carcinoma cells and the portion of PES activity that is represented by 
PGE2 production in the different cell lines. These studies will require 
maximizing our yield of cells proliferated attached to microcarriers. Second, 

206 



ZOl CM 07162-02 LETM 

the synthesis of PGE2 and other eicosanoids in primary cultures of human lung 
carcinomas and prostanoid production in selected human lung cancer patients 
will be determined. An essential component of the latter proposal will include 
the classification of human lung carcinomas upon the basis of ultrastructural 
features. These latter studies will allow us to determine whether prostaglandin 
synthesis is a unique characteristic of certain types of human lung carcinomas. 

In order to determine the role of PGE2 and related eicosanoids in the patho- 
physiology of human lung cancer, it is essential to have the analytical capa- 
bilities for the identification and quantitation of prostaglandins and related 
eicosanoids. These compounds are normally present in body fluids in subnano- 
gram quantities. Three methods for quantitation of prostaglandins and related 
eicosanoids have been developed. These are: 1) bioassay, 2) radioimmunoassay 
(RIA) and 3) combined gas chromatography-mass spectrometry (GCMS). Of these 
three methods of quantitation of these compounds, GC-MS analysis is the most 
selective. When negative ion chemical ionization of electron-capture deriva- 
tives of prostaglandins and related eicosanoids is employed, mass spectrometry 
is also the most sensitive method for their quantitation with amounts of less 
than 100 femtograms being reliably quantitated. Thus, mass spectrometry is 
recognized as the analytical method with which other methods of quantitation 
are compared. 

Four quadrupole mass spectrometer systems with a capillary gas chromatograph 
interface and operable in the negative ion detection mode were evaluated for 
sensitivity in the detection and quantitation of electron-capture derivatives of 
prostanoids. After extensive evaluation of each of the four instruments for 
sensitivity in the measurement of prostaglandin standards, it was determined 
that the Finnigan MAT 4610B system was 50-200 times more sensitive than the 
other instruments at the time of the evaluations. Thus, a recommendation for 
purchase of the Finnigan MAT 4610B system was made. This instrument is 
presently on order from Finnigan MAT (Requisition No. 196225; Contract No. 
263-85 C-0089) with an anticipated shipment date from the manufacturer of May 
31, 1985. 

The GC/MS system will be employed for quantitation of prostanoids synthesized 
by human lung carcinoma cells and for the assessment of prostanoid production 
in selected human lung cancer patients. The methods for quantitation of PGE2, 
other primary prostaglandins and related eicosanoids are similar to those 
described above (see Methods Employed). 

Because of the rapid metabolism of the prostaglandins and related eicosanoids 
and their formation in cellular elements ex vivo , direct measurement of 
eicosanoids in either plasma or serum does not provide a reliable index 
of the production of prostanoids in vivo . For example, it has been shown that 
the most reliable index of PGE2 production in vivo is via measurement of either 
a circulating metabolite or a urinary metabolite of PGE2. PGE2 is transformed 
by 15-dehydrogenation and A^-^.^gduction to 15-keto-13,14-dihydro-PGE (15K- 
H2-PGE2). Further degradation of I5K-H2-PGE2 via two beta-oxidative steps and 
omega-oxidation yields (-)-7a-hydroxy-5,ll-diketotetranoprostane-l,16-dioic 
acid, a compound more frequently referred to as PGE-M. PGE-M is the most abun- 
dant characteristic metabolite of PGE2 excreted in urine of several species 
including humans. We propose to employ the excretion rate of PGE-M as an index 



207 



ZOl CM 07162-02 LETM 

Of PGE^ production in patients with lung carcinomas. Similar approaches of 
analysis will be employed for the assessment of the in vivo synthesis of other 
prostanoids if appropriate. 



208 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07163-02 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line Ixtween the borders.) 

Xenobiotic Metabolism by Prostaglandin Endoperoxide Synthetase (PES) 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: S. S. Lau Sr. Staff Fellow LETM, NCI 



Others: J. B. McMahon 
M. R. Boyd 
M. G. McMenamin 
K. E. Greene 



Cancer Expert 
Associate Director 
Biologist 
Bio. Lab. Tech. 



LETM, NCI 
DTP, NCI 
LETM, NCI 
LETM, NCI 



COOPERATING UNITS (H any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacol ogy and Toxicology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

0.3 



PROFESSIONAL: 
0.6 



0.3 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The site of toxicity of a number of xenobiotics and antitumor agents requiring 
metabolic activation is distributed in tissues containing very little P-450 mono- 
oxygenase activity. Because of the ubiquitous distribution of the prostaglandin 
endoperoxide synthase (PES) system and the ability of this system to catalyze the 
cooxidation of xenobiotics to carcinogenic, mutagenic or other reactive species, 
it has been implicated as playing a significant role in the metabolism of xenobio- 
tics. We have initiated studies on the metabolism of a variety of compounds by 
ram seminal vesicle (RSV) microsomes (a rich source of PES). These compounds 
include: a) anticancer agents, such as NMF, Cytoxan, PCNU, AZQ, caracemide 
and mitoxantrone; b) a pulmonary toxin, ipomeanol; and c) nephrotoxins such as 
o-bromophenol and 2-bromohydroquinone. During prostaglandin biosynthesis, mito- 
xantrone, o-bromophenol and 2-bromohydroquinone were cooxidized to reactive metab- 
olites which then bound to RSV microsomal protein. Furthermore, two of the human 
lung carcinoma cell lines containing high PES activity are susceptible to mito- 
xantrone induced cytotoxicity. The degree of cytotoxicity induced by mitoxantrone 
correlates with the PES activity in these two cell lines. 

The mechanism(s) of PES-mediated cooxidation and toxicity of antitumor agents and 
other xenobiotics in human lung carcinoma-derived cells are currently under inves- 
tigation. The utilization of homogenous human lung carcinoma cells systems for 
systematic screening of anticancer drugs directed at the PES system may provide 
hope for achieving target cell mediated antineoplastic effectiveness in lung 
cancer therapy. 



209 



PHS 6040 (Rev. 1/84) 



GPO 9l4>eiS 



ZOl CM 07163-02 LETM 

Objectives : 

The importance of bioactivation of xenobiotics to toxic intermediates is well 
recognized. These bioactivations have been shown to be critical for the expres- 
sion of the cytotoxic and/or carcinogenic potential of many compounds. Work 
from several laboratories indicates that a variety of compounds undergo oxida- 
tive metabolism during prostaglandin biosynthesis in vitro (Marnett and Elnig, 
Rev. Biochem. Toxicol . 5: 135, 1983). The term cooxidation has been applied to 
the description of PES catalyzed metabolism of xenobiotics. Among the compounds 
oxidized are chemical carcinogens that require oxidative activation to express 
their carcinogenic potential such as polycyclic aromatic hydrocarbons, aromatic 
amines, aflatoxin and nitrofurans. Because PES is widely distributed in mam- 
malian tissues, further investigation is required to determine whether the PES 
system can serve as an important oxidative pathway in the metabolism of xeno- 
biotics especially in tissues that have a low content of cytochrome P-450. Due 
to the lack of availability of homogeneous cell populations rich in the PES 
system, the vast majority of studies investigating the role of cooxidation as a 
pathway of bioactivation have been conducted in subcellular tissue preparations. 
In the past year, we have characterized several human lung carcinoma cell lines 
which differ extensively in their PES activity (see Annual Reports ZOl 07173-01 
LETM and ZOl CM 07174-01 LETM). The selected human lung carcinoma cell lines 
for this study have varying PES activities listed in ascending order: NCI-H69, 
NCI-H128, NCI-H322, NCI-H358 and NCI-H460 (see Annual Reports ZOl CM 07173-01 
LETM and ZOl CM 07174-01 LETM). Both NCI-H69 and NCI-H128 are non-small cell 
carcinoma lines, NCI-H322 and NCI-H358 are well differentiated adenocarcinomas, 
and NCI-H460 is derived from a large cell carcinoma. These human lung car- 
cinoma cell lines were employed as models to explore the ability of the dif- 
ferent human lung cancers to activate xenobiotics, especially antitumor agents, 
directed against the PES system in the hope of achieving a more selective 
therapeutic effectiveness. 

Methods Employed : 

Chemical . Anticancer agents employed in this study included NMF (NSC 3051), 
Cytoxan (NSC 26271), PCNU (NSC 95466), AZQ (NSC 182986), Caracemide (NSC 
253272) and mitoxantrone (NSC 301739). Other compound used were ipomeanol, 
o-bromophenol and 2-bromohydroquinone. 

Preparation of ram seminal vesicle (RSV) microsomes . Frozen ram seminal vesicu- 
lar glands were thawed slowly at 4°C overnight. The tissue was minced and homo- 
genized with a Potter-El vehjem type homogenizer in 4 volumes (w/v) of 0.1 M 
phosphate buffer containing 1 mM EDTA, pH 7.8. The homogenates were centri- 
fuged at 15,000 g for 20 min and the supernatant was centrifuged at 100,000 g 
for 1 hr to harvest the microsomes. 



Enzymatic incubation with ram seminal vesicle microsomes . Various ^^C-labeled 
substrates, NMF, cytoxap, PCNU, AZQ, ceracemide, mitoxatrone, (at a conc6 
:ion of 15 yM, 1.5 x 10^ dpm/nmole), -^H-ipomeanol , ^*C-o-bromophenol and 



substrates, NMF, cytoxap, PCNU, AZQ, ceracemide, mitoxatrone, (at a concentra- 
tion of 15 yM, 1.5 X 10^ dpm/nmole), -^H-ipomeanol , ^*C-o-bromophenol and 
'*C-2-bromohydroquinone (at a concentration of 100 yM, 2200 dpm/nmole), were 



incubated with 0.08 mM arachidonic acid, 2 mg of RSV microsomes, 0.1 M phosphate 
buffer, 1 mM EDTA, pH 7.4 in a final volume of 1.5 ml at 37''C for 10 min. 
Incubations were terminated by transferring the mixture into 1 ml of 10% TCA. 



210 



ZOl CM 07163-02 LETM 

The covalent binding of the radio-labeled reactive metabolites of the above 
substrates to RSV microsomal protein was determined. 

Cytotoxicity study in human lung carcinoma cell lines . Human non-small cell 
carcinoma cell lines, NCI-H322, NCI-H358 and NCI-H460 were grown as mono- 
layers in 35 cm^ disk. The small cell carcinoma cell lines, NCI-H69 and 
NCI -128 were grown as suspension cultures. The culture medium, RPMI 1640 was 
removed from cell suspensions and monolayers and the cells washed 2 times with 
5 ml of the same medium. Mitoxantrone, in concentrations ranging from 0.25- 
1.0 nW was added to the wells and the final volume was adjusted to 2 ml with 
RPMI 1640. The cell mixtures was incubated at 37 "C in an atmosphere of 95% 
air/5% COg for 6 hr. At each hour, 0.1 or 0.2 ml of the incubation medium was 
removed for LDH leakage determination. 

Major Findings : 

It has been demonstrated that certain types of reactions can be catalyzed by 
PES. These include N- and 0-demethylation, epoxidation, S- and N-oxidation, 
C-hydroxy-lation and dehydrogenation (Marnet and Eling, Rev. Biochem. Toxicol . 
5: 135, 1983). In the search for appropriate substrates for the PES system, a 
variety of compounds, different in chemical structure, were incubated with RSV 
microsomes (which contain high levels of PES). Of all the compounds tested 
(see above "Chemicals" section), only three showed significant covalent binding. 
These were mitoxantrone, o-bromophenol and 2-bromohydroquinone (specific activi- 
ties: 8.8 ± 1.3, 21.8 ± 3.7, and 46.4 ± 8.4 nmole/mg protein/lO min, respec- 
tively). These results suggest that PES may play a role in the activation of 
mitoxantrone (an anticancer agent), o-bromophenol and 2-bromohydroquinone (both 
nephrotoxins) to reactive species. 

Toxicity Study . When NCI-H322 and NCI-H358 cells were exposed to mitoxantrone 
in vitro , a time and dose dependent cytotoxicity measuring leakage of the cyto- 
solic enzyme LDH was evident. Furthermore, the NCI-H358 cell line which posses- 
ses higher PES enzyme activity (2-fold) than the NCI-H322 cell line is more 
susceptible to the cytotoxic effect of mitoxantrone. This effect was both dose 
and time dependent. For example, 6 hr from the beginning of exposure to 1.0 mM 
of drug, >66% of LDH leakage in NCI-H358 cells and >38% of LDH leakage in 
NCI-H322 cells were observed. Furthermore, the dosage required to produce a 
similar LDH leakage over a period of 6 hr was twice as high for NCI-H322 cells 
as for NCI-H35a cells. Although the mechanism of cytotoxicity produced by 
mitoxantrone in these cell lines is not clear, these results suggest a correl- 
ation between the PES activity and the cytotoxicity of mitbxantrone. Appro- 
priate inhibitors, such as aspirin, indomethacin, propylthiouracil, and stimu- 
lators such as calcium ionophore A23187 will also be used to further examine 
the possible role of PES in bioactivating mitoxantrone to its cytotoxic species. 

Proposed Course : 

Studies are planned to continue the characterization of the role of PES in 
xenobiotic metabolism, especially in the human lung carcinoma cell lines. The 
metabolism and the cytotoxic effects of mitoxantrone, o-bromophenol and 2-bromo- 
hydroquinone in NCI-H322 and NCI-H358 cells as well as in other human lung 
carcinoma cell lines including NCI-H460 (very high in PES activity), NCI-H69 

211 



ZOl CM 07163-02 LETM 

and NCI-H128 (both with negligible PES activity) will be examined. The 
effects of PES inhibitors on the metabolism and cytotoxicity of mitoxantrone, 
o-bromophenol , and 2-bromohydroquinone, as well as the mechanisms of toxicity 
induced by these agents in these human lung carcinoma cell lines will be 
investigated. 

Significance to Biomedical Research and the Program of the Institute : 

We have demonstrated that the PES system isolated from ram seminal vesicles is 
capable of activating the anticancer agent, mitoxantrone and the nephrotoxins, 
o-bromophenol and 2-bromohydroquinone to covalently bound material during 
prostaglandin biosynthesis. Furthermore, two of the human lung carcinoma cell 
lines containing high PES activity are susceptible to mitoxantrone induced 
cytotoxicity. A systematic screening of anticancer drugs as well as model 
compounds directed at the PES system may provide the hope of achieving more 
selective therapeutic effectiveness in lung cancer. 



212 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07168-01 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line befiveen the borders.) 



Biochemical Toxicology of the Nitrosoureas and the Thiol Modulating Agent, BSO 



PRINCIPAL INVESTIGATOR (List other professional personnel tjelow the Principal investigator) (Name, title, laboratory, and Institute affiliation) 

P. I.: R. A. Kramer Staff Fellow LETM, NCI 



Others: M. R. Boyd 
D. J. Reed 
H. M. Schuller 
J. H. Dees 
M. 6. McMenamin 
K. E. Greene 



Associate Director 

IPA 

Vet. Med. Officer 

Cancer Expert 

Biologist 

Bio. Lab. Tech. 



DTP, NCI 
LETM, NCI 
LETM, NCI 
LETM, NCI 
LETM, NCI 
LETM, NCI 



COOPERATING UNITS (If any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

1.0 



PROFESSIONAL: 

0.5 



0.5 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues B (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The objective of this project is to understand the biochemical mechanisms for the 
target organ toxicity of anticancer drugs. These studies have focused on two main 
areas of research: 1) the first project deals with the mechanism for the renal 
toxicity of MeCCNU and related nitrosoureas (i.e., CCNU, BCNU, chlorozotocin); and 
2) the second study involves investigations on the use of buthionine sulfoximine 
as a thiol modulating agent in cancer chemotherapy. 

Studies completed this year have demonstrated that: a) carbamylati on-mediated 
reactions may not be necessary for nephrotoxicity to develop following the ad- 
ministration of nitrosourea anticancer agents; b) the alkylation of kidney DNA 
may be an important cellular target for nitrosourea nephrotoxicity; c) gluta- 
thione plays an important protective role against MeCCNU-induced renal toxicity; 
d) hepatic drug metabolizing enzymes can activate MeCCNU and CCNU, but not PCNU, 
to more polar and more reactive alkylating intermediates which can bind macro- 
molecules in vitro ; e) a liver- derived metabolite of MeCCNU may be responsible 
for the reTial toxicity of the parent nitrosourea; f) in vivo administration of 
buthionine sulfoximine markedly reduced normal tissue, and L-PAM resistant LI 210 
tumor glutathione levels following a single dose of BSO, or after a multiple 
dosing regimen; g) of the normal tissues, the kidney was the most sensitive, and 
the bone marrow the most resistant, to thiol depletion by BSO; h) BSO increased 
the renal and hepatic toxicity of MeCCNU in F344 rats and increased the lethality 
of L-PAM in BDF mice. 



213 



PHS 6040 (Rev. 1/84) 



spo st4*«te 



ZOl CM 07168-01 LETM 
Objectives : 

1. Utilize the knowledge gained from an understanding of the biochemical 
mechanisms for the target organ toxicity of anticancer agents to aid in the 
rational design of better, less toxic, chemotherapeutic agents. 

2. Develop improved methods for predicting, monitoring or treating such toxic 
reactions in patients. 

3. Develop strategies for targeting tumor tissue, and minimizing host toxicity, 
to anticancer agents by the selective manipulation of tissue GSH levels. 

I. Nephrotoxicity of the Nitrosoureas . Nephrotoxicity severely limits the 
therapeutic usefulness of a number of anticancer drugs (e.g., nitrosoureas, cis- 
platinum, mitomycin C). Renal toxicity was noted in preclinical studies for 
virtually all the nitrosoureas in clinical use. However, only recently have 
certain of these agents (i.e., MeCCNU) been shown to result in a delayed, 
irreversible and often fatal renal toxicity. The goals of this project have 
been to: a) develop an in vivo model for the study of nitrosourea nephro- 
toxicity (R.A. Kramer and M.R. Boyd, J. Pharmacol. Exp. Ther. 22: 409, 1983; 
ZOl CM 07139-02 LETM); and to b) elucidate the biochemical mechanisms for the 
renal toxicity of this class of antiatumor drugs (ZOl CM 07138-02 LETM). 

Major Findings ; 

Nephrotoxicity of the Chi oroethyl nitrosoureas is Independent of Carbamylat- 
ing activity . Most chl oroethyl nitrosoureas can degrade in solution to form 
carbonium ion alkylating agents in addition to isocyanates that are capable of 
carbamylation reactions. Whereas, antitumor activity is believed to be due to 
the aklylation of nucleophilic sites in DNA, carbamylation reactions have been 
implicated in mediating the host toxicity of the parent nitrosourea. Chlorozo- 
tocin is a glucopyranosyl conjugated chl oroethyl nitrosourea that represents 
the first clinically studied analog designed for low carbamylating activity. 
At equimolar doses, chlorozotocin was found to be a much more potent nephro- 
toxicant than MeCCNU. At low doses, both drugs produced a similar chronic and 
progressive nephropathy which was delayed in onset and resulted in karyomegaly 
to the collecting ducts of the renal medulla. 

DNA Alkylation and Nephrotoxicity of the Chl oroethyl nitrosoureas . Renal DNA 
was found to be the major target for reactive alkylating intermediates derived 
from MeCCNU. Approximately 30% of the total drug in kidney was covalently 
bound to DNA; this compares to only 7% being protein bound. Kidney DNA bound 
2.5- and 30-fold more MeCCNU than in the liver or the lung, respectively. The 
administration of MeCCNU resulted in an increase in kidney DNA (yg DNA/g 
kidney) which was proportional to the initial levels of drug bound covalently 
to DNA. Moreover, treatment with MeCCNU, or chlorozotocin, produced extensive 
karyomegaly to the collecting ducts of the renal medulla. These studies suggest 
that it may not be possible to dissociate the antitumor activity of the nitroso- 
ureas (i.e., DNA alkylation), from their potential for nephrotoxicity. 

Protective Role of GSH Against MeCCNU-induced Renal Toxicity . Treatment of 
F344 rats with MeCCNU resulted in a time- and dose-related decrease of GSH 



214 



ZOl CM 07168-01 LETM 



preferentially in liver. Administration of MeCCNU to rats pretreated with 
buthionine sulfoximine (a selective inhibitor of glutathione biosynthesis) 
resulted in: a depletion of hepatic and renal GSH which was additive to the 
effects of either of these treatments alone; an increase in the concentration 
of reactive intermediates of MeCCNU which became irreversibly bound to renal 
and hepatic macromolecules; increased the nephrotoxicity of MeCCNU, and caused 
hepatotoxicity that was not ordinarily seen when MeCCNU was administered alone. 
MeCCNU was not found to effect glutathione reductase activity, or the ratio of 
GSH/GSSH, in any tissue tested. This differs considerably from the pneumotoxic 
nitrosourea, BCNU, which selectively effects these parameters within its target 
organ, the lung. 

Metabolic Activation of MeCCNU . MeCCNU was shown to be metabolized by the 
hepatic monooxygenase system to reactive alkylating intermediates which bound 
irreversibly to macromolecules in vitro . This reaction met the criteria for a 
cytochrome P-450 dependent reaction, however, kidney microsomes were not found 
to catalyze this alkylation reaction. CCNU, but not PCNU, also was found to be 
activated by the liver monooxygenase system. Phenobarbital pretreatment 
resulted in a 20-fold increase in the alkylation of microsomal protein by 
MeCCNU; the alkylating metabolite accounting for up to 25% of the total metab- 
olite profile of MeCCNU. In contrast, the carbamylating activity of MeCCNU was 
not significantly affected by the monooxygenase system. Liver microsomes were 
found to metabolize MeCCNU to a metabolite that was toxic to renal slices in 
vitro (i.e., renal slice organic ion transport; see Annual Report ZOl CM 07T39- 
01 LETM). Addition of GSH to this in vitro reaction protected against the 
metabolite-dependent decrease in renal slice organic ion uptake and completely 
inhibited covalent binding to liver microsomes. GSH and other soluble nucleo- 
philes (N-acetyl cysteine, cysteine) also were found to inhibit covalent binding, 
and three N-acetylcysteine-MeCCNU metabolite adducts have been separated by 
HPLC. 

Relationship Between Hepatic Metabolism and the Renal Toxicity of MeCCNU . In 
vivo studies demonstrated that the same enzyme systems involved with the 
metabolic activation of MeCCNU in vitro , may be involved in the metabolic 
activation and renal toxicity oT~MeCCNU in vivo . Inducers and inhibitors of 
these enzymes markedly altered both the in vivo covalent binding and the renal 
toxicity of MeCCNU. Pretreatment with pTienobarbital , an inducer of the 
monooxygenase system in liver but not in kidney, increased both the alkylation 
and toxicity of MeCCNU in the kidney. Moreover, an in yiyo/ in vitro toxicity 
assay demonstrated the presence of a metabolite in the bile o? a MeCCNU treated 
rat which was toxic to cells in vitro . 

Chloroethyl-GSH Adducts . The exact chemical nature of the putative liver- 
derived nephrotoxic metabolite is not known. However, it is possible that 
hepatic metabolism of MeCCNU could lead to the formation of a 2-chloroethyl-GSH 
adduct (see Annual Report ZOl CM 07159-01 LETM). Studies initiated this year 
have shown that 2-chloroethyl-GSH is a potent nephrotoxicant, presumably via 
the formation of a reactive episulfonium ion. Moreover, we have found that 
this reactive GSH adduct may act by a different mechanism than other known 
nephrotoxic GSH-adducts (e.g., dichlorovinyl-GSH; bromohydroquinone-GSH). 
These compounds are dependent upon the enzyme y-glutamyl transpeptidase (y-GT) 

215 



Z01 CM 07168-01 LETM 

for transport into proximal tubule cells where they ultimately exert their 
toxicity. In contrast, 2-chloroethyl-GSH was nephrotoxic by a y ST indepen- 
dent mechanism, providing evidence that the kidney can process GSH-adducts to 
form their corresponding mercapturic acids by more than one mechanism. 

II. Thiol Modulation . GSH plays a crucial role in the cellular protection 
against a variety of toxicologic insults, including reactive oxygen intermedi- 
ates, free radicals, lipid peroxides and reactive electrophiles. The overall 
goal of this project is to develop strategies that would maximize thiol deple- 
tion in tumor tissue, and minimize the possible host toxicity resulting from 
combination therapies involving thiol depletion. These studies have initially 
focused on buthionine sulfoximine (BSO), a selective inhibitor of the rate- 
limiting enzyme in GSH biosynthesis (i.e., y-glutamyl cysteine synthetase). 

Major Findings : 

1. Adminisration of BSO to F344 rats or BDF mice dramatically reduced tissue 
GSH concentrations. 

2. Large differences were observed between tisues with respect to the rate of 
GSH depletion and recovery, and the maximum level of thiol depletion obtained 
following a single dose of BSO. In this regard, the kidney was the most sen- 
sitive organ for thiol depletion by BSO (20% of control), and the bone marrow 
was the most resistant (85% of control). 

3. In vivo administration of a single dose of BSO led to a 75% decrease in GSH 
levels in L-PAM resistant L1210 in BDF mice. Multiple dosing regimens of BSO 
led to an even further decrease in tissue and tumor GSH levels. Tumor GSH 
levels being the most depleted following multiple doses of BSO (lO-fold de- 
crease). 

4. BSO pretreatment increased the renal and hepatic toxicity of MeCCNU in F344 
rats and increased the lethality of L-PAM in BDF mice. 

Publications : 

1. Kramer, R.A., Schuller, H.M., Smith, A.C. and Boyd, M.R.: Effects of 
buthionine sulfoximine on the nephrotoxicity of l-(2-chloroethyl )-3-(trans- 
4-methylcyclohexyl)-l-nitrosourea (MeCCNU). J. Pharmacol. Exp. Ther. , in 
press. 

2. Kramer, R.A., McMenamin, M.G. and Boyd, M.R.: Differential distribution 
and covalent binding of two labeled forms of l-(2-chloroethyl )-3-(trans-4- 
methylcyclohexyl )-l-nitrosourea (Methyl CCNU) in the Fischer 344 rat. 
Cancer Chemother. Pharmacol . 14: 150-155, 1985. 

3. Kramer, R.A., McMenamin, M.G. and Boyd, M.R.: Mechanism of chloroethyl- 
nitrosourea nephrotoxicity: Studies with MeCCNU. In Bach, P.H. and Lock, 
E.A. (Eds.): Renal Heterogeneity and Target Cell Toxicity . John Wiley & 
Sons, in press. 



216 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07169-01 LETM 



PERIOD COVERED 

October 1, 1984 to September 30. 1985 

TITLE OF PROJECT (80 characters or less. TWe must tit on one line behveen the borders.) 



Tetraolatin Toxicity: Comparative Nephrotoxicity of Platinum Antic ancer Agents 

1- :* : : . . ........ .L- n^ > • i-..._>i««>,.r l lUam^ H»a lahnratnrv anrl InstllUtB affiliation) 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, me. laboratory, and Institute affiliation) 

P. I.: J. H. Smith PRAT Staff Fellow NIGMS 

Others: M.A. Smith Staff Fellow LETM. NCI 
C. L. Litterst Pharmacologist LPEi. nli 
J. Uozumi Visiting Fellow LPET. NCI 
S. J. Lovett . Bio. Lab. Aid LETM. NCI 
T. MacLain Bio. Lab. Tech. LPET, NCI 
M. R. Boyd Associate Director DTP, NCI 

COOPERATING UNITS (if any) 

U.S. Environmental Protection Agency (M. P. Copley) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics & Metabolism 

SECTION 

Pharmacology and Toxicology Section 

INSTITUTE AND LOCATION 

NCI. NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 

1.0 



PROFESSIONAL: 

0.5 



OTHER: 

0.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects D (b) Human tissues H (c) Neither 

n (a1) Minors 
n (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Tetraplatin (NSC 363812) is a new anticancer platinum drug analog targeted for 
clinical development because of its effectiveness against cisplatin-resistant 
tumor cell lines and its improved formulation. The toxicity of tetraplatin was 
assessed in Fischer 344 rats and compared to equimolar doses of cisplatin and 
CHIP (NSC 256927). This emphasis of this investigation was the comparison of the 
relative nephrotoxicity of these platinum analogs and the relative sensitivity of 
various tests used to monitor renal function. At equimolar doses tetraplatin was 
less nephrotoxic than cisplatin. Furthermore, the characteristics of tetraplatin 
nephrotoxicity differed markedly from cisplatin. Most notably, a severe diuresis 
occurred at the dose of tetraplatin where indications of nephrotoxicity were first 
detected. In contrast to cisplatin. effects on proximal tubular cell function 
assessed in renal cortical slices were less severe. Histologically, the tetra- 
platin lesion extended throughout the renal cortex and no frank tubular necrosis 
was observed. Cisplatin lesions were characteristically localized in the cortico- 
medullary junction and were more severe. Urinary excretion of proteins, glucose 
and enzymes was the most sensitive indicator for the onset of nephrotoxicity. 
BUN and serum creatinine were not elevated until the renal damage was more severe 
at higher doses. There appear to be other differences between the toxicity ot 
tetraplatin and cisplatin relating to hematologic disorders and gastrointestinal 
toxicity. 



217 



GPO 914-aiS 

PHS 6040 (Rev. 1/84) 



ZOl CM 07169-01 LETM 
Objectives : 

Nephrotoxicity is the major dose-limiting factor for the therapeutic usefulness 
of cisplatin. A multitude of cisplatin analogs have been evaluated for 
effectiveness against cisplatin-resistant tumor cell lines and for improved 
formulation (purity, solubility, and stability). Tetrachloro(d,l-trans)l,2- 
diaminocyclohexane platinum (IV) (NSC 363812, Tetraplatin) was recommended for 
development to clinical trial by the Platinum Analog Working Group at the March 
20, 1984 meeting of the Decision Network Committee. It was recommended that 
the nephrotoxicity of tetraplatin be evaluated before passing this drug to the 
Decision Point 2B Status. 

The purpose of this investigation was to evaluate the nephrotoxic potential of 
tetraplatin. As a basis for the comparison, equimolar doses of cisplatin and 
CHIP (NSC 256927) were compared to tetraplatin in the Fisher 344 rat. A second 
goal of this investigation was to evaluate the sensitivity of noninvasive 
parameters of renal function and toxicity in order to better monitor for the 
onset of nephrotoxicity in the clinic. 

Methods : 

Adult male Fischer 344 rats (10 wk) received a single, i.v. bolus injection of 
saline, cisplatin, CHIP or tetraplatin in a volume of 4 ml/kg at equimolar 
doses of drugs. The doses were 6.7, 13.4, 26.7 or 53.3 pmol/kg and were equiv- 
alent to 2, 4, 8 and 16 mg/kg of cisplatin, respectively. Injections were 
made between 0800 to 0930 hr. Rats were killed on days 1, 3, 5, 8 and 15 for 
assessment of toxicity. There were 4 replicate rats per dose and time point. 
The emphasis of this investigation was the comparison of the relative nephro- 
toxicity of platinum analogs and the relative sensitivity of various tests 
used to monitor renal function. Therefore, rats to be killed on day 15 were 
housed in stainless steel metabolism cages and urine was collected daily on 
ice. Urine was analyzed for total daily urine volume, osmolality, pH, protein, 
Pt concentration and urinary enzymes. Lactate dehydrogenase (LDH) excretion 
was measured as an example of a cytosolic enzyme and N-acetyl-3-glucosamindase 
(NAG) excretion was measured as a representative lysosomal enzyme. Enzyme 
excretions monitored as representatives of brush border damage (and thus 
proximal tubular damage) included alanine aminopeptidase (AAP), alkaline phos- 
phatase (AP), Y-glutamyl transpeptidase (y-GT) and maltase. In addition, the 
following parameters were assessed in rats killed on day 1, 3, 5, 8 and 15. 

1 - The change of body weight from the day of injection. 

2 - Blood was collected for the vena cava for: 

a - complete blood counts on whole blood; and for 

b - serum analysis of urea (BUN), creatinine, electrolytes, (Ca^"*", 
Cl~, K"*", Mg^"*", Na"*") and total Pt concentration. 

3 - An aliquot of urine was likewise analyzed for electrolytes. 

4 - Kidneys were analyzed for Pt and total glutathione concentration. 



218 



ZOl CM 07169-01 LETM 

Renal cortical slices were prepared for in vitro assessment of proximal 
tubular function which included organic ion accumulation and gluco- 
neogenesis. Additionally, samples of kidney, liver and duodenum were 
prepared for histopathological examination. 

Major Findings : 

At equimolar doses, tetraplatin was less nephrotoxic than cisplatin. At a 
dose of 6.7 umol/kg there was no evidence of tetraplatin nephrotoxicity, in 
contrast to rats receiving this dose of cisplatin. 

Furthermore, the characteristics of tetraplatin nephrotoxicity differed marked- 
ly from those of cisplatin. Most notably, 13.3 ymol/kg of tetraplatin produced 
a striking 10-20 fold elevation of daily urine volume; rats v^ere voiding up to 
100 ml of urine per 24 hr. This was accompanied by a corresponding increased 
excretion of protein, glucose, urinary enzymes and K"*". Proximal tubular cell 
function assessed in renal cortical slices was not markedly affected in rats 
treated with tetraplatin. In contrast, 13.3 ymol/kg of cisplatin decreased 
organic ion accumulation and gluconeogenesis and resulted in small elevations 
of glutathione concentrations in renal cortical slices. Urinary excretion of 
brush border-derived enzymes after both cisplatin and tetraplatin indicated 
damage to the proximal tubular portion of the nephron. Histopathological 
examination of kidneys confirmed differences between cisplatin and tetraplatin 
nephrotoxicity. Renal lesions after cisplatin primarily were localized to the 
corticomedullary junction and were much more severe than those of tetraplatin, 
where the lesion occurred more diffusely throughout the renal cortex. The 
extent of cellular and tubular degeneration and necrosis was much more severe 
after cisplatin. In contrast to cisplatin, no giant cells nor frank tubular 
necrosis was observed with tetraplatin, even at the highest dose of 53.3 ymol/ 
kg. No indication of nephrotoxicity was detected in rats treated with CHIP, 
another Pt(IV) analog, except at this highest dose of 53.3 ymol/kg. 

Urinary analysis for excretion of protein, glucose and enzymes was the most 
sensitive indicator for the onset of nephrotoxicity for all platinum drugs 
evaluated. BUN and creatinine were elevated slightly (3-fold) 5 d after 26.7 
ymol/kg of tetraplatin and by 10-fold after 53.3 ymol/kg of tetraplatin. 
Marked elevations of urinary protein and enzyme excretion were detected after 
13.3 umol/kg of tetraplatin. This sequence was observed in cisplatin and 
CHIP treated rats as well. 

There appear to be other differences between the toxicity of tetraplatin and 
cisplatin. Decreases of white blood cell count and platelets were detected 
with the lowest doses of tetraplatin, in contrast to cisplatin. Furthermore, 
characteristics of gastrointestinal toxicity differed with tetraplatin. Little 
or no feces was apparent in metabolism cages, food consumption appeared to be 
decreased, and symptoms of a hemorrhagic stomach were observed on kill dates 
in rats treated with 26.7 ymol/kg tetraplatin. At the highest dose, rats 
exhibited a severe watery diarrhea and stomachs were noted to be enlarged and 
fluid-filled. 



219 



ZOl CM 07169-01 LETM 

Significance to Biomedical Research and the Program of the Institute : 

The nephrotoxic potential of a new and promising platinum analog anticancer 
agents was evaluated in a novel protocol. The nephrotoxicity was compared at 
depth with cisplatin, the platinum drug most widely used in the clinic to date. 
Data obtained from this investigation will be useful in designing further 
toxicologic investigations of tetraplatin and other platinum drug analogs, and 
in monitoring and reducing the incidence of nephrotoxicity if this drug is 
evaluated clinically. These data strongly indicate that monitoring of BUN or 
creatinine is inappropriate for assessing the onset of nephrotoxicity after 
this class of anticancer drugs. 

Proposed Course : 

Tetraplatin has been pased to Decision Point 2B by the Decision Network com- 
mittee and will now enter full-scale preclinical toxicity evaluation. These 
platinum drug analogs are being evaluated currently in several in vitro models 
for assessing nephrotoxicity, including renal cortical slices (see Annual 
Report ZOl CM 07160-02 LETM) and isolated renal epithelial cells. Upon the 
availability of human renal tissue, these compounds will be evaluated in similar 
in vitro systems in an attempt to predict the nephrotoxic potential of tetra- 
platin to humans. 

Publications ; 

1. Litterst, C, Smith, J.H., Smith, M.A., Uozumi, J. and Copley, M.: 
Sensitivity of urinary enzymes as indicators of renal toxicity of the 
anticancer durg cisplatin. In Bianchi, C. and Blaufox, D.M. (Eds.): 
Proceedings from Newer Diagnostic Methods in Nephrology and Urology. 
Basel, Switzerland, Karger Pub! . Co., in press. 



220 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07171-01 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Thiol -Promoted Lipid Peroxidation in Kidney Microsomes 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: E. G. Mimnaugh Chemist LETM, NCI 



COOPERATING UNITS (If any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 
0.1 



PROFESSIONAL: 
0.1 



0.0 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



n (b) Human tissues B (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Although reduced glutathione (GSH) is known to have an inhibitory (antioxidant) 
effect on lipid peroxidation in microsomes from liver in vitro and is thought 
to play an important role in controlling lipid peroxidation in vivo through 
the action of glutathione peroxidase, it was observed that, in contrast, GSH 
dramatically enhanced NADPH-dependent lipid peroxidation in rodent kidney 
microsomal fractions. This unexpected result was studied further with the aim 
of understanding the mechanism by which GSH and other thiols promoted lipid 
peroxidation. This process could have toxicological importance in kidney in that 
it may amplify NADPH-dependent lipid peroxidation. 



221 



PHS 6040 (Rev. 1/84) 



GPO 814-918 



ZOl CM 07171-01 LETM 

Objectives : 

The enzymatic diversion of reducing equivalents from thiols to promote the 
peroxidation of membrane lipids appears to be a previously unrecognized mecha- 
nism of lipid peroxidation. The objectives of this study are to characterize 
the biochemistry of this process through the use of inhibitors and altered 
incubation conditions, to investigate the apparent organ-specificity of thiol - 
promoted peroxidation, to attempt to identify the enzyme, and to determine if 
the process has toxicological relevancy. 

Methods : 

Microsomal fractions are isolated from kidney homogenates by differential 
centrifugation. Lipid peroxidation is assayed by the thiobarbituric acid 
method. The conversion of GSH to GSSG or other oxidative products will be 
determined by HPLC. The participation of oxyradicals in thiol -promoted lipid 
peroxidation will be assessed by the use of specific scavengers of reactive 
oxygen species or by direct assay. 

Major Findings : 

Lipid peroxidation supported by GSH was concentration (1 to 10 mM) and time- 
dependent, correlated with GSH oxidation, and was synergistic with either NADPH 
or NAOH. The process appeared to be enzymatic in that the effect could be 
completely blocked by heat-inactivating the kidney microsomes, and to be metal 
ion-dependent (Cu^'*', Fe^"*" and Fe^"*"). Other thiols could substitute for GSH and 
these included: cysteine, N-acetyl cysteine, cysteamine, dithiothreitol and 
dimercaprol, but S-methyl glutathione could not support peroxidation. Lipid 
peroxidation in the presence of GSH and NADPH could be diminished by scavengers 
of oxyradicals and by metal ion chelators as well as by KCN. It was noted that 
adriamycin-stimulated kidney NADPH-dependent microsomal lipid peroxidation 
could be further enhanced by GSH. These preliminary results suggest that in 
kidney, GSH-enhanced lipid peroxidation is an enzyme-mediated process which may 
utilize reducing equivalents derived from thiol oxidation. 

Significance to Biomedical Research : 

This process may have toxicological importance in that it can amplify NADPH- or 
NADH-dependent peroxidation of unsaturated membrane lipids. 

Proposed Course : 

Glutathione-promoted peroxidation appears to be kidney-specific and enzyme- 
mediated. The identity of this thiol -dependent enzyme is unknown. Future 
experiments will be designed to characterize the enzyme. 



222 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07172-01 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Role for Reactive Oxyradicals and Lipid Peroxidation in Anthracycline Cytotoxicity 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



P. I.: E. G. Mimnaugh 



Chemist 



LETM, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

0.2 



PROFESSIONAL: 



0.1 



0.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues Q (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Recent reports support the hypothesis that a mechanism by which anthracyclines 
cause cardiotoxicity involves the formation of drug free radical and enhanced 
oxyradical production. It is possible that at least a portion of the antitumor 
activity of anthracyclines may also result from anthracycline-mediated oxyradical 
production leading to damage to intracellular membranes through the process of 
lipid peroxidation. Information relevant to this possibility is presently 
lacking, and, therefore, this project was designed to evaluate the oxyradical 
hypothesis of cytotoxicity with isolated microsomal and mitochondria subcellular 
fractions from several model tumors. 



223 



PHS 6040 (Rev. 1/84) 



GPO SI 4-9 IB 



ZOl CM 07172-01 LETM 

Objectives ; 

Previous studies from this and other laboratories have shown that either 
microsomes or mitochondria from various tissues, in the presence of a suitable 
reducing agent, are capable of metabolically activating Adriamycin by a one 
electron reduction to an unstable semiquinone free radical. The Adriamycin 
semi qui none intermediate subsequently reacts with molecular oxygen to generate 
superoxide radical, hydrogen peroxide, and ultimately hydroxyl radical, which 
initiate and propagate the peroxidation of polyenoic membrane phosphoglycerides. 
Membrane lipid peroxidation is a destructive process in that it disrupts mem- 
brane structure and function, inactivates membrane-bound enzymes, generates 
reactive lipid-derived aldehyde products, and ultimately, leads to cell death. 
The objectives of this study are to determine whether the above described 
biochemial process contributes to the antitumor activity of anthracyclines, and 
to describe those factors which participate in the process. 

Methods Employed ; 

Tumors are grown either in cell culture or in vivo in mice, and microsomal and 
mitochondrial subcellular fractions are isolated from tumor homogenates. These 
fractions contain the enzymes capable of metabolically activating anthracyclines 
(NADPH- cytochrome P-450 reductase and NADH-dehydrogenase, respectively) and the 
membrane unsaturated lipids, which are targets for oxyradical attack. Incuba- 
tions are conducted in the presence of Adriamycin and lipid peroxidation is 
assessed by the thiobarbituric acid method. Enzymatic assays are conducted, 
and oxyradical scavengers are employed to delineate the mechanism of lipid 
peroxidation. 

Major Findings ; 

Adriamycin stimulated NADPH-dependent lipid peroxidation 4 to 7-fold in micro- 
somes isolated from EMT6 mammary carcinoma cells and from B-16 solid tumors. 
The maximal enhancement of lipid peroxidation in microsomes from B-16 melanoma 
was observed with adriamycin at 100 uM, with exogenously added ferrous iron at 
10 yM and in incubations conducted under a 100% oxygen atmosphere. Microsomal 
NADPH-cytochrome P-450 reductase activities, which could contribute reducing 
equivalents for lipid peroxidation, were 56 ± 67 and 28 ± 6 nmoles of cytochrome 
c reduced/mg protein/mi n for EMT6 and B-16 tumors, respectively. Adriamycin 
Tailed to enhance NADPH-dependent lipid peroxidation in either EMT6 or B-16 
melanoma mitochondria. 

Significance to Biomedical Research and the Program of the Institute ; 

Elucidation of the mechanisms of tumor cell killing by anthracyclines will 
result in a better understanding of the biomedical toxicology of this class of 
antitumor drugs and provide a basis for the rational design of drugs which 
utilize reactive oxyradical s in cytotoxic chemotherapy. 

Proposed Course ; 

Further work is planned to investigate the participation of superoxide, hydrogen 
peroxide and hydroxyl radical in adriamycin-stimulated tumor microsomal lipid 

224 



ZOl CM 07172-01 LETM 

peroxidation, and to extend the study to include other anthracycline-sensitive 
tumors. 

Publications : 

1. Sinha, B.K., Trush, M.A., Kennedy, K.A. and Mimnaugh, E.G.: Enzymatic 
activation and binding of adriamycin to nuclear DNA. Cancer Res. 44: 
2892-2986, 1984. 

2. Mimnaugh, E.G., Trush, M.A., Bhatnagar, M. and Gram, T.E.: Enhancement of 
reactive oxygen-dependent mitochrondrial membrane lipid peroxidation by 
the anticancer drug, adriamycin. Biochem. Pharmacol . 34: 847-856, 1985. 

3. Mimnaugh, E.G., Kennedy, K.A., Trush, M.A., and Sinha, B.K.: Adriamycin- 
enhanced membrane lipid peroxidation in isolated nuclei. Cancer Res. , in 
press. 



225 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07173-01 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) 

Arachidonic Acid Metabolism in Human Lung Adenocarcinoma 



PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alflliatlon) 

PI: S. S. Lau Sr. Staff Fellow LETM, NCI 



Others: J. B. McMahon 
H. M. Schuller 
M. R. Boyd 
M. G. McMenamin 
K. E. Greene 



Cancer Expert 
Vet. Med. Officer 
Associate Director 
Chemi st 
Bio. Lab. Tech. 



LETM, NCI 
LETM, NCI 
DTP, NCI 
LETM, NCI 
LETM, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 

0.4 



PROFESSIONAL: 



0.4 



0.7 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

To date, there have been no reports linking arachidonic acid (AA) metabolism with 
the pathobiology of specific human tumor cell populations in pulmonary adenocar- 
cinoma (PAC). The correlation of prostaglandin (PG) biosynthesis with the patho- 
biology of PAC was studied by exploring the metabolism of AA in human lung car- 
cinoma cell lines. Two PAC derived cell lines (NCI-H322 and NCI-H358) and two 
small cell carcinoma derived cell lines {NCI-H69 and NCI-H128) were used. No 
metabolism of AA occurred in either unstimulated or calcium ionophore A23187- 
stimulated NCI-H69 and NCI-H128 cells. The major metabolite of AA isolated from 
both NCI-H322 and NCI-H358 cells was identified as prostaglandin E2 {P6E2). The 
prostaglandin endoperoxide synthase (PES) activity expressed as immunoreactive 
PGE2, (pmole/min/mg protein) was found to be 10.3 ± 0.28 and 4.8 ± 0.48 in NCI- 
H358 and NCI-H322 cells, respectively. Production of PGE2 in both NCI-H358 and 
NCI-H322 cells was linear up to 10 min with apparent Km of 8.3 and 5.5 yM and 
Vmax of 12.5 and 5.3 pmole/min/mg protein for NCI-H358 and NCI-H322 cells, respec 
tively. Moreover, PGE2 did not undergo further metabolism by either the NCI-H358 
nor the NCI-H322 cells. Aspirin, inhibited PGE2 production by 78% and 60% in 
NCI-H358 and NCI-H322 cells, respectively. In the presence of exogenous AA, 
A23187 stimulated PGE2 production in NCI-H322 cells by 76% without affecting the 
production of PGE2 in the NCI-H358 cells. In contrast, A23187 stimulated the 
endogenous production of PGE2 to a greater extent in both NCI-H322 and NCI-H358 
cells. These results suggest that certain types of tumors within the category of 
human PAC may preferentially synthesize and release PGE2. This finding may be 
useful diagnostically since elevated PGE2 synthesis may be a characteristic unique 
to a group or subclass of PAC in patients. Furthermore, the human PAC lines can 
be employed as models to explore the ability of the different human lung cancers 
to activate xenobiotics, especially antitumor agents directed at the PES system 
in hopes of achieving more selective therapeutic effectiveness. 

226 



PHS 6040 (Rev. 1/84) 



ePO BM.»ia 



ZOl CM 07173-01 LETM 

Objectives : 

The pathobiology of human lung cancer is often associated with the biosynthesis 
and release of various biologically active substances by malignant cells. A 
number of studies have indicated that prostaglandins (PGs) and other eicosanoids 
may play a role in some aspects of the pathobiology of malignant disease, such 
as tumor promotion, cellular proliferation and differentiation. This study is 
initiated to determine whether cancer cells produce high levels of PGs, and if 
specific cell types are responsible for such reactions. We have used various 
well established and morphologically well characterized human lung cancer cell 
lines (see Annual Report ZOl CM 07175-01 LETM). For these studies, four human 
lung carcinoma cell lines were chosen. Two are well differentiated PAC, NCI- 
H322 and NCI-H358. The other two are established small cell carcinoma lines 
(SCCL), NCI-H69 and NCI-H128. Ultrastructurally, NCI-H322 possesses rough and 
smooth endoplasmic reticulum and electron-dense secretion granules which are 
characteristic of bronchiolar Clara cells. The NCI-H358 cells possess endo- 
plasmic reticulum and well-defined lamellar bodies, hallmark structures of 
alveolar type II cells. Both SCCL, NCI-H69 and NCI-H128, are essentially 
devoid of endoplasmic reticulum and express features of endocrine cells. In 
this report, we present details of our findings on the metabolism of AA in 
these human lung carcinoma cell lines. 

Methods Employed : 

Cell culture . The two PAC (NCI-H322 and NCI-H358) were grown as monolayers in 
75 cm^ plastic flasks. They were maintained in RPMI 1640 medium supplemented 
with glutamine (2 mM), FBS (10% v/v) and gentamycin (50 mg/ml ) at 37°C in an 
atmosphere of 95% air/5% 002- The cells were subcultured weekly and the medium 
was replaced by fresh medium every 3 days. The cultures were used when the 
monolayer had reached near confluency (about 6-8 days). The two SCCL (NCI-H69 
and NCI-H128) were grown in suspension and maintained as described above. 

Incubation . Prior to the incubation the cells were washed three times with 
20 ml of Hank's balanced salt solution (without Ca"*"*" and Mg"*"*"). Three ml of 
Hank's balanced salt solution containing calcium (1.5 mM) were then added to 
either suspend the cells or to cover the monolayers. Three yCi of l-^^-^C-AA 
(60 mCi/mmol) were added to each flask and incubated for 35 min. In other 
studies in which the calcium ionophore A23187 (20 yM) was added, the cells 
were incubated at 37''C with or without ^^C-labeled AA for 10 min before the 
addition of the ionophore and 15 minutes subsequent to the addition of the iono- 
phore. In studies in which aspirin was added to the cells, the cells were 
incubated with 0.1 mM aspirin for 10 min before the ^^C-labeled AA was added. 
In two other groups of studies, exogenous AA (1.4 to 33.2 yM) was added to 
monolayers of NCI-H322 and NCI-H358 cells and incubations were performed at 
37 "C for periods of time ranging from 0-30 min. The incubations were ter- 
minated by removal of the incubation medium from the suspensions or monolayers 
of cells. 

Analysis of products : The incubation medium was removed from suspensions of 
SCCL {NCI-H69 and MCI-H128) and monolayers of PAC (NCI-H322 and NCI-H358) and 
analyzed by reversed phase high performance liquid chromatography (HPLC) for 
the presence of ^*C-labeled metabolites of AA. The medium removed from the 

227 



ZOl CM 07173-01 LETM 

cells was evaluated for the oresence of fatty acid cyclooxygenase and lipoxy- 
genase products of AA. The •'■^C-labeled products resolved by HPLC were directly 
compared with known standards of fatty acid cyclooxygenase and lipoxygenase 
products of AA. The identity of I'+C-labeled metabolites of AA isolated from the 
cell suspensions and monolayers was established via combined gas chromatography- 
mass spectrometry. Quantitation of the metabolite isolated from monolayers of 
NCI-H322 and NCI-H358 cells was achieved by radioimmunoassay (RIA). 

Major Findings ; 

The major cyclooxygenase-peroxidase catalyzed ^^C-AA metabolite in these human 
carcinoma cell lines was identified as PGE2. The lipoxygenase pathways of 
^*C-AA in these human cell lines was also assessed by HPLC assay but no metab- 
olite(s) could be detected under our assay conditions. NCI-H358 and NCI-H322 
cells, both PAC, are capable of metabolizing ^^C-AA to PGEo. In contrast, 
levels of PGE2 in both the NCI-H69 and NCI-H128 cells, both SCCL, were nonde- 
tectable. Formation of PGE2 in both the NCI-H322 and NCI-H358 cells was linear 
up to 10 min in the presence of exogenous AA (18 \M). The kinetic parameters 
for the metabolic transformation of AA were investigated in the NCI-H358 and 
NCI-H322 cell lines. Similar apparent Michaelis-Menten affinity constant (k^) 
values for the formation of PGE2 were obtained in NCI-H358 and NCI-H322 (8.3 
and 5.5 ^M, respectively). However, the rate of formation of PGE2 was signi - 
ficantly higher in the NCI-H358 cells than in the NCI-H322 cells. Their appar- 
ent Vmax values were 12.5 and 5.3 pmole/min/mg protein, respectively. The PES 
activity expressed as immunoreactive PGE2 in pmole/min/mg protein was found to 
be 10.3 ± 0.28 and 4.8 ± 0.48 in NCI-H358 and NCI-H322, respectively. Aspirin, 
a cyclooxygenase inhibitor, at 0.1 mM, inhibited PGE2 production by 78% and 60% 
in NCI-H358 and NCI-H322 cells, respectively. In the presence of endogenous AA, 
the calcium ionophore A23187, at 20 yM, stimulated PGE2 production in NCI-H322 
cells by 76%, while it did not affect the production of PGE2 in the NCI-H358 
cells. In contrast, A23187 stimulated the endogenous production of PGE2 to 
a greater extent in both NCI-H322 and NCI-H358 cells (3- and 8-fold, respec- 
tively). Furthermore, ^H-PGEg was incubated with NCI-H358 and NCI-H322 cell 
lines to determine if they were capable of metabolizing PGE2. No disappearance 
of ^H-P6E2 in the incubation medium in either of these two cell lines was 
observed. 

Significance to Biomedical Research and the Program of the Institute : 

We have demonstrated in the study that two PAC preferentially metabolize AA 
primarily to PGE2. Moreover, these results show that within the category of 
human PAC, AA metabolism exhibits significant differences in individual cell 
types. This finding provides a tool for a more detailed examination of biochem- 
ical functions of these tumor cells in relation to the pathobiology of human 
lung cancer. The preferential biosynthesis of P6E2 by certain types of lung 
cancer cell lines may be useful diagnostically, and in the determination of the 
effectiveness of antitumor therapy in human subjects. Furthermore, the human 
PAC lines can be employed as models to explore the ability of the different 
human lung cancers to activate xenobiotics, especially antitumor agents directed 
at the PES system, in hopes of achieving more selective therapeutic effective- 
ness. 



228 



ZOl CM 07173-01 LETM 

Proposed Course : 

It is well estabished that a variety of compounds undergo oxidative metabolism 
during prostaglandin biosynthesis in vitro . The ability of certain types of 
human lung carcinomas to synthesize prostaglandins may convey significant 
capability to such tumors for the metabolic activation of antitumor agents and 
other xenobiotics via P-450 monooxygenase-independent mechanism. Thus, studies 
of PES-mediated cooxidation of antitumor agents and other xenobiotics will be 
carried out in these human lung carcinoma-derived cells. Furthermore, the role 
of AA metabolism in tumor differentiation as well as metastases development in 
vivo will be examined in tumor-bearing nude mice after injection of homogenous 
carcinoma cells of early and late passages from individual cell lines. 



229 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07174-01 LETM 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) 

Arachidonic Acid in Pulmonary Large Cell Carcinoma 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aftiliation) 

PI: S. S. Lau Sr. Staff Fellow LETM, NCI 



Others: H. M. Schuller 
J. B. McMahon 
M. G. McMenamin 
K. E. Greene 



Vet. Med. Officer 

Expert 

Biologist 

Bio. Lab. Tech. 



LETM, NCI 

LETM, NCI 

LETM, NCI 

LETM, NCI 



COOPERATING UNITS (if any) 

Laboratory of Chemical Pharmacology, NHLBI, NIH (T. J. Monks) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pharmacology and Toxicology Section 



INSTITUTE AND LOCATION 

NCI. NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS; 

0.3 



PROFESSIONAL: 

0.6 



0.3 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



B (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Clinically, large cell carcinoma (LCC) is by far the least treatable lung cancer. 
Characterization of this subclass of carcinoma by an exclusive cell type is not 
possible even under electron microscopic examination. Thus, investigation of the 
biochemical and morphological features of human LCC is warranted. The pathobiol- 
ogy of human lung cancer is often associated with the biosynthesis and release of 
various biologically active substances by malignant cells. A number of studies 
have indicated that prostaglandins (PGs) and other eicosanoids may play a role in 
the pathobiology of malignant disease, i.e., tumor promotion, cellular prolifera- 
tion and differentiation. One metabolite of arachidonic acid (AA), prostaglandin 
E2 (PGE2), has been implicated as a mediator of hypercalcemia associated with 
certain lung cancers (Seyberth at al., N. Engl. J. Med. 239: 1228, 1975). The 
leukotrienes participate in the initiation of immune and inflammatory responses 
(Lewis and Austen, J. Clin. Invest. 73:889, 1984) and thus could play a role in 
host defense mechanisms in human lung cancer. Studies of the metabolism of AA 
were undertaken to determine the pathways of AA metabolism and the relevance of 
this metabolism in human LCC. Our preliminary studies demonstrated that a homog- 
enous human LCC line, NCI-H460, unlike any other known human lung carcinoma cell 
line maintained in this laboratory possesses significantly higher PGE2 synthesis 
(6 fold) in the early passage (15) as compared to that in the late passage (93). 
Furthermore, the morphology of this cell line (see Annual Report ZOl CM 07175-01 
LETM) has changed. Originally diagnosed as LCC (containing large amounts of 
endoplasmic reticulum), it was reclassified as a small cell carcinoma (exhibiting 
endocrine cell features) in passage 15 and it is now (passage 93) described as an 
oncocytoma (containing large numbers of mitochondria). The mechanism for such an 
alteration is unknown. To examine the role of AA metabolism in tumor development 
in vivo , cells from early and late passages of NCI-H460 will be injected into 
nude mice. The profile of AA metabolism in the primary tumor as well as in the 
metastases formation will be investigated. 



PHS 6040 (Rev. 1/84) 



230 



GPO S14.»IS 



ZOl CM 07174-01 LETM 

Objectives : 

The pathophysiology of human lung cancer has been attributed in part to the 
biosynthesis and release of different mediators by malignant cells. Because 
the products of AA possess potent biological activities and are preferentially 
synthesized by human non-small cell carcinoma of the lung, arachi donate metabo- 
lism may be relevant to human lung cancer-associated pathobiology. Seyberth 
and coworkers (N. Engl. J. Med. 239:1228, 1975) have demonstrated that produc- 
tion of PGE2 is significantly elevated in certain populations of hyercalcemic 
cancer patients with primary pulmonary malignancies classified as squamous cell 
carcinomas. Bennett et. al . (Br. J. Cancer 46:888, 1982) reported that 
levels of prostaglandin-like material, bioassayable as P6E2 activity, is signif- 
icantly higher in human lung carcinoma tissue than in adjacent normal lung 
tissue after incubation in vitro . While these studies indicate that eicosanoid 
production is elevated in human lung carcinoma and fibrosarcoma tissues, and in 
squamous cell carcinoma of the lung, attempts to correlate a role of PGs and 
other eicosanoids in the pathobiology of human lung cancer have proven unreward- 
ing. A major impediment in determining the role of PGs in the pathobiology of 
human lung cancer cell in the studies cited is the lack of availability of 
homogenous cell populations defined by cell type derived from human lung car- 
cinomas. As a result, there is a paucity of information related to the metab- 
olism of AA and other fatty acid precursors of eicosanoids in human lung car- 
cinomas. To determine whether cancer cells produce high levels of PGs and 
whether specific cell types are responsible for such production, we have used 
various well established and morphologically well characterized human lung 
cancer cell lines (see Annual Report ZOl CM 07175-01 LETM). Some of the results 
of the metabolism of AA in various human lung cancer lines has been summarized 
in a separate report (see Annual Report ZOl CM 07173-01 LETM). Among these 
cell lines, NCI-H460 demonstrates a unique characteristic. Histopathologically, 
its tumor of origin was diagnosed as large cell carcinoma while the cell line 
derived from it demonstrated features of endocrine cells at early passage which 
subsequently changed to oncocytoma at late passage. The objectives of the 
present study are: 1) to compare the metabolism of AA in early and late 
passages in culture and 2) to investigate the role of AA metabolism in tumor 
development in nude mice after injection of homogenous NCI-H460 LCC. 

Methods Employed : 

Cell culture and incubations: NCI-H460 cells from passage 15 and passage 93 
were grown as monolayers in 75 cm^ plastic flasks. They were maintained in 
RPMI 1640 medium supplemented with glutamine, FBS and gentamycin, at 37°C in an 
atmosphere of 95% air/5% CO^. Prior to the incubation with AA, cells were 
washed three times with 20 ml of Hank's balanced salt solution (without Ca"*"*" 
and Mg''"*'). Three ml of Hank's balanced salt solution (HBSS) containing calcium 
(1.5 mM) were then added to cover the monolayers. Cells were incubated at 37°C 
for 10 minutes in the presence or absence of aspirin (0.1 mM); ^^C-AA (1 yCi, 
40 )iM) was then added and the cells were incubated at 37°C for 10 minutes. 
In studies in which the calcium ionophore A23187 (20 yM) was added, the cells 
were incubated at 37°C with or without exogenous AA for 10 minutes before the 
addition of ionophore and for 15 minutes subsequent to the addition of the 
ionophore. The incubations were terminated by removal of the HBSS from the 
monolayer cells. 



231 



ZOl CM 07174-01 LETM 



Analysis: 



The HBSS removed from tlie monolayers of the two passages of NCI-H460 cells was 
analyzed by HPLC for the presence of cyclooxygenase products. Quantation of 
PGE2 was achieved by radioimmunoassay (RIA). 

Major Findings : 

The predominant cyclooxygenase product of AA was found to be PGE2 in both early 
(15) and late (93) passages of NCI-H460 cell lines. The profile of AA metab- 
olism measured as PGE2 formation in the early (passage 15) and late (passage 
93) passages of cell line NCI-H460 is substantially different in both rate and 
response to stimulator, such as A23187. These results are summarized in the 
following table. 





PGE2 (p mole/mi n/mg protein) 




Passage 15 


Passage 93 


Additions 


+ AA (40 yM) 


-AA 


+AA (40 pM) 


-AA 


none 


37.5 ± 0.5(100%) 


7.6 ± 0.2(100%) 


6.3 ± 0.9(100%) 


0.43 ± 0.03(100%) 


+Asp(.lmM) 


15.2 ± 1.7(40%) 


N.D. 


1.7 ± 0.1(22%) 


N.D. 


+A23187 
(20 uM) 


50.3 ± 2.4(134%) 


40.1 ± 2.0(528%) 


7.2 ± 0.4(115%) 


0.86 ± 0.1(200%) 



PGE2 production is significantly higher in passage 15 than in passage 93 in the 
presence and absence of exogenously added AA (6 fold in the presence and 18 fold 
in the absence of AA). Furthermore, cells from passage 15 are far more suscept- 
ible to A23187 stimulation in the endogenous P6E2 synthesis (528% over control) 
than that from passage 93 (200% over control). 

Proposed Course : 

Our preliminary results demonstrate that the alteration of biochemical proper- 
ties in PG biosynthesis occurred during continuous cell subculture. Concom- 
itant with this study, the morphology of this cell line was found to change 
from LCC (initial tumor) to small cell carcinoma which exhibits endocrine cell 
features (passage 15) and subsequently to oncocytoma (passage 93, containing 
large numbers of mitochondria). The existence of such a unique cell line 
provides us an opportunity to study the interrelationship of AA metabolism and 
the pathobiology of lung carcinoma. Further in vitro studies are planned to 



232 



Z01 CM 07174-01 LETM 

evaluate the lipoxygenase pathways of AA metabolism in these cells. Homogenous 
cells of NCI-H460 with early as well as late passages will be injected into nude 
mice, the profile of AA metabolism and degree of tumor development will be 
determined. Inhibitors of AA metabolism such as indomethacin, aspirin, flu- 
fenamic acid and nordihydroguaiaretic acid will be administered before or at 
different times after injection of the tumor cells from different passages. The 
effect of these compounds on the overall metabolism of AA and tumor and/or 
metastases formation will be evaluated. Moreover, additional representative 
cell lines derived from large cell carcinomas will be investigated. The above 
proposed studies together with those proposed in Annual Report ZOl CM 07173-01 
LETM will allow us to better assess the role of AA metabolism in this hetero- 
genous class of human lung cancer. 

Significance to Biomedical Research and the Program of the Institute ; 

It is clear that a selective therapeutic attack on cancer cells is possible 
only when the basic differences between cancer and normal cells are well under- 
stood. The PGs and related eicosanoids have been implicated as important fac- 
tors in a number of pathobiological aspects of malignant disease. In addition, 
they have been implicated as mediators in certain aspects of metastatic dis- 
semination of human primary tumors. Due to the fact that pulmonary LCC is the 
least treatable lung cancer, investigations of the biochemical function of LCC 
is warranted. The proposed studies should allow us to evaluate the role of AA 
in tumor differentiation and/or metastases formation in experimental animals 
after injection of the homogenous cells from different passages, especially 
those from the initial passage. Such investigation should also allow us to 
determine whether or not the profile of AA metabolism in human lung cells in 
vi tro accurately reflects AA metabolism in vivo in different stages of the 
disease. 



233 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07152-02 LETM 



PERIOD COVERED 



October 1, 1984 to September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) 

Studies on Clara cell mediated 1unq carcinogenesis 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: H. M. Schuller Vet. Med. Officer LETM, NCI 



Others: 



M. Falzon 

J. B. McMahon 

M. Gregg 

S. Walton 



Visiting Fellow 
Cancer Expert 
Bio. Lab. Tech. 
Bio. Lab. Aid 



LETM, NCI 

LETM, NCI 

LETM, NCI 

LETM, NCI 



COOPERATING UNITS (it any) 



LAB/BRANCH 



Laboratory of Experimental Therapeutics and Metabolism 



Pathology and Ultrastructural Oncology Section 



INSTITUTE AND LOCATION 



NCI, NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 

1.0 



PROFESSIONAL: 

0.5 



0.5 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues H (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

A number of N-nitrosamines are powerful respiratory tract carcinogens which re- 
quire metabolic activation in the host organism. This metabolic activation is 
believed to be mediated by cytochrome P-450 enzymes although unequivocal evidence 
for this hypothesis has not yet been achieved. An experiment was conducted to 
investigate the effect of the P-450 enzyme inhibitor, piperonyl butoxide, on the 
induction of lung tumors which originate from Clara cells in hamsters treated with 
N-nitrosodi ethyl amine (DEN). The effect of piperonyl butoxide on covalent binding 
and distribution of the parent nitrosamine was examined in vivo after 1 dose of 
^^C-DEN. Moreover, the effect of piperonyl butoxide on tFfe tumor incidence in- 
duced by DEN in a chronic study was investigated by histopathology. Piperonyl 
butoxide significantly inhibited metabolism of DEN in the respiratory tract and 
inhibited the induction of lung tumors. These data provide the first experimental 
evidence for cytochrome P-450 enzymes being a crucial factor to the metabolic 
conversion of DEN into a carcinogenic metabolite in vivo . 

As a comparative approach, human lung cancer cell lines (HCCL) with and without 
morphological features of pulmonary Clara cells were tested for their ability to 
metabolize DEN in vitro . It was found that the cell line with Clara cell features 
metabolized DEN to a significantly greater extent than cell lines derived from 
other types of lung cancers and that such metabolism was significantly inhibited 
by cytochrome P-450 inhibitors. Moreover, an HCCL with morphological features of 
alveolar type II cells was found to metabolize DEN to a great extent via prosta- 
glandin endoperoxide synthetase. This is the first experimental evidence for dif- 
ferent cell types in the lung to metabolize a nitrosamine via different pathways 
and may be of potentially great importance in explaining the cell type-specific 
effects of nitrosamines. 

234 



PHS 6040 (Rev. 1/84) 



sPo OM'Ste 



ZOl CM 07152-02 LETM 

Objectives : 

N-nitr OS amines are ubiquitous in man's environment and are formed endogenously 
by reaction between amine precursors and nitrosating agents. They require 
metabolic activation in the host organism which is believed to be mediated by 
cytochrome P-450 enzymes. However, proof for this hypothesis has not yet been 
reached. N-nitrosodi ethyl amine (DEN) is a powerful respiratory tract carcino- 
gen in hamsters. Studies on the pathogenesis of the lung tumors and auto- 
radiographic studies on the site of covalent binding in the lungs have given 
evidence for the pulmonary Clara cell as being the origin of lung tumors and an 
important site of covalent binding in the lungs. The objective of this study 
was to examine if cytochrome P-450 enzymes are an important factor for the 
activation of DEN to carcinogenic metabolites in this in vivo system and if 
parallel data could be obtained using well -differentiated human lung cancer 
cell lines of defined cell type. 

Methods Employed : 

In vivo studies . Initial studies were undertaken to determine the time of 
maximum covalent binding in trachea, lung and liver after injection of l^C-DEN. 
The time-point of maximum covalent binding (4 hr) was then selected to study 
the effect of pretreatment with the cytochrome P-450 inhibitor, piperonyl 
butoxide, on covalent binding as well as distribution of ^^C-DEN. In another 
experiment, hamsters were s.c. injected 2 x per week for 20 weeks with DEN 
while others received pretreatment with piperonyl butoxide before each DEN 
injection. One group injected with piperonyl butoxide alone, and one injected 
with saline (vehicle of DEN) served as controls. All animals were sacrificed 
after 20 weeks on test at which time DEN alone had induced tumors in lung and 
trachea in about 70% of the animals as shown by earlier experiments. Complete 
autopsies were done on all animals, and lungs and livers were processed for 
histopathology from paraffin sections stained with hematoxylin/eosin and PAS- 
reaction. Tissue samples of these organs were also processed for electron 
microscopic investigation. 

In vitro studies . The metabolism of ^^C-DEN was studied in the following cell 
lines: NCI-H322 derived from human pulmonary adenocarcinoma and exhibiting 
ultrastructural features of Clara cells; NCI-H358 derived from human pulmonary 
adenocarcinoma and exhibiting ultrastructural features of alveolar type II 
cells; NCI-H128 and NCI-H69 both derived from human pulmonary small cell cancer 
and demonstrating ultrastructural features of poorly differentiated endocrine 
cells. Metabolism of the nitrosamine was monitored by CO2 production and 
covalent binding of radiolabel from ^*C-DEN to cell proteins and DNA. The 
effect of pre-exposure to nitrogen or a carbon monoxide/oxygen mixture on such 
metabolism was studied as \^as the effect of preincubation with aspirin and 
indomethacin. 

Major Findings : 

In the hamster model in vivo , pretreatment with piperonyl butoxide 2 hrs before 
injection of ■'•^C-DEN resulted in a 90% inhibition of covalent binding in lung 
and liver. In the chronic experiment, 9 out of 10 hamsters treated with DEN 
alone had multiple tumors in the lungs. In the animals pretreated with 
piperonyl butoxide, no tumors developed in the lungs. 

235 



ZOl CM 07152-02 LETM 

Human lung cancer cell lines in vitro . ^^C-DEN was metabolized by adeno- 
carcinoma-derived NCI-H322 (wTEh Clara cell features) to a significantly greater 
extent than by NCI-H358 (with features of alveolar type II cells) while NCI-H69 
and NCI-H128 (derived from small cell carcinoma) were unable to metabolize the 
compound. Metabolism was markedly inhibited by heat denaturation of the cell 
protein. ^^C-DEN metabolism by NCI-H322 was greatly decreased when the incuba- 
tion was carried out under (i) anaerobic conditions and (ii) in the presence of 
a carbon monoxide enriched atmosphere. These results suggested the involvement 
of a cytochrome P-450-dependent monooxygenase enzyme system. Metabolism by NCI- 
H358 was also decreased in the absence of oxygen or presence of carbon monoxide 
although the effects were relatively small compared to the results with NCI- 
H322. On the other hand, aspirin or indomethacin, which are inhibitors of the 
fatty acid cyclooxygenase component of prostaglandin endoperoxide synthetase, 
preferentially inhibited ^^C-DEN metabolism by NCI-H358. There were little or 
no effects of these inhibitors on the metabolism of DEN in NCI-H322. The data 
suggest that DEN metabolism in different lung cell types may be carried out by 
different enzyme systems which in turn may contribute to the selective effect 
of DEN in the lung. 

Significance to Biomedical Research and the Program of the Institute; 

The identification of in vivo mediators of respiratory tract carcinogenesis 
induced by xenobiotics is an important step towards an understanding of the 
mechanism of carcinogenesis in this organ system. However, to extrapolate 
such data generated in an animal model to man, it is vital to obtain com- 
parative data using the respective human organs and cell types. In the 
absence of isolated systems of normal human cell types, well differentiated 
early passage lung cancer cell lines comprised of one clearly identifiable 
cell type are a valuable tool for this approach. Such knowledge is an impor- 
tant basis for the development of target oriented inhibition and treatment 
of respiratory tract cancer which may lead to the development of anticancer 
drugs with high target specificity. 

Proposed Course : 

Further in vivo studies will focus on the comparison of ultrastructural changes 
induced in this model system during DEN carcinogenesis with and without pre- 
treatment with piperonyl butoxide. Also, autoradiographic studies will be 
applied to investigate the effect of piperonyl butoxide on the distribution of 
bound radioactivity in the respiratory organs after injection of l^C-DEN. 
Moreover, comparative studies with inhibitors of other enzyme systems will be 
conducted. The planned complementary in vitro studies include the identification 
of DNA adducts of DEN-exposed HCCL as well as identification of the metabolites 
formed. Moreover, isolated rat and hamster lung cell types will be used for 
the same assays. 

Publications : 

1. Reznik-Schiiller, H.M.: Experimental carcinogenesis of bronchiolo- 

alveolar neoplasms. In McDowell, E.M. (Ed.): Current Problems in Tumor 
Pathology. Vol. III. Lung Tumor Pathobiology . Edinburgh, Churchill- 
Livingston, in press. 



236 



ZOl CM 07152-02 LETM 

2. Pour, P. and Reznik-Schiiller, H.M.: Squamous cell carcinomas of the 
bronchi in Syrian golden hamsters. In Jones, T.C. and Mohr, U. (Eds.): 
ILSI Monographs on Pathology of Laboratory Animals. Vol. 2, Respiratory 
System, in press. 

3. Schuller, H.M., McMahon, J.B.: Inhibition of N-nitrosodiethylamine-induced 
respiratory tract carcinogenesis by piperonyl butoxide in hamsters. Cancer 
Res. , in press. 

4. Falzon, M., McMahon, J.B., Gazdar, A.F., and Schuller, H.M.: Preferential 
metabolism of N-nitrosodi ethyl amine by two cell lines derived from human 
pulmonary adenocarcinomas. Carcinogenesis , in press. 

5. Schuller, H.M., Stinson, S.F., Ward, J.M., McMahon, J.B., Singh, G., Katayl . 
S.L.: Loss of Clara cell antigens from neoplastic Clara cell derived 
lesions induced in the hamster lung by N-nitrosodi ethyl amine. Am. J. 
Pathol., in press. 



237 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07153-02 LETM 



PERIOD COVERED 



October 1. 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must tit on one ilne between the borders.) 

Biology of Human Lung Cancer Cell Lines In Vitro 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: J. B. McMahon Cancer Expert LETM, NCI 



Others: M. Falzon 

H. M. Schuller 

A. C. Smith 

A. A. del Campo 

M. R. Boyd 



Visiting Fellow 
Vet. Med. Officer 
Staff Fellow 
Bio. Lab. Tech. 
Associate Director 



LETM, NCI 
LETM, NCI 
LETM, NCI 
LETM, NCI 
DTP, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Experimental Therapeutics and Metabolism 



SECTION 

Pathology and Ultrastructural Oncology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 

1.0 



PROFESSIONAL: 

0.5 



0.5 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
n (a2) Interviews 



H (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Lung cancer is one of the most common, most lethal, but least treatable diseases. 
All currently available anticancer drugs are essentially ineffective against 
most human lung cancers. For the purpose of therapy, lung cancers are generally 
classified into small cell and non-small cell cancers. With respect to the many 
different tumor types found in the category of non-small cell cancer, it is diffi- 
cult to imagine that any one anticancer drug can be effective against all of them. 
This project utilizes the subclassifi cation of cell lines by cell type and dif- 
ferentiation generated by Annual Report ZOl CM 07175-01 LETM to further define 
biological and functional aspects of these defined cell lines in vitro . Such 
studies include scanning and transmission electron microscopy, quantitative image 
analysis, detailed analysis of cytoplasmic enzymes, antigenic expression, endo- 
crine and/or secretory activity, as well as competence for binding and metabolism 
of 4-ipomeanol and other candidate anticancer drugs. We found clear cut cell type 
specific differences for almost every parameter studied. Moreover, we found that 
some of the well differentiated tumor cell lines have retained biochemical path- 
ways which are considered functional markers of their normal cells of origin. 
Among those were: expression of monooxygenase and other xenobi otic-metabolizing 
enzyme activities (markers of normal pulmonary Clara cells) in an adenocarcinoma 
line with the ultrastructure of Clara cells; phospholipid biosynthesis (marker 
for normal alveolar type II cells) in an adenocarcinoma line with ultrastructure 
of alveolar type II cells; neuroendocrine activity in several small cell cancer 
lines and carcinoid lines. We also found cell type specific susceptibility to 
drug-induced toxicity, selectively high levels of prostaglandin synthetase 
activity which were cell type and differentiation dependent in collaboration with 
other investigators within the LETM. 

238 



PHS 6040 (Rev 1/84) 



GPO SI 4-ai8 



ZOl CM 07153-02 LETM 

Objectives : 

Although human lung cancer is usually divided into two broad histopatholog- 
ical categories, namely small cell lung cancer (SCLC) and non-small cell lung 
cancer (NSCLC), these tumors are far more complex. Often human tumor lines are 
comprised of a mixture of functionally and morphologically different cell 
types. In order to effectively design therapy for these tumors, the cell 
biology of the individual cell types must be studied in detail. The avai - 
lability of a large number of human lung cancer cell lines derived from tumors 
of specific cell types may provide a means to this end. By carefully charac- 
terizing biological, functional and pharmacological aspects of cell lines of 
defined cell type and differentiation (generated by ultrastructural analysis; 
see Annual Report ZOl CM 07175-01 LETM), we may be able to discover individual 
traits expressed by different tumor cell types which will allow us to tailor 
therapy. 

Methods Employed : 

Human lung cancer cell lines were obtained from Dr. Adi Gazdar, NCI-Navy 
Clinical Oncology Branch. The culture conditions varied according to the 
growth characterization of the cells. SCLC cell lines and carcinoids were 
grown as suspension cultures while NSCLC cell lines were maintained as monolayer 
cultures. All cells were grown in RPMI 1640 medium supplemented with 10% fetal 
bovine serum. All cell lines and tumor xenographs were diagnosed at the ultra- 
structural level by Dr. H.M. Schuller upon arrival at LETM (see Annual Report 
ZOl CM 07175-01 LETM). Further ultrastructural analysis under various experi- 
mental conditions was done with an analytical electron microscope using the 
transmission mode at 80 KV. SCLC cell lines and carcinoids were analyzed 
for amine precursor uptake and decarboxylation (APUD) features such as 
formaldehyde-induced fluorescence (FIF); L-dopadecarboxylase (DDC) activity; 
cytoplasmic dense-cored granules; production of the polypeptide hormone, 
calcitonin and bombesin, and intracellular 5-HT content assessed by immuno- 
peroxidase staining. These cell lines were also analyzed for the modulation 
of these APUD cell features by biogenic amines. The ability of the cell lines 
to produce prostglandins from arachidonic acid as well as to use arachidonic 
acid as a substrate for cooxidation reactions was carried out as part of a 
collaborative project with Dr. S. Lau (see Annual Report ZOl CM 07173-01 LETM). 
In order to assess whether the lung cancer cell lines possessed the metabolic 
machinery known to exist in specific normal lung cell types a variety of assays 
were performed. 4-Ipomeanol, a candidate anticancer drug with known target 
cell specificity toward normal rodent Clara cells, was used as a model substrate 
for cytochrome P-450 activity (a marker of pulmonary Clara cells). The metab- 
olism of 4-ipomeanol was monitored by covalent binding of labeled 4-ipomeanol. 
Known metabolic inhibitors and inducers were used along with these assays. 
Assays for specific enzymes involved in xenobiotic metabolism were carried out 
using standard techniques modified for cultured cells. The cytotoxicity of 4- 
ipomeanol as well as other candidate anticancer drugs, was monitored by colony 
forming assays and soft agar clonigenic assay systems. Analysis of phospholipid 
biosynthesis by the cell lines was monitored by ^^C-choline incorporation into 
cellular lipids and also secreted into the extracellular media. 



239 



ZOl CM 07153-02 LETM 
Major Findings : 

When the human lung cancer cell lines were analyzed for a wide variety of 
properties, the following results and observations were found: 

1. Human lung carcinoid cell lines and SCLC lines had both retained a number 
of functional properties which are the markers of their cell types of origin 

iE"jT?p'\'"''?-''"V^I]^- ^^^^^ ^'"^^"^^^ induction of dense-cored granules 
by 5-HTP, kinetics of FIF expression, and production of polypeptide hormones. 

?• ^°^^ well differentiated cell lines, both of the SCLC and NSCLC types were 
able to synthesize prostaglandins (see Annual Report ZOl CM 07173-01 LETM). 

If Killer *°i^^i^^ °^ ^•'^"J^ ^5^^ ^"""^^ Report ZOl CM 07122-05 LPET) to a panel 
of NSLC cell lines correlated with the cellular deoxycyti di ne kinase activity. 

4. Two NSCLC cell lines NCI-H322 and NCI-H358, which retain many properties of 
Clara cells and alveolar type II cells respectively, possess cytochrome Puf^ 
dependent monooxygenase activity. Furthermore, other microsomal enzymes such 
as cytochrome bg. NADPH cytochrome c reductase and epoxide hydrolase arl llso 
expressed. 

5. The metabolism-dependent covalent binding of 4-ipomeanol to cellular pro- 
teins was evident only in bronchi olo-alveolar carcinoma derived cell lines and 
was absent in the SCLC cell lines. The metabolism to the reactive intermediate 
was oxygen dependent, inducible, substrate concentration and time-dependent. 

6. Cytotoxicity of 4-ipomeanol to NSCLC cell lines was greater in the cell 
line with Clara cell features than in other cell lines. 

7. The NCI-H358 cell line which has characteristics of alveolar type II cells 
had retained the marker function of its cell of origin to convert choline into 
phosphatidyl choline, an essential component of lung surfactant. 

Significance to Biomedical Research and the Program of the Institute ; 

Lung cancer in man presents vast problems to the scientist both from a clinical 
standpoint and from basic cell biology. Therapeutic responses are difficult to 
predict in part because most human lung tumors are heterogeneous and often con- 
tain malignant cells of different functional and morphological types. Although 
recent studies on the various cell types in mammalian lung during normal dif- 
ferentiation and in response to injury have added much to our knowledge, the 
cellular mechanism responsible for the generation of heterogeneity and the 
interconversions of cell types seen in malignancy are poorly understood. De- 
tailed studies on the cell biology of human cancer cell lines under controlled 
in mro conditions may aid in our understanding of these complex processes and 
help design more appropriate therapies. Moreover, well differentiated cell 
lines at early passages represent a valuable tool to study cell -type specific 
biology, biochemistry and pharmacology of identified lung cell types. In the 
absence of in vitro systems to study individual normal types of lung cells, 
such data will much improve our understanding of functional aspects mediated by 
cell type in the normal lung. 

240 



ZOl CM 07153-02 LETM 

Proposed Course : 

The acquisition and characterization of more human cell lines of specific cell 
types is the key component in the proposed future course of this project. We 
plan to use as many early passage cell lines as possible established from human 
tumors which have been diagnosed and characterized at the electron microscope 
level (see Annual Report ZOl CM 07175-01 LETM). In this way we can more ac- 
curately correlate our observations in vitro with those encountered in vivo . 
Studies on the chemical and biochemical properties of these cell lines will be 
expanded both in our laboratory as well as through collaborative projects 
inside and outside of NIH. This project will work closely with the LCDDP, as 
well as the new DCT lung cancer drug discovery project, in order to provide 
some basic science support for the mechanisms of action of newly acquired 
cancer chemotherapeutic agents. 

Publication : 

1. McMahon, J.B., Reznik-Schii'ller, H.M., Gazdar, A.F. and Becker, K.L.: 

Influence of priming with 5-hydroxy tryptophan on APUD-characteri sties in 
human small cell lung cancer cell lines. Lung 162: 261-269, 1984. 



241 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07154-02 LETM 



PERIOD COVERED 



October 1, 1984 to September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) 

Isolation, Characterization & Selective Growth of Rodent Luna Cell Type<; Tn Vitrn 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atllllaSonJ' 

PI: J. B. McMahon Cancer Expert LETM, NCI 



Others: A. C. Smith 

A. A. del Campo 

D. Gronski 

H. M. SchuHer 



Staff Fellow 
Bio. Lab. Tech. 
Bio. Lab. Aid 
Vet. Med. Officer 



LETM, NCI 

LETM, NCI 

LETM, NCI 

LETM, NCI 



COOPERATING UNITS (it any) 



LAB/BRANCH 



Laboratory of Experimental Therapeutics and Metabolisr 



SECTION 

Pathology and Ultrastructural Oncology Section 

INSTITUTE AND LOCATION 

NCI, NIH. Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

1.0 



PROFESSIONAL: 
0.5 



0.5 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



n (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

It is well established that certain pulmonary toxins and carcinogens act on spe- 
cific cell types of the rodent lungs. Metabolism studies in vivo , or experiments 
using whole organ homogenates and fractions, may therefore not be suited to detect 
metabolic pathways operative in such specific cell types. It is the objective of 
this project to isolate and selectively grow the major epithelial cell types of 
the rat and hamster lung for comparative studies on their biology and response to 
toxins, carcinogens and anticancer drugs. These defined cell populations will be 
used for comparative studies on the effects of various pulmonary agents on human 
lung cancer cell lines (see Annual Report ZOl CM 07152-02 LETM). Immunoperoxidase 
staining of isolated rat lung epithelial cells with an antibody preparation speci- 
fic for rat surfactant apoproteins has revealed that rat alveolar type II cells 
can be purified to 95% by selective plating and proliferation. These cells which 
proliferated in vitro were shown to retain their epithelial morphology and con- 
tained osmiophi lie bodies for 5 days in culture. This specific antisera prepara- 
tion revealed that the isolated type II cells retained the capability to produce 
surfactant-like material. Using immunoperoxidase method, two patterns of staining 
were observed, one being a diffuse cytoplasmic stain and the other consisting of 
intensely stained cytoplasmic structures. These different patterns may reflect 
different stages of maturation of lamellar structures during the production and 
secretion of lung surfactant apoprotein. Analysis of phospholipid biosynthesis 
revealed that the isolated alveolar type II cells actively converted choline into 
phosphatidylcholine and that the prosynthetic activity increased with time in 
culture. The morphology of the isolated type II cells as well as other isolated 
cell types was characterized by a newly established technique which allows for 
correlative morphological characterization by light and electron microscopy. 

242 



PHS 6040 (Rev. 1/84) 



GPO 9l4.«ta 



ZOl CM 07154-02 LETM 

Objectives : "^ 

Many pulmonary toxins and carcinogens act on specific cell types in the lung. 
These cells are capable of activating potentially toxic or carcinogenic com- 
pounds present in the circulation and thus can mediate the toxic and/or car- 
cinogenic responses. Due to the heterogeneous nature of the lung, investiga- 
tions on the metabolic activation of endogenous and exogenous substrates by 
specific cell types in vivo have often been difficult to interpret. The 
objectives of this study are to develop methods for the isolation and pur- 
ification of specific cell types from the lungs of animals that have known 
susceptibility to certain toxins and carcinogens. Modification of existing 
technologies such as immunocytochemistry and correlative light and electron 
microscopy are being utilized to clearly identify the isolated cell popula- 
tions. Once established, these defined cell populations will allow the in- 
vestigation of the biochemical mechanisms of selected compounds at a cellular 
and subcellular level. The principal cells of interest include alveolar type 
II cells, Clara cells and endocrine cells, and in rat and hamster lungs. 
Compounds of known in vivo specificity for certain cell types such as BCNU, 
4-ipomeanol and di etRy 1 ni trosami ne are initially studied in these in vitro 
systems in order to establish if the isolated cells are functionally comparable 
with their counterparts in vivo . 

Methods Employed : 

Lung cell types are isolated using both published as well as newly developed 
techniques. Rat and hamster alveolar type II cells are isolated by intratracheal 
elastase digestion. Macrophages from both species are isolated by lavage and 
primary adherence. Interstitial fibroblasts are isolated by collagenase 
digestion of lung tissue that had been pretreated with elastase to remove the 
epithelial cell component. Clara cells are purified from elastase treated 
lungs by differential plating and density gradient centrifugation. A variety 
of techniques are used to identify and quantitate the isolated lung cell 
preparations. These include the use of antibody preparations obtained through 
a collaborative effort with Dr. Singh at the University of Pittsburg School fo 
Medicine VA Medical Center. One of these immunological reagents is specific 
for the surfactant apoprotein secreted by type II cells while another reacts 
specifically with secretory proteins of Clara cells. Immunological staining 
utilizes the peroxidase antiperoxidase method using diaminobenzodine as the 
indicator. The metabolism of 4-ipomeanol was monitored by covalent binding to 
cell macromolecules. Phospholipid biosynthesis was monitored by the incorpora- 
tion of ^*C-choline into cellular and secreted phosphatidylcholine. Correlative 
light and electron microscopy were done as follows: Cells were grown on Aclar 
film (Allied Chemical) on which a locator grid was drawn with India ink. After 
photomicroscopical documentation of colonies of interest under the inverted 
photomicroscope, the same film with the attached cells is processed for trans- 
mission electron microscopy. The resulting EPON replicas are stained with 
Toluidine Blue for high resolution bright field light microscopy, allowing 
the precise localization of specific cell colonies previously photographed. 
These areas are then trimmed and thin sectioned for further evaluation by 
transmission electron microscopy. 



243 



ZOl CM 07154-02 LETM 
Major Findings : 

Using isolated lung cell preparations under various experimental conditions, 
the following results and observations were found: 

1. Alveolar type II cells can be isolated from rat lung with a viability of 

96 ± 3% (n=20). After primary plating (24 hr), 75% of the attached cells react 
with the anti-rat surfactant apoprotein antiserum, the attached cells prolifer- 
ate for approximately 7 days and by the 4th day, 94% of the cells present are 
type II cells as shown by both immunoreactivity and electron microscopy. 

2. The isolated rat alveolar type II cells incorporated ^'^C-choline into 
cellular phosphatidylcholine (PC) and some of the l^C-PC was secreted into the 
media. There was a 200% increase in PC biosynthesis after 48 hr in culture and 
a 20% increase after 72 hr while the secretion rate of ^^C-PC remained constant 
for 72 hr in culture. 

3. The metabolic activation of ^H-ipomeanol was studied in vitro in isolated 
lung cell preparation from rats and hamsters. Covalent binding of ^H-ipomeanol 
was significantly higher in hamster lung cells than in rat lung cells. The 
binding in both species was found to be dependent on the cytochrome P-450 
monooxygenase system. 

Significance to Biomedical Research and the Program of the Institute : 

The development of procedures for the isolation and characterization of speci- 
fic lung cell types is essential for the study of the mechanisms of toxicity 
and carcinogenesis involving epithelial cell types of the lungs. Specific lung 
cell populations isolated from animals of various species may provide insight 
into the species and tissue specificities often displayed by pulmonary toxins 
and carcinogens. Manipulating the cells under controlled conditions may allow 
for the development of strategies to intervene in the toxic or carcinogenesis 
process. Preparations of isolated rodent lung cell types which have been 
recognized as targets of xenobiotics through in vivo experimentation present a 
useful tool to elucidate critical cell type-specific pathways responsible for 
the selective effects. A typical example for this approach is presented in 
a collaborative study (see Annual Report ZOl CM 07140-03 LETM) which reports 
the effect of BCNU on rat alveolar type II cells in vitro , a model system for 
BCNU-induced fibrosis in man. 

Proposed Course : 

The future efforts of this project will include improving the techniques for 
lung cell isolation and identification. A large portion of the efforts will be 
devoted to the purification of hamster and rat Clara cells and APUD cells and 
methods to clearly identify these cell types in vitro . Comparative studies 
will be undertaken to examine the possible causes for the species and tissue 
specificities of model compounds such as BCNU, DEN and 4-ipomeanol. Specific 
immunological reagents will be used in future studies for characterization and 
identification especially with respect to Clara cells and type II cells. 
Another important direction for this project involves the development of methods 



244 



ZOl CM 07154-02 LETM 

to facilitate the morphological characterization of the isolated lung cell 
preparations. This is critical in that techniques that attempt to isolate 
specific cell types from organ systems, i.e., lung, depend on reliable methods 
of ultrastructural identification in addition to biochemical and/or histochemical 
investigations. 

Therefore, we will develop correlative techniques that will combine the conven- 
ience and large sampling capability of light microscopy with ultrastructure 
evaluation to aid in the identification of particular cell types. 

Publication : 

1. McMahon, J.B., Smith, A.C., del Campo, A. A., Sing, G., Katyal , S., and 
Schuller, H.M.: Characterization of proliferating rat alveolar type II 
cells in vitro by immunological biochemical and morphological criteria. 
Exp. Lung Res. , in press. 



245 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PERIOD COVERED ' " 

October 1, 1984 to September 30. 1985 

TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) 



PROJECT NUMBER 

Z01 CM 07175-01 LETM 



Ultrastructural Classification of Human Lung Cance rs and Cell Lines 

^RINCIPAl IWVPRTiRATnn /; ,>. «,m„ ......_., ._,_ — 7. — — — : — :^ ■ ' — *- ""=^ 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: H. M. Schuller Vet. Med. Officer LETM, NCI 



Others: A. A. del Campo 
M. Gregg 
S. Walton 



Bio. Lab. Tech. 

Chemist 

Bio. Lab. Aid 



LETM, NCI 
LETM, NCI 
LETM. NCI 



COOPERATING UNITS (if any) 



Clinical Oncology Branch, NCI-Navy (A. F. Gazdar) 



LAB/BRANCH " ' 

Laboratory of Experimental Therapeutics and Metabol i sm 



SECTION 

Pathology and Ultrastructural Oncology Section 

20205 



INSTITUTE AND LOCATION 

NCI, NIH. Bethesda, Maryland 



TOTAL MAN-YEARS: 

1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



PROFESSIONAL: 

0.5 



OTHER: 



0.5 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) " 

The histopathology classification of lung cancers and cell lines is based on cri- 
teria detectable by light microscopy such as cell size and shape as well as growth 
patterns. Attempts to characterize the tumors by cell type are generally beyond 
the resolution of light microscopy. This is the reason Sy tumors of a given 
histopathology type may be comprised of a large variety of different cell types 
rhi?!^.r'*";;'5 °^ '•^\'- ^^' ^^^^tio^ of cens and tissues including tumors to 
chemicals and drugs is largely dictated by their biochemical machinery which in 
turn IS cell type specific. Efforts to study the response of lung tumors and cell 
lines to chemicals and drugs require, therefore, that the tumors be classified by 
cell type and differentiation using electron microscopy. 

We therefore investigate surgical samples of lung cancers as well as the cell 
lines derived from them by electron microscopy and classify them by cell type and 
differentiation. Since it is presently not known to what extent the HCCL change 
during prolonged life in vitro , we also monitor the cell lines at early and late 
kL-^^^^-' '^° i^^^l "^ ^?^® processed 31 surgical samples which are currently 
being diagnosed. A total of 25 cell lines has thus fSr been investigated which 
resulted in modified or altered diagnosis in many cases. Some of the lines were 
examined at early and late passages and there is evidence that, in particular, 
well differentiated tumors tend to change morphologically after several passages 
in mro. We are using the ultrastructural classification of tumors and cell 
lines as a basis for the design of comparative biochemical and pharmacological 
experiments (within LETM and in collaboration with other institutes) aimed at 
elucidating cell type specific reactions. 



246 



PHS 6040 (Rev. 1/84) 



GPo ei4-ei8 



ZOl CM 07175-01 LETM 

Objectives : 

The histopathology diagnosis of lung cancers and cell lines derived from them 
is insuficient in that it does not allow for a classification by cell type and 
differentiation. However, the response of cells including tumor cells is 
largely mediated by their biochemical machinery which in turn is cell type and 
differentiation dependent. We therefore subclassify surgical samples of human 
lung tumors and cell lines derived from them by electron microscopy to provide 
this vital information. The ultrastructural classification is then used as a 
basis to select tumors and cell lines for experiments on biology, metabolic 
competence and response to drugs (including anticancer drugs). It is antici- 
pated that by comparing such data derived from material of known cell type and 
degree of differentiation cell type-specific behavior and reactions will be 
detected. This information is vital for all research within LETM on various 
biological and biochemical aspects of lung cancers and cell lines. Moreover, 
these data represent a much needed asset for the interdisciplinary DCT lung 
cancer drug discovery project which is using the very same cell lines for drug 
screening. 

Methods Employed : 

Surgical samples (supplied by the NCI-Navy Clinical Oncology Branch, Dr. Gazdar) 
are processed for high resolution light-microscopy and transmission electron 
microscopy according to standard procedures. All samples are divided into 
osmicated and non-osmicated material to allow for immunocytochemistry and/or 
special stains, if required. Cell lines are processed similarly with slight 
modifications to enhance the contrast. All samples (surgical and cell lines) 
are diagnosed histopathologically by high resolution light microscopy and 
photomicrographs of representative areas are taken for recording. Such 
representative areas are then thin sectioned for diagnosis at the electron 
microscopic level. EM diagnosing and photography are done using a JEOL 100 CX 
electron microscope operated at 60 KV. The ultrastructural morphology of 
surgical samples is compared with the cell lines derived from them as are 
early and late passages of individual cell lines. 

Major Findings : 

Of the surgical samples investigated to date, one bronchiolo-alveolar carcinoma 
was subclassified as an endocrine tumor due to the presence of dense-cored 
endocrine- type cytoplasmic granules. In another case, a large cell carcinoma 
was reclassified as oncocytoma with possible steroid hormone production because 
of abundant mitochondria with tubular christae. Of the 25 cell lines studied 
to date, seven resulted in an ultrastructural diagnosis different from the 
original histopathology diagnosis of their tumors of origin. Moreover, ultra- 
structural classification added the subclassification of adenocarcinomas by 
cell type which lead to the identification of a line with features of pulmonary 
Clara cells (NCI-H322) and alveolar type II cells (NCI-H358), respectively, 
which were used for studies on metabolism of di ethyl nitrosamine and 4-ipomeanol 
as well as phospholipid synthesis and prostaglandin synthetase activity in 
collaboration with other investigators within LETM and within PUOS. 



247 



Z01 CM 07175-01 LETM 

Studies of early and late passages of a cell line (NCI-H460) derived from a 
large cell carcinoma resulted in reclassification of the early passage into 
small cell cancer because of the presence of dense-cored endocrine- type 
granules and demonstrated the gradual transition of such cells into oncocytes 
in the later passages. Electron microscopic diagnosis of early passages - 
derived from four suspected carcinoid- tumors confirmed the tentative histo- 
pathology diagnosis and resulted in the identification of one unique well 
differentiated carcinoid cell line (NCI-H727) with features of normal pulmonary 
endocrine cells. This cell line is of much interest to study the physiological 
role of normal pulmonary endocrine cells and of their reaction to chemicals and 
drugs. 

Significance to Biomedical Research and the Program of the Institute : 

The outlined ultrastructural classification of lung cancers and cell lines by 
cell type and differentiation is the vital basis for all ongoing and future 
research on lung cancer biology and therapy within LETM and to the OCT lung 
cancer drug discovery project. It makes it possible to detect and compare cell 
type and differentiation dependent biological, biochemical and pharmacological 
properties. Such data will substantially further our understanding of func- 
tional aspects of normal cell types and of the cancers derived from them and 
will pave the way towards a disease-oriented "custom-tailored" cancer therapy. 

Proposed Course : 

This is a long-term project that will have to run over several years in order 
to collect statistically significant data. While during the current initial 
period the major emphasis is on diagnostic aspects, future studies will also 
include electron microscopic investigations on the subcellular effects of 
chemicals and drugs. Such data are anticipated to generate insight into intra- 
cellular targets at the organelle level which can then be used for experiments 
focused on biochemical mechanisms of chemicals and drugs. 

Publication : 

1. Schuller, H.M., del Campo, A., McMahon, J.B., Oie, H.B., Boyd, M.R. and 
Gazdar, A.F.: Ultrastructural classification of human lung cancer cell 
lines. JNCI, in press. 



248 



ANNUAL REPORT OF THE LABORATORY OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

The Laboratory of Pharmacology and Experimental Therapeutics (formerly the 
Laboratory of Medicinal Chemistry and Biology) was established in 1975 in order 
to provide a facility capable of antitumor drug development from the stage of 
design and synthesis through biochemical and pharmacological characterization 
to Phase I clinical trial. The organizational components are the Medicinal 
Chemistry Section, concerned with the rational design and synthesis of new 
agents, and the Biochemical Pharmacology Section, concerned primarily with 
fundamental mode of action studies. In addition, the Laboratory as a whole 
develops analytical methodology for the detection of antitumor drugs and their 
metabolites, and also participates actively in the clinical pharmacology /Phase 
I trials of new agents which either originated within the Laboratory or which 
were derived from extramural sources but in whose preclinical development the 
Laboratory has played a major role. 

During the past year, the design and synthesis of potential differentiating 
agents has continued within the Medicinal Chemistry Section of the Laboratory. 
At least 12 recently synthesized compounds have proven to be as active as 1- 
methyl-2-pyridone (MP), the prototype reference compound, while several from 
among this group have been found to be significantly more potent than MP in 
terms of differentiating activity, utilizing the HL-60 test system. The most 
active compound found to date is a cyclopropyl analog of 2-pyridone. 

We have recently shown that arabinosyl-5-azacytosine (ara-5-AC), a compound 
originally synthesized within LPET and presently under active development toward 
clinical trial, exhibits unexpectedly high anticerebral antitumor activity in 
the IC LI 210 murine test system (i.e., activity greater than that of ara-C, and 
comparable to that of the prototype reference compound, BCNU). In addition, we 
have recently developed a rapid and sensitive HPLC assay for the quantitation 
of ara-5-AC in biological fluids. These findings will be of direct applicability 
in the projected Phase I/I I clinical trials of this compound shortly to be 
initiated by OCT. 

The LPET compound, 2-oxypyrimidine riboside (zebularine), shows significant, 
although not outstanding, activity in the LI 210 and 816 melanoma test systems. 
We have recently found this compound to exhibit unusually high activity against 
both DNA virus (herpes simplex and adenovirus) and RNA virus (poliovirus Type 
III and dengue virus) test systems. The mechanism for this unanticipated 
activity is now being explored. 

Analytical methodology has been developed for the determination of the LPET- 
developed compound spirohydantoin mustard (spiromustine), and clinical pharma- 
cology studies are being actively pursued in collaboration with COP, OCT, with 
NINCDS, NIH and also with extramural institutions participating in the Phase I 
trial of this agent. 



249 



In last year's Annual Report, a total synthesis of the antitumor antibiotic 
Neplanocin A was described. This synthetic scheme has now been shortened and 
improved, and further adapted for utilization in the synthesis of other purine 
and pyrimidine cyclopentenyl nucleoside analogs of the neplanocin class. This 
series is of interest because of the antitumor activity of many of these 
compounds, and also because of their other biological properties such as 
antiviral activity and inhibition of the enzyme uridine-cytidine kinase. 

In another area of synthesis, all the possible phosphonate analogs of the ac- 
tive metabolite of TAD (the active anabolite of tiazofurin) have now been 
prepared. One of these {3-methylene TAD) was equipotent to TAD as an IMPD 
inhibitor and possesses the advantage of being resistant toward cleavage by 
TAD-inactivating enzymes. 

In studies of differences between metastatic cells and their non-metastatic 
counterparts, an L1210 system utilizing tumor cells isolated from murine liver 
has been utilized. Marked differences have been observed between liver meta- 
static L1210 tumor cells and their ascitic counterparts, e.g., a 2-fold in- 
crease in glutathione concentration and a 5-fold greater level of surface 
SH-groups in the liver metastatic cells. The metastatic cells are more resis- 
tant to melphalan, but can be resensitized by blocking the surface sulfhydryls 
with 6,6-dithionicotinic acid. 

Studies have continued within LPET on the process of tubulin assembly and on 
the effect of known and potential antimitotic drugs on this process. One of 
the "microtubule-associated proteins" (MAPS), has now been purified to homo- 
geneity; this protein causes the formation of microtubule "bundles" (distinct 
microtubules which aggregate laterally). The active component adheres to 
hydroxyl apatite but not to DEAE-cellulose, is heat stable, and can be recon- 
stituted after denaturation in 8 M urea. The fraction has now been purified by 
preparative HPLC, resulting in an electrophoretically homogeneous protein made 
up of two subunits with molecular weights ca. 25,000. Extensive studies on new 
tubulin inhibitors (combretastatin, 6-benzyl-l ,3-benzodioxole derivatives, 5,6- 
diarylpyridazin-3-one derivatives) have also been initiated, with a view to 
establishing their site and mechanism of action at the molecular level. 

In the area of drug resistance, studies have been carried out on both natural 
and acquired resistance to ara-5-AC, an LPET compound soon to enter Phase I 
clinical trial. In most test systems, resistance was most commonly accompanied 
by a decreased level or in some cases, by a complete deletion of deoxycy ti di ne 
kinase, the enzyme responsible for anabolic activation of the drug. In terms 
of catabolism, ara-5-AC, unlike ara-C, is not inactivated by mammalian cytidine 
deaminase, an observation which may be of relevance in initial clinical dose- 
scheduling of the drug. 

The new agent, merbarone (NSC 336629), has been extensively examined in terms 
of possible mechanism of action. The compound is an effective but atypical 
inhibitor of DNA synthesis; among a number of factors examined, this inhibition 
appears to correlate most closely with the ability of the compound to generate 
single-strand DNA breaks which do not, however, appear to be protein-associated. 
Investigation of the molecular pharmacology of this unusual wide-spectrum 
antitumor agent is continuing. 



250 



In conjunction with its research output in fields related to drug development, 
the Laboratory of Pharmacology and Experimental Therapeutics continued its 
active publication record in 1984-85. A total of 52 papers describing these 
and related studies appeared or were accepted for publication during the cur- 
rent year; these publications are listed in the following section of this 
report. 

Publications : 

1. Batra, O.K., Jurd, L. and Hamel , E.: Structure-function studies with 
derivatives of 6-benzyl-l,3-benzodiazole, a new class of synthetic compounds 
which inhibit tubulin polymerization and mitosis. Mol. Pharmacol ., 27: 
94-102, 1985. 

2. Batra, J.K., Lin, CM., Hamel, E., Jurd, L. and Powers, L.J.: New anti- 
neoplastic agents with anti tubulin activity. Ann. N.Y. Acad. Sci . , in 
press. 

3. Bender, R.A. and Hamel, E.: Vinca alkaloids. In: Pinedo, H.M. and Chabner, 
B.A. (Eds.): Cancer Chemotherapy Annual, Vol. 6 , Amsterdam, Elsevier 
Science Publishers, 1984, pp. 100-110. 

4. Carlson, J. A., Litterst, C.L., Greenberg, R.A., Day, T.G. and Masterson, 
B.J.: Platinum tissue concentrations folowing intra-arterial and intravenous 
cis-diamminedichloroplatinum-II in New Zealand white rabbits. Am. J. 
Obstet. Gynecol . 148: 313-317, 1984. 

5. Carney, D.N., Ahluwalia, G.S., Jayaram, H.N., Cooney, D.A. and Johns, 
D.G.: Relationships between the cytotoxicity of tiazofurin and its 
metabolism by cultured human lung cancer cells. J. Clin. Invest . 75: 175- 
182, 1985. 

6. Cooney, D.A., Jayaram, H.N., Ahluwalia, G., Dion, R.L., Zwelling, L.A., 
Kerrigan, D. and Johns, D.G.: Pharmacologic and physiologic consequences 
of resistance to tiazofurin. In Spitzy, K.H. and Karrer, K. (Eds.): 
Proc. 13th International Congress of Chemotherapy . Verlag H. Egermann, 
Vienna, 1984, pp. 261/46-261/52. 

7. Cooney, D. and Kutzmits, R.: Pharmacology of anticancer drugs. Cancer 
Treat. Rev . 11, 1984. 

8. Cooney, D.A., Stergis, G., Jayaram, H.N.: Enzymes, Therapeutic. In Kirk, 
R.E. and Othmer, D.F. (Eds.): Concise Encyclopedia of Chemical Technology . 
New York, John Wiley & Sons, 1985, pp. 429-430. 

9. Curt, G.A., Kelley, J. A., Find, R.L., Huguenin, P.N., Roth, J.S., Batist, 
6., Jenkins, J. and Collins, J.M.: A phase I and pharmacokinetic study of 
dihydro-5-azacytidine {NSC-26488). Cancer Res ., in press. 

10. Farris, F.F., King, F.G., Dedrick, R.L. and Litterst, C.L.: Physiological 
model for the pharmacokinetics of cis-dichlorodiammineplatinum (II) (DDP) 
in the tumored rat. J. Pharmacokin. Biopharm., in press. 



251 



11. Gebeyehu, G., Marquez, V.E., Van Cott, A., Cooney, D.A., Kelley, J. A., 
Jayaram, H.N., Ahluwalia, G.S., Dion, R.L., Wilson, Y.A. and Johns, D.G.: 
Ribavirin, tiazofurin and selenazofurin: Mononucleotides and NAD dinucleo- 
tide analogs. Synthesis, structure and interactions with IMP dehydro- 
genase. J. Med. Chem. 28: 99-105, 1985. 

12. Gram, T.E.: The pulmonary mixed function oxidase system. In Witschi, 
H.P. and Brain, J.D. (Eds.): The Toxicology of Inhaled Materials. Part 
I: General Principles of Inhalation Toxicology . Berlin, Springer-Verlag, 
1985, pp. 421-470. 

13. Green, J. A., Vistica, D.T., Young, R.C., Hamilton, T.C., Rogan, A.M., 
Whang-Peng, J. and Ozols, R.F.: Melphalan resistance in human ovarian 
cancer: Characterization of drug resistant cell lines and potentiation of 
melphalan cytotoxicity by glutathione depletion. Cancer Res . 44: 5427-31, 
1984. 

14. Hamel , E.: Antimitotic drugs and tubulin-nucleotide interactions. In 
Glazer, R.I. (Ed.): Developments in Cancer Chemotherapy , Boca Raton, FL, 
CRC Press, 1984, pp. 131-164. 

15. Hamel, E. and Lin, CM.: Separation of active tubulin and microtubule- 
associated proteins by ultracentrifugation and isolation of a component 
causing the formation of microtubule bundles. Biochemistry 23: 4173-4184, 
1984. 

16. Hamel, E. and Lin, CM.: Guanosine 5 '-£-(3-thiotri phosphate): a potent 
nucleotide inhibitor of microtubule assembly. J. Biol . Chem. 259: 11060- 
11069, 1984. 

17. Hamel, E. and Ling, CM.: Dideoxy guanosine nucleotides and microtubule 
assembly. Ann. N.Y. Acad. Sci. , in press. 

18. Hamel, E., Lustbader, J. and Lin, CM.: Deoxy guanosine nucleotide analogs: 
potent stimulators of microtubule nucleation with reduced affinity for the 
exchangeable nucleotide binding site of tubulin. Biochemistry 23: 5314- 
4325, 1984. 

19. Huang, A.B., Lin, CM. and Hamel, E.: Maytansine inhibits nucleotide 
binding at the exchangeable site of tubulin. Biochem. Biophys. Res. 
Commun ., in press. 

20. Huguenin, P.N., Jayaram, H.N. and Kelley, J. A.: Reverse phase HPLC 
determination of 5,6-dihydro-5-azacytidine in biological fluids. J. Liquid 
Chromatogr . 7: 1433-1453, 1984. 

21. Huguenin, P.N. and Kelley, J. A.: The synthesis of spiromustine-da. A 
general approach to octadeuterated nitrogen mustards. J. Labelled Compds. 
Radiopharm. , in press. 



252 



22. Jayaram, H.N. and Johns, D.G.: Metabolic and mechanistic studies with 
oncolytic C-nucleoside, tiazofurin (2-3-D-ribofuranosylthiazole-4-carbox- 
amide, NSC-286193). In Glazer, R.I. (Ed.): Developments in Cancer Chemo- 
therapy . Boca Raton, FL, CRC Press, 1984, pp. 115-130. 

23. Kelley, J. A., Roth, J.S. and Litterst, C.L.: Gas chromatographic 
determination of hexamethylene bisacetamide in plasma and urine. Anal . 
Letters , in press. 

24. Krijgsheld, K.R. and Gram, T.E.: Selective induction of renal microsomal 
cytochrome P-450-llnked monooxygenases by 1,2-dlchloroethylene in mice. 
Biochem. Pharmacol ., 33: 1951-1956, 1984. 

25. Krijgsheld, K.R., Lowe, M.C., Mimnaugh, E.G., Trush, M.A., Ginsburg, E. 
and Gram, T.E.: Selective damage to nonciliated bronchiolar epithelial 
cells in relation to impairment of pulmonary monooxygenase activities by 
1,1-dichloroethylene in mice. Toxicol. Appl. Pharmacol . 74: 201-213, 1984. 

26. Lim, M-I., Moyer, J.D., Cysyk, R.L. and Marquez, V.E.: Cyclopentenyl- 
uridine and cyclopentenylcytidine as inhibitors of uridine-cytidine kinase. 
J. Med. Chem. 27: 1536-1538, 1984. 

27. Litterst, C.L.: Cisplatinum: A review, with special reference to cellular 
and molecular interactions. Agents and Actions 15: 520-525, 1980. 

28. Litterst, C.L.: Toxicology of the antineoplastic agents with special 
reference to reproductive toxicology, carcinogenesis and teratogenesis. 
In Haley, T. and Berndt, W. (Eds.): Handbook of Toxicology . New York, 
Hemisphere Publ., in press. 

29. Litterst, C.L. and Copley, M.: Cisplatin and Doxorubicin: Comparative 
evaluation of nephrotoxicity using serum chemistry, urinalysis and his- 
tology. In Bach, P. (Ed.): Renal Heterogeneity and Target Cell Toxicity . 
New York, Wiley & Sons, in press. 

30. Litterst, C.L. and Reed, E.: Platinum Compounds. In Kaiser, H.E. (Ed.): 
Profressive Stages in Neoplastic Growth . Chapter 196, Oxford, Pergamon 
Press, in press. 

31. Litterst, C.L., Roth, J.S. and Kelley, J. A.: Distribution, elimination, 
metabolism and bioavailability of hexamethylenebisacetamide in rats. Inv. 
New Drugs , in press. 

32. Litterst, C.L., Smith, M., Smith, J., Uozumi , J. and Copley, M.: Sensitiv- 
ity of urinary enzymes as indicators of renal toxicity of the anticancer 
drug cisplatinum. In Bianchi, C. and Blaufox, D.M. (Eds.): Newer Diag - 
nostic methods in nephrology and urology . Basel, Switzerland, Karger 
Publ. Co., 1985. 

33. Mao, D.T. and Marquez, V.E.: Synthesis of 2-3-D-ara and 2-3-D-xylo- 
furanosylthiazole-4-carboxamide. Tetrahedron Lett. 25: 2111-2114, 1984. 



253 



34. Marquez, V.E.: Inhibition of cytidine deaminase: Mechanism and effects on 
the metabolism of antitumor agents. In Glazer, R.I. (Ed.): Developments 
in Cancer Chemotherapy . Boca Raton, FL, CRC Press, 1984, pp. 91-114. 

35. Marquez, V.E. and Lim, M-I.: Carbocyclic nucleosides. Medicinal Research 
Reviews, in press. 

36. Mohindru, A., Fisher, J.M. and Rabinovitz, M.: Endogenous copper is 
cytotoxic to a lymphoma in primary culture which requires thiols for 
growth. Experientia , in press. 

37. Okine, L.K., Goochee, J.M. and Gram, T.E.: Studies on the distribution 
and covalent binding of 1,1-dichloroethylene in the mouse: Effect of 
various pretreatments on covalent binding in vivo . Biochem. Pharmacol ., 
in press. 

38. Okine, L.K., Lowe, M.C., Mimnaugh, E.G., Goochee, J.M. and Gram, T.E.: 
Protection by methyl prednisolone against butyl ated hydroxytoluene-induced 
pulmonary damage and impairment of microsomal monooxygenase activities in 
the mouse: lack of effect on fibrosis. Exp. Lung Res . , in press. 

39. Osman, N.M., Copley, M.P. and Litterst, C.L.: Amelioration of cisplatinum- 
induced nephrotoxicity by the diuretic acetazolamide in F344 rats. Cancer 
Treat. Rep . 68: 999-1004. 

40. Osman, N.M., Copley, M.P. and Litterst, C.L.: Effects of the diuretics 
mannitol or acetazolamide on nephrotoxicity and physiological disposition 
of cisplatinum in rats. Cancer Chemother. Pharmacol . 13: 58-62, 1984. 

41. Pierson, H.F.: Pharmacological perturbation of murine melanoma growth by 
copper chelates. Cancer Lett . 26: 221-233, 1985. 

42. Pierson, H.F.: Administration of copper chelates enhances tumorgenicity 
of B16 melanoma in heterogenetic mice. Cancer Treat. Rep . , in press. 

43. Pierson, H.F. and Meadows, G.G.: Nutritional and pharmacological modula- 
tion of peroxidation in murine melanoma. J. Natl. Cancer Inst . , in press. 

44. Poirier, M.C., Reed, E., Zwelling, L.A., Ozols, R.F., Litterst, C.L. and 
Yuspa, S.H.: The use of polyclonal antibodies to quantitate cis- diammine- 
dichloroplatinum (II)-DNA adducts in cancer patients and animal models. 
Environ. Health Perspect. , in press. 

45. Somfai-Relle, S., Suzukake, K., Vistica, B.P. and Vistica, D.T.: Gluta- 
thi one-conferred resistance to antineoplastics: Approaches toward its 
reduction. Cancer Treat. Rev . 11: 43-54, 1984. 

46. Sporn, M.B., Roberts, A.B. and Driscoll, J.S.: Growth factor and differ- 
entiation . In De Vita, V.T., Hellman, S. and Rosenberg, S.A. (Eds.): 
Cancer Principles and Practice of Oncology , Second Ed., Philadelphia, 
Lippincott, 1985, pp. 49-66. 



254 



47. Tong, S., Hirokata, Y., Litterst, C.L. and Gram, T.E.: Interaction of the 
oncolytic drug, l-(2-chloroethyl )-3-cyclohexyl-l-nitrosourea (CCNU) with 
the mixed function oxidase system in rats. Chem. Biol. Interact. 49: 105- 
120, 1984. 

48. Tono-oka, S, Tone, Y., Marquez, V.E., Cooney, D.A., Sekikawa, I., Azuma, 
I.: Enzymic synthesis and biochemical activity of various indazole adenine 
di nucleotides. Bull. Chem. Soc. Jpn . 58: 309-315, 1985. 

49. Trush, M.A., Reasor, M.J. and Van Dyke, K.: Oxidant-mediated electronic 
excitation of imipramine. Biochem. Pharmacol . 33: 3523-3526, 1984. 

50. Tyagi, A.K. and Cooney, D.A.: Biochemical pharmacology, metabolism and 
mechanism of action of L-alanosine, a novel natural antitumor agent. Adv. 
Pharmac. Chemother. 20: 69-121, 1984. 



51. Uozumi, J. and Litterst, C.L.: Effect of cisplatin on renal ATPase activ- 
ity in vivo and in vitro. Cancer Chemother. Pharmacol ., in press. 

52. Zaharko, D.S., Covey, J.M. and Kelley, J. A.: Plasma kinetics and effects 
of 5,6-dihydro-5-azacytidine in mice and L1210 tumor. Inv. New Drugs 3: 
35-41. 1985. 



255 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



Z01 CM 03580-16 LPET 



PERIOD COVERED 



October 1. 1984 to September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) 

Chemical Research in the Development of New Anticancer Drugs 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: J. S. Driscoll Head, Medicinal Chemistry LPET, NCI 

Section 



Others: V. E. Marquez 
A. Haces 



Visiting Scientist 
Visiting Fellow 



LPET, NCI 
LPET, NCI 



COOPERATING UNITS (U any) 

Laboratory of Biological Chemistry, Drug Evaluation Branch 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Medicinal Chemistry Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

1.7 



PROFESSIONAL: 

1.7 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues Q (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The objective of this work is the discovery of new drugs which are clinically 
useful against cancer. The following topics are of current interest: 1) synthe- 
sis and evaluation of differentiating agents , 2) plasma stability of spiromustine 
analogs, 3) ara-AC intracerebral antitumor activity, 4) antitumor, antiviral and " 
cytidine deaminase inhibition of zebularine and 5) potentiation of AZQ CNS anti- 
tumor activity. 



256 



PHS 6040 (Rev. 1/84) 



GPO »I4-9IS 



ZOl CM 03580-16 LPET 

Project Description : 

General Objective : 

The objective of this project is the discovery of new types of drugs which are 
clinically useful against cancer. Medicinal chemical research is directed 
toward the synthesis of new compounds which have potential as useful agents. 
Leads for this program are generated from structure-activity studies, the DTP 
screening program, the literature, and biochemical rationale. 

Specific Objectives : 

1. Synthesis of differentiating agents 

2. Synthesis of spiromustine analogs 

3. Ara-AC activity against IC LI 210 leukemia 

4. Activity of Zebularine 

5. Potentiation of AZQ CNS antitumor activity 

Synthesis of Differentiating Agents (Drs. Haces, Breitman, Driscoll) . A number 
of chemicals have been shown to cause certain murine or human tumor cells to 
differentiate into more mature cells with reduced proliferative properties. 
The mechanism of action of these materials is unknown. Hexamethylene bis- 
acetamide (HMBA) is one of these chemicals is presently the subject of an NCI 
sponsored Phase I clinical trial. It now appears that the concentration of 
HMBA which can be attained in humans may be marginal with respect to the 
concentrations anticipated necessary for differentiation. Therefore, new 
differentiating agents with higher potencies are required. Structure-activity 
studies were initiated in two series of compounds known to possess differen- 
tiating properties - the bisamides and the pyridones. 

HMBA Analogs . Shortly after the discovery of the differentiating proper- 
ties of HMBA, Marks and co-workers ( J.B.C. 253: 4214, 1978) published the only 
structure-activity study ever carried out for this series of compounds. It was 
confined mainly to amides, acids and amines and showed that relatively minor 
structural modifications (e.g., NMF to HMBA) could increase compound potency by 
a factor of 50 in the Friend erythroleukemia system. When it became apparent 
from our preclinical studies that attaining the sustained blood levels of HMBA 
that were required in vitro might be a problem in humans, we began a a further 
structure-activity study to see if unexplored structural modifications could 
produce an even more potent compound. Because considerable expertise already 
existed in the use of the HL-60 cell line for differentiation studies of this 
type (Dr. Breitman), this line was chosen as the in vitro test system. 

It was soon discovered, contrary to what might be expected from the literature, 
that HMBA (used as a positive control in all experiments) killed ca. 90% of the 
test cells at concentrations which caused 99% differentiation of the remaining 
10% viable cells. For this reason, a differentiation factor (DF) was devised 
which measures both differentiation and viability at a given concentration 
(DF = differentiation x viability). An ideal compound [producing 100% viability 
(1.0) and 100% differentiation (1.0)] would have a DF = 1.0. For HMBA (standard 
4 day exposure), DF is maximum (0.40) at a concentration of 3 mM, giving 59% 
differentiation with 67% viability. At 5 mM, although 96% differentiation of 



257 



Z01 CM 03580-16 LPET 



viable cells was achieved, 87% of the total cell population was dead, resulting 
in a DF value of only 0.12. 

Seventeen HMBA analogs were synthesized and chemically characterized. In 
addition to bis-amides, these included bis-imides and hydrazides. Five of 
these compounds produced DF values in the HMBA range (0.3-0.5) but at concentra- 
tions approximating that of the parent {1-3 mM) so no significant increase in 
potency was achieved. The bis-hydantoin (1) was one of the more structurally 
interesting actives discovered. Work on fWis series has been suspended. 



^-w-w 






Pyridone Analogs . Early work by Marks showed that 2-pyridone would cause 
differentiation in Friend cells. More recent work by Sartorelli has shown that 
certain analogs of this compound are as potent as HMBA in the Friend cell line 
and appear to be just as active. Because of a long-standing interest in the 
antitumor properties of 2- and 4-pyridones, seven new compounds were synthe- 
sized and a total of 24 of these materials evaluated for differentiating 
properties in the HL-60 system. 1 -Methyl -2-pyridone (MP), a compound shown by 
the Sartorelli group to produce 89% differentiation at 4 mM concentration, was 
used as a positive control in all experiments. In our studies, the DF value 
(see above) for MP remained approximately constant for 3, 4 and 5 mM concentra- 
tions (DF = 0.3) with percent differentiation increasing from 42% to 78% but 
with cell viability declining from 66% to 42%. 

At least 12 of the pyridones tested from our series proved to be as good as MP 
from the point of maximum DF value and potency. Several compounds were 
significantly better than either MP or HMBA. The most interesting compound 
discovered thus far is the cyclopropyl analog, 2, with DF = 0.7 at 30 \M and 
0.1 mM concentrations. Several 3-acyloxy-2-pyrTdones have proven active and 
this series will be explored further to find the optimum analog. 







1^ 



3 



258 



ZOl CM 03580-16 LPET 

Ara-AC Activity Against Intracerebral Tumor Models (Drs. Driscoll , Plowman, 
Johns). Ara-AC is an LPET compound currently in preclinical toxicology. Ft 
has a hybrid structure (3) containing the sugar of ara-C and the heterocyclic 
base of 5-azacytidine (5-AC). Ara-AC was chosen as a clinical candidate based 
on DN2 level activity against all three of the human tumor xenografts in the 
NCI tumor panel. Because of known ara-C activity against intracerebral (IC) 
tumor models, ara-AC was evaluated in direct comparison with its two parent 
drugs against IC L1210 leukemia in mice (IC tumor implant; IP drug treatment). 
Although 5-AC was active (ILS = 70%) in this system, it was much less active 
than the other two compounds. Ara-AC and ara-C were most active on an "around- 
the-clock" treatment schedule. While each compound produced 60 day survivors, 
ara-AC did so over a greater dosage range (Table 1). This general superiority 
was also observed: a) on the qd 1-9 treatment schedule, b) when a greater 
tumor burden was used, and c) in the IC P388 model. 

Table 1 

Comparison of Ara-C and Ara-AC Activity Against IC L1210 
Using the Q3HX8; Dl,5,9 Treatment Schedule 





Experiment 1 


Experiment 2 




7o ILS 


(LTS/6)* 


% ILS 


(LTS/6)* 


Dose 










(mg/kg/injection) 


Ara-C 


Ara-AC 


Ara-C 


Ara-AC 


75 


2 





Toxic 


48 


37.5 


11 


28 


11 


>640 (3) 


18.75 


>581 (5) 


>581 (5) 


>640 (5) 


>640 (4) 


9.38 


218 (2) 


>581 (5) 


196 


>640 (3) 


4.69 


115 


263 (1) 


146 (1) 


251 (1) 


2.34 


70 


193 


107 (1) 


>640 (3) 


1.17 


56 


161 (1) 


65 


134 


0.59 


5 


51 


39 


60 




[BCNU>581% (5)] 


[BCNU>640% (4)] 



*10^ cells IC implant. IP drug treatment, 
measured on day 60. 



Long term survivors 



259 



ZOl CM 03580-16 LPET 

Zebularine and its Analogs (Drs. Driscoll, Marquez. Plowman. Haces, Johns) . 
Zebulan'ne (4_) is a previously known compound which the LPET found had a number 
of interesting biological properties. 




HO OH 




"^'V"'^*^"*^^'^^*"*'-^"^*^'^'- 



Although 4^ has the simplest possible pyrimidine nucleoside structure, the com- 
pound possesses a wide spectrum of activities. Zebularine has: a) DN2 level 
LI 210 and B16 melanoma activity, b) cytidine deaminase (Cdase) inhibitory 
activity and c) as good or better antiviral activity as ribavirin against 
both RNA and DNA viruses. 

The Cdase activity of tetrahydrouridine (THU) is abolished in the presence of 
acid. Compound 4, as an acid stable compound, is being evaluated for oral 
administration wTth ara-C in an attempt to determine whether an oral ara-C 
formulation might be feasible. Initial in vivo experiments against L1210 
leukemia indicate that zebularine is at least as efficacious as THU in poten- 
tiating oral ara-C activity. Syntheses of the ara- and 2'-deoxy analogs are in 
progress. 

Potentiation of CNS Antitumor Activity (Drs. Driscoll and Cooney) . AZQ and the 
nitrosoureas are drugs active against CMS tumors but whose limiting toxicities 
are peripheral. If a rescue agent could be devised which would destroy peri- 
pheral AZQ (or nitrosourea) but was not able to cross the blood-brain-barrier 
(BBB), it is possible that the CNS concentrations of these antitumor drugs 
might be raised relative to that achieved with currently administered doses. 
While this would shift the limiting toxicity to the CNS, this should be accept- 
able and even desirable for brain tumor patients. Our prototype rescue agent, 
MESNA, is an ionic sulfhydryl nucleophile which should not cross the BBB. 
Initial in vitro studies showed no effect of MESNA on AZQ toxicity so addi- 
tional, more nucleophilic (but still ionic) agents are being sought. 

Spiromustine Analogs (Drs. Haces, Driscoll). Spiromustine (5^, n = 2) is an 
LPET compound undergoing both adult and pediatric Phase I brain tumor studies 
in the NIH Clinical Center. 



260 



ZOl CM 03580-16 LPET 

This compound is very hydrolytically unstable and a limited synthetic analog 
study was undertaken to determine the effect of the number of hydantoin-mustard 
methylene spacer groups on: a) compound stability in human plasma and b) anti- 
tumor activity. Analogs of 5 with n = 2-5 were prepared. As n increased, the 
compounds became more unstabTe. Methylene number had essentially no effect 
P388 antitumor activity. The effect of methylene number on neurotoxicity 
(spiromustine limiting toxicity) will be evaluated as soon as an appropriate 
model is devised. The synthetic aspects of this project have been terminated. 

Publication : 

1. Sporn, M.B., Roberts, A.B. and Driscoll, J.S.: Growth factor and dif- 
ferentiation. In De Vita, V.T., Hellman, S. and Rosenberg, S.A. (Eds.): 
Cancer Principles and Practice of Oncology , Second Ed., Philadelphia, 
Lippincott, 1985, pp. 49-66. 



261 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 03581-16 LPET 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line tietween the borders.) 

The Analytical Chemistry of New Anticancer Drugs 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alfiliation) 

PI: James A. Kelley Research Chemist LPET, NCI 



Others: 



J. Driscoll 
R. Heideman 
J. Roth 



Section Head 
I. P. A. 
Chemist 



LPET, NCI 
LPET, NCI 
LPET, NCI 



COOPERATING UNITS (if any) 

Medicine Branch, Clinical Pharmacology Branch, Pediatric Branch, COP, 
OCT; Surgical Neurology Branch, NINCDS 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Medicinal Chemistry Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 



20205 



TOTAL MAN-YEARS: 

2.5 



PROFESSIONAL: 
1.5 



1.0 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
n (a2) Interviews 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The objective of this project is the research and development of analytical 
methods which are used to: (1) establish the structure and purity of new anti- 
tumor agents and their metabolites, (2) determine physical and chemical proper- 
ties of new anticancer drugs, (3) quantitate drugs and their metabolites in 
biological samples to elucidate pharmacology and to determine pharmacokinetics , 
and (4) study reaction mechanisms of potentially useful sythetic transformations. 
Mass Spectrometry , gas chromatography and high-performance liquid chromatography , 
either alone or in combination, are emphasized techniques. Compounds of current 
interest are cy ti di ne analogs, cytidine deaminase inhibitors , modified nucleo- 
sides , oligonucleotides , nitrogen mustards and differentiating agents. The 
kinetics and products of the acid-catalyzed isomerization of reduced pyrimidine 
ribosides and tetrahydrouri di ne have been determined. 



262 



PHS 6040 (Rev. 1/84) 



GPO BI4-91B 



ZOl CM 03581-16 LPET 

Project Description : 

General Objective : 

The objective of this project is the research and development of analytical 
techniques for establishing the structure and purity of new anticancer drug 
candidates, determining their important physical and chemical properties, 
elucidating structures of metabolites of new antitumor agents, measuring these 
drugs and their metabolites in physiological samples and studying reaction 
mechanisms. Mass spectrometry (MS), gas chromatography (GC), high-performance 
liquid chromatography (HPLC) and the combination of these techniques are the 
emphasized methods. Other analytical methods such as NMR, UV and IR spec- 
troscopy are also employed. 

Specific Objectives : 

1. Analytical methods development for spiromustine and determination of human 
pharmacokinetics. 

2. Central nervous system pharmacokinetics of hexamethylene bisacetamide in 
non-human primates. 

3. Analytical methods development and preclinical pharmacology for arabinosyl- 
5-azacytosine (Ara-AC). 

4. Synthetic and collaborative project support. 

Major Find'ings : 

1 . Analytical Methods Development for Spiromustine and Determination of Human 
Pharmacokinetics : 

a. Analytical Methods Development (Drs. Kelley, Heideman) : Spiromustine 
(spirohydantoin mustard, NSC 1/2112, la), a new candidate antitumor agent with 
apparent activity against central nervous system (CMS) tumors, is currently 
undergoing Phase I clinical trial. Because of the low dose that is administered 
to human patients and because of this compound's extreme plasma instability 
(ti/2 = 6.5 min, pH 7.4, 37°), very sensitive and specific analytical methods 
are required to measure this drug in biological samples and define plasma 
elimination and CNS penetration. We have developed both a GC/MS method employ- 
ing an isotopically labelled internal standard with selected ion monitoring, 
and a capillary GC method using a wide-bore fused silica capillary column with 
a nitrogen-phosphorus detector (NPD). Spiromustine-d3 (j^) is used as an 




/CX2CX2CI la Ri,R2= -(CHz)^ ;X = H 

lb Ri,R2= -(CH2)- ; X = D 
l£ Ri,R2= E*;X = H 



263 



ZOl CM 03581-16 LPET 

internal standard for the GC/MS procedure, while di ethyl hydantoin mustard (Ic) 
is the internal standard for the 6C-NPD method. The same extractive workupTs 
carried out for both methods. Because of the plasma instability of spiromus- 
tine, biological samples must be rapidly extracted as they are obtained at the 
patient's beside. A single 2 min extraction with hexane- toluene (7:3, v/v) 
gives an average recovery of 86% for the 25-255 ng/ml range. Subsequent 
analysis by either analytical method allows reliable mesurement of 10 ng/ml 
levels of spiromustine in human plasma. Work is currently in progress to 
increase the sensitivity of the GC/MS method by substituting the more inert 
wide-bore capillary columns for the packed columns now used. A ten-fold in- 
crease in detection limits is anticipated. 

b. Human Pharmacokinetics of Spiromustine in an NCI/NINCDS Phase I Clinical 
Trial (Drs. Kelley, Heideman, Curt, Collins and Kufta) : Spiromustine has been 
administered to 22 patients with high grade primary CNS malignancy. It was 
given as an i.v. bolus q. 4 hr x 3 every 28 days at doses ranging from 3.0 - 
15.6 mg/m2. A fractionation of the dose has been employed in the hopes of 
minimizing the dose-limiting neurotoxicity reported by other clinical inves- 
tigators. Dose fractionation places an added demand on the sensitivity required 
in the analysis of biological samples. Plasma samples from 8 different patients 
have been analyzed and the plasma concentration (Cp) versus time data from 5 of 
these were sufficient to define pharmacokinetic parameters (Table 1). A two- 
compartment open model where the ti/2 for the terminal phase is only 12.5+ 3.3 
min (n=5) adequately defines the plasma elimination curve. All measured~plasma 
levels are in the nanogram per ml range with peak plasma levels exceeding 200 
ng/ml (6 x 10~'M) in some patients. Administration of spiromustine as a short 
(10-15 min) infusion is planned in order to validate this pharmacokinetic model 
since extrapolation to zero time appears to involve considerable uncertainty. 
An estimate of Cp at steady-state will allow determination whether the clearance 
of this rapidly eliminated drug has been overestimated. 

Table 1. Pharmacokinetic Summary of Spiromustine Phase I Clinical Trial 



Patient Dose 



(mg/m^) 



Fractionated 

Dose 

(mg/m^) 



Peak** 
Cp 
(ng/ml) 



n/2 
(min 



(a) 



TB 



'dss 



ti/2(3) CL 

(min) (ml/minXm2) (l./m^) 



S.B 


9.9 


3.3 


246 


1.0 


J.S. 


9.9 


3.3 


313 


1.0 


J.A. 


12.3 


4.1 


667 


1.3 


D.H. 


12.3 


3.1<1 


168 


0.6 


K.V. 


12.3 


4.1 


766 


0.8 



** measured 

^ total body clearance 

^ volume of distribution at steady-state 

^ administered 75% of normal dose 



13.7 1096 11.7 

14.8 1679 12.8 
6.6 1201 6.0 

13.5 2895 33.9 

13.9 1383 10.0 



264 



ZIO CM 03581-16 LPET 

c. Plasma and CNS Pharmacokinetics of Spiromustine in a Pediatric Phase I Clin- 
ical Trial (Drs. Heideman, Kelley, Poplack) : A pediatric Phase I clinical trial 
is also currently underway in which patients (_< 19 yrs) are administered the 
drug as an i.v. bolus weekly x 3 on a 28 day cycle. Five patients have been 
treated at 4.5 or 5.5 mg/m^ per i.v. bolus with either manageable or no apparent 
neurotoxicity. One patient treated at the higher dose has had plasma levels of 
spiromustine measured after two courses of drug on the same cycle. The result- 
ing plasma elimination curves and the measured Cp's were similar to those ob- 
served on the adult Phase I study (see above), although mean total body clear- 
ance was somewhat higher (2740 versus 1650 ml/min x m^). The plasma kinetics 
of additional patients will be examined to determine whether this difference is 
real. Also planned are studies to document spiromustine CNS penetration and 
elimination kinetics in those patients where an Ommaya reservoir or implanted 
shunt allows serial cerebrospinal fluid sampling. 

2. Central Nervous System Pharmacokinetics of Hexamethyl ene Bisacetamide (HMBA) 
In Non-human Primates (Drs. Kelley, PoplackTl HMBA, a synthetic polar-planar 
compound with potent differentiating activity in selected tm vitro model 
systems, is the first compound of this type chosen by the NCI to undergo clin- 
ical trial. Analytical methods for measuring HMBA in plasma, urine and cere- 
brospinal fluid (CSF) have been further refined. The combination of solid-phase 
extraction (Sep-Pak) for isolation from the biological matrix, and wide-bore 
fused silica polar (OV-17) capillary column chromatography together with a 
nitrogen-phosphorus detector for GC analysis allows determination of less than 
100 ng/ml HMBA in a 0.2 ml sample. Since the initial Phase I trials with of 
this agent Indicate that metabolism may be more pronounced than that observed 
In preclinical studies, the developed analytical methods will be modified to 
concurrently measure partially and completely deacylated HMBA. This methodology 
will then be applied to a bioavailability and metabolism study of orally admin- 
istered HMBA in the context of an NCI Phase I clinical trial. 

A study to assess the ability of HMBA to penetrate the central nervous system 
in rhesus monkeys was completed. This study was prompted by the report that 
the major toxicity of HMBA in dogs was neurologic. Plasma and lumbar CSF 
kinetics were determined after both i.v. bolus and continuous infusion doses of 
HMBA In the same animals (Table 2). It can be seen that this compound rapidly 
and readily penetrates the CNS with a mean CSF: plasma ratio of 0.36 (n=6). 
Also Interesting to note are that peak and steady-state CSF and plasma levels 
are Inversely proportional to total body clearance, suggesting non-linear 
kinetics (or saturation of elimination mechanisms) in this species at these 
doses. 



265 



ZOl CM 03581-16 LPET 
Table 2. CSF and Plasma Kinetics of HMBA in Rhesus Monkeys 
IV "Bolus" (short infusion) 



Monkey Dose Cp(peak) 

(mg/kg) (mcg/ml ) 



^1/2 C^TB Crsp(peak) CSF: plasma 

(min) (ml/min/kg) (mcg/ml) ratio 



1 


226 


357 


59 


6.5 


53 


0.24 


2 


240 


745 


103 


3.8 


124 


0.55 


3 


485 


1755 


171 


2.5 


164 


0.27 



8 Hr Continuous Infusion 



Monkey 



Dose 



ss 



CI 



TB 



(mg/kg/min) (mcg/ml) (ml/min/kg) 



Css(CSF) 
(mcg/ml ) 



CSF: plasma 
ratio 



3 


0.42 


1 


0.60 


2 


1.5 



44 



599 



9.5 


10 


6.8 


28 


2.5 


356 



0.23 
0.32 
0.58 



3. Analytical Methods Development and Preclinical Pharmacology for Arabi nosy 1- 
5-azacytosine (Ara-AC) (Drs. Kelley and Heideman) : Ara-AC (NSC 281272) is a 
new synthetic nucleoside which combines the structural elements of two estab- 
lished antitumor agents, Ara-C (the arabi nose sugar) and 5-AC (the triazine 
base). Chemically Ara-AC behaves like 5-AC, while it most closely resembles 
Ara-C in its antitumor and other biological properties. A sensitive and speci- 
fic HPLC assay suitable for measurement of Ara-AC in clinical samples and for 
determination of human pharmacokinetics is currently under development. This 
assay utilizes reverse phase HPLC with UV detection at 240nm and 2'-deoxy-5- 
azacytidine, an analog with similar aqueous stability (ti/2 (a) =26 min 
versus 24 min for Ara-AC at pH 7.4, PBS, 37"), as an internal standard. A 
major effort is being devoted to sample cleanup since removal of interfering 
endogenous materials will be the major determinant of sensitivity. On-line 
solid-phase extraction with column switching is anticipated as a solution to 
this problem. The developed analytical method will then be validated and 
applied to a preclinical pharmacology study in a rhesus monkey model to deter- 
mine the extent of Ara-AC CMS penetration. This will be of considerabl interest 
because of this compound's high activity in the murine intracerebral L1210 
leukemia model . 



266 



ZOl CM 03581-16 LPET 



4. Synthetic and Collaborative Project Support 



a. Furanose - Pyranose Isomerization (Drs. Kelley, Marquez, Driscoll) : 
Unsaturated pyrimidine, diazepinone and cyclic urea ribosides undergo an acid- 
catalyzed isomerization from a 3-ribofuranose to a predominantly 3-ribopyran- 
ose form. Cytidine deaminase inhibitors which belong to the above classes of 
compounds can be almost completely deactivated by this isomerization. The pH 
dependent isomerization kinetics were investigated using 2 as a model compound. 
The resulting decomposition products were isolated by semT-p reparative HPLC and 
chemically characterized. At pH 1 (37°), ribofuranoside 2^ underwent rapid iso- 
merization (ti/2 = 10 min) to the g-pyranose 3^, which was slowly reductively 
formylated by the formaldehyde preservative in the buffer. It was also demon- 
strated that 3^ eventually achieves equilibrium with 2. This acid-catalyzed 
equilibrium was also investigated for tetrahydrouridTne (THU) under conditions 
approximating those seen after oral administration. THU underwent isomeriza- 
tion with biphasic kinetics (ti/2 (a) = 30 min) to two major products in 
simulated gastric fluid (pH 1.2, 37°). These THU isomers are still in the 
process of being characterized. 




HO 




H 



HO 4 



o, ^^P' 



OH 



b. Spirohydatoin Mustard Analogs (Drs. Kelley, Heideman, Haces, Driscoll) : 
The human plasma stability and the electron impact mass spectral characteristics 
of a series of spirohydantoin mustard analogs (5^) have been determined and 
related to structure. The in vitro plasma half-lives of analogs n=2-5 were 
measured at 37° at clinically" achieveable concentrations of Imcg/ml using the 
capillary GC-NPD analysis developed for spiromustine (see above). Spiromustine 
itself was the most stable homolog (ti/2 = 6.5 min). As the methylene unit 
spacer increased, the stability of these analogs approached that of nitrogen 



267 



ZOl CM 03581-16 LPET 

mustard (ti/2 = 1.6 tnin). Substitution at the 5-posit1on of the hydantoin ring 
had little effect on mustard stability for n=2. A unique bi cyclic ion (6^) was 
observed in the mass spectra of all of these analogs. This fragment ion 
predominated for n=3, but its abundance could only be correlated with ring size 
and not mustard stability. 





c. Applications of Fast Atom Bombardment (FAB) Mass Spectrometry to Nucleoside 
and Nucleotide Analysis (Dr. KelleyT : The MCS continues to have an extensive 
program in the synthesis of new nucleosides and nucleotides. Rapid and simple 
methods employing FAB mass spectrometry continue to be investigated and utilized 
for the characterization of underivatized nucleic acid constituents. Negative 
ion FAB mass spectrometry has been applied to determine the structure and 
purity of chemically and enzymatically synthesized NAD analogs. Chemical 
methods to enhance the sequence-indicating fragment ions in FAB mass spectra of 
oligoribonucleotides are currently under investigation. The use of FAB mass 
spectrometry to quantitate selected compounds isolated from a biological matrix 
also remains an interest. 

d. Miscellaneous : Numerous samples which cannot be categorized as coming from 
any one project area were also analyzed by the appropriate mass spectral and 
chromatographic techniques on an individual basis. Included in this group were 
S-adenosyl methionine derivatives, inositol phosphates, neplanocin analogs, 
4-pyridone derivatives, sugar phosphonates and photo-affinity labelled vinblas- 
tine derivatives. 

Publications : 

1. Huguenin, P.N., Jayaram, H.N. and Kelley, J. A.: Reverse phase HPLC 
determination of 5,6-dihydro-5-azacytidine in biological fluids. 

J. Liquid Chromatogr. 7:1433-1453, 1984. 

2. Gebeyehu, 6., Marquez, V.E., Van Cott, A., Cooney, D.A., Kelley, J. A., 
Jayaram , H.N., Ahluwalia, G.S., Dion, R.L., Wilson, Y.A. and Johns, D.G.: 
Ribavirin, tiazofurin and selenazofurin: Mononucleotides and adenine 

di nucleotide analogues. Synthesis, structure and interactions with IMP 
dehydrogenase. J. Med. Chem. 28:99-105, 1985. 

3. Zaharko, D.S., Covey, J.M. and Kelley, J. A.: Plasma kinetics and effects 
of 5,6-dihydro-5-azacytidine in mice and L1210 tumor. Inv. New Drugs 
3:35-41, 1985. 

268 



ZOl CM 03581-16 LPET 

4. Litterst, C.L., Roth, J.S. and Kelley, J. A.: Distribution, elimination, 
metabolism and bioavailability of hexamethylenebisacetamide in rats. 
Inv. New Drugs , in press. 

5. Huguenin, P.N. and Kelley, J. A.: The synthesis of spiromustine-ds. A 
general approach to octadeuterated nitrogen mustards. J. Label el led. Compds . 
Radiopharm. , in press. 

6. Curt, G.A., Kelley, J. A., Fine, R.L., Huguenin, P.M., Roth, J.S., Batist, G., 
Jenkins, J. and Collins, J.M.: A phase I and pharmacokinetic study of 
dihydro-5-azacytidine (NSC-264880). Cancer Res. , in press. 

7. Kelley, J. A., Roth, J.S. and Litterst, C.L: Gas chromatographic determina- 
tion of hexamethylene bisacetamide in plasma and urine. Analyt. Lett. , in 
press. 



269 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07167-01 LPET 



PERIOD COVERED 



October 1, 1984 to September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must lit or\ one line between the borders.) 

Enzyme Inhibitors as Potential Anticancer Drugs 



PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: V. E. Marquez Visiting Scientist LPET, NCI 



Others: J. S. Driscoll 

C. H. Kim 
C. K. H. Tseng 
R. Fuller 
S. Petraglia 



Head, Medicinal Chemistry LPET, NCI 

Section 
Visiting Fellow LPET, NCI 
Visiting Fellow LPET, NCI 
Chemist LPET, NCI 
Chemist LPET, NCI 



COOPERATING UNITS (it any) 



Laboratory of Biological Chemistry 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Medicinal Chemistry Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 



20205 



TOTAL MAN-YEARS: 

5.2 



PROFESSIONAL 

3.2 



2.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



D (b) Human tissues Q (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The objective of this project is to design and synthesize mechanism-based 
inhibitors of enzymatic reactions that are critical for the growth of neoplastic 
cells. The resulting compounds are intended to be used as therapeutic agents and 
research probes. The following topics are of current interest: 1) Synthesis of 
cyclopen1:eny"l nucleosides ( Neplanocin A analogs), 2) Pi nucleotide analogs of 
NAD as IMPD inhibitors , 3") Transition-state inhibitors of cytidine triphosphate 
(CTP) synthetase, 4) Synthesis of diazepinone nucleosides as fradulent uridine 
and cytidine analogs, and 5) Synthesis of stable analogs of the 2' ,5'-o1igo- 
adenylate trimer as inducers of interferon production. 



270 



PHS 6040 (Rev. 1/84) 



GPO 914-916 



ZOl CM 07167-01 LPET 
Objectives : 

The objective of this project is the exploitation of recent biochemical findings 
concerning enzymatic mechanisms that are enhanced in rapidly proliferating 
cells, in order to design appropriate inhibitors for key enzymes. A suitably 
altered substrate (mechanism-based inhibitor) is selected as a candidate target 
and synthesized. The selected candidates are designed as reversible transition- 
state inhibitors or irreversible (K^at^ suicide inhibitors. A correlation 
between structure and activity in the series of inhibitors prepared serves to 
delineate the course of future synthetic work. 

Major Findings : 

Synthesis of Cyclopentenyl Nucleosides (Neplanocin A Analogs) (Drs. Tseng and 
Marquez )^ The original synthetic schemes for neplanocin A (la) and the cyclo- 
pentenyl (CPE-) pyrimi dines lb and 2£ were shortened considerably by the use of 
a direct displacement approacTT that employed the cyclopentenyl a-tosylate 2 
and the alkali metal salts of the corresponding purine and pyrimi dine bases. 



HP 

\ 

1 


B 

X1 


la. 
b, 
c, 
'5, 
e, 
f, 

g. 


B = adenine (neplanocin A) 

B = uracil (CPE-Uracil) 

B = cytosine (CPE-cytosine) 

B = thymine 

B = 5-FU 

B = 5-IU 

B = 3-deazaadenine 


PhCHp 
2 








HO OH 





This new approach is currently being extended to the syntheses of other 
pyrimidines (e.g., l_d - ]f) and to the important target, 3-deazaneplanocin 
(Ig). Conversion of the cyclopentenyl riboside isosteres into the arabinosyl 
and 2'-deoxy analogs will follow. 

Biological studies (Drs. J. Moyer and R. Cysyk, LBC) with CPE-uracil (lb) and 
CPE-cytosine (Ic) indicated that both compounds behaved as inhibitors of the 
enzyme uridine~Tinase. Although the CPE-uracil was the less potent of the two, 
it was attractive for its almost total lack of cytotoxicity. A marked decrease 
in labeled uridine uptake from intestine, liver, and spleen was observed in 
vivo following the administration of CPE-uracil at doses ranging from 100 to 
300 mg/kg. In vitro studies conducted by Dr. Glazer (LBC) and confirmed in 
vivo by Dr. Moyer demonstrated that CPE-cytosine is a very potent inhibitor of 
CTP synthesis whose mechanism appears to be consistent with the irreversible 
inactivation of the enzyme. CPE-cytosine is a powerful differentiator of HL-60 
cells in vitro (Dr. Glazer, LBC) (10"'M). Additionally, following a qd 1-5 
treatment schedule at a dose of 1 mg/kg in mice bearing LI 210 leukemia, an ILS 
of 67% was observed (Dr. Moyer, LBC). These and other biological results have 
provided incentive to continue the syntheses of other purine and pyrimi dine 
analogs bearing the novel cyclopentenyl ring. 

Dinucleotide Analogs of NAD (Drs. Tseng and Marquez ). The preparation of all 
possible phosphonate analogs of the active metabolite of tiazofurin, thiazole-4- 
carboxamide adenine dinucleotide (TAD), was completed with the synthesis of g- 
methylene TAD (3b). 



271 




ZOl CM 07167-01 LPET 

A = adenine 

T = thiazole-4-carboxamide 



HO OH 

3a . a-methylene TAD 
b, g-methylene TAD 
£, Y-methylene TAD 

Synthesis of 3-methylene TAD was accomplished by the coupling of adenosine 5'- 
{ a, e-methylene) diphosphate with protected tiazofurin (2' ,3'-acetonide) 
in the presence of dicyclohexylcarbodiimide (DCC). All the analogs (3a-c) re- 
tained their inosine monophosphate dehydrogenase (IMPD) activity, pluT~tWey 
were more resistant than TAD towards enzymatic cleavage. In particular, 
the e methylene TAD (3b), was equi potent to TAD as an IMPD inhibitor and was 
totally resistant to phosphodiesteric cleavage by both venom phosphodiesterase 
and TAD-phosphodi esterase (Dr. Cooney, LPET). Since it is believed that TAD 
breakdown is responsible in part for either the lack of response or the develop- 
ment of resistance to tiazofurin, this compound offers a unique opportunity to 
test this hypothesis. Additionally, 3b was cytotoxic to P388 cells in vitro 
(IC50 = 45 uM) which meant that despite being a charged molecule it was 
transported across the cell membrane. Efforts are now directed towards 
preparing a more lipid-soluble form of this compound with the intent of 
improving its penetrability into the cell. 

Transition-State Inhibitors of Cytidine Triphosphate Synthetase (Drs . Kim 
and Ma rquezi: ihe key intermediate tor this project, (6^), was obtained by 
Hilbert-Johnson reaction employing a protected halogenosugar and the heter 
cycle 5 which was prepared in four steps from compound 4. 



hetero- 
r— OCPh 



COOH 



CH,S 






CH,0 




CPh 





HO OH 



272 



ZOl CM 07167-01 LPET 

The deblocking of 6^ produced compound _7, whereas compounds 8 and 10 were ob- 
tained from 6^ in a three step sequence which involved a parTial rMuction with 
sodium borohydride, separation of isomers, and deblocking. The 4-hydroxymethyl 
substituent in compounds ]_ and 9^ is really a masked aldehyde group that can be 
generated through an intramolecular redox reaction. In 7^, however, the stabil- 
ity of the aromatic ring prevents aldehyde formation whereas in the partially 
reduced compound, 9, the aldehyde is readily unmasked and is immediately 
hydrated to give tTTe hemiacetal ^0. 

Compounds 7, 8 and 10 have been submitted for testing (Dr. Cooney) to see 
whether CTF pools wTTl be affected or not. Transformation of the pyrimidine 
hydroxyl group in ]_ and 8^ into halogens, phosphonate groups, etc., is planned 
for the neair future. 

Synthesis of Diazepinone Nucleosides as Potential Antitumor Agents (Drs. Kim 
and Marquez). The synthesis of ring-expanded cytidine (16) was accomplished 
from the nucleoside Vl_ in seven steps. After oxidation with phenyl seleninic 
anhydride, compound 12 was obtained; following a two-step oxidation-elimination 
of the phenyl selenium substituent as PheSeOH, the ring-expanded uridine analog 
13a was generated. Thiation of the 13a and treatment of this compound with 
methyl iodide, produced a mixture of 24 and 15, which was readily aminated and 
deblocked in one operation to give 26, the desired compound. 

O 



I I I 

R R R 13a,X=0 

11 12 b,X=S 




R = 2' ,3' ,5'-tri-0-benzoyl-g-D-ribofuranosyl 
R' = 3-D-ribofuranosyl 

Ring-expanded cytidine was a poor inhibitor of uridine-cytidine kinase (Dr. J. 
Moyer) and showed poor growth inhibitory properties against LI 210 cells in 
vitro (37% inhibition at 1 mM). This compound will be evaluated as substrate 
or inhibitor of cytidine deaminase. It is expected that this project will 
terminate soon. 

Synthesis of Phosphonate Analogs of the 2' ,5'-01igoadenylate Trimer Core 
(Drs. Tseng and Marquez) . Progress towards the target (2'-5')A3 analog in 
which the phosphate ester linkages between the 2' -hydroxyl group and the adja- 
cent 5 '-phosphate of the trimer are replaced by hydrolytically stable methylene 
linkages, has centered around the synthesis of the monomer 21. Two independent 
approaches, which led to the syntheses of precursors 20 and~Z"2, were initiated. 
Compund 22 offers the attractive feature that the resulting a-stereochemistry 
of the phosphonate group in 21 will be uniquely established. However, several 
attempts to displace the bromine atom of 22 by lithium dimethyl methyl phos- 
phonate, or even by the smaller cyanide anion, have failed. 



273 



ZOl CM 07167-01 LPET 



0CH2O 




OCH, 



0CH2O \pr0CH3), 

20 \r ^^ 
o 



3 steps 




19 \r ^ 




A = adenine 
Bz= benzoyl 
Tfstriflate 




OCH, 




23a, R=OH 
b, R=OTf 



OCH, 



— ribose 



In the second approach, compound ^ has been prepared from D-(-) -ribose in 
seven steps. Although in this approach the selective stereochemical control is 
lost, the chemistry is more favorable and efforts to condense adenine with the 
chlorosugar derived f rom ^ are being pursued. In view of the lack of 
controlled stereochemistry characteristic of this route, separation and 
identification of isomers is anticipated. Relative to the naturally occurring 
material, the target compound is expected to yield an enzymatically more stable 
trimer core capable of inducing production of interferon in treated cells. 

Publications : 

1. Marquez, V.E.: Inhibition of cytidine deaminase: mechanism and effects on 
the metabolism of antitumor agents. In Glazer, R.I. (Ed.): Developments 
in Cancer Chemotherapy , CRC Press, Inc., 1984, pp. 91-114. 

2. Mao, D.T., Marquez, V.E.: Synthesis of 2-3D-ara and 2-e-D-xylofuranosyl- 
thiazole-4-carboxamide: Tetrahedron Lett., 25: 2111-2114, 1984. 



274 



ZOl CM 07167-01 LPET 

3. Lim, M-I., Moyer, J.D., Cysyk, R.L. and Marquez, V.E.: Cyclopenteneyl- 
uridine and cyclopentenylcytidine as inhibitors of uridine-cytidine kinase. 
J. Med. Chem. 27: 1536-1538, 1984. 

4. Gebeyehu, G., Marquez, V.E., Van Cott, A., Cooney, D.A., Kelley, J. A., 
Jayaram, H.N., Ahluwalia, G.S., Dion, R.L., Wilson, Y.A. and Johns, D.6.: 
Ribavirin, tiazofurin and selenazofurin: Mononucleotides and NAD di nucleo- 
tide analogs. Synthesis, structure and interactions with IMP dehydrogenase. 
J. Med. Chem. 28: 99-105, 1985. 

5. Tono-oka, S., Tone, Y., Marquez, V.E., Cooney, D.A., Sekikawa, I., Azuma, 
I.: Enzymic synthesis and biochemical activity of various indazole adenine 
di nucleotides. Bull. Chem. Soc. Jpn. 58: 309-315, 1985. 

6. Monks, A., Marquez, V.E., Mao, D.T., Cysyk, R.L.: Inhibition of nucleoside 
transport by 2-e-D-ribofuranosylthiazole-4-carboxamide (tiazofurin) and 
related analogues. Cancer Lett. , in press. 

7. Glazer, R.I., Knode, M.C., Lim, M-I. and Marquez, V.E.: Cyclopentenyl 
cytidine analogue: an inhibitor of cytidine triphosphate synthesis in human 
colon carcinoma cells. Biochem. Pharmacol. , in press. 

8. Marquez, V.E. and Lim, M-I.: Carbocyclic Nucleosides. Medicinal Research 
Reviews, in press. 



275 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07102-10 LPET 



PERIOD COVERED 



October 1, 1984 - September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) 

Tubulin Structure and Function as Sites for Pharmacologic Attack 



PRINCIPAL INVESTIGATOR (List other prolessional personnel beiow the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: E. Hamel Senior Investigator LPET, NCI 



Others: 



J. K. Batra 
C. Duanmu 
C. M. Lin 



Visiting Fellow 
Visiting Fellow 
Biologist 



LPET, NCI 
LPET, NCI 
LPET, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



Biochemical Pharmacology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, MD 20205 



TOTAL MAN-YEARS: 

4.0 



PROFESSIONAL: 



3.0 



1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
n (a2) Interviews 



D (b) Human tissues Q, (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The rational development of new antineoplastic agents directed against tubulin, 
a protein critical for cell division, requires greater understanding of the 
interactions between the polypeptide subunits of tubulin, its two tightly bound 
guanine nucleotides, and drugs which inhibit mitosis. Substitution of ATP and 
ATP analogs for GTP in glycerol -induced polymerization was demonstrated. This 
will permit structure-function studies of nucleotide interactions at the ex- 
changeable site with more readily available ATP analogs. Degradation of the 
nonexchangeable GTP of tubulin by phosphofructokinase was observed, representing 
the first approach to this nucleotide which does not require the denaturation of 
the protein. Studies on the interactions of a large group of antimitotic agents 
with tubulin continued. The classes of compounds examined were combretastatin 
congeners; derivatives of 6-benzyl-l,3-benzodioxole and related compounds; deri- 
vatives of 5,6-diarylpyridazin-3-one; 2,4-dichlorobenzylthiocyanate; and alkyl 
carbamates of aromatic amines. A microtubule-associated protein which causes 
the formation of microtubule bundles was purified to homogeneity. Microtubule- 
associated protein(s) with nucleoside monophosphate kinase activity were resolved 
into components with different substrate specificity, and one component was 
purified to homogeneity. Maytansine was found to inhibit entry and exit of GDP 
and GTP at the exchangeable nucleotide binding site. Efforts to separate the 
a and 3 subunits of tubulin continued, and efforts to prepare GTP analogs 
derivatized at the y-phosphate were initiated. 



276 



PHS 6040 (Rev. 1/84) 



ZOl CM 07102-10 LPET 

Microtubules, cellular organelles critical for cell division, are sensitive to 
a number of antineoplastic drugs. Their major constituent is an acidic protein 
known as tubulin, which consists of two different polypeptide chains and two 
molar equivalents of GTP. Half this GTP (the exchangeable nucleotide) is 
hydrolyzed during microtubule assembly from tubulin and microtubule-associated 
proteins (MAPs — minor, but essential, components of the microtubule). The 
remainder of the GTP (the nonexchangeable nucleotide) is not altered during 
tubulin polymerization and can only be removed from tubulin by destroying the 
protein. Its function is unknown. 

We have confirmed that in glycerol -induced tubulin polymerization (a reaction 
condition in which MAPs are not required) high concentrations of ATP can 
directly replace GTP in supporting tubulin polymerization. Contrary to inter- 
pretations in the literature, we have found that this polymerization reaction 
requires an interaction of ATP at the exchangeable nucleotide site and probably 
involves hydrolysis of ATP to ADP. In ATP-supported polymerization, GDP in 
the exchangeable site is displaced by ATP; and GDP is a potent inhibitor of 
the reaction. In addition, we have found that ribose- and polyphosphate- 
modified ATP analogs closely mimic comparable GTP analogs in their effects on 
tubulin polymerization. This implies that other ATP analogs may also be used 
to define in further detail structural requirements for the nucleotide at the 
exchangeable site. This is of importance since ATP analogs are much more 
common than GTP analogs and should save a considerable amount of synthetic 
work. 

We have observed that when phosphofructokinase and fructose-6-phosphate are 
added to microtubules, even when GTP in the reaction mixture has been exhausted, 
there is an immediate rapid and extensive depolymerization of the microtubules. 
Since both the sugar and enzyme are required for this to occur, nucleotide 
breakdown is implied. This in turn suggests that it is the nonexchangeable GTP 
which may be involved in the disassembly process catalyzed by phosphofructokin- 
ase, and we are attempting to document whether nonexchangealbe site GTP break- 
down is occurring. 

New antimitotic agents continue to be an active area of interest in the labor- 
atory. We are currently studying the following classes of drugs: 

1) Analogs of combretastatin. Combretastatin (NSC 348103) is a natural product 
isolated by G. R. Petti t of Arizona State University from the South African 
tree Combretum caff rum ; and we have demonstrated that combretastatin is a potent 
inhibitor of tubulin polymerization. Dr. Pettit's group has now purified, but 
not yet fully characterized, a number of additional compounds from Combretum 
caffrum . We have established that at least two of these as yet unidentified 
agents are considerably more potent than combretastatin itself as tubulin 
inhibitors. 

2) A large number of derivatives of 6-benzyl-l,3-benzodioxole have been pre- 
pared by Dr. L. Jurd of the Department of Agriculture as potential insect 
sterilants. A significant number of these compounds have antineoplastic activ- 
ity and inhibit tubulin polymerization. They are most analogous to podophyl- 
lotoxin structurally, and like podophyllotoxin, inhibit both tubul in-dependent 



277 



ZOl CM 07102-10 LPET 

GTP hydrolysis and the binding of colchicine to tubulin. Initial studies 
established minimal structural requirements for the simplest benzyl benzodioxole 
derivatives (a 1-3 carbon substituent at position 5, and a methoxy group at 
position 4' in the benzene ring) v^hich have either an unsubstituted one carbon 
bridge between the benzene and benzodioxole rings or one or two methyl groups 
at this bridge position. Additional methoxy substituents on the benzene ring 
at the 3' and 5' positions, which seemingly increase the structural analogy to 
podophyllotoxin, resulted in almost complete loss of activity. 

Dr. Jurd has more recently prepared a large series of derivatives with a 
morpholino group at the bridge carbon and no substituent at the 5 position. 
The variation is in the substituents on the benzene ring. The most active 
compounds in this group are also potent tubulin inhibitors, and they too seem 
most analogous to podophyllotoxin in their interactions with the protein. 
These compounds permit a more detailed structure-function analysis than is 
possible with the much more complicated natural product podophyllotoxin, and 
provide promising leads for further synthetic work with this group of drugs. 
The morpholino compounds themselves, however, have limited cytotoxicity and do 
not appear to cause mitotic arrest in cultured cells. 

Dr. Jurd has also prepared a group of compounds with a third ring (of variable 
structure) fused to the benzodioxole moiety. The benzene ring is attached 
directly to this third ring. Several of these agents have potent anti tubulin 
activity, and most of the compounds active against tubulin in vitro also cause 
mitotic arrest. All active compounds have three methoxy groups, attached at 
positions 3', 4' and 5', on the benzene ring. Although both the third fused 
ring and the trimethoxy structure appear to substantially increase their anal- 
ogy to podophyllotoxin, these new agents are more comparable to colchicine in 
their effects on tubul in-dependent GTP hydrolysis; for, like colchicine, they 
stimulate rather than inhibit this reaction even while inhibiting the micro- 
tubule assembly reaction normally coupled to GTP hydrolysis. 

3) Or. L. Powers of SDS Biotech Corporation has prepared over 100 derivatives 
of 5,6-diarylpyridazin-3-one as potential antihypertensive agents. Some of 
these compounds were found to be potent herbicides, and this seemed to be a 
consequence of inhibition of mitosis in plant tissues. Several members of this 
class were then submitted to the NCI for screening, and some of these were 
found to have antineoplastic activity. We have found that a number of these 
drugs inhibit mitosis of mammalian cells in culture and the polymerization of 
tubulin in vitro. They potently stimulate tubul in-dependent GTP hydrolysis; 
but they probably bind at a previously undescribed site on tubulin for they do 
not inhibit the binding of either colchicine, vinblastine or maytansine to the 
protein. Most active compounds possess a nitrile group at position 4; and in 
vitro interactions with tubulin are significantly enhanced by chloride substi- 
tuents on the phenyl rings. There is little overlap between compounds most 
active against mammalian tubulin and those which are most active in inhibiting 
mitosis in plant cells. 

4) The compound 2,4-dichlorobenzylthiocyanate (NSC 145813) has been found to 
inhibit mitosis in murine leukemia cells and, in collaboration with other 
investigators, to cause clumping or bundling of microtubules in CHO cells. 



278 



ZOl CM 07102-10 LPET 

In addition, certain lines of CHO cells with mutants in tubulin genes are 
resistant to NSC 145813. The effects of this agent on in vitro tubulin poly- 
merization are highly unusual. There is minimal change in the reaction unless 
drug and tubulin are preincubated (in the absence of GTP to avoid polymeriza- 
tion). With a preincubation at 37° lasting at least one hour, however, low 
concentrations of the drug completely inhibit tubulin polymerization. This 
inhibition can be prevented with dithiothreitol . We believe that NSC 145813 
is acting as a highly specific alkylating agent, attacking one or more of the 
sulfhydral groups of tubulin, and that this reaction produces its antimitotic 
and anti tubulin effects. We are in the process of obtaining radiolabeled 
drug to use to prove this hypothesis. We will also attempt to document which 
one of the subunits of tubulin acts as the target of the drug. 

5) A number of compounds with very different structures have been found to have 
antineoplastic and antimitotic properties and to inhibit tubulin polymerization. 
Their only common feature is that they are alkyl carbamates of aromatic amines. 
We have obtained a computer search of the NCI drug collection, and there are 
at least 140 compounds with structural features which suggest possible anti- 
mitotic activity. We are currently screening these compounds in an effort to 
identify additional drugs with antitubulin properties. 

Another area of active interest in the laboratory is microtubule-associated 
proteins. We are currently purifying two components from this large group 
of proteins. The first of these causes the formation of microtubule bundles 
(distinc microtubules which aggregate laterally). The active component does 
not bind to DEAE-cellulose, does adhere to hydroxyapatite, is heat-stable, and 
can be reconstituted after denaturation in 8 M urea. Most recently the protein 
has been further purified on a sizing column by high pressure liquid chromato- 
graphy. This last step appears to result in an electrophoretically homogeneous 
protein consisting of two subunits of molecular weights of approximately 20,000 
and 30,000. This last step will now be attempted on a preparative scale. 

The second MAP of interest to us is the enzyme nucleoside monophosphate kinase 
(NMK). Our interest in this enzyme began when we observed that extensive 
nucleotide interconversions occurred in microtubule protein preparations if 
both radiolabeled GTP and adenosine 5'-[6,Y-imido]triphosphate were included in 
the reaction mixture. Similar results were obtained when AMP was substituted 
for the ATP analog, probably indicating the letter's contamination with AMP. 
(Repurification of the analog resulted in the disappearance of the nucleotide 
interconversions observed with the impure compound.) NMK does not bind to 
DEAE-cellulose or hydroxyapatite. When NMK is applied to cibacron blue-agarose, 
active components which are purified to electrophoretic homogeneity are ob- 
tained. The initial protein preparation accepts as substrates AMP, ADP, ATP, 
GMP, GDP, and GTP, with A-G mixtures preferred to AMP-ATP or GMP-GTP. Following 
cibacron blue-agarose chromatography substrate specificity changes, suggesting 
the resolution of the NMK into two or more different components. 

Additional areas of activity in the laboratory. 

1) The antimitotic compound maytansine was found to inhibit completely the 



279 



ZOl CM 07102-10 LPET 

entry or exit of GDP or GTP at the exchangeable nucleotide binding site of 
tubulin. 2) Initial, unsuccessful attempts were made to prepare GTP analogs 
derivatized at the y-phosphate with hydrophobic groups. Previous studies from 
this laboratory have indicated that such compounds might be potent inhibitors 
of polymerization through interactions at the exchangeable site. 3) We have 
observed that under certain reaction conditions tubulin with GDP in the ex- 
changeable site will co-polymerize with tubulin bearing GTP. We are attempting 
to define these conditions in detail. 4) We are continuing to attempt the 
preparative separation of the two subunits of tubulin and reconstitution of the 
protein's activity from its subunits. 

Publications : 

1. Hamel , E. and Lin, CM.: Separation of active tubulin and microtubule- 
associated proteins by ultracentrifugation and isolation of a component 
causing the formation of microtubule bundles. Biochemistry 23: 4173-4184, 
1984. 

2. Hamel, E., Lustbader, J. and Lin, CM.: Deoxyguanosine nucleotide analogs: 
potent stimulators of microtubule nucleation with reduced affinity for the 
exchangeable nucleotide binding site of tubulin. Biochemistry 23: 5314- 
5325, 1984. 

3. Hamel, E. and Lin, CM.: Guanosine 5'-0-(3-thiotriphosphate): a potent 
nucleotide inhibitor of microtubule assembly. J. Biol . Ch em. 259: 11060- 
11069, 1984. 

4. Bender, R.A. and Hamel, E.: Vinca alkaloids. In: Pinedo, H.M. and Chabner, 
B.A. (Eds.): Cancer Chemotherapy Annual, Vol. 6 , Amsterdam, Elsevier 
Science Publishers, 1984, pp. 100-110. 

5. Batra, J.K., Jurd, L. and Hamel, E.: Structure-function studies with 
derivatives of 6-benzyl-l,3-benzodioxole, a new class of synthetic com- 
pounds which inhibit tubulin polymerization and mitosis. Mol . Pharmacol . , 
27: 94-102, 1985. 

6. Hamel, E. and Lin, CM.: Di deoxyguanosine nucleotides and microtubule 
assembly. Ann. N.Y. Acad. Sci. , in press. 

7. Batra, J.K., Lin, CM., Hamel, E., Jurd, L. and Powers, L.J.: New anti- 
neoplastic agents with antitubulin activity. Ann. N.Y. Acad. Sci. , in 
press. 

8. Huang, A.B., Lin, CM. and Hamel, E.: Maytansine inhibits nucleotide 
binding at the exchangeable site of tubulin. Biochem. Biophys. Res. 
Commun. , in press. 



280 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07104-10 LPET 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 chamclars or less. Title must fit on one line between the tmrders.) 

L-Phenyl alanine Mustard Cytotoxicity and Therapy 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: D. T. Vistica Pharmacologist LPET, NCI 



Others: S. Ahmad 
H. Schalch 
J. Hill 



Visiting Fellow 

Biologist 

Biologist 



LPET, NCI 
LPET, NCI 
LPET, NCI 



COOPERATING UNITS (If any) 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Biochemical Pharmacology Section 



INSTITUTE AND LOCATION 

NCI. NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

3.4 



PROFESSIONAL: 

2.0 



1.4 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



[E (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Metastatic migration of the murine LI 210 leukemia and an L-phenyl alanine mustard 
resistant variant (L1210/L-PAM) from the peritoneal cavity and infiltration into 
the liver results in a 2-fold increase in the glutathione (GSH) content of both 
tumor cells. These increases in the cellular content of GSH are accompanied by a 
2-fold increase in the resistance of these tumor cells to L-phenyl alanine mustard 
Cell surface labeling studies indicate that cells isolated from the liver have a 
5-fold greater content of surface sulfhydryl groups as compared to their ascitic 
counterparts. The former can be sensitized to L-phenyl alanine mustard by blockinji 
these sulfhydryl groups with 6-6' dithiodi nicotinic acid. 

These results demonstrate a determinant role of the hepatic microenvironment in 
the maintenance of tumor cell GSH, cell surface sulfhydryl groups and the sen- 
sitivity of these tumor cells to L-phenyl alanine mustard. 



281 



PHS 6040 (Rev. 1/84) 



OPO 0I4-9IS 



ZOl CM 07104-10 LPET 

Objectives : 

This project is designed to examine the role of the environment of the host 
organ on tumor cell proliferation and sensitivity to chemotherapeutic regimens. 

Methods Employed : 

A. Maintenance of Tumors 

The murine L1210 leukemia and a line developed for resistance to L-phenyl alanine 
mustard (L1210/L-PAM) were maintained in male CDF-) mice by weekly intraperi- 
toneal injection of 1 x 10^ and 1 x 10° cells respectively. 

B. Isolation of Tumor Cells from the Ascites and Liver 

Animals bearing 7 day tumors were anesthesized with 5% chloral hydrate. Tumor 
cells were removed from the peritoneal cavity and used for determination of 
cellular glutathione and sensitivity to L-phenyl alanine mustard as described 
below. 

Tumor cells were isolated from the liver following sequential perfusion calcium 
and magnesium free Hank's balanced salt solution containing 1mm EGTA followed 
by 100 units/ml collagenase in William's Medium E. The liver was removed and 
teased apart in Dulbecco's phosphate buffered saline (without calcium and mag- 
nesium) containing 0.1mm sulfhydryl modified bovine serum albumin and 10mm 
glucose. The cell mixture, containing tumor cells and hepatocytes, was centri- 
fuged at 300 x g and the cells washed twice in Dulbecco's phosphate buffered 
saline containing 0.1mm sulfhydryl modified bovine serum albumin and 10mm 
glucose. The cells were then resuspended in 50ml of the same buffer system in 
a 50ml Falcon conical centrifuge tube and allowed to stand undisturbed at ZS'C 
for 60 minutes. The top 30ml containing tumor cells was removed and utilized 
for determination cellular glutathione and sensitivity to L-phenyl alanine 
mustard as described below. 

C. Determination of Cellular Glutathione Content 

Tumor cells from the ascites and liver (2x10^) were washed twice in Dulbecco's 
phosphate buffered saline containing 0.1mm bovine serum albumin and red blood 
cells removed by hypertonic lysis in 0.87% ammonium chloride for 5 minutes at 
room temperature. Cells were then washed once in Dulbecco's Phosphate buffered 
saline, pelleted by centrifugation at 300 x g for 10 minutes and lysed in dis- 
tilled water. Sulfosalicylic acid was added to a final concentration of 3% and 
protein removed by centrifugation at 12,000 x g for 2 min. Glutathione (GSH) 
and its disulfide (GSSG) were assayed by the standard spectrophotometric assay 
of Griffith. 

D. Evaluation of L-phenyl alanine Mustard Cytotoxicity 

Cells, isolated from the ascites and liver, as described above, were suspended 
in RPMI 1630 medium containing 16% heat-inactivated fetal calf serum, 40 yg/ml 
gentamicin and 50 pM B-meraptoethanol . Cells were exposed to L-phenyl alanine 



282 



ZOl CM 07104-10 LPET 

mustard for 2 days and surviving cells estimated by clonal growth in soft- 
nutrient agar for 2 weeks. 

E. Determination of Membrane Sulfhydryl Content of Cells Isolated 
from the Ascites and Live7 

Cells, in Dulbecco's phosphate buffered saline containing 0.1mm sulfhydryl 
modified bovine serum albumin were exposed to 100 ym [^^C] 6,6'-dithiodinico- 
tinic acid for 15 minutes at 37°C. Cells were washed twice in the same buffer 
system and treated with 1mm glutathione to release the bound radiolabel. Cells 
were then removed by centrifugation thru versilube F-50 silicone oil and the 
supernatant was counted by liquid scintillation spectrometry. 

Major Findings : 

1. Metastatic migration from the peritoneal cavity and infiltration into the 
liver results in a 2-fold increase in the GSH content of both L1210 and 
L1210/L-PAM tumor cells. 

2. These increases in the cellular content of GSH are accompanied by a 2-fold 
increase in the resistance of these tumor cells to L-phenyl alanine mustard. 

3. These observed differences in GSH content and sensitivity to L-phenyl alanine 
mustard in cells isolated from the ascites and liver are observed only in 
vivo since short term culture results in cells with similar GSH content and 
sensitivity to L-phenyl alanine mustard. 

4. Cell surface labeling studies indicate that cells isolated from the liver 
have a 5-fold greater content of surface sulfhydryl groups as compared to 
their ascitic counterparts. The former can be sensitized to L-phenyl alanine 
mustard by blocking these sulfhydryl groups with 6-6' dithiodinicotinic 
acid. 

5. These results demonstrate a determinant role of the hepatic microenvironment 
in the maintenance of tumor cell GSH, cell surface sulfhydryl groups and 
the sensitivity of these tumor cells to L-phenyl alanine mustard. 

Publications : 

1. Somfai-Relle, S., Suzukake, K., Vistica, B.P., and Vistica, D.T.: Gluta- 
thi one-conferred resistance to antineoplastics: Approaches toward its 
reduction. Cancer Treat. Rev . 11: 43-54, 1984. 

2. Green, J. A., Vistica, D.T., Young, R.C., Hamilton, T.C., Rogan, A.M., and 
Ozols, R.F.: Potentiation of melphalan cytotoxicity in human ovarian cancer 
cell lines by glutathione depletion. Cancer Res. 44: 5427-31, 1984. 



283 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07119-06 LPET 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must lit on or)e llrye between the borders.) 

Studies on the Biochemical Toxicology of Oncolytic Platinum Compounds 



PRINCIPAL INVESTIGATOR (Ust other professional personnel tmlow the Principal Investigator.) (Name, title, laboratory, and Institute atflllatlon) 

PI: C. L. Litterst Pharmacologist LPET, NCI 



Others: J. Uozumi 



Vi sting Fellow 



LPET, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Biochemical Pharmacology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 

2.5 



PROFESSIONAL: 

1.5 



1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues Eil (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Although the toxic effects of cisplatinum on kidney have been appreciated for 
some time, the renal handling of cisplatinum and the mechanism by which the renal 
toxicity occurs are still incompletely understood. These mechanisms could be 
more easily defined if the molecular sites of interaction of cisplatin were 
recognized. This project is designed to define how the kidney handles cisplatin 
under normal conditions and after various pretreatments or other experimental 
conditions. Inherent in this study is an attempt to localize the sites of inter- 
action of cisplatin and its intracellular binding sites. This section reports 
the sex and tissue specificity of platinum binding to DNA, the effect of cis- 
platinum on renal ATPase function in vivo and in vitro and the blockage of 
cisplatin-induced renal toxicity by sodium thiosulfate. 



284 



PHS 6040 (Rev. 1/84) 



6PO 014.9II 



Z01 CM 07119-06 LPET 

Studies on the mechanism of action of cisplatin : 

Much work has been done to document the DMA binding of cisPt and to establish 
this as the probable mechanism of the antitumor effect. Similarly, much effort 
has been invested in documenting the various toxicities produced by cisPt, 
although the mechanisms of these effects have not yet been satisfactorily 
established. There is an obvious interest in attempting to establish whether 
there is any relation between DMA binding and renal toxicity. We have therefore 
investigated the relation between DMA binding as measured by an ELISA technique 
which is specific for the N-6 DMA adduct with cisPt, and various parameters of 
platinum toxicity and pharmacokinetics. We have documented an apparent sex- 
related specificity in the uptake of cisPt into renal nuclei between male and 
female rats. Although the total renal concentration of Pt is greater in female 
rats, the DMA binding in females is less than in males. This finding is consis- 
tent with clinical response of ovarian and testicular cancer patients and with 
the differential response observed between older (post-menopausal ) and younger 
(pre-menopausal ) ovarian cancer patients. The implication is that there is a 
hormonal influence on Pt binding or transport which is responsible for the 
lesser amount of binding in females. A related organ-specificity also was 
noted, with testicles accumulating substantially greater amounts of DMA adducts 
that did ovaries. This is again consistent with the greater clinical response 
seen in testicular cancer patients than in ovarian patients. Finally, in both 
kideys and gonads of males and females, DMA binding but not whole tissue 
platinum content, appears to be dose dependent, with a dramatically elevated 
increase in binding at high cisplatin doses. This suggests, among other things, 
that the transport of platinum into the nucleus, or the cytosolic binding of 
platinum, may be saturable. These early results are currently in manuscript 
form, and experiments are continuing to investigate the mechanism behind these 
findings. 

The effect of cisplatin on content and function of renal ATPase was investigated 
as a possible explanation for the renal toxicity of the drug. When purified 
ATPase or when ATPase from tissue homogenates were incubated in vitro with 
cisplatin decreases in ATPase activity were observed. However, either very 
high concentrations of cisplatin (400-600 uM) or very long times of incubation 
(2-3 hrs.) were required before any changes were noted. In vivo, cisplatin 
produced a decrease in ATPase that occurred several days after drug administra- 
tion and 1-2 days after significant elevations of BUN occurred. Renal platinum 
concentrations were below the concentrations required to produce significant 
decreases in activity when tested in vitro. It was concluded that cisplatin 
probably does not interact with ATPase as a primary site of toxicity, but that 
cisplatin does have the ability to decrease the activity of this enzyme as a 
secondary effect. 

Sodium thiosulfate (STS) was shown to completely block the cisplatin induced 
rise in BUN and to prevent cisplatin-induced deaths. The mechanism of this 
interaction was investigated by examining the subcellular distribution of 
platinum in rats receiving cisplatin and in rats receiving cisplatin plus STS. 
Renal platinum concentrations in STS pretreated rats were less in nuclei, 
mitochondria, and microsomes during the first 8 hours after cisplatin 
administration. Similar decreases in platinum content were not consistently 
observed in liver. The differences in distribution were statistically signif- 



285 



Z01 CM 07119-06 LPET 

leant but were of a relatively small magnitude, so the biological significance 
is questionable. Because of this small changes in distribution, it is probably 
not realistic to ascribe the dramatic blockage of renal toxicity entirely to 
changes in subcellular distribution of platinum. 

Pharmacokinetics and toxicity of experimental drugs : 

Tetraplatin is a new analog of cisplatin that has shown activity in animal 
tumor systems which are resistent to cisplatin. We have conducted extensive 
preclinical toxicity studies of this compound in comparison with cisplatin and 
CHIP, another cisplatin analog with chemical similarity to tetraplatin. We 
evaluated the effect of 4 equimolar doses of the drugs on renal histology, 
urinary and plasma enzymes, and renal slice function over a 15 day time course. 
It appears from early inspection of the data, as if tetraplatin is less nephro- 
toxic, but that the toxicity is not dramatically less. In addition, the histo- 
pathologic distribution of the lesion is generally throughout the cortex, 
rather than being restricted to the corticomedullary junction, as is the case 
with cisplatin. We are continuing to evaluate our data so that we we will be 
able to draw more definite conclusions regarding the relative toxicity of this 
analog. 

Publications : . 

1. Litterst, C.L. and Reed, E.: Platinum Compounds. In Kaiser, H.E. (Ed.): 
Progressive Stages in Neoplastic Growth . Chapter 196, Oxford, Pergamon 
Press, in press. 

2. Litterst, C.L.: Cisplatinum: A review, with special reference to cellular 
and molecular interactions. Agents Actions 15: 520-525, 1985. 

3. Litterst, C.L.: Toxicology of the Antineoplastic Agents, with Special 
Reference to Reproductive Toxicology, Carcinogenesis, and Teratogenesis. 
In Haley, T. and Berndt, W., (Eds.): Handbook of Toxicology , New York, 
Hemisphere Publishing Corp., in press. 

4. Litterst, C.L. and Copley, M.: Cisplatin and Doxorubicin: Comparative 
evaluation of nephrotoxicity using serum chemistry, urinalysis and histolo- 
gy. In Bach, P. (Ed.): Renal heterogeneity and Target Cell Toxicity . 

New York, John Wiley & sons, in press. 

5. Poirier, M.C., Reed, E., Zwelling, L.A., Ozols, R.F., Litterst, C.L. and 
Yuspa, S.H.: The use of polyclonal antibodies to quantitate cis- diammine- 
dichloroplatinum (II)-DNA adducts in cancer patients and animal models. 
Environ. Health Perspect ., in press. 

6. Farris, P.P., King, P.G., Dedrick, R.L. and Litterst, C.L.: Physiological 
model for the pharmacokinetics of cis-dichlorodiammineplatinum (II) (DDP) 
in the tumored rat. J. Pharmacokin. Biopharm. , in press. 

7. Uozumi, J. and Litterst, C.L.: Effect of cisplatin on renal ATPase activity 
in vivo and in vitro. Cancer Chemotherapy & Pharmacology, in press. 



286 



ZOl CM 07119-06 LPET 

Kelley, J. A., Roth, J.S. and Litterst, C.L.: Gas chromatographic determina- 
tion of hexamethylene bisacetamide in plasma and urine. Anal. Letters , in 
press. 

Litterst, C.L., Smith, \A., Smith, J., Uozumi , J. and Copley, M.: Sensi- 
tivity of urinary enzymes as indicators of renal toxicity of the anticancer 
drug cisplatinum. In Bianchi, C. and Blaufox, D.M. (Eds.): Newer Diagnos- 
tic Methods in Nephrology and Urology . Basel, Switzerland, Karger Publish- 
ing Co., in press. 



287 



DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE 
NOTrCE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07120-06 LPET 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title musf //( on one line befween the borders.) 

Role of Drug Metabolism in Modulating Toxicological Responses 



PRINCIPAL INVESTIGATOR (Ust other prolessional personnel below the Principal Investigator) (Name, title, laboratory, and Institute affiliation) 

PI: T. E. Gram Pharmacologist LPET, NCI 



Others: J. Goochee 
L. K. Okine 



Chemist 
Visiting Fellow 



LPET, NCI 
LPET, NCI 



COOPERATING UNITS (If any) 

National Heart, Lung and Blood Institute, NIH (M. C. Lowe) 



UB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Biochemical Pharmacology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS; 

2.5 



PROFESSIONAL: 

1.5 



1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues 13 (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The existence and biochemical mechanisms of organ-specific toxicity are the sub- 
ject of heightened interest among toxicologists. Earlier studies in this labor- 
atory described selective necrosis of pulmonary non-ciliated bronchiolar (Clara) 
cells following administration of naphthalene to mice. No damage to other lung 
cells was noted and no pathologic changes, as evidenced by histology or enzymic 
alterations were observed. The work described in this section describes condi- 
tions under which 1 ,1-dichloroethylene (DCE) produces selective damage to mouse 
lung without morphologic or enzymatic evidence of nephro- or hepatotoxicity. 
Accompanying the lung damage there was a significant impairment of pulmonary 
cytochrome P-450 linked monooxygenase activities. Simultaneous with these changes 
there was a paradoxical increase in certain of these activities in kidney; these 
increases were found to be the result of enzyme induction. 



288 



PHS 6040 (Rev. 1/84) 



GPO 814>Sie 



ZOl CM 07120-06 LPET 
Methods Employed : 

Standard enzymatic and analytical techniques have been utilized. 
Major Findings : 

1 . In vivo distribution of [^^C] 1 ,1-dichloroethylene, covalent binding, and 
effects of drugs in the mouse , rhe distribution and covalent binding of a 
single dose of [1 ,2-l^C]l ,1-dichloroethylene (DCE; 125 mg/kg, I. P.) were studied 
in male C57B1/6N mice. Total radioactivity was distributed in whole homogenates 
of all tissues studied with peak levls occurring within 6 hours. Covalent 
binding of radioactive material peaked at 6-12 hours in all tissues and highest 
levels were found in kidney, liver and lung with smaller amounts in skeletal 
muscle, heart, spleen, and gut. Covalent binding in kidney, liver and lung 
fell to 50% of peak levels in about 4 days. Between 12 hours and 4 days after 
DCE administration, 70-100% of total radioactivity present in homogenates of 
kidney, liver and lung was covalently bound. The three tissues showed similar 
spread in total radioactivity in subcellular fractions 24 hr after exposure to 
DCE; most of the radioactivity was covalently bound (60-100% and distributed 
fairly uniformly with a slight tendency to concentrate in the mitochondrial 
fraction. Phenobarbital (PB) and 3-methylcholanthrene (3-MC) pretreatments 
increased the covalent binding in the liver and lung but had no effect in 
kidney. Piperonyl butoxide and SKF-525A decreased the covalent binding in 
liver and lung but the latter increased binding in the kidney while the former 
decreased it. Di ethyl maleate administration increased the covalent binding (2- 
to 3-fold) in all three tissues as well as increasing lethal toxicity. These 
results are consistent with the view that DCE is metabolized to some reactive 
intermediate(s) which may be detoxified by conjugation with glutathione. 

2. Effect of steroid pretreatment on the pulmonary damage produced by butyl ated 
hydroxytoluene in mice . The effects of the synthetic corticosteroid methyl- 
prednisolone (MP; 30 mg/kg, s.c. given twice daily for 3 days), on the pneumo- 
toxic effects of a single dose of butylated hydroxytoluene (BHT; 400 mg/kg, 
i.p.) over a 10 day experimental period was investigated in male C57B1/5N 

mice. BHT alone caused time-dependent alveolar hypercellularity, inflammatory 
infiltration, alveolar septal thickening with some degree of recovery by day 
10. The pulmonary monoosygenase activities reflected the degree of alveolar 
damage and Clara cell abnormality with time; reductions in monooxygenase 
activities occurred and minimum levels (7-15% of control) were reached by day 5 
and again a trend towards recovery by day 10. MP administered 0, 24 and 48 hr 
after BHT treatment partially protected mice from these effects of BHT in a 
distinctly time-dependent fashion; the degree of protection decreased as the 
time between BHT challenge and MP treatment increased. Although MP alone did 
not morphologically affect Clara and alveolar cells, it increased, decreased or 
had no effect on the monooxygenase activities. About 25% of the mice that 
received BHT alone died by day 5 and 50% by day 10. MP completely blocked the 
lethal effects of BHT by day 5 and reduced the deaths to between 15% and 25% by 
day 10. Interestingly, MP did not protect against the BHT-induced pulmonary 
fibrosis, measured as total lung hydroxyproline content, irrespective of the 
time between BHT challenge and MP treatment. MP alone did not cause any deaths 
nor increase lung hydroxyproline content. 



289 



ZOl CM 07120-06 LPET 
Publications : 

1. Trush, M.A., Reasor, M.J. and Van Dyke, K.: Oxidant-mediated electronic 
excitation of imipramine. Biochem. Pharmacol. 33: 3523-26, 1984. 

2. Krijgsheld, K.R., Lowe, M.C., Mimnaugh, E.G., Trush, M.A., Ginsburg, E. 
and Gram, T.E.: Selective damage to nonciliated bronchi olar epithelial 
cells in relation to imapirment of pulmonary monooxygenases activities by 
1,1-dichloroethylene in mice. Toxicol. Appl . Pharmacol. 74: 201-213, 1984. 

3. Krijgsheld, K.R. and Gram, T.E.: Selective induction of renal microsomal 
cytochrome P-450-linked monooxygenases by 1 ,2-dichloroethylene in mice. 
Biochem. Pharmacol . 33: 1951-1956, 1984. 

4. Gram, T.E.: The pulmonary mixed function oxidase system. In Witschi, H.P. 
and Brain, J.D. (Eds.): The Toxicology of Inhaled Materials. Part I: 
General Principles of Inhalation Toxicology . Berlin, Springer-Verlag, 
1985, pp. 421-470. 

5. Okine, L.K., Goochee, J.M. and Gram, T.E.: Studies on the distribution and 
covalent binding of 1,1-dichloroethylene in the mouse: Effect of various 
pretreatments on covalent binding in vivo . Biochem. Pharmacol ., in press. 

6. Okine, L.K., Lowe, M.C., Mimnaugh, E.G., Goochee, J.M. and Gram, T.E.: 
Protection by methyl prednisolone against butylated hydroxytoluene-induced 
pulmonary damage and impairment of microsomal monooxygenase activities in 
the mouse: lack of effect on fibrosis. Exp. Lung Res. , in press. 



290 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM-07122-05 LPET 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on or\e line betweer\ the borders.) 



Biochemical and Pharmacological Studie'; with nnrnlytir MurlPnciHoc 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

P. I.: D. A. Cooney Senior Investigator LPET, NCI 



Others: 



Y. A. Wilson 

M. Dalai 

E. B. Gregory 

G. Kang 

V. E. Marquez 



Chemist 
Microbiologist 
Bio. Lab. Tech. 
Visiting Fellow 
Visiting Scientist 



LPET, NCI 

LPET, NCI 

LPET, NCI 

LPET, NCI 

LPET, NCI 



COOPERATING UNITS (If any) 



Laboratory of Biological Chemistry (R. Glazer and S. Arnold) 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Biochemical Pharmacology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

2.5 



PROFESSIONAL: 



1.0 



1.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



Q (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Biochemical and Pharmacologic Studies with Arabinosyl-5-Azacytosine (Ara-AC) 

Ara-AC is a hybrid nucleoside combining the structural elements of arabinosyl 
cytosine and 5-azacytidine. During the past year, systematic studies were carried 
out to determine the mechanisms by which tumor cells acquire resistance to this 
agent. Three models were adopted: 1) spontaneous resistance, exemplified by the 
transplantable colon carcinoma 38; 2) acquired resistance provoked by cultivation 
of P388 cells in the presence of incremental concentrations of Ara-AC over 100 
generations; 3) resistance provoked by mutagenesis with MNNG. In all three cases, 
the resistance was accompanied by a fall, and in some cases a deletion of deoxycy- 
tidine kinase, the enzyme responsible for inaugurating the anabolism of the drug. 
In the case of the colon carcinoma 38, a second enzymatic factor was identified, 
namely an abundant endowment with a cytosolic nucleoside triphosphatase, which 
prevents the accumulation of adequate levels of the proximate antimetabolite, 
Ara-AC-triphosphate. These studies were next extended to a panel of cultured 
human tumor cells. In these lines there was a positive correlation (r = 0.63) 
between deoxycytidine kinase levels and the IC50 of Ara-AC. It is therefore 
proposed that measurement of this kinase might well be of prognostic value in 
patients scheduled to receive Ara-AC in its forthcoming Phase I trials. 



291 



PHS 6040 (Rev. 1/84) 



GPO ai4-SIB 



ZOl CM-07122-05 LPET 

Studies with Arabinosyl-5-Azacytosine (Ara-AC) 

Ara-AC is a hybrid nucleoside, synthesized in the Division of Cancer Treatment 
and being readied for Phase I trials with a high priority. In last year's 
Report, studies on the mechanism of action of this drug were recapitulated: 
Ara-AC undergoes extensive anabolism to the 5 '-triphosphate which in turn 
inhibits the synthesis of DNA, but is also incorporated into that macromolecule, 
where it undergoes ring-opening and engenders inhibition of methyl ati on. During 
the present year an extensive series of experiments were conducted to determine 
the mechanism(s) by which tumor cells become resistant to Ara-AC. This study 
is rendered all the more relevant by virtue of the apparently facile emergence 
of resistance to Ara-C and 5-AC in patients receiving these agents. Three 
categories of resistance were examined: spontaneous or native resistance, 
exemplified by the colon carcinoma 38; resistance provoked by cultivation of 
P388 cells in the presence of incremental concentrations of Ara-AC over 100 
transplant generations; and resistance provoked by exposure to MNNG followed by 
cloning in agarose. This procedure yielded 8 separate clones, all capable of 
growing normally in concentrations of the drug ~1000 times those tolerated by 
the parental strain. Resistance in all of aforementioned cases was found to be 
associated with a depression in or deletion of the specific activity of deoxy- 
cytidine kinase the enzyme which inaugurates the metabolism of Ara-AC. An 
identical mechanism is very frequently associated with resistance to Ara-C. 
Interestingly, a second factor contributing to resistance was uncovered in 
Colon Carcinoma 38: namely, a rich endowment with a cytosolic nucleotidase, 
which preferentially degrades nucleoside triphosphates. This activity would 
of course preclude the maintenance of effective levels of the putative prox- 
imate antimetabolite of Ara-AC, namely Ara-AC- triphosphate. Pursuant to these 
studies with rodent cells and tumors, a prospective examination was conducted 
of the relationship between the concentration of deoxycytidine kinase and the 
cytotoxicity of Ara-AC towards a panel of cultured human tumor cells. 
Although the correlation between these two parameters was positive (r = 0.633) 
it was not perfect. Nevertheless it was clear from the results that tumor 
cells deficient in deoxycytidine kinase were more refractory to Ara-AC than 
their well -endowed counterparts. 

Publications : 

1. Gebeyehu, G., Marquez, V.E., Van Cott, A., Cooney, D.A., Kelley, J. A., 
Jayaram, H.N., Ahluwalia, G.S., Dion, R.L., Wilson, Y.A. and Johns, D.G.: 
Ribavirin, tiazofurin, and selenazofurin: mononucleotides and nicotinamide 
adenine di nucleotide analogs. Synthesis, structure and interactions with 
IMP dihydrogenase. J. Med. Chem. 28: 99-105, 1985. 

2. Tono-Oka, S., Tone, Y., Marquez, V.E., Cooney, D.A., Sekikawa, I. and Azuma, 
I.: Enzymatic synthesis and biochemical activity of various indazole 
adenine di nucleotides. Bulletin of the Chemical Society of Japan 58: 309- 
315, 1985. 

3. Carney, D.N., Ahluwalia, G.S., Jayaram, H.N., Cooney, D.A. and Johns, D.6.: 
Relationships between the cytotoxicity of tiazofurin and its metabolism by 
cultured human lung cancer cells. J. Clin. Invest. 75: 175-182, 1985. 



292 



ZOl CM-07122-05 LPET 

4. Cooney, D.A., Stergis, G., Jayaram, H.N.: Enzymes, Therapeutic. In Kirk, 
R.E. and Othmer, D.F. (Eds.): Concise Encyclope dia of Chemical Technology. 
New York, John Wiley & Sons, 19Bb, pp. 429-430. 

5. Cooney, D. and Kutzmits, R.: Pharmacology of anticancer drugs. Cancer 
Treat. Rev. , in press. 

6. Tyagi, A.K. and Cooney, D.A.: Biochemical pharmacology, metabolism and 
mechanism of action of L-alanosine, a novel natural antitumor agent. Adv. 
Pharmac. Chemother. 20; 69-121, 1984. 



293 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07129-04 LPET 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) 

Copper and Its Chelates in Cytotoxicity, Chemotherapy and Melanoma Promotion 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: M. Rabinovitz Research Chemist LPET, NCI 



Others: H. F. Pierson 
J. M. Fisher 



PRAT Fellow 
Chemist 



LPET, NCI 
LPET, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Biochemical Pharmacology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

0.75 



PROFESSIONAL: 



0.5 



0.25 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



n (b) Human tissues \Jk (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The cytotoxicity of the copper chelate of the copper-specific chelating agent 
2,9-dimethyl-l,10-phenanthroline to L1210 murine lymphoma cells in vitro had been 
shown to be due to its marked uptake by these cells. Such uptake was dependent 
upon the tetrahedral bi dentate structure of the copper chelate and resulted in a 
small but significant in vivo chemotherapeutic activity. Accordingly, we had 
synthesized the corresponding halomethyl analogs in order to investigate their 
possible activity as copper-dependent, intercalating alkylating agents. These 
comprised the series: 2-chloromethyl-9-methyl-l,10-phenanthroline, NSC 382655; 
2-bromomethyl-9-methyl-l,10-phenanthroline, NSC 382656; 2,9-bis-(chloromethyl )-l, 
10-phenanthroline; and 2,9-bis-(bromomethyl )-l,10-phenanthroline. Copper de- 
pendency for cytotoxicity was found only in the monohalomethyl compounds in the 
range 1 to 3 pM. They were submitted for chemotherapeutic evaluation with a 
procedure for formulation as cuprous chelates. 

Copper chelates modulate 816 melanoma growth and metastasis in both a murine 
strain and copper chelate specific manner. Tumors failed to grow in CDF^ and 
Swiss mice as subcutaneous transplants unless the mice were pretreated with cop- 
per chelates. Thus the cuprous chelate of neocuproine ( 2, 9-dimethyl-l, 10-phen- 
anthroline) promoted testicular metastasis in NIH Swiss mice but inhibited them 
in CDFi mice, while the nitrilotriacetic acid cupric chelate permitted such 
metastases in both strains. 



294 



PHS 6040 (Rev. 1/84) 



GPO BI4-91B 



ZOl CM 07129-04 LPET 



Objectives; 



Our studies are directed toward understanding the basis of copper-mediated 
cytotoxicity and the sensitivity to copper and its chelates to tumor cells. 
This information is applied to the design of regimens and new agents for 
chemotherapy. 

Methods Employed : 

The principal methods employed involve techniques such as primary and estab- 
lished cell culture, viability estimates by clonal growth, cell size distri- 
bution and chemotherapeutic evaluation with tumor bearing mice. Studies with 
isotopically labelled components are also carried out in experiments designed 
to ascertain mechanisms of action. Information gained from such biochemical 
studies is applied to the design of new agents, a route for their synthesis is 
proposed and after preparation, the new compounds are evaluated for chemo- 
therapeutic efficacy and postulated mechanisms of action. 

Major Findings : 

A. A New Class of Halomethylphenanthrolines as Potential Copper-Dependent, 
Chemotherapeutic Agents : 

The copper-specific chelating agent, 2,9-dimethyl-l,10-phenanthroline had 
significant but limited chemotherapeutic activity against the P388 lymphoma 
when administered in vivo together with copper (Biochem. Pharmacol. 32: 362, 
1983). Cytotoxici'Cy was due to marked uptake of the copper chelate "Sy the 
cells, which in turn was dependent upon the unique tetrahedral ring system 
formed only with the 2- and 9-dimethyl derivative. In order to exploit this 
property more fully, we had four members of a new class of 2,9-halomethyl- 
phenanthrolines synthesized as potential copper-dependent, DNA-intercalating, 
alkylating agents. These were prepared by Drs. Vinod Gupta and George Newkome 
of the Department of Chemistry, LSU, Baton Rouge, LA. 




CH2C1 




CH2Br CIH2C 





CH2CI BrH2C 



HI 



IV 



CH2Br 



I 2-chloromethyl-9-methyl-l,10-phenanthroline; NSC 382655 

II 2-bromomethyl-9-methyl-l,10-phenanthroline; NSC 382656 

III 2,9-bis-(chloromethyl )-l,10-phenanthroline 

IV 2,9-bis-(bromomethyl )-l,10-phenanthroline 

All 4 componds formed bright yellow cuprous chelates and were found to be 
cytotoxic to P388 cells in primary culture in the range 1 to 3 yM. However, 
copper dependency for cytotoxicity was observed only in the monohalogenated 
compounds, and these were submitted for chemotherapeutic evaluation with a 
procedure for formulation as cuprous chelates. 



295 



ZOl CM 07129-04 LPET 

B. Copper Chelates Promote Melanoma Tumorigenicity (by Herbert Pierson) 

This study was aimed at clarifying the role of exogenous copper on B16 melanoma 
in vivo . Groups of BDFi, DBA, CDFi and NIH Swiss mice were treated ip with 
either the cupric chelate or nitrilotriacetic acid (NTA: Cu"*"^, 20:10 pmoles/kg) 
or the cuprous chelate of neocuproine (NC:Cu''"S 10:5 pmoles/kg) every other 
day for 7 days prior to tumor inoculation and every other day for 15 days 
thereafter. Treatment with NC:Cu"''^: promoted tumor growth; increased body 
weight gain; stimulated tumor growth rate; inhibited tumor encapsulation; 
permitted tumor growth as unrestrained ascites; enhanced tumor pigmentation; 
and increased the final weight of tumors despite hastened host mortality. 
Tumors failed to grow in CDFi and NIH Swiss mice as sc transplants unless 
mice were pretreated with copper chelates. NTA:Cu''"2 had little influence on 
metastasis of rapidly growing tumors (BDFI), partially inhibited pulmonary and 
testicular metastasis, but enhanced hepatic metastasis of slowly growing tumors 
(DBA); and permitted pulmonary, hepatic, and testicular metastasis in CDFI and 
NIH Swiss mice. NC:C^1 enhanced pulmonary metastasis of rapidly growing tumors 
(BDFI); partially inhibited pulmonary metastasis of slowly growing tumors 
(DBA); inhibited testicular metastasis in CDFI mice, and enhanced pulmonary, 
hepatic and testicular metastasis in NIH Swiss mice. These results suggest 
that exogenous copper modulates B16 melanoma growth and metastasis in both a 
murine strain and copper chelate-specific manner. Possible mechanisms include 
enhanced copper nutriture of tumors and inhibition of immune responses. 

Publications ; 

1. Pierson, H.F.: Pharmacological perturbation of murine melanoma growth by 
copper chelates. Cancer Lett. 26: 221-233, 1985. 

2. Pierson, H.F.: Administration of copper chelates enhances tumorgenicity 
of B16 melanoma in heterogenetic mice. Cancer Treat. Rep. , in press. 

3. Pierson, H.F. and Meadows, G.G.: Nutritional and pharmacological 
modulation of peroxidation in murine melanoma. J. Natl . Cancer Inst ., 
in press. 



296 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07164-01 LPET 



PERIOD COVERED 



October 1. 1985 to September 30. 1985 



TITLE OF PROJECT (80 characters or lass. Title must fit on one line beftveen ttie borders.) 



Differential Competition between Metabolites and Cytotoxic Analogs 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: M. Rabinovitz Research Chemist LPET, NCI 



Others: 



H. F. Pierson 
J. M. Fisher 



PRAT Fellow 
Chemi st 



LPET, NCI 
LPET. NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Biochemical Pharmacology Section 



INSTITUTE AND LOCATION 

NCI, NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 

0.75 



PROFESSIONAL: 

0.5 



0.25 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues Q (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Taurine mustard, N-bis-(2-chloroethyl)aminoethane sulfonic acid, had significant 
chemotherapeutic activity against the P388 and L1210 lymphocytic leukemias, and 
the pigmented and unpigmented B16 melanomas. Its cytotoxicity toward the P388 in 
primary culture (IC50 = 50 uM) was not reduced by a high taurine concentration (5 
mM). Taurine also did not reduce its efficacy in vivo . However, in vivo , taurine 
protected against neurotoxicity. Intestinal necrosis, pulmonary emBSITTormation 
and tail vein necrosis when administered i.v. with taurine mustard. 



297 



PHS 6040 (Rev. 1/84) 



GPO St4-ei8 



ZOl CM 07164-01 LPET 

Objectives : 

The principal objective of this ongoing series of studies is to evaluate the 
competitive interaction between a metabolite and a newly designed cytotoxic 
analog. Information obtained in this manner is applied to the tumor bearing 
animal in attempts to limit toxicity to the host. 

Methods Employed : 

The principal methods employed involve techniques such as primary and estab- 
lished cell culture, viability estimates by clonal growth, cell size distribu- 
tion and chemotherapeutic evaluation with tumor bearing mice. Studies with 
isotopically labelled components are also carried out in experiments designed 
to ascertain mechanisms of action. Information gained from such biochemical 
studies is applied to the design of new agents, a route for their synthesis is 
proposed and after preparation, the new compounds are evaluated for chemothera- 
peutic efficacy and postulated mechanisms of action. 

Major Findings : 

Taurine Protects Host Tissues from the Toxicity of Taurine Mustard : 

The high intracellular concentration of taurine in tumors, its unique uptake 
characteristics, together with its suggested role as a displacer of hi stone and 
non-hi stone protein from DNA as a mechanism for its effect on cellular prolifer- 
ation made an investigation of taurine mustard, N-bis(2-chloroethyl )amino- 
ethanesulfonic acid (TM) of interest. 

H2N CH2 CH2 SO3 H CI CH2 CH2. 

N CH2 CH2 SO3 H 




TAURINE CI CH2 CH2 TAUMUSTINE 



The antitumor activity of taurine mustard (taumustine) was evaluated in the 
murine P388 and L1210 lymphocytic leukemias, and the pigmented and nonpigmented 
B16 melanoma systems. Antitumor activity was found to be dependent upon the 
route of administration, treatment schedule and anatomical placement of tumor 
inoculum. Treatment with a single i.p. dose of taumustine (40 mg/kg) resulted 
in a 130% increase in survival for mice bearing P388 (i.p.), 93% increase in 
survival for mice bearing L1210 (i.p.), and about 80% increase in survival for 
mice bearing B16 melanoma (i.p.). Treatment with a single dose of taumustine 
increased the survival of mice bearing s.c. melanomas by 45% and decreased 
final tumor weight, but did not increase survival of mice bearing s.c. L1210 
leukemia despite a reduction of final tumor weight. Repeated low doses (10 
mg/kg) of taumustine resulted in 250% increase in survival for mice bearing 
P388 (i.p.), absence of ascites from day 4 onward, and the presence of pulmon- 
ary emboli from day 5 onward. 

The inclusion of taurine (5 mM) in the culture medium of P388 cells in primary 
culture for 45 hr did not alter the cytotoxicity of taumustine (IC50 = 50 pM). 
However, pretreatment of the tumor bearing host with taurine (250 mg/kg) 30 min 



298 



ZOl CM 07164-01 LPET 

before daily treatments with taumustine for up to 8 days did not interfere with 
antitumor activity (140-160% increased survival), promoted ascites formation 
independent of the number of treatments, and afforded protection against the 
formation of pulmonary emboli. Treatment of tumor-bearing mice with taurine 
also abrogated neurotoxicity, intestinal necrosis, pulmonary emboli formation, 
and tail vein necrosis due to the administration of a toxic dose of taumustine. 
The modulation by taurine of taumustine activity suggests that the combination 
of these agents may offer an advantage of selectivity and host protection during 
chemotherapy. 



299 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07165-01 LPET 



PERIOD COVERED 



October 1, 1985 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must tit on one line beftveen the borders.) 

The Sulfhydryl Group in Cancer Cell Growth and in Chemotherapy 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute affiliation) 

PI: M. Rabinovitz Research Chemist LPET, NCI 



Other: 



H. F. Pierson 
J. M. Fisher 



PRAT Fellow 
Chemist 



LPET, NCI 
LPET, NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Biochemical Pharmacology Section 



INSTITUTE AND LOCATION 

NCI. NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 

1.5 



PROFESSIONAL: 

1.0 



0.5 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



D (b) Human tissues [jl (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Many tumors in primary culture, both murine and human, cannot utilize cystine as 
a source of cysteine for growth. Attempts were therefore made to maintain an 
oxidative stress in vivo which would convert available cysteine to cystine and 
thus inhibit tumor growth. The mixture of hydroxocobalamin (vit. B-12) and 
ascorbate were very efective in oxidizing cysteine in vitro and were shown to 
increase the life span of P388 lymphoma-bearing mice by 70 percent. 

Cells which will not grow with cystine as a source of cysteine require mercapto- 
ethanol or its oxidized form, hydroxyethyl disulfide, as a growth factor. These 
facilitate the uptake by the cell of the cysteine moiety as the mixed disulfice. 
A similar interaction with inverse purpose, the promotion of cytotoxicity of homo- 
cystine, selenocystine and selenocystamine by dimethyl disulfide was discovered. 
This reaction increased the cytotoxicity of selenocystamine in vitro by two orders 
of magnitude while reducing its host toxicity in vivo . 



300 



PHS 6040 (Rev. 1/84) 



OPO •I4<«ia 



ZOl CM 07165-01 LPET 

Objectives : 

These studies are directed toward understanding the critical role of mercapto- 
ethanol in support of growth of tumor cells in primary culture and exploiting 
this role to chemotherapeutic advantage. 

Methods Employed : 

The principal methods employed involve techniques such as primary and estab- 
lished cell culture, viability estimates by clonal growth, cell size distribu- 
tion and chemotherapeutic evaluation with tumor bearing mice. Studies with 
isotopically labelled components are also carried out in experiments designed 
to ascertain mechanisms of action. Information gained from such biochemical 
studies is applied to the design of new agents, a route for their synthesis is 
proposed and after preparation, the new compounds are evaluated for chemother- 
apeutic efficacy and postulated mechanisms of action. 

Major Findings ; 

1. Depletion of Extracellular Cysteine with Hydroxocobalamin and Ascorbate in 
Experimental Cancer Chemotherapy : 

Treatment of mice bearing P388 leukemia with combined hydroxocobalamin (0.10 
mmole/kg) and sodium ascorbate (1.0 mmoles/kg) for 10 consecutive days resulted 
in 70% increase in survival. Treatment with dehydroascorbate (1.0 mmole/kg) 
produced similar increased survival, but treatment for 15 days did not improve 
survival. P388 cells incubated in primary culture with cystine as a source of 
cysteine require exogenous thiols or disulfides for growth; and also grow with 
cysteine, but only when trace copper is sequestered with bathocuproine sulfon- 
ate, a copper specific chelate which prevents copper catalyzed oxidation of 
cysteine to cystine. Cells grew in medium preincubated for 8 hr with cysteine 
and bathocuproine sulfonate, but lysed when the preincubated medium included 
hydroxocobalamin (10 yM) and sodium ascorbate (100 yM). This defect was 
corrected by resupplementation with cysteine or cystine and bishydroxyethyl 
disulfide (25 yM) after addition of the cells. Further support for cysteine 
depletion in therapy is obtained by therapeutic interference with 4-thiamethio- 
nine (25.0 ymoles/kg). This mixed disulfide of cysteine and methyl mercaptan 
enters cells as a methionine analog, is reduced to supply cysteine, and thus 
withstands the oxidative environment that limits utilization of cysteine as a 
nutrient. Ultimate failure of therapy may be due to increased availability of 
cysteine produced by host tissues, in particular, infiltrating macrophages. 

2. Adduct Formation in Selective Toxicity: Methyl thio-capping of Selenocyste- 
amine and its Potentiation of Antitumor Activity and Reduction of Host Toxicity 

Low molecular weight sulfhydryl compounds or disulfides like mercaptoethanol or 
hydroxyethyl disulfide are known to be required for growth of murine lymphomas 
and human tumors in primary cell culture. This is due to the formation and 
facilitated transport of mixed disulfides of cysteine and low molecular weight 
sulfhydryl compounds. An analogous reaction between mercaptans and diselenides 
or selenols and disulfides forms unstable thioselenates. We have found that 
dimethyl disulfide was the most effective of several mercaptans and disulfides 

301 



ZOl CM 07165-01 LPET 

for potentiating the cytotoxicity of homocystine I, selenocystine II, and 
selenocystamine III, in culture. A proposed mechanism by which dimethyl disul- 
f oxide increases the cellular uptake is indicated below: 



>. 




HOOCCCHjCHjSSCHjCH,C-COOH —^ CHjSSCH^CHiC-COOH 
NH, £ H it 

H NH, NH, 

HOOC-C-CH,S«SeCH2C-COOH — » CHjSSeCHi-C-COOH 
NH, IT H H 

HjNCHiCHiSeSeCHiCHiNH, — ^ CHjSSeCHiCHjNH, 

Cytotoxicity of all 3 compounds was accompanied by marked cell swelling and 
lysis. Such lysis was dependent on unbalanced growth, for homocystine treated 
cells did not lyse in the absence of methionine. High concentrations of leucine 
(5 mM) partially prevented the cytotoxicity of the mixture dimethyl disulfide 
and selenocystine, indicating that the methyl thio-adduct of selenocysteine 
entered cells via the leucine transport system. However, both homocystine and 
selenocystine were too insoluble to formulate for in vivo experiments and our 
attention was therefore direct toward selenocystamTne, a water soluble compound. 

Dimethyl disulfide markedly increased the cytotoxicity of selenocystamine by 2 
orders of magnitude (40 pM to 0.5 yM for 50% inhibition of growth in 45 hr). 
When mice were injected ip with 22 ymoles/kg of selenocystamine on Day 0, all 
were dead on Day 4. If dimethyl disulfide (500 ymoles/kg) was either mixed 
with selenocystamine or injected immediately prior to it, the mice survived 
treatment for at least 21 days afterwards, suggesting that methyl thio-capping 
of selenocysteamine abrogated host toxicity. Such combined treatment may be 
of value in chemotherapy. 

Publications : 

1. Mohindru, A., Fisher, J.M. and Rabinovitz, M.: Endogenous copper is 

cytotoxic to a lymphoma in primary culture which requires thiols for growth. 
Experientia , in press. 



302 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 07166-01 LPET 



PERIOD COVERED 



October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) 

Biochemical and Pharmacologic Studies with Oncolytic Barbituric Acid Derivatives 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

P. I.: D. A. Cooney Senior Investigator LPET, NCI 



Others: 



Y. A. Wilson 

M. Dalai 

E. B. Gregory 

G. Kang 



Chemist 

Microbiologist 
Bio. Lab. Tech. 
Visiting Fellow 



LPET, NCI 

LPET, NCI 

LPET, NCI 

LPET, NCI 



COOPERATING UNITS (if any) 

Laboratory of Biological Chemistry (R. Glazer) 
Laboratory of Experimental Therapeutics and Metabolism (J 
Clinical Pharmacology Branch, COP (B. Sinha) 



McMahon) 



LAB/BRANCH 

Laboratory of Pharmacology and Experimental Therapeutics 



SECTION 

Molecular Pharmacology Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 



20205 



TOTAL MAN-YEARS: 

2.5 



PROFESSIONAL: 



1.0 



1.5 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



n (b) Human tissues E (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Biochemical and Pharmacologic Studies with Oncol oy tic Barbituric Acid Derivatives 

Merbarone is a hybrid molecule, consisting of thiobarbituric acid conjoined to 
aniline in an amide linkage; the drug is curative of the L1210 leukemia. In last 
year's Annual Report it was documented that merbarone promptly arrested DNA 
synthesis in cultured L1210 cells. In the present year, the ability of merba- 
rone to produce damage to DNA was examined. These studies used cultured L1210 
cells as their principal tool. Exposure of these cells to an IC50 concentration 
of the drug for 16 hours resulted in a highly significant rate of single-strand 
breaks. These breaks did not, however, appear to be protein-associated. Approx- 
imately 24 hours were required for the repair of 50% of the SSB's. When merbarone 
was incubated with hepatic microsomes and NADPH, ESR signals suggestive of oxygen- 
centered radicals were generated. As a working hypothesis, it is suggested that 
such radicals may be mediating the DNA damage described here. 



303 



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ZOl CM 07166-01 LPET 
Initial Mechanistic Studies with Merbarone (NSC 336628) : 

The structure of merbarone [5-{N-phenylcarboxaniido)-2-thiobarbituric acid 
(NSC 336628) is shown in Fig. 1. Originally synthesized as a potential herb- 
icide, the compound was discovered to exhibit curative activity against L1210 
leukemia, when administered to mice by either the intraperitoneal or oral 
routes. Because of this activity, merbarone is now being readied for clinical 
trials. As an adjunct to those trials, our laboratory has instituted studies 
into the mechanism of action of this structurally novel molecule. L1210 cells, 
cultivated in RPMI 1630 medium, served as the principal experimental tools for' 
our studies. Exposure of logarithmically growing cultures of these murine 
lymphoblasts to graduated concentrations of merbarone (1-100 yM, dissolved in 
DMSO) resulted in progressive inhibition of proliferation. The median inhibi- 
tory concentration (IC50) was 10 ijM (range 9-13 yM). Cloning experiments 
revealed that a twenty-four hour exposure to 10 yM merbarone produced a 0.5 log 
decrease in viability, higher concentrations being progressively more cytocidal. 
Preliminary pharmacokinetic studies established that a single intraperitoneal 
injection of L^'^CJmerbarone, at its optimal therapeutic dose (50 mg/kg) produced 
plasma concentrations of drug-derived radioactivity which exceeded or approx- 
imated 10 yM for -A hr. This result establishes that the concentrations of 
merbarone used j_n vitro can be achieved jn^ vivo . 

Flow cytometry revealed that a 16 hr exposure of L1210 cells to merbarone 
(10 yM) markedly slowed progression of cells from S phase. Parallel studies 
of the influence of merbarone on macromolecular syntheses revealed that a 2 hr 
exposure to an IC50 concentration (10 yM) of the drug failed to alter nucleic 
acid or protein synthesis, but that a 24 hr exposure reduced the rate of DNA 
synthesis by 30-50%. High concentrations of merbarone (>IC99) promptly and 
totally blocked the incorporation of L-valine, uridine and thymidine into 
macromolecules. Transformation of human lymphocytes in the presence of 20 yM 
merbarone did not lead to prominent chromosomal aberrations. Mechanistic 
studies with merbarone were initially guided by its bipartite structure - a 
molecular conjugate of thiobarbituric acid and aniline, joined in amide linkage. 
Thiobarbituric acid has two major biologically significant properties: its 
reactivity with di aldehydes, notably malonaldehyde, and its ability to inhibit 
the membrane enzyme, y-glutamyl transpeptidase. Merbarone, tested at 100 yM 
under published conditions failed to function as a substrate or inhibitor in 
either system. Aniline is known to react with erythrocytes producing methemo- 
globinemia in susceptible subjects. Merbarone incubated in vitro with human 
red cells (10-100 yM) failed to produce methemoglobin. Parenthetically, it is 
relevant that merbarone was also nitrobenzyl pyridine-negative and so, is 
unlikely to function as an alkylating species - at least in its native state. 
The drug also was incapable of inhibiting the respiration of intact L1210 cells 
to a significant degree in vitro (at concentrations up to 50 yM) and so is 
unlikely to function as a straightforward respiratory inhibitor. 

The pyrimidine moiety of merbarone prompted studies of its possible role as an 
antimetabolite. However, exposure of L1210 cells to 10 yM drug for 24 hr 
failed to engender significant perturbation of their nucleotide or amino acid 
pools; a 5 hr exposure to 5 and 10 yM merbarone also was without influence on 
the cellular content of PRPP. In addition, none of the customary preformed 
purines or pyrimi dines (singly, and in combination) or amino acids, tested at 

304 



ZOl CM-071 66-01 LPET 

their maximally tolerated concentrations, proved capable of overcoming mer- 
barone's cytotoxicity, thus making it unlikely that the drug was behaving as a 
classical antimetabolite. 

Attempts to determine whether merbarone might bind to DNA were complicated by 
its avid affinity for conventional cellulose dialysis tubing, and a variety of 
other polymeric materials. When nucleic acids were extracted from L1210 
cells exposed to 10 pM [^^Clmerbarone for 16 hr, using SDS phenol extraction, 
only negligible radioactivity was associated with these macromolecules. The 
drug also appeared not to displace the intercalating compound, ethidium bromide, 
from calf thymus DNA as adjudged by fluorescence spectrometry. However, when 
L1210 cells were exposed to merbarone (10-50 pM) for 16-24 hr and their DNA 
examined by alkaline elution. Significant numbers of dose-related single 
strand breaks (SSB's) were demonstrable. SSBs were produced in both [^^C]- 
thymidine prelabeled cells and in cells exposed to [ '^]thymidine and merbarone 
simultaneously, although the latter protocol resulted in higher single strand 
break frequencies. Proteinase-K did not materially alter the elution pattern 
of DNA damaged by merbarone, suggesting that the SSB's are not protein- 
associated. Moreover, initial experiments demonstrated no formation of DNA- 
protein crosslinks by this compound. 

In order to examine the rate at which cells repaired the merbarone-induced 
lesions in their DNA, alkaline elution analyses were conducted at representative 
time-points after removal of the drug. These experiments demonstrated that about 
12 hr were required for the repair of 50% of these lesions. When merbarone was 
incubated with hepatic microsomes and NAOH, ESR signals suggestive of the 
presence of oxygen-centered radicals were generated. 

Although it is not clear from these experiments whether merbarone itself, or a 
metabolite, produces the DNA damage observed by alkaline elution, this damage 
does seem to be implicated in the therapeutic action of the drug inasmuch as it 
is seen at comparatively low concentrations of merbarone (from the IC20 upward). 
It will remain for future studies to pursue the lines of investigation suggested 
by the data reported herein. 



305 



ANNUAL REPORT OF THE LABORATORY OF TUMOR CELL BIOLOGY 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

The objectives of the Laboratory of Tumor Cell Biology are to develop, implement, 
and analyze data obtained from studies of cellular proliferation, cell differen- 
tiation, and biochemical growth characteristics of normal and malignant mammalian 
cells both TM vivo and in vitro . Particular attention is given to hematopoietic 
cells, their normal behavior and especially changes seen during leukemogenesis. 
Because of unusual access to human blood cells and because of the interest of 
this group, there is special focus on human leukemias and lymphomas, and acquired 
immune deficiency syndrome. It is anticipated that an enhanced understanding of 
cell regulatory mechanisms will permit the optimal use of anti-tumor agents in 
the therapy of cancer and the development of new approaches. 

The Laboratory of Tumor Cell Biology is concerned with several biological and 
biochemical problems: (1) Studies on the cellular and molecular origin and path- 
ogenesis of human leukemia. Biochemical control mechanisms involved in cell 
differentiation and neoplastic transformation are examined. Tumor viruses of 
animals are used both as tools (to define and isolate genes and gene products 
important for growth in man) as well as for help in understanding mechanisms of 
naturally occurring animal leukemias. Also, studies designed to determine the 
distribution of human T-cell leukemia virus (HTLV) in T-cell leukemia patients, 
patients with acquired immune deficiency syndrome (AIDS), and normals in differ- 
ent parts of the world are being intensively studied. (2) Studies on the bio- 
chemical events preceding mitosis. An understanding of these events appears 
essential to the control of proliferation, information derived from such studies 
may lead to more effective inhibitors of neoplastic cell growth. Events leading 
to mitosis are also of interest since many of the effective antitumor agents are 
useful only when cells are in DNA replication or in mitosis. Phytohemagglutinin 
stimulated human lymphocytes and tissue culture cells are the principal tools in 
these studies. (3) Attempts to develop new approaches to cancer chemotherapy 
using Information gained from basic cellular studies. (4) Studies on the devel- 
opment of biochemical and immunological markers for malignant cells are carried 
out. Biochemical and immunological studies are also conducted in individuals 
with disorders associated with an increased incidence of neoplasia. (5) Controls 
regulating cellular growth and differentiation, and the process of malignant 
transformation in hematopoietic cells. (6) Growth factors (and their receptors) 
that control the growth and differentiation of blood cells have been isolated and 
are under intensive study, e.g., T-cell growth factor (TCGF), CSF, and related 
hematopoietic growth effecting molecules. 

During the past year a number of findings were reported by investigators from 
the Laboratory. 

Major Findings : 

1. More than 100 isolates of HTLV-III have been obtained from patients with 
acquired immune deficiency syndrome (AIDS) and pre-AIDS. 



307 



2. HTLV-III has been isolated from semen or saliva of AIDS patients. 

3. Seroepidemiological studies with HTLV-III show that 95% of the AIDS and 
pre-AIDS patients and approximately 45% of healthy homosexuals carry HTLV- 
III antibodies. 

4. The sera from AIDS and pre-AIDS patients precipitates proteins of 41,000, 
120,000 and 160,000 daltons. 

5. Serological evidence for high prevalence (65%) of HTLV-III antibody in 
Ugandan children surveyed in 1972. 

6. Development of an assay for detection of neutralizing antibodies to HTLV- 
III in sera of individuals exposed to the virus. 

7. Demonstration of heterosexual transmission of HTLV-III by serologic de- 
tection of antibodies in sexual partners of AIDS and ARC cases in New York. 

8. Seroepidemiological studies show that populations at risk for development 
of AIDS include Canadian and Japanese hemophiliacs, Haitian immigrants to 
New Guinea, Zairians, Rwandese, and male prostitutes in Singapore. 

9. A monoclonal antibody against a 41,000 dal ton protein induced by HTLV-III 
infection has been prepared and characterized. 

10. Demonstration of an 18% prevalence of HTLV-III antibodies among black drug 
users in New York. 

11. HTLV-I antibodies have been found in sickle cell anemia patients in 
Martinique, Venezuela, several villages of Alaskan Aleuts and Swedish Lapps. 

12. Studies on the development of an animal model indicate chimpanzees to be a 
suitable animal. Seroconversion and HTLV-III isolation was demonstrated in 
chimps infected with HTLV-III. 

13. Development of an in situ hybridization technique to detect HTLV-III se- 
quences in several tissues, including brain, from patients with AIDS and ARC. 

14. Several drugs, including foscarnet, cis- plantinum and lapachone derivatives 
have been shown to inhibit HTLV-III repl i cation. 

15. Molecularly cloned and sequenced several HTLV-III isolates. Showed HTLV-III 
to be similar to LAV and ARV. 

16. Determined the splice acceptor site for the pX^ gene of HTLV-I and sequenced 
the £X region of HTLV-Ib- 

17. Developed a biologically active plasmid clone by using molecular clones of 
HTLV-III and Sprotoplast fusion technique. 

18. Determined the nucleotide sequence of the envelope gene of HTLV-III (RF). 

19. Demonstration of trans- acting transcriptional activation for HTLV-III. 



308 



20. Demonstration of HTLV-III to visna virus. 

21. Demonstration of HTLV-III infection in brains of AIDS patients with 
encephalopathy. 



309 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

701 CM 06117-13 LTCB 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line befween the borders.) 

Molecular and Physiological Control Mechanisms in Normal and Neoplastic Cells 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

Robert C. Gallo Chief LTCB NCI 

Marvin S. Reitz Chemist 

Prem S. Sarin 

W. Carl Saxinger 

Flossie Wong-Staal 

Karen Chayat 

Lee Ratner 



Leonard Seigel 



Chemist 

Microbiologist 
Microbiologist 
Clinical Associate 
Clinical Associate 



Clinical Associate 



LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 



COOPERATING UNITS (if any) 

Stu Aaronson, Viral Carcinogenesis Branch, National Cancer Institute; Rolf Neth, 
University of Hamburg; Robin Weiss, Imperial Cancer Research Fund, London, 
England; Dani Bolognesi and Bart Haynes, Duke University; Ken McCredie, M. D. 



LAB/BRANCH 

Laboratory of Tumor Cell Biology 

SECTION Sections on Hematopoietic cellular Control Mechanisms, Hematopoietic Cell 
Biochemistry and Immunology, and Molecular Genetics of Hematopoietic Cells. 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



56 



PROFESSIONAL: 



33 



23 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



(b) Human tissues 



D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

This Laboratory is concerned with five areas of research: (1) molecular and 
physiological control mechanisms in normal and neoplastic cells, designed to 
obtain information on the molecular mechanisms involved in neoplastic transfor- 
mation, including a search for and cloning of viral genomes and genome products 
in human tumor tissues; (2) the identification, isolation and demonstration of 
biological activity of viral information in human leukemic cells and cells from 
patients with acquired immune deficiency syndrome (AIDST] (T5 search for bio- 
chemical markers of minimal neoplastic disease and the development of practi- 
cally useful microtests for the detection of such markers; (4) cell differentia- 
tion vn_ vitro"! (This relates to a major interest of the Laboratory: Does the 
phenotypic abnormality of leukemia in man result from a block in leukocyte 
maturation ?) (5) Based on new information in the literature and from studies 
within this laboratory, new approaches to cancer chemotherapy are evaluated in 
in vitro and in vivo systems. This is the uTtiinate goal of the Laboratory. 



310 



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ZOl CM 06117-13 LTCB 

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL 
INVESTIGATORS AND ALL OTHER PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 



Suresh Arya 
S. Zaki Salahuddin 
Ann SI i ski -Mark 
Suzanne Gartner 
Mary Harper 

Marjorie Robert-Gurof f 
Genoveffa Franchini 
Mikulas Popovic 
Anna Aldovini 
Chan Guo 
Hong-Guang Guo 
Beatrice Hahn 
Ruth Jarrett 
Jorg Jendis 
Takashi Okamoto 
Yoshitaka Taguchi 
Enrico Cal lalti 
Sandra Colombini 
Amanda Fisher 
Corrado Gurgo 
Shuji Nakamura 
Jorg Schupbach 
Bruno Starcich 
Petr Stockbauer 
George Shaw 

COOPERATING UNITS 



Cancer Expert 
Cancer Expert 
Cancer Expert 
Staff Fellow 
Staff Fellow 
Staff Fellow 
Visiting Associate 
Visiting Scientist 
Visiting Fellow 
Visiting Fel low 
Visiti ng Fellow 
Visiting Fel low 
Visiti ng Fellow 
Visiting Fel low 
Visiting Fellow 
Visiting Fellow 
Guest Researcher 
Guest Researcher 
Guest Researcher 
Guest Researcher 
Guest Researcher 
Guest Researcher 
Guest Researcher 
Guest Researcher 
IPA 



LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 



Anderson Hospital and Tumor Institute; Yohei Ito, University of Kyoto; Myron 
Essex, Harvard University; Bill Haseltine, Harvard University; Jack Strominger, 
Harvard University; Roger Monier, Cancer Institute, Villejuif; Volker Erfle, 
Munich; Fernando deNoronha, Cornell University; Ivor Royston, University of 
California at San Diego; Bill Blattner, Epidemiology Branch, National Cancer 
Institute; Mark Smulson, Georgetown University; Isaac Witz, Tel Aviv University. 



311 



ZOl CM 06117-13 LTCB 
Project Description: 
Objectives 

1. It is anticipated that a greater understanding of the processes involved in 
the molecular control of cellular growth, differentiation, and carcinogenic 
transformation, including the pathogenesis of human neoplasias, will lead 
to the ultimate goal of developing improved approaches to therapy of human 
neoplasia. Special focus is on the leukmias and lymphomas. 

2. The development of "markers" of neoplastic cells may lead to (a) quantita- 
tion of residual tumor cells after therapy and (b) determining whether 
cells (e.g., in leukemia) of patients in remission are really normal. 

3. To develop new concepts of chemotherapy and apply them to animal model 
systems as rapidly as possible as new information is derived from basic 
experimental studies. 

These objectives have primarily been pursued by the following approaches: 

1. Biochemical studies on the properties of the RNA of type-C viruses and 
on the overall pathway of replication of these viruses. Purposes: 

a. To obtain more information on the mechanism of transcription of 
this RNA to DNA via reverse transcriptase. 

b. To determine if diagnostic probes can be obtained, i.e., is their 
structure specific enough that we can use this information to find 
viral RNA in cells? 

c. In understanding the mechanisms involved in integration and ex- 
pression of viral genes, we can plan approaches to interfere with 
this expression and then evaluate the overall biological effect of 
this interference. We particularly wish to know if viral expres- 
sion is required to maintain the cell in the neoplastic state. 

2. Pursuing studies leading to an understanding of the origin of tumor 
viruses, how they acquire their oncogenic potential, how they interact 
with cells, and how they are transmitted throughout nature. These 
studies are primarily carried out with techniques of molecular hybridi- 
zation, restriction enzyme analysis and gene cloning. 

3. Leukocyte differentiation in vitro . The soft agar technique for in- 
vestigating maturation and proliferation of normal and leukemic human 
bone marrow cells were recently set up in our laboratory. Attempts are 
made to study exogenous and endogenous (released from feeder layers of 
normal cells) factors which affect these processes. Attempts have been 
made here and in other laboratories to differentiate human leukemic 
blast cells with apparent success. The implications of this to under- 
standing leukemogenesis and for potential therapeutic approaches are 
obvious. The mechanisms involved in the maturation process are under 
study. 

312 



ZOl CM 06117-13 LTCB 

4. Growth of leukemic myeloblasts in liquid suspension under the stimulus 
of a conditioned media factor produced by human embryonic culture cells. 

5. Markers: (a) Immuno-chemical technique for finding reverse transcript- 
ase and other viral macromolecules in intact cells are being developed, 
(b) Techniques for detecting viral specific nucleic acids in intact 
cells are also being developed. 

6. Cell separation studies are being carried out to enrich subpopulation 
of leukemic cells which may contain the type-C RNA tumor virus related 
markers and other biological markers. 

7. Techniques are being developed to use monoclonal antibodies, prepared 
against cell surface antigens for subtyping and separation of peripheral 
blood and bone marrow cells with the help of a fluorescence activated 
cell sorter. 

9. Recombinant DNA technique is being utilized to obtain molecular DNA 
clones of defective and non-defective primate and human viruses. DNA 
from these clones will be utilized to carry out transfection experiments 
and for generation of subgenomic fragments for probes and functional 
analysis. 

9. Human T cell growth factor (TCGF) has been purified to homogeneity for 
further characterization. Studies are in progress to determine recep- 
tors on activated T cells for TCGF. 

10. The distribution of HTLV in human T cell leukemia patients (HTLV-I, 
HTLV-II) and patients with acquired immune deficiency syndrome (AIDS) 
(HTLV-III) from various parts of the world is being actively pursued. 

Major Findings 

1. More than 100 isolates of HTLV-III have been obtained from patients with 
acquired immune deficiency syndrome (AIDS) and pre-AIDS. 

2. HTLV-III has been isolated from semen or saliva of AIDS patients. 

3. Seroepidemiological studies with HTLV-III show that 95% of the AIDS and 
pre-AIDS patients and approximately 45% of healthy homosexuals carry HTLV- 
III antibodies. 

4. The sera from AIDS and pre-AIDS patients precipitates proteins of 41,000, 
120,000 and 160,000 daltons. 

5. Serological evidence for high prevalence (65%) of HTLV-III antibody in 
Ugandan children surveyed in 1972. 

6. Development of an assay for detection of neutralizing antibodies to HTLV- 
III in sera of individuals exposed to the virus. 

7. Demonstration of heterosexual transmission of HTLV-III by serologic de- 
tection of antibodies in sexual partners of AIDS and ARC cases in New York. 

313 



ZOl CM 06117-13 LTCB 

8. Seroepidemiological studies show that populations at risk for development 
of AIDS include Canadian and Japanese hemophiliacs, Haitian immigrants to 
New Guinea, Zairians, Rwandese, and male prostitutes in Singapore. 

9. A monoclonal antibody against a 41,000 dalton protein induced by HTLV-III 
infection has been prepared and characterized. 

10. Demonstration of an 18% prevalence of HTLV-III antibodies among black drug 
users in New York. 

11. HTLV-I antibodies have been found in sickle cell anemia patients in 
Martinique, Venezuela, several villages of Alaskan Aleuts and Swedish Lapps. 

12. Studies on the development of an animal model indicate chimpanzees to be a 
suitable animal. Seroconversion and HTLV-III isolation was demonstrated in 
chimps infected with HTLV-III. 

13. Development of an in situ hybridization technique to detect HTLV-III se- 
quences in several tissues, including brain, from patients with AIDS and ARC. 

14. Several drugs, including foscarnet, cis-plantinum and lapachone derivatives 
have been shown to inhibit HTLV-III replication. 

15. Molecularly cloned and sequenced several HTLV-III isolates. Showed HTLV-III 
to be similar to LAV and ARV. 

16. Determined the splice acceptor site for the pX gene of HTLV-I and sequenced 
the pX region of HTLV-Ib* 

17. Developed a biologically active plasmid clone by using molecular clones of 
HTLV-III and a protoplast fusion technique. 

18. Determined the nucleotide sequence of the envelope gene of HTLV-III (RF). 

19. Demonstration of trans- acting transcriptional activation for HTLV-III. 

20. Demonstration of HTLV-III to visna virus. 

21. Demonstration of HTLV-III infection in brains of AIDS patients with encepha- 
1 opathy . 

Significance to Biomedical Research and the Program of the Institute 

As outlined in the Objectives, these studies are designed to obtain fundamental 
information on molecular and physiological control mechanism and the patho- 
genesis of neoplasia with the ultimate goal of developing new and improved 
approaches for anti- tumor therapy. In addition, some studies are designed to 
develop biochemical "markers" of neoplastic cells. 

Proposed Course 

As described above, some projects will terminate and others will continue to be 
actively pursued. 

314 



ZOl CM 06117-13 LTCB 
Methods Employed 

1. Human leukocytes were isolated and purified as previously aescrioed {J_. 
Clin. Invest . 48: 105-116, 1969; Science 165: 400-402, 1969). PHA stimu- 
lation of purified lymphocytes has also been described ( biological Lffect 
on Polynucleotides , Springer-Verlag, New York, 1971, pp. 303-334; Blood 
37: 282-292, 1971). 

2. DMA polymerase activities were purified and characterized as reported 
( Nature New Biology 240: 67-72; Proc. Nat. Acad. Sci . 69: 2879-2884, 1972; 
Proc. Nat. Acad. Sci . 69: 3228-3232, 1972; ONA Synthesis jji vitro . Pro- 
ceedings of the Second Annual Steenbock Symposium , 1972). 

3. Viral reverse transcriptase was purified and studied as described ( Nature 
234: 194-198, 1971; J. Virol . 12: 431-439, 1973; Biochim. Biophys. Acta 
454: 212-221, 1976, 479: 198-206, 1977, 564: 235-245, 1979; Virology 112: 
355-360, 1981). 

4. Macromolecular synthesis, viability, mitosis in leukemic and normal cells 
and the effects of specific agents were evaluated as described before (J_. 
Natl. Cancer Inst . 46: 789-795, 1971; Science 165: 400-402, 1969). 

5. Iji vitro leukopoiesis is studied by the soft agar technique developed by 
Paran and Sachs. In addition human myelogenous leukemic leukocytes are 
propagated in liquid suspension culture ( Science 187: 350, 1975). 

6. Induction of type-C virus from "non-producer" cells by iododeoxyuridine is 
carried out essentially as originally described oy Rowe and colleagues. 
Infectious units, focus formation and plaque assays for virus are carried 
out by conventional techniques. 

7. Molecular cloning and molecular hybridization studies are carried out by 
conventional and by newly evolved techniques. These include: (a) filter 
technique with DNA; (b) filter techique with RNA covalently attached ( Proc. 
Nat. Acad. Sci . 70: 3219-3224, 1973); (c) Cesium sulfate gradient analyses; 
(d) Si nuclease treatment; (e) RNA-DNA hybridization by competition analyses 
( Methods in Cancer Research , Vol. XI). 

8. Tissue culture, virus production, cell viability estimates, cloning of 
cells are all carried out by standard techniques. Established procedures 
for titering infectious, leukemic viruses (XC test) and transforming sar- 
coma viruses (focus formation) are routinely performed. Also, virus neu- 
tralization procedures are performed by standard procedures. 

9. Virus quantitation, virus specific molecules, metabolism of viral RNA and 
proteins are studied by conventional techniques. 

10. Cell separation studies are carried out using fluorescence activated cell 
sorter (FACS), f icol 1-hypaque gradients, sucrose density gradients, free 
flow electrophoresis and centrifugal elutriation. (Lancet 1: 508-509, 
1976). 



315 



ZOl CM 06117-13 LTCB 
Publications 

1. Alter, H.J., Eichberg, J.W., Masur, H., Saxinger, W.C., Gallo, R., Macher, 
A.M., Lane, H.C., and Fauci, A.S.: Transmission of HTLV-III infection from 
human plasma to chimpanzees: an animal model for AIDS. Science 226: 549- 
552, 1984. 

2. Aoki, T., Hamada, C, Ohno, S., Miyakoshi, H., Koide, H., Robert-Guroff, M., 
Ting, R.C., and Gallo, R.C.: Location of Human T-Cell Leukemia Virus (HTLV) 
pi 9 and a Role of Natural Antibodies to HTLV in Antibody-Dependent Cell- 
Mediated Cytotoxicity of HTLV-Producing Cells. In Gallo, R.C., Essex, M., 
and Gross, L. (Eds.): Human T-Cell Leukemia/Lymphoma Virus . New York, Cold 
Spring Harbor Press, 1984, pp. 307-312. 

3. Aoki, T., Miyakoshi, H., Usuda, Y., Chermann, J.-C, Barre-Sinoussi, F., 
Ting, R.C., and Gallo, R.C.: Antibodies to HTLV-I and III in sera from two 
Japanese patients, one with possible AIDS. Lancet ii: 936-937, 1984. 

4. Arya, S.K., and Gallo, R.C.: Transcriptional modulation of human T-cell 
growth factor gene by phorbol ester and interleukin 1. Biochemistry 23: 
6685-6690, 1984. 

5. Arya, S.K., Gallo, R.C., Hahn, B.H., Shaw, G.M., Popovic, M., Salahuddin, 
S.Z., and Wong-Staal , F.: Homology of genomes of AIDS-associated virus with 
genomes of human T-cell leukemia viruses. Science 225: 927-930, 1984. 

6. Arya, S.K., Wong-Staal, F., and Gallo, R.C.: Dexamethasone-mediated inhib- 
ition of human T-cell growth factor and y interferon messenger RNA. J. 
Inwunol . 133: 273-276, 1984. ~ 

7. Arya, S.K., Wong-Staal, F., and Gallo, R.C.: Transcriptional regulation of 
a tumor promoter and mitogen-inducible gene in human lymphocytes. Mol . 
Cell. 

8. Biggar, R.J., Mel bye, M., Kestems, L., Sarngadharan, M.G., DeFeyter, M., 
Blattner, W.A., Gallo, R.C., and Gigase, P.L.: Kaposi's sarcoma in Zaire is 
not associated with HTLV-III infection. N. Engl. J. Med . 311: 1051-1052, 
1984. 

9. Blattner, W.A., Clark, J.W., Gibbs, W.N., Jaffe, E.S., Robert-Guroff, M., 
Saxinger, W.C, and Gallo, R.C.: Human T-Cell Leukemia/Lymphoma Virus: 
Epidemiology and Relationship to Human Malignancy. In Gallo, R.C., Essex, 
M., and Gross, L. (Eds.): Human T-Cell Leukemia/Lymphoma Virus . New York, 
Cold Spring Harbor Laboratory, 1984, pp. 267-274. 

10. Blick, M., Westin, E., Gutterman, J., Wong-Staal, F., Gallo, R.C., McCredie, 
K., Keating, M., and Murphy, E.: Oncogene expression in human leukemia. 
Blood 64: 1234-1239, 1984. 

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12. Clark, J.W., Blattner, W.A. , and Gallo, R.C.: HTLV: Epidemiology and 
Disease Relationship. In Aoki , T., Tsubura, E., and Urushizaki, I. (Eds.): 
Manipulation of Host Defence Mechanisms. Hong Kong, Excerpta Medica, 1984, 
pp. 124-136. 

13. Clarke, M.F., Trainor, CD., Mann, D.L., Gallo, R.C., and Reitz, M.S.: 
Methylation of human T-cell leukemia virus proviral DNA and viral RNA 
expression in short- and long-term cultures of infected cells. Virology 
135: 97-104, 1984. 

14. Franchini, G., Wong-Staal , P., and Gallo, R.C.: Molecular studies of human 
T-cell leukemia virus and adult T-cell leukemia. J. Invest. Derm . 83: 63s- 
66s, 1984. 

15. Franchini, G., Wong-Staal, P., and Gallo, R.C.: Human T-cell leukemia virus 
(HTLV-I) transcripts in fresh and cultured cells of patients with adult T- 
cell leukemia. Proc. Nat. Acad. Sci . USA 81: 6207-6211, 1984. 

16. Prazer, I.H., Sarngadharan, M.G., Mackay, I.R., and Gallo, R.C.: Antibody 
to human T cell leukaemia virus type III in Australian homosexual men with 
lymphadenopathy. Med. J. Australia Sep 1: 274-276, 1984. 

17. Gallo, R.C.: Human T-Cell Leukemia-Lymphoma Virus and T-Cell Malignancies 
in Adults. In Wyke, L.M., and Weiss, R.A. (Eds.): Cancer Surveys . Oxford, 
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Lymphoma Virus . New York, Cold Spring Harbor Laboratory, 1984, pp. 1-8. 

19. Gallo, R.C., Arya, S.K., Lindner, S.G., Wong-Staal, P., and Sarngadharan, 
M.G.: Human T-Cell Growth Factor, Growth of Human Neoplastic T-Cells, and 
Human T-Cell Leukemia-Lymphoma Virus. In Goldstein, A.L. (Ed.): Thymic 
Hormones and Lymphokines . New York, Plenum Press, 1984, pp. 1-17. 

20. Gallo, R.C., and Blattner, W.A.: Human T-Cell Leukemia/Lymphoma Viruses: 
ATL and AIDS. In DeVita, V.T., Jr., Hellman, S. , and Rosenberg, S.A. 
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Lymphoma Virus . New York, Cold Spring Harbor Laboratory, 1984, 398 pp. 

22. Gallo, R.C., Salahuddin, S.Z., Sarngadharan, M.G., Popovic, M., and Markham, 
P.D.: Human T-Lymphotropic Retroviruses (HTLV) and AIDS. In Gottlieb, M.S. 
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23. Gallo, R.C., and Wong-Staal, P.: Current thoughts on the viral etiology of 
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24. Gelmann, E.P., Franchini, G. , Manzari, V., Wong-Staal, F., and Gallo, R.C.: 
Molecular Cloning of a New Unique Human T-Cell Leukemia Virus (HTLV-IImq). 
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Lymphoma Virus . New York, Cold Spring Harbor Laboratory, 1984, pp. 189-195. 

25. Gelmann, E.P., Wong-Staal, F., and Gallo, R.C.: The Etiology of Acute 
Leukemia: Molecular Genetics and Viral Oncology. In Wiernik, P.H., Canellos, 
G.P., Kyle, R.A., and Schiffer, C.A. (Eds.): Neoplastic Diseases of the 
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26. Goedert, J.J., Sarngadharan, M.G., Biggar, R.J., Weiss, S.H., Winn, D.M., 
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and immunodeficiency conditions in homosexual men. Lancet ii : 711-716, 1984. 

27. Greaves, M.F., Verbi, W., Tilley, R., Lister, T.A., Habeshaw, J., Guo, 
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28. Greaves, M.F., Verbi, W., Tilley, R. , Lister, T.A., Robert-Guroff, M., 
Blattner, W.A. , Reitz, M.S., Jr., and Gallo, R.C.: Human T-Cell Leukemia 
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Spring Harbor Laboratory, 1984, pp. 297-306. 

29. Greene, W.C. , Robb, R.J., Depper, J.M., Leonard, W.J., Drogula, C. , Svetlik, 
P.B., Wong-Staal, F., Gallo, R.C., and Waldinann, T.A.: Phorbol diester 
induces expression of Tac antigen on human acute T lymphocytic leukemic 
cells. J. Immunol . 133: 1042-1047, 1984. 

30. Groopman, J.E., Salahuddin, S.Z. , Sarngadharan, M.G., Markham, P.D., Gonda, 
M., Sliski, A., and Gallo, R.C.: HTLV-III in saliva of people with AIDS- 
related complex and healthy homosexual men at risk for AIDS. Science 226: 
447-449, 1984. 

31. Groopman, J.E., Salahuddin, S.Z., Sarngadharan, M.6., Mullins, J. I., 
Sullivan, J. L., Mulder, C. , O'Hara, C.J., Cheeseman, S.H. , Haverkos, H., 
Forgacs, P., Riedel, N., McLane, M.F., Essex, M., and Gallo, R.C.: Viro- 
logic studies in a case of transfusion- associated AIDS. N. Engl. J. Med . 
311: 1419-1422, 1984. 

32. Hahn, B., Gallo, R.C., Franchini, G., Popovic, M., Aoki , T., Salahuddin, 
S.Z., Markham, P.D., and Wong-Staal, F.: Clonal selection of human T-cell 
leukemia virus- infected cells vn vivo and vn vitro . Mol . Biol . Med . 2: 
29-36, 1984. 

33. Hahn, B.H., Popovic, M., Kalyanaraman, V.S., Shaw, 6.M., LoMonico, A., 
Weiss, S.H., Wong-Staal, F., and Gallo, R.C.: Detection and Characteriza- 
tion of an HTLV-II Provirus in a Patient with AIDS. In Gottlieb, M.S. and 
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F.: Molecular cloning and characterization of the HTLV-III virus associated 
with AIDS. Nature 312: 166-169, 1984. 

35. Hahn, B.H., Shaw, G.M., Popovic, M., LoMonico, A., Gallo, R.C., and 
Wong-Staal, F.: Molecular cloning and analysis of a new variant of human T- 
cell leukemia virus (HTLV-Ib) from an African patient with adult T-cell 
leukemia-lymphoma. Int. J. Cancer 34: 613-618, 1984. 

36. Haynes, B.F., Palker, T.J., Robert-Guroff , M., Kalyanaraman, V.S., Gallo, 
R.C., Bolognesi, D.P., and Scearce, R.M.: Monoclonal Antibodies Against 
Human T Cell Leukemia/Lymphoma Virus pi 9 and p24 Internal Core Proteins: 
Spectrum of Normal Tissue Reactivity and Use as Diagnostic Probes. In 
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37. Jaffe, E.S., Blattner, W.A., Blayney, D.W., Bunn, P. A., Jr., Cossman, J., 
Robert-Guroff, M., and Gallo, R.C.: The pathologic spectrum of adult T- 
cell leukemia/lymphoma in the United States. Am. J. Surg. Pathol . 8: 263- 
275, 1984. 

38. Josephs, S.F., Wong-Staal, F., Manzari , V., Gallo, R.C., Sodroski, J.G., 
Trus, M.D., Perkins, D., Patarca, R., and Haseltine, W.A.: Long terminal 
repeat structure of an American isolate of type I human T-cell leukemia 
virus. Virology 139: 340-345, 1984. 

39. Kleinerman, E.S., Zicht, R., Sarin, P.S., Gallo, R.C., and Fidler, I. J.: 
Constitutive production and release of a lymphokine with macrophage-activat- 
ing factor activity distinct from y-interferon by a human T-cell leukemia 
virus-positive cell line. Cancer Res . 44: 4470-4475, 1984. 

40. Krohn, K., Ranki, A., Antonen, J., Valle, S.-L., Suni, J., Vaheri, A., 
Saxinger, C, and Gallo, R.C.: Immune functions in HTLV-III antibody posi- 
tive homosexual men without clinical AIDS. Lancet ii: 746-747, 1984. 

41. Kronke, M., Leonard, W.J., Depper, J.M., Arya, S.K., Wong-Staal, F., Gallo, 
R.C., Waldmann, T.A., and Greene, W.C: Cyclosporin A inhibits T cell 
growth factor gene expression at the level of mRNA transcription. Proc. 
Nat. Acad. Sci. USA 81: 5214-5218, 1984. 

42. Lange Wantzin, G., Saxinger, W.C, Woods, A., Larsen, J.K., Thomsen, K., and 
Gallo, R.C.: Human T-cell leukemia virus in cutaneous T-cell lymphoma in 
Denmark. Acta Derm. Venereol . (Stockholm) 64: 395-399, 1984. 

43. Lee, T.H., Coligan, J.E., McLane, M.F., Sodroski, J.G., Popovic, M., 
Wong-Staal, F., Gallo, R.C., Haseltine, W. , and Essex, M.: Serological 
cross-reactivity between envelope gene products of type I and type II human 
T-cell leukemia virus. Proc. Nat. Acad. Sci. USA 81: 7579-7583, 1984. 

44. Lee, T.H., Coligan, J.E., Sodroski, J.G., Haseltine, W.A. , Salahuddin, S.Z., 
Wong-Staal, F., Gallo, R.C., and Essex, M.: Antigens encoded by 3'-terminal 
region of human T-cell leukemia virus: evidence for a functional gene. 
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45. Longo, D.L., Gelmann, E.P., Cossman, J., Young, R.A. , Gallo, R.C., O'Brien, 
S.J., and Matis, L.A.: Isolation of HTLV-transformed B-lymphocyte clone 
from a patient with HTLV-associated adult T-cell leukaemia. Mature 310: 
505-506, 1984. 

46. Mann, D.L., Clark, J., Clarke, M., Reitz, M., Popovic, M., Franchini, G., 
Trainor, CD., Strong, D.M., Blattner, W.A. , and Gallo, R.C.: Identifica- 
tion of the human T cell lymphoma virus in B cell lines established from 
patients with adult T cell leukemia. J. Clin. Invest . 74: 56-62, 1984. 

47. Markham, P.D., Gallo, R.C., and Salahuddin, S.Z.: In Vitro Transformation 
of Human Cord Blood and Bone Marrow T Lymphocytes bylTTLTi In Wright, D.G., 
and Greenberger, J.S. (Eds.): Long-Term Bone Marrow Culture . New YorK, 
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48. Markham, P.O., Salahuddin, S.Z., and Gallo, R.C.: In Vitro Cultivation of 
Normal and Neoplastic Human T-Lymphocytes. In McCulloch, E.A. (Ed.): 
Clinics in Haematol ogy . London, W. B. Saunders, Ltd., 1984, Vol. 3., pp. 
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49. Markham, P.O., Sarngadharan, M.G., Salahuddin, S.Z., Popovic, M., and Gallo, 
R.C.: Correlation Between Exposure to Human T-Cell Leukemia-Lymphoma Virus- 
Ill and the Development of AIDS. In Selikoff, I.J., Teirstein, A.S., and 
Hirschman, S.Z. (Eds.): Acquired Immune Deficiency Syndrome . New York, 
The New York Acaderny of Sciences, 1984, pp. 106-109. 

50. Melbye, M., Bigyar, R.J., Ebbesen, P., Sarngadharan, M.G., Weiss, S.H., 
Gallo, R.C., and Blattner, W.A.: Seroepidemiology of HTLV-III antibody in 
Danish homosexual men: prevalence, transmission, and disease outcome. Br. 
Med. J . 289: 573-575. 1984. 

51. Melbye, M., Froebel , K.S., Madhok, R., Biggar, R.J., Sarin, P.S., Stenbjerg, 
S., Lowe, G.D.O., Forbes, CD., Goedert, J.J., Gallo, R.C, and Ebbesen, P.: 
HTLV-III seropositivity in European haemophiliacs exposed to factor VII con- 
centrate imported from the USA. Lancet ii: 1444-1446, 1984. 

52. Merino, F., Robert-Guroff , M., Clark, J., Biondo-Bracho, M., Blatter, W.A., 
and Gallo, R.C: Natural antibodies to human T-cell leukemia/lymphoma virus 
in healthy Venezuelan populations. Int. J. Cancer 34: 501-506, 1984. 

53. Mitsuya, H., Popovic, M., Yarchoan, R. , Matsushita, S., Gallo, R.C, and 
Broder, S.: Suramin protection of T cells in vitro against infectivity and 
cytopathic effect of HTLV-III. Science 226 :"~T72^174, 1984. 

54. Nowell, P.C, Finan, J.B., Clark, J., Sarin, P.S., and Gallo, R.C: Karyo- 
typic differences between primary cultures and cell lines from tumors with 
the human T-cell leukemia virus. JNCI 73: 849-852, 1984. 

55. Oroszlan, S. , Copeland, T.D., Kalyanaraman, V.S. , Sarngadharan, M.G. , 
Schultz, A.M., and Gallo, R.C: Chemical Analysis of Human T-Cell Leukemia 
Virus Structural Proteins. In Gallo, R.C, Essex, M., and Gross, L. (Eds.): 
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56. Popovic, M., Flornenberg, N., Volkman, D.J., Mann, D., Fauci, A.S., Dupont, 
B., and Gallo, R.C.: Alteration of T-cell functions by infection with HTLV- 
I orHTLV-II. Science 226: 459-462, 1984. 

57. Popovic, M., Kalyanaraman, V.S., Mann, D., Richardson, E., Sarin, P.S., and 
Gallo, R.C.: Infection and Transformation of T Cells by Human T-Cell Leu- 
kemia/Lymphoma Virus of Subgroups I and II (HTLV-I, HTLV-II). In Gallo, 
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New York, Cold Spring Harbor Laboratory, 1984, pp. 217-227. 

58. Popovic, M. , Read-Connole, E., and Gallo, R.C: T4 positive human neoplastic 
cell lines susceptible to and permissive for HTLV-III. Lancet ii: 1472-1473, 
1984. 

59. Popovic, M., Wong-Staal , P., Sarin, P.S., and Gallo, R.C: Biology of Human 
T-Cell Leukemia/Lymphoma Virus: Transformation of Human T-cells In Vivo and 
In Vitro . In Klein, G. (Ed.): Advances in Viral Oncology . NewTbrk, Raven 
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60. Ratner, L., Sarin, P.S., Wong-Staal, F., ana Gallo, R.C: Human and Primate 
T-Lymphotropic Retroviruses (HTLV and PTLV): Subtypes, Biological Activity, 
and Role in Neoplasias. In Rigby, P.W.J., and Wilkie, N.M. (Eds.): Viruses 
and Cancer . CamDridge, Cambridge University Press, 1984, pp. 261-289. 

61. Reitz, M.S., Jr., Clarke, M.F., Mann, D.L., and Gallo, R.C: Human T-Cell 
Leukemia/Lymphoma Virus and Class I Major Histocompatability Antigens. In 
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62. Robert-Guroff, M., Blayney, D.W., Safai , B., Lange, M., Gelmann, E.P., 
Gutterman, J.W., Mansell, P.W.A., Goedert, J.L., Groopman, J.E., Steigbigel, 
N.H., Sidhu, G.S., Johnson, J.M., Friedman-Kien, A.E., Downing, R. , Bayley, 
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at risk. Lancet ii: 128-131, 1984. 

63. Robert-Guroff, M., Coutinho, R.A., Zadelhoff, A.W., Vyth-Dreese, F.A., and 
Rumke, P.: Prevalence of HTLV-specific antibodies in Surinam emigrants to 
the Netherlands. Leu. Res . 8: 501-504, 1984. 

64. Robert-Guroff, M. , Schupbach, J., Blayney, D.W., Kalyanaraman, V.S., Merino, 
F., Sarngadharan, M.G., Clark, J.W. , Saxinger, W.C, Blattner, W.A., and 
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Virus, Type I. In Gallo, R.C, Essex, M., and Gross, L. (Eds.): Human T- 
Cell Leukemia/Lymphoma Virus . New York, Cold Spring Harbor Laboratory, 
1984, pp. 285-295. 

65. Roth, J. A., Scuderi , P., Westin, E., and Gallo, R.C: A novel approach to 
production of antitumor monoclonal antibodies: antibody to a cell surface 
glycoprotein associated with transformation oy a human oncogene. Surgery 
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66. Rowley, J.D., Haren, J.M., Wong-Staal, F. , Franchini, G., Gallo, R.C., and 
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man T-Cell Leukemia/Lymphoma Virus. In Gallo, R.C., Essex, M., and Gross, 
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67. Safai , B., Sarngadharan, M.G., Groopman, J.E., Arnett, K., Popovic, M., 
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human T-lymphotropic retrovirus type III in acquired immunodeficiency syn- 
drome. Lancet i: 1438-1440, 1984. 

68. Salahuddin, S.Z., Groopman, d.E., Markham, P.O., Sarngadharan, M.G., 
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69. Sarin, P.S., and Gallo, R.C.: Role of Viruses in the Etiology of AIOS. 
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70. Sarin, P.S., and Gallo, R.C.: Retroviruses in human T-cell malignancies. 
Cancer Invest . 2(6): 467-478, 1984. 

71. Sarin, P.S., and Gallo, R.C.: Chronic Leukemia. In Notkins, A.L., and 
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72. Sarin, P.S., and Gallo, R.C.: Human T-lymphomtropic retroviruses in adult 
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73. Sarin, P.S., Taguchi, Y., and Gallo, R.C.: Human T Cell Leukemia Virus, T 
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74. Sarngadharan, M.G., Kalyanaraman, V.S., Schupbach, J., Bruch, L., and 
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75. Saxinger, W. , Blattner, W.A. , Levine, P.H., Clark, J., Biggar, R. , Hoh, M., 
Moghissi, J., Jacobs, P., Wilson, L., Jacobson, R. , Crookes, R., Strong, M., 
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Human T-cell leukemia virus (HTLV-I) antibodies in Africa. Science 225: 
1473-1476, 1984. 

76. Saxinger, W.C., Lange-Wantzin, G., Thomsen, K., Lapin, B., Yakovleva, L., 
Li, Y.-W., Guo, H.-G., Robert-Guroff , M. , Blattner, W.A. , Ito, Y. , and 
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77. Schupbach, J., Sarngadharan, M.G. , and Gallo, R.C.: Antigens on HTLV- 
infected cells recognized by leukemia and AIDS sera are related to HTLV 
viral glycoprotein. Science 224: 607-610, 1984. 

78. Shaw, G.M., Broder, S., Essex, M., and Gallo, R.C.: Human T-Cell Leukemia 
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79. Shaw, 6.M., Gonda, M.A., Flickinger, G.H. , Hahn, B.H., Gallo, R.C., and 
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T-cell leukemia virus family (HTLV-I and HTLV-II) are highly conserved, 
especially in pX. Proc. Nat. Acad. Sci . USA 81: 4544-4548, 1984. 

80. Shaw, G.M., Hahn, B.H., Arya, S.K., Groopman, J.E., Gallo, R.C., and 
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tropic) virus type III in the acquired immune deficiency syndrome. Science 
226: 1165-1171, 1984. 

81. Shaw, G.M., Hahn, B.H., Popovic, M., LoMonico, A., Wong-Staal, F., and 
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82. Sodroski, J., Patarca, R., Perkins, D., Briggs, D., Lee, T.-H., Essex, M., 
Coligan, J., Wong-Staal, P., Gallo, R.C., and Haseltine, W.A.: Sequence of 
the envelope glycoprotein gene of type II human T lymphotropic virus. 
Science 225: 421^24, 1984. 

83. Sodroski, J., Trus, M., Perkins, D., Patarca, R., Wong-Staal, P., Gelmann, 
E., Gallo, R.C. , and Haseltine, W.A.: Repetitive structure in the long- 
terminal repeat element of a type II human T-cell leukemia virus. Proc. 
Nat. Acad. Sci. USA 81: 4617-4621, 1984. 

84. Suciu-Poca, N., Rubinstein, P., Popovic, M., Gallo, R.C, and King, D.W.: 
Reactivity of HTLV-transformed human T-cell lines to MHC class II antigens. 
Nature 312: 275-277, 1984. 

85. Waldmann, T.A., Greene, W.C, Sarin, P.S., Saxinger, C, Blayney, D.W., 
Blattner, W.A., Goldman, C.K., Bongiovanni, K. , Sharrow, S., Depper, J.M., 
Leonard, W., Uchiyama, T., and Gallo, R.C: Functional and phenotypic com- 
parison to human T cell leukemia/lymphoma virus positive adult T cell leu- 
kemia with human T cell leukemia/lymphoma virus negative Sezary leukemia, 
and their distinction using anti-Tac. J. CI. Invest . 73: 1711-1718, 1984. 

86. Williams, C.K.O., Alabi, G.O., Junaid, T.A., Saxinger, C, Gallo, R.C, 
Blayney, D.W., Blattner, W.A. , and Greaves, M.P.: Human T cell leukaemia 
virus-associated lymphoproliferative disease: a report of two cases in 
Nigeria. Br. Med. J. 288: 1495-1496, 1984. 



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87. Wong-Staal, F. , Franchini, G., Hahn, B., Arya, S.K., Gelmann, E.P., Manzari , 
v., and Gallo, R.C.: Human T-Cell Leukemia/Lymphorna Virus, In Vitro Trans- 
formation, and Leukemia: Some Molecular Studies. In Gallo, RTT. , Essex, M., 
and Gross, L. (Eds.): Human T-Cell Leukemia/Lymphoma Virus . New York, Cold 
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88. Wong-Staal, F., Franchini, G., Hahn, B., Arya, S., Gelmann, E.P., Manzari, 
v., Popovic, M., Aoki , T., and Gallo, R.C.: Molecular Studies of HTLV, Leu- 
kemia and In Vitro Transformation. In Aoki, T., Tsubura, E., and Urushizaki, 
I. (Eds.): Manipulation of Host Defence Mechanisms . Hong Kong, Excerpta 
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89. Wong-Staal, F., and Gallo, R.C.: The Family of Human T-Cell Tropic Retro- 
viruses Called HTLV and Its Role in Adult T-Cell Leukemia. In Ford, R.J., 
Fuller, L.M., and Hagemeister, F.B. (Eds.): New Perspectives in Human 
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90. Yi-Wan, L., Saxinger, W.C, Blattner, W.A., and Gallo, R.C.: Detection of 
human T-cell leukemia/lymphoma virus (HTLV) antibody in the normal subjects 
in China. J. Chinese One. (China) 6: 98-100, 1984. 

91. Yi-Wan, L. , Saxinger, W.C, Shu-Fan, C. , Ming-Yi, L., Blattner, W.A. , and 
Gallo, R.C.: Nature infection of human T-cell leukemia/lymphoma virus 
(HTLV) in Rhesus monkeys in China. Cancer (China) 3: 164-165, 1984. 

92. Zagury, D., Bernard, D., Leibowitch, J., Safai , B., Groopman, J.E., Feldnan, 
M., Sarngadharan, M.6., and Gallo, R.C.: HTLV-III in cells cultured from 
semen of two patients with AIDS. Science 226: 449-451, 1984. 

93. Aoki, T. , Miyakoshi , H., Koide, H., Yoshida, T., Ishikawa, H., Sugisaki, Y., 
Mizukoshi, M., Tamura, K., Misawa, H., Hamada, C, Ting, R.C., Robert- 
Guroff, M., and Gallo, R.C.: Seroepidemiology of human T-lymphotropic 
retrovirus type I (HTLV-I) in residents of Niigara Prefecture, Japan. 
Comparative studies by indirect immunofluorescence microscopy and enzyme- 
linked immunosorbent assay. Int. J. Cancer 35: 301-306, 1985. 

94. Blattner, W.A. , and Gallo, R.C.: Human T-Cell Leukemia/Lymphoma Viruses: 
Clinical and Epidemiologic Features. In Vogt, P.K. (Ed.): Current Topics 
in Microbiology and Immunology . Berlin-Heidelberg, Springer-Verlag, 1985, 
Vol. 115, pp. 67-88. 

95. Broder, S., and Gallo, R.C.: Human T-cell leukemia viruses (HTLV): A unique 
family of pathogenic retroviruses. Ann. Rev. Immunol . 3: 321-336, 1985. 

96. Chang, N.T., Chanda, P.K., Barone, A.D., McKinney, S., Rhodes, D.P., Tam, 
S.H., Shearman, C.W., Huang, J., Chang, T.W., Gallo, R.C. , and Wong-Staal, 
F.: Expression of Escherichia Coli of open reading frame gene segments of 
HTLV-III. Science 228: 93-96. T^H^ 

97. Gajdusek, D.C., Amyx, H.L., Gibbs, C.J., Jr., Asher, D.M., Rodgers-Johnson, 
P., Epstein, L.G., Sarin, P.S., Gallo, R.C, Maluish, A., Arthur, L.O., 
Montagnier, L., and Mildvan, D.: Infection of chimpanzees by human T-lympho- 
tropic retroviruses in brain and other tissues from AIDS patients. Lancet 
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98. Gallo, R.C., Manzari, V., Horneff, J., and Wong-Staal, F.: The Role of 
Retroviruses and One Genes in Some Human Leukemias and Lymphomas. In 
Bertazzoni, U., BoTTum, F.J., and Ghione, M. (Eds.): Contributions of 
Modern Biology to Medicine . New York, Raven Press, 19851 pp^ 133-137. 

99. Gallo, R.C, , Robert-Guroff , M.: The HTLV family of human retroviruses 
and their association with T cell suppression. In Nakamura, R.M. (Eds.): 
Clinical Laboratory Molecular Analyses . San Diego, Grune and Stratton 
Publishers, 1985, pp. 257-266. 

100. Gallo, R.C., Shaw, G., Hahn, B., Wong-Staal, P., Popovic, M., Schupbach, 
J., Sarngadharan, M.G., Arya, S. , Salahuddin, S.Z., and Reitz, M.S., Jr.: 
The Family of Human T-Cell Leukemia Viruses and Their Role in the Cause of 
T-Cell Leukemia and AIDS. In Furmanski, P., Hager, J.C, and Rich, M.A. 
(Eds.): RNA Tumor Viruses, Oncogenes, Human Cancer and AIDS: On The 
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101. Gallo, R.C., Wong-Staal, F., Markham, P.D., Popovic, M., and Sarngadharan, 
M.G.: Human Leukemia Viruses: The HTLV "Family" and Their Role in Human 
Malignancies and Immune Deficiency Disease. In Wahren, B., Holm., G., 
Hammarstrom, S. , and Perlmann, P. (Eds.): Molecular Biology of Tumor Cells . 
New York, Raven Press, 1985, pp. 183-213. 

102. Goedert, J.J., Sarngadharan, M.G., Eyster, M.E., Weiss, S.H., Bodner, A.J., 
Gallo, R.C., and Blattner, W.A.: Antibodies reactive with human T cell 
leukemia viruses in the serum of hemophiliacs receiving factor VIII concen- 
trate. Blood 65: 492-495, 1985. 

103. Gonda, M.A., Wong-Staal, F., Gallo, R.C., Clements, J.E., Narayan, 0., and 
Gilden, R.V.: Sequence homology and morphologic similarity of HTLV-III and 
visna virus, a pathogenic lentivirus. Science 227: 173-177, 1985. 

104. Groopman, J.E., Sarngadharan, M.G. , Salahuddin, S.Z., Buxbaum, R. , Huberman, 
M.S., Kinniburgh, J., Sliski, A., McLane, M.F., Essex, M., and Gallo, R.C.: 
Apparent transmission of human T-cell leukemia virus type III to a hetero- 
sexual woman with the acquired immunodeficiency syndrome. Ann. Int. Med . 
102: 63-66, 1985. 

105. Hirsch, M.S., Wormser, G.P., Schooley, R.T., Ho, D.D., Felenstein, D., 
Hopkins, C.C. , Joline, C, Duncanson, F, , Sarngadharan, M.G. , Saxinger, C. , 
and Gallo, R.C.: Risk of nosocomial infection with human T-cell lympho- 
tropic virus III (HTLV-III). N. Engl. J. Med . 312: 1-4, 1985. 

106. McDougal, J.S., Jaffe, H.W., Cabridilla, CD., Sarngadharan, M.G., 
Nicholson, J.K.A., Kalyanaraman, V.S. , Schable, C.A., Kilbourne, B. , Evatt, 
B.L., Gallo, R.C., and Curran, J.W.: Screening tests for blood donors pre- 
sumed to have transmitted the acquired immunodeficiency syndrome. Blood 
65: 772-775, 1985. 

107. Ratner, L., Gallo, R.C., and Wong-Staal, F.: HTLV-III, LAV, ARV are vari- 
ants of the same AIDS virus. Nature 313: 636-637, 1985. 



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108. Ratner, L., Gallo, R.C., and Wong-Staal, F.: Cloning of Human Oncogenes. 
In Becker, Y. (Ed.): Recombinant DMA Research and Virus . Boston, Marti nus 
Nijhoff, 1985, pp. 15-35. 

109. Ratner, L., Haseltine, W. , Patarca, R., Livak, K.J., Starcich, B., Josephs, 
S.F., Doran, E.R. , Raf al ski , J. A., Whitehorn, E.A. , Baumeister, K., 
Ivanoff, L., Petteway, S.R., Jr., Pearson, M.L., Lautenberger, J. A., Papas, 
T.S., Ghrayeb, J., Chang, N.T., Gallo, R.C., and Wong-Staal, P.: Complete 
nucleotide sequence of the AIDS virus, HTLV-III. Nature 313: 277-id84, 1985. 

110. Redfield, R.R. , Markham, P.O., Salahuddin, S.Z., Sarngadharan, M.6., 
Bodner, A. J., Folks, T.M., Ballou, W.R., Wright, D.C., and Gallo, R.C.: 
Frequent transmission of HTLV-III among spouses of patients with AIUS- 
related complex and AIDS. JAMA 253: 1571-1573, 1985. 

111. Robert-Guroff, M., and Gallo, R.C.: The Family of Human T Cell Lymphotropic 
Type C Retroviruses Known as HTLV and Their Role in Human T Cell Diseases. 
In Nakamura, R.M. (Eds.): Clinical Laboratory Molecular Analyses . San 
Diego, Grune and Stratton, 1985, pp. 109-127. 

112. Robert-Guroff, M., Markham, P.D., Popovic, M., and Gallo, R.C.: Isolation, 
Characterization, and Biological Effects of the First Human Retroviruses: 
The Human T-Lymphotropic Retrovirus Family. In Vogt, P.K. (Ed.): Current 
Topics in Microbiology and Immunology. Berlin-Heidelberg, Springer-Verlag, 
1985, Vol. 115, pp. 7-31. 

113. Sarin, P.S., and Gallo, R.C.: Cellular transformation by human T cell 
leukemia retroviruses (HTLV). Pharmac. Ther . 27: 49-62, 1985. 

114. Saxinger, W.C, Levine, P.H., Dean, A.G., deThe, G., Lange-Wantzin, G., 
Moghissi, J., Laurent, F. , Hoh, M. , Sarngadharan, M.G., and Gallo, R.C.: 
Evidence for exposure to HTLV-III in Uganda prior to 1973. Science 227: 
1036-1038, 1985. 

115. Schupbach, J., Haller, 0., Vogt, M., Luthy, R., Joller, H., Oelz, 0., 
Popovic, M., Sarngadharan, M.G., and Gallo, R.C.: Antibodies to HTLV-III 
in Swiss patients with AIDS and pre-AIDS and in groups at risk for AIDS. 
N. Engl. J. Med . 312: 265-270, 1985. 

116. Shaw, G.M., Harper, M.E., Hahn, B.H., Epstein, L.G., Gajdusek, D.C., Price, 
R.W., Navia, B.A., Petito, C.K., O'Hara, C.J., Groopman, J.E., Cho, E.-S., 
Oleske, J.M., Wong-Staal, F., and Gallo, R.C.: HTLV-III infection in brains 
of children and adults with AIDS encephalopathy. Science 227: 177-182, 
1985. 

117. Sodroski , J., Rosen, C, Wong-Staal, F., Salahuddin, S.Z., Popovic, M., 
Arya, S., Gallo, R.C., and Haseltine, W.A.: Trans- acting transcriptional 
regulation of human T-cell leukemia virus type III long terminal repeat. 
Science 227; 171-173, 1985. 

118. Starcich, B., Ratner, L., Josephs, S.F., Okamoto, T., Gallo, R.C., and 
Wong-Staal, F.: Characterization of long terminal repeat sequences of 
HTLV-III. Science 227: 538-540, 1985. 

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119. Stevens, C.E. , Taylor, P.E., Rubinstein, P., Ting, R.C.Y., Bodner, A.J., 
Sarngadharan, M.G., and Gallo, R.C.: Safety of the hepatitis B vaccine. 
N. Engl. J. Med . 312: 375-376, 1985. 

120. Weiss, S.H., Goedert, J.J., Sarngadharan, M.G., bodner, A.J., Biggar, R.J., 
Clark, J.W., Dodd, R.Y., Gelmann, E.P., Giron, J.S., Greene, M.H., Melbye, 
M., Popovic, M., Robert-Guroff, M., Saxinger, W.C, Simberkoff , M., Winn, 
D.M., Gallo, R.C., and Blattner, W.A.: Screening test for HTLV-III (AIDS 
agent) antibodies. JAMA 253: 221-225, 1985. 

121. Whang-Peng, J., Bunn, P. A., Knutsen, T., Kao-Shan, C.S. , Broder, S., Jaffe, 
E.S., Gelmann, E., Blattner, W., Lofters, W. , Young, R.C., and Gallo, R.C.: 
Cytogenic studies in human T-cell lymphoma virus (HTLV) -positive leukemia- 
lymphoma in the United States. J. Natl. Cancer Inst . 74: 357-369, 1985. 

122. Wong-Staal, F. , Franchini, G., Hahn, B. , Arya, S., Gelmann, E.P., Manzari, 
v., and Gallo, R.C.: Molecular Biology of HTLV. In Aaronson, S.A., Frati, 
L., and Verna, R. (Eds.): Genetic and Phenotypic Markers of Tumors . New 
York, Plenum Press, 1985, pp. 337-344. 

123. Wong-Staal, F., and Gallo, R.C.: The family of human T-lymphotropic leu- 
kemia viruses: HTLV-I as the cause of adult T cell leukemia and HTLV-III 
as the cause of acquired immunodeficiency syndrome. Blood 65: 253-263, 
1985. 

124. Abe, T., Kinoshita, T., Matsuda, J., Ryu, T., Miyakoshi , H., Aoki , T., 
Ting, R.C. , Robert-Guroff, M., Gallo, R.C.: Detection of antibodies to 
HTLV-I and III in sera from Japanese hemophiliacs. Cancer kes . In press. 

125. Blattner, W.A. , Clark, J.W., Gibbs, W.N., Williams, C.K.O., Nomura, A., 
Mann, u., Saxinger, C, Robert-Guroff, M., Gallo, R.C: HTLV: Epidemiology 
ana relationship to disease. Human Retroviruses in Lymphoma/Leukemia . 

In press. 

126. Bronson, D.L., Saxinger, W.C, Ritzi, D.M., Fraley, E.E.: Production of 
virions with retrovirus morphology by human embryonal carcinoma cells in 
vitro . J. Gen. Virology . In press. 

127. Clarke, M.F., Mann, D.L., MUrray, C. , Reitz, M.S.: Differential methylation 
of class I histo-compatibility antigen genes in T-cell lines derived from 
two different types of T-cell malignancies. Leukemia Res . In press. 

128. DeRossi, A., Aldovini, A., Franchini, G., Mann, D., Gallo, R.C, Wong- 
Staal, F.: Clonal selection of T-lymphocytes infected by cell-free human 
T-cell leukemia/lymphoma virus type I: Parameters of virus integration and 
expression. Virology . In press. 

129. DeVecchis, L., Graziani, G., Macchi, B., Grandori , C, Pastore, S., 
Popovic, M., Gallo, R.C, Bonmassar, E.: Decline of natural cytotoxicity 
of human lymphocytes following infection with human T-cell leukemia/ 
lymphoma virus (HTLV). Leukemia Res . In press. 



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130. Di Marzo Veronese, F. , Sarngadharan, M.G., Rahman, R. , Markham, P.O., 
Popovic, M., Badner, A.J., Gallo, K.C.: Monoclonal antibodies specific 
for p24, the major core protein of HTLV-III. PNAS. In press. 

131. Fisher, A.G., Collalti, E., Ratner, L., Gallo, R.C., Wong-Staal , F.: A 
molecular clone of HTLV-III with biological activity. Nature . In press. 

132. Franchini, G., Gallo, R.C.: Viruses, ONC genes and leukemia. In press. 

133. Franchini, G., Mann, D.L., Popovic, M., Zicht, R.R., Gallo, R.C., Wong- 
Staal, F.: HTLV-I infection of T- and B-cells of a patient with adult 
T-cell leukemia/lymphoma (ATLL) and transmission of HTLV-I from B-cells to 
normal T-cell s. Leukemia Res . In press. 

134. Gallo, R.C.: The family of human lymphotropic retroviruses called HTLV: 
HTLV-I in adult T-cell leukemia (ATL), HTLV-II in hairy cell leukemias, 
and HTLV-III in AIDS. Human Retroviruses in Lymphoma/Leukemia . In press. 

135. Gallo, R.C.: The human T-cell leukemia/lymphotropic retroviruses (HTLV) 
family: past, present and future. Cancer Res . In press. 

136. Gallo, R.C.: Human T-cell leukemia (lymphotropic) retroviruses and their 
causative role in T-cell malignancies and acquired immune deficiency syn- 
drome. Accomplishments in Cancer Research . In press. 

137. Gallo, R.C., Clarke, M.F., Guo, H.G., Wong-Staal, F. , Reitz, M.S., Jr.: 
Recent observations on human T-cell leukemia viruses (HTLV) and T-cell 
malignancies: possible mechanisms of leukemogenesis. Biomedicine A 
Pharmacotherapie . In press. 

138. Gallo, R.C., Ratner, L., Popovic, M., Salahuddin, S.Z., Sarngadharan, M.G., 
Wong-Staal, F. , Shaw, G. , Hahn, B. , Markham, P.O., Groopman, J., Safai , B., 
Reitz, M.S., Robert-Guroff, M.: The family of human T-lympho tropic retro- 
viruses called human T-cell leukemia/lymphoma virus (HTLV): their role in 
lymphoid malignancies and lympho-suppressive disorders (AIDS). Genetics 
Cell Differentiation and Cancer . In press. 

139. Gallo, R.C., Reitz, M.S., Jr.: The first human retroviruses: are there 
others? Microbiol. Sci. In press. 

140. Gallo, R.C., Sarngadharan, M.G., Popovic, M., Shaw, G.M., Hahn, B., Wong- 
Staal, F., Robert-Guroff, M., Salahuddin, S.Z., Markham, P.D.: HTLV-III 
and the etiology of AIDS. Progress in Allergy 37 . In press. 

141. Gallo, R.C., Westin, E.H., Franchini, V.: Retroviruses and ONC genes in 
human leukemia and lymphoma. Biomedicine and Pharmacotherapie . In press. 

142. Gallo, R.C., Wong-Staal, F. , Clarke, M.F., Guo, H.G., Westing, E., Saxinger, 
W.C, Blattner, W.A., Reitz, M.S., Jr.: Human T-cell leukemia viruses and 
oncogenes and the origin of some human leukemias and lymphomas. Theories 
and Models in Cellular Transformation. In press. 



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143. Gold, J.W.M., Weikel, C.L., Goldbold, J., et al . : Unexplained persistent 
lymphadenopathy in homosexual men and the acquired immune ueficiency syn- 
drome. Medicine . In press. 

144. Gonda, M.A., Wony-Staal , F., Gallo, R.C., Clements, J.E., bilden, R.V.: 
Heteroduplex mappin in the molecular analysis of the human T-cell leukemia 
(lympho tropic) viruses. Cancer Res. In press. 

145. Gootenberg, J.E., Ruscetti, F.W., Gallo, R.C.: A biochefnical variant of 
human T-cell growth factor produced by a cutaneous T-cell lymphoma cell 
line. 3rd International Lymphokine Workshop . In press. 

146. Groopman, J.E., Mayer, K.H., Sarngadharan, M.G., Ayotte, D., et al.: 
Seroepidemiology of HTLV-III among homosexual men with AIDS, generalized 
lymphadenopathy & asymptomatic case controls in Boston. Annals Int. Med. 
In press. 

147. Hahn, B.H., Gonda, M.A., Shaw, G.M., Popovic, M., Hoxie, J. A., Gallo, R.C., 
Wong-Staal, F.: Genomic diversity of the AIDS virus HTLV-III: different 
viruses exhibit greatest divergence in their envelope genes. PMAS. In 
press. 

148. Joseph, S.: Simian sarcoma virus oncogenes. Biomedicine . In press. 

149. Josephs, S.F. , Wong-Staal, F., Gallo, R.C.: Oncogenes, growth factors, 
and transformation: our lessons from the sis gene. Cancer Surveys . In 
press. 

150. Krohn, K., Ranki , A., Antonen, J., Valle, S.L., Suni , j., Vaheri , A., 
Saxinger, C, Gallo, R.C.: Immune functions in homosexual men with anti- 
bodies to HTLV-III in Finland. CI. Exp. Immunol. In press. 

151. Markham, P.D., Salahuddin, S.Z., Popovic, M., Patel , A., Veren, K. , 
Fladager, A., Orndorff, S., Gallo, R.C.: Advances in the isolation of 
HTLV-III from patients with AIDS, ARC, and donors at risk. Cancer Res. 
In press. 

152. Markham, P.D., Salahuddin, S.Z., Popovic, H., Sarngadharan, M.G., Gallo, 
R.C.: Retrovirus infections in man: human T-cell leukemia (lymphotropic) 
viruses. Viral Mechanism of Immune Suppression . In press. 

153. Markham, P.D. , Shaw, G.M., Gallo, R.C.: The etiology of AIDS. AIDS. In 
press. 

154. Markham P.D., Shaw, G.M., Salahuddin, S.Z., Hahn, B., Sarngadharan, M.G., 
Gallo, R.C.: Etiology of AIDS: biological and biochemical characteristics 
of HTLV-III. International Conference on AIDS-Associated Syndrome . In 
press. 

155. Okamoto, T., Josephs, S.F., Kawanishi, M., Wong-Staal, F.: Determination 
of a splice acceptor site of pX gene in HTLV-I infected cells. Virology . 
In press. 



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156. Popovic, M., Gallo, R.C.: Human T-cell lymphotropic retroviruses (HTLV) 
associated with T-cell malignancy and immunodeficiency: in vitro studies. 
16th Intl. Leucocyte Conference . In press. 

157. Ratner, L., Josephs, S.F., Starcich, B., Hahn, B., Shaw, G.M., Gallo, R.C., 
Wong-Staal, F.: Nucleotide sequence analysis of a variant human T-cell 
leukemia virus (HTLV-lb) provirus with a deletion in pX-I. J. Virology . 
In press. 

158. Ratner, L., Wong-Staal, P., Shaw, G.M., Sodroski , J.G., Haseltine, W.A., 
Gallo, R.C.: Mechanism of Leukemogenesis by human T-cell leukemia virus 
types I and II - role of the lor gene. 6th International Symposium on 
Prevention and Detection of Cancer . In press. 

159. Reitz, M.S., Jr.: Non- human primates and humans. Veterinary Cancer Medi- 
cine 2nd Ed. In press. 

160. Reitz, M.S., Jr., Gallo, R.C.: Human T-cell leukemia viruses. Molecular 
Basis of Blood Diseases . In press. 

161. Robert-Guroff, M., Clark, J.W.: Antigenicity of HTLV-associated gp52: 
Greater response in leukemia patients compared to healthy donors exposed 
to the virus. Cancer Res . In press. 

162. Robert-Guroff, M., Gallo, R.C.: HTLV and leukemia in man. UCLA Symposium 
Leukemia 1985 . In press. 

163. Robert-Guroff, M., Gallo, R.C.: Human T-cell leukemia (lymphotropic) retro- 
viruses: The HTLV family and their role in leukemias, lymphomas and AIDS. 
Retroviruses and Human Pathology . In press. 

164. Robert-Guroff, M., Gallo, R.C.: Immunology of Human T-cell leukemia/ 
lymphoma (lymphotropic) viruses (the HTLV "family"). Basic and Clinical 
Tumor Immunology Vol. 2. In press. 

165. Salahuddin, S.Z., Gallo, R.C.: Viral leukemias. Encyclopedia of Science 
and Technology . In press. 

166. Salahuddin, S.Z., Markham, P.D., Popovic, M., Sarngadharan, M.G., Orndorff, 
S. , Fladagar, A., Pat el , A., Gold, J., Gallo, R.C.: Une-hundred-one infec- 
tious HTLV-III isolates from AIDS and ARC Pa-patients, and clinically 
normal donors at risk for AIDS. PNAS . In press. 

167. Sarin, P.S.: HTLV in adult T-cell leukemia and acquired immune deficiency 
syndrome. Cell Transformation . In press. 

168. Sarin, P.S., Gallo, R.C.: Characteristics of a human T-cell leukemia virus 
isolated from patients with T-cell malignancies. Fifth International 
Symposium on Bovine Leukemia Virus . In press. 

169. Sarin, P.S., Gallo, R.C.: Human T-cell leukemia (lymphotropic) retroviruses 
(HTLV): HTLV-I, HTLV-II, and HTLV-III. Revue De L'Institut Pasteur De Lyon . 
In press. 

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170. Sarngadharan, M.G., Bruch, L., Popovic, M., Gallo, R.C.: Immunological 
properties of the GAG protein p24 of the AIDS retrovirus (HTLV-III). 
PNAS. In press. 

171. Sarngadharan, M.T., Devico, A.L., Bruch, L., Schupbach, J., Gallo, R.C., 
HTLV-III: The etiologic agent of AIDS. Human Retroviruses in Lymphoma/ 
Leukemia . In press. 

172. Sarngadharan, M.G., Markham, P.D., Gallo, R.C.: Human T-cell leukemia 
viruses: Primary etiological agents for adult T-cell leukemia and acquired 
iimuno-deficiency syndrome. Virology . In press. 

173. Sarngadharan, M.G., Schupbach, J., Bruch, L., DeVico, A., Markham, P.D., 
Wong-Staal , F., Gallo, R.C., Seroepidemological evidence for HTLV-III 
infection as the primary etiologic factor for acquired immunodeficiency 
syndrome. Infection Immunity and Transfusion . In press. 

174. Saxinger, W.C. , Blayney, D.W., Postal, M. , Blattner, W.A. , Moghissi, J., 
Gallo, R.C.: Risk of infection of laboratory workers with the human T-cell 
leukemia virus (HTLV). Lancet . In press. 

175. Saxinger, W.C. Gallo, R.C.: Human T-cell growth factor (TCGF): its dis- 
covery, properties and some basic and applied uses in the long term propa- 
gation of human mature T-cell s. Human Cancer Immunology . In press. 

176. Saxinger, C. , Lange-Wantzin, G., Levine, P.H., Dean, A., Gallo, R.C.: 
Unique pattern of HTLV-III (AIDS-related) antigen recognition by sera from 
african children: Uganda (1972). Cancer Res . In press. 

177. Volkflian, D.J., Popovic, M., Gallo, R.C., Rauci, A.S.: Human T-cell 
leukemia/ lymphoma virus- infected antigen-specific T-cell clones: Indis- 
criminant helper function and lymphokine production. J. Immunology . In 
press. 

178. Westin, E.H., Wong-Staal, P., Gallo, R.: Retroviruses ana ONC genes in 
human leukemias and lymphomas. Adult Leukemias . In press. 

179. Wong-Staal, P.: Molecular Diology of viruses of the HTLV family. Retro- 
viruses and Human Pathology . In press. 

180. Wong-Staal, P., Hahn, B.H., Shaw, G.M., Arya, S.K., Harper, M., Ratner, L., 
Starcich, B., Okamoto, T., Josephs, S.P., Gallo, R.C.: Molecular charac- 
terization of numan T-lymphotropic leukemia virus type III associated with 
the acquired immunodeficiency syndrome. Human Retroviruses in Lymphoma/ 
Leukemia . In press. 

181. Wong-Staal, P., Ratner, L., Shaw, G., Hahn, B., Harper, M., Pranchini, G., 
Gallo, R.: Molecular biology of human T-lymphotropic retroviruses. Cancer 
Res. In press. 



331 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07148-02 LTCB 



PERIOD COVERED , 

October T, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. We musf fit on one line between ttie lx)rdera.) 

Cellular Biological Studies on T-Cell Malignancies and Lymphomas 



PRINCIPAL INVESTIGATOR (Ust other prolesslonel personnel tielow the Principal Investigator.) (Name, title, laboratory, and Institute atfUlaUon) 

Prem S. Sarin Chemist LTCB NCI 

S. Zaki Salahuddin Cancer Expert LTCB NCI 

Suzanne Gartner Staff Fellow LTCB NCI 

Mikulas Popovic Visiting Scientist LTCB NCI 

Jorg Jendis Visiting Fellow LTCB NCI 

Yoshitaka Taguchi Visiting Fellow LTCB NCI 

Shuji Nakamura Guest Researcher LTCB NCI 

Petr Stockbauer Guest Researcher LTCB NCI 



COOPERATING UNITS (it any) 

Robin Weiss, Imperial Cancer Research Fund, London, England; Bart Haynes, Duke 
University; Ken McCredie, Anderson Hospital and Tumor Institute; l^^ron Essex, 
Harvard University. 



LAB/BRANCH 

Laboratory of Tumor Cell Biology 



SECTION 

Hematopoietic Cellular Control Mechanisms 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

15 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects HI (b) Human tissues D (c) Neither 

D (a1) Minors 
n (a2) Interviews 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The cell biology studies have been focused on the role of human T lymphotroplc 
retroviruses on human T cell malignancies and acquired immune deficiency syndrome 
(AIDS). More than 100 isolates of HTLV-I have been obtained from patients with 
adult T cell leukemia- lymphoma, and over 100 isolates have also been obtained 
from patients with AIDS and pre-AIOS. HTLV-I has been shown to be a transforming 
virus whereas HTLV-III is cytopathic. Both HTLV-I and HTLV-I II have specificity 
for 0KT4 positive T helper cells. HTLV-III has been transmitted to a cell line 
which is productively infected with HTLV-III thus allowing the production of 
large quantities of virus for cell biology, molecular biological and Diochemlcal 
studies. HTLV-III has also been isolated from saliva or semen from AIDS or pre- 
AIDS patients. HTLV-III isolates obtained from different patients show some 
genetic variations. An HTLV-III from a Haitian AIDS patient has been found to be 
the most divergent compared with other HTLV-III isolates. The virus probably 
attaches to the T cells through the 0KT4 receptor since treatment of the virus 
with anti-T4 blocks infection of the receipient cells. Drugs that block HTLV-III 
replication are being tested in in vitro system. Preliminary studies indicate 
foscarnet, cis platinum and lapacFone derivates may be useful In treatment of 
patients with AIDS or pre-AIDS. 



332 



PHS 6040 (Rev. 1/84) 



OPO CM.*!! 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07149-02 LTCb 



PERIOD COVERED 

October 1, 1984 to September 3u, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line t>etween the txirders.) 

Molecular Biological Studies on HTLV and Oncogenes 



PRINCIPAL INVESTIGATOR (Ust other professional personnel tjelow the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 



Flossie Wong-Staal 
Lee Ratner 
Suresh Arya 
Marvin Reitz 
Mary Harper 
Genoveffa Franchini 
Anna Al dovini 
Chan Quo 



licrobiologist 
Clinical Associate 
Cancer Expert 
Cancer Expert 
Staff Fellow 
Visiting Associate 
Visiting Fellow 
Visiting Fellow 



LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 
LTCB NCI 



COOPERATING UNITS (if any) 

Stu Aaronson, Viral Carcinogenesis Branch, National Cancer Institute; Rolf Neth, 
University of Hamburg; Robin Weiss, Imperial Cancer Research Fund, London, 
England; Dani Bolognesi and Bart Haynes, Duke University; Ken McCredie, M.Q. 



LAB/BRANCH 

Laboratory of Tumor Cell Biology 



SECTION 

Molecular Genetics of Hematopoietic Cells 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 202u5 



TOTAL MAN-YEARS: 



22 



PROFESSIONAL: 



15 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
n (a2) Interviews 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Studies on human retroviruses and oncogenes have been pursued with particular 
emphasis on their role in human disease. Two subgroups of a human T cell leu- 
kemia virus, designated HTLV-I and HTLV-II, have the unique capacity to transform 
human T cells Jjt^ vitro , leading to cloned cell populations. Molecular cloning 
and comparative analysis of the genomes of HTLV-I and HTLV-II, revealed sequence 
conservation throughout, but particularly in a coding region designated pX^ and an 
enhancer sequences in the viral LTR. These results have direct relevance in the 
possible mechanism or transformation by these viruses. Recently HTLV-I II has 
been postulated to be the etiologic agent of acquired immune deficiency syndrome. 
We have molecularly cloned and sequenced the HTLV-III genomes. A comparative 
analysis of the multiple HTLV-III isolats has shown divergence in the env gene. 
HTLV-III has been found to be similar to LAV or ARV and shows sequence homology 
to visna virus. HTLV-III infection has also been found in brain tissues of AIDS 
patients with encephalopathy. This was made possible by the recently developed 
in situ hybridization technique. It is now possible to express various HTLV-III 
proteins in prokaryotic systems and show transfection of biologically active 
HTLV-III DNA and demonstration of cytopathic effect. 



333 



PHS 6040 (Rev. 1/84) 



GPO SI4-Sia 



ZOl CM 07149-02 LTCB 

NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL 
INVESTIGATORS AND ALL OTHER PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT 

Hong-Guang Guo Visiting Fellow LTCB NCI 

Beatrice Hahn Visiting Fel low LTCB NCI 

Ruth Jarrett Visiting Fellow LTCB NCI 

Takashi Okamoto Visiting Fel low LTCB NCI 

Amanda Fisher Guest Researcher LTCB NCI 

Bruno Starcich Guest Researcher LTCB NCI 

George Shaw JPA LTCB NCI 

COOPERATING UNITS 

Anderson Hospital and Tumor Institute; Bill Haseltine, Harvard University Takis 
Papas, NCI-FCRF; Volker Erfle. Munich. umversixy, laKis 



334 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 

ZOl CM 07150-02 LTCB 



PERIOD COVERED 

October 1. 1984 to September 30, 



1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 



Seroepidemiological Studies on Human T-Lymphotropic Retroviruses 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Nanrte, title, laboratory, and institute affiliation) 

Prem S. Sarin Chemist LTCB NCI 

W. Carl Saxinger Microbiologist LTCB NCI 

Marjorie Robert-Guroff Staff Fellow LTCB NCI 

Jorg Schupbach Guest Researcher LTCB NCI 



COOPERATING UNITS (if any) 

Dani Bolognesi, Duke University; Yohei Ito, University of Kyoto; Bill Haseltine, 
Harvard University; Volker Erfle, Munich; Bill Blattner, Environmental Epidemi- 
ology Section, NCI; Mark Smulson, Georgetown University; Isaac Witz, Tel Aviv. 



LAB/BRANCH 

Laboratory of Tumor Cell Biology 



Hematopoietic Cell Biochemistry and Immunology 



INSTITUTE AND LOCATION 

NCI. NIH. Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 



PROFESSIONAL: 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



(b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The worldwide distribution of HTLV infection, the mechanism of its transmission 
and its role in various types of T cell malignancies and patients with acquired 
immune deficiency syndrome (AIDS) and pre-AIDS has been extensively studied. A 
highly sensitive ELISA technique has been developed and extensively used for de- 
tection of HTLV infection. Seroepidemiological studies on the distribution of 
HTLV-III show that 95% of the AIDS and pre-AIDS patients, and approximately 45% 
of healthy homosexuals carry HTLV-III antibodies. High incidence (65%) of HTLV- 
III antibodies has also been found in sera of Ugandan children collected in 1972. 
Detection of HTLV-III antibodies in sexual partners of AIDS and ARC cases in New 
York suggest heterosexual transmission of HTLV-III. Other seroepidemiological 
studies show that populations at risk for development of AIDS include Canadian 
and Japanese hemophiliacs, Haitian immigrants to New Guinea, Zairians, Rwandese 
and male prostitutes in Singapore. The sera from AIDS and pre-AIDS patients has 
been shown to precipitate HTLV-III envelope proteins of 41,000, 120,000 and 
160,000 daltons. HTLV-III inoculations into chimpanzees show that these animals 
seroconvert and virus has been reisolated from their peripheral blood indicating 
infection of these animals. None of these animals have so far developed AIDS- 
like syndrome. 



335 



PHS 6040 (Rev. 1/84) 



GPo SI 4-sia 



ANNUAL REPORT OF THE LABORATORY OF MOLECULAR PHARMACOLOGY 

DEVELOPMENTAL THERAPEUTICS PROGRAM 

DIVISION OF CANCER TREATMENT 

October 1, 1984 to September 30, 1985 

The Laboratory of Molecular Pharmacology is carrying out studies in 3 major 
areas: (1) studies of the molecular mechanisms of action of alkylating agents 
such as chloroethylnitrosoureas and nitrogen mustards as well as platinum 
complexes; (2) studies of topoisomerase II as a target of action of DNA inter- 
calating agents and epipodophyllotoxins; (3) studies of the regulation of 
chromosomal protein biosynthesis in relation to the control of cell prolif- 
eration. A major goal is to develop strategies for the selective killing of 
particular human tumor cell types. 

DNA Chloroethylating Agents 

A major continuing aim of the Laboratory has been to elucidate the mechanisms of 
the extraordinary activity of the chloroethylnitrosoureas (ClEtNUs) against a 
broad spectrum of murine tumors, and to work towards the optimized application 
of this mechanism to the treatment of human cancer. We had previously obtained 
evidence for a selective toxic mechanism against some human tumor cell strains 
that is based on the chemical addition of chloroethyl groups to guanine-06 
positions on DNA. The sensitive tumor cells had been reported to be deficient 
in the activity of an enzyme that removes alkyl groups, including chloroethyl 
groups, from guanine-06 positions thus repairing these DNA lesions. Our data 
had suggested that the enhanced cell killing in the sensitive tumor cells was 
due to spontaneous chemical conversion of unrepaired 06-chloroethyl guanines to 
possibly lethal interstrand crosslinks. These conclusions have now been sup- 
ported by data from other laboratories. This selective cytotoxic mechanism is 
an attractive basis for cancer chemotherapy because we know the chemical lesion 
that probably is responsible for the selective killing of tumor cells, and a 
biochemical test can be devised to determine whether a given tumor has the re- 
quired sensitivity characteristic: low guanine-06-alkyltransferase activity. 

The ClEtNU drugs used in clinical chemotherapy (BCNU, CCNU and MeCCNU) produce 
diverse chemical reactions with macromolecules in cells, and most of these re- 
actions probably do not contribute to any therapeutic result. Our strategy is 
to reduce or eliminate unnecessary chemical reactivities by identifying other 
chloroethylating compounds that retain the high antitumor activities but lack 
some of the chemical side reactions. 

We had previously focussed our attention on the carbamoyl ati on reactions of CCNU 
and BCNU. We had presented evidence that carbamoylation reactions do not con- 
tribute to antitumor activity, but do interfere with the selective action 
against human tumor cells, probably on the basis of an observed inhibition of 
the ligase reaction in DNA nucleotide excision repair. We therefore argued 
for the development of a known non-carbamoylating analog to replace CCNU in 
the clinic. 



33: 



At the same time, we proposed the development of a new type of chloroethylatinq 
agent, 2-chloroethyl(methanesulfony1)methanesulfonate ("CIEtSoSo") which had 
recently been prepared at the Southern Research Institute and which was found 
to equal the ClEtNUs in its action against murine tumors. We reported last 
year that CIEtSoSo is similar to ClEtNUs in its crosslinking effects on DNA in 
human tumor cells and in its selective action against cells deficient in 
guanine-06-alkyltransferase. These results strongly indicated that CIEtSoSo 
IS a new chemical class of chloroethylating agent. 

From its chemistry, it was clear that CIEtSoSo cannot carbamoylate In 
addition, it seemed possible that CIEtSoSo would cause much less hydroxylation 
an unnecessary side reaction that ClEtNUs prominently produce. 

Our work during the current year addressed the questions: (1) whether CIEtSoSo 
does in fact exhibit the anticipated lower hydroxyethylation/chloroethylation 
ratio relative to ClEtNUs; (2) whether CIEtSoSo differs from ClEtNUs in regard 
to the base-sequence selectivity of its reactions with DNA. 

Chloroethyl and hydroxyethyl adducts of guanine in DNA were isolated by means of 
HPLC and the chemical structures were verified by mass spectrometry in collabo- 
ration with Dr. John Strong, Laboratory of Medicinal Chemistry & Biology OCT 
It was found that, whereas a ClEtNU produced somewhat more 7-hydroxyethyiquanine 
than 7-chloroethylguanine, CIEtSoSo produced 7-chloroethylguanine but no detect- 
able hydroxyethyl products. Thus CIEtSoSo lacks two prominent unnecessary side- 
reactions of ClEtNUs: carbamoylation and hydroxyethyl at ion. 

The base-sequence selectivity for reaction of these compounds at guanine-N7 
positions of DNA was determined by minor modifications of standard DNA 
sequencing methods. ClEtNUs were found to react with markedly different 
intensities with different guanines in a DNA fragment. Of particular interest 
was the finding of disproportionately strong reactions at runs of 4 or more 
G s. This suggests that ClEtNUs may react with strong preference for G-rich 
regions of DNA strands. 

CIEtSoSo, on the other hand, exhibited little or no selectivity among different 
G s. This however refers to the guanine-N7 position whereas the major cytotoxic 
reaction more likely is at guanine-06. Guanine-06 adducts unfortunately cannot 
be detected by standard DNA sequencing techniques. We plan to study possible 
procedures that would allow this determination. We plan then to study modifi- 
cations of the CIEtSoSo structure that may alter the base sequence selectivity 
as well as the selectivity between the 06 and N7-guanine positions. 

Studies were also carried out on human guanine-06-alkyltransferase to improve 
the methods of isolation and assay. In addition, attempts were made to transfer 
this enzyme activity by means of purified DNA, as part of a continuing effort to 
isolate the gene and to determine the genetic cause of the deficiency in some 
human tumor. 

A collaborative project with the Surgical Neurology Branch, NINCDS, is under way 
to test for a correlation between clinical response of malignant gliomas treated 
with azindinylbenzoquinone, cisplatin or BCNU and the DNA interstrand cross- 
linking response to these drugs of cell lines derived from the tumors. 



338 



DNA Base Sequence Selectivity for Alkylation py Nitrogen Mustards 

The nitrogen mustards are the oldest class of anti-cancer drugs and are still 
among the most effective available clinical drugs. Although large numbers of 
active nitrogen mustards were prepared In the 1950's and 1960's, there was 
during that time period no adequate molecular basis for optimizing the action 
of this class of compounds. We have during the past year initiated studies to 
apply modern techniques and concepts of DNA chemistry to this problem. 

Current information, much of it from this Laboratory, indicates that the major 
cytotoxic lesions produced by nitrogen mustards are covalent crosslinks between 
the N7 positions of 2 guanine residues located on opposite DNA strands or 
adjacent to each other on the same strand. This suggeted that GC-rich regions 
in DNA may constitute hot-spots for alkylation and crosslinking by these drugs. 
We searched the DNA sequence data base to determine where in the human genome 
highly GC-rich regions may occur. Included among the most GC-rich regions 
were regions in some oncogenes, most notably in the 5' flank of H-ras. We 
therefore focussed our studies on a pBR plasmid containing a human H-ras 
insert. 

After investigating several possible procedures for localizing DNA interstrand 
crosslinks in a DNA sequence, we concluded that this difficult problem is best 
deferred, and we concentrated our studies on guanine-N? alkylations which are 
easily quantitated by standard DNA sequencing techniques and which are pre- 
requisite for the formation of both inter and intrastrand crosslinks. 

The major findings to date are the following: (1) there are marked differences 
in reactivity among guanines in a DNA sequence; (2) the differences are not 
markedly dependent on the solvent conditions of reactions; (3) the guanine 
selectivity pattern is different for different nitrogen mustards; (4) the 
potential Interstrand crosslinking sites -- e.g. 5'-GC-3' -- show relatively 
low reactivity for HN2, melphalan and spirohydantoin mustard; (5) these sites 
however show a markedly enhanced reaction with uracil mustard; (6) runs of G's 
(potential sites of intra-strand crosslinking) exhibited disproportionately 
high reactivity with most nitrogen mustards; (7) quinacrine mustard exhibited a 
uniquely different pattern of reactions. 

We are beginning now to analyse the reaction intensity patterns quantitatively 
by means of densitometry and computer analysis. We will attempt to identify 
the elements of DNA sequence and structure that determine reactivity at specific 
sites. Molecular graphics systems will be utilized to formulate hypotheses on 
the basis of which nitrogen mustard structures with new or enhanced DNA sequence 
preferences could be synthesized. 

Topoisomerase II as Target of Anticancer Drug Action 

On the basis of previous work in this Laboratory, we had hypothesized an action 
of DNA intercalating agents on a DNA topoisomerase enzyme. This hypothesis was 
formulated as an explanation for the observed formation of protein-associated 
DNA strand breaks in mammalian cells treated with several DNA intercalating 
drugs, including m-AMSA (amsacrine), adrlamycin, 5-1minodaunorubicin, ellip- 
ticine and 9-hydroxy-l -methyl el lipticini urn. We had developed the methodology 
and the conceptual framework for the analysis of these DNA effects in mammalian 

339 



cells. Recent work in severdl laboratories has confirmed and extended this 
work, and another class of drugs, the epipodophyllotoxins, has been 
found to produce similar effects. 

As reported in the previous Annual Report, we had isolated a fraction from cell 
nuclei that stimulated the intercalator-dependent production of protein- 
associated ONA strand breaks. In the meantime Dr. Leroy Liu and his coworkers 
at Johns Hopkins University, who had been studying purified mammalian topo- 
isomerase II, made anologous observations on the effects of DNA intercalating 
agents and epipodophyllotoxins. Our active fraction from cell nuclei had the 
properties expected for topoisomerase II. Thus there is now strong evidence 
that the analysis of protein-associated strand breaks in mammalian cells that 
we had developed does in fact measure an effect on one or more topoisomerase 
enzymes. 

During the current year we have investigated several aspects of the mechanisms 
of the drug effects on topoisomerase II and the consequent effects on cells. 

Studies of a multi-drug resistant Chinese hamster cell line disclosed a reduced 
protein-associated strand break response to intercalators. In view of the 
possibility that this cell line might haye developed several independent re- 
sistance mechanisms, an attempt was made to transfer the resistance to mouse 
3T3 cells by means of DNA from the resistant cells. Two resistant clones of 
3T3 cells have been obtained which show reduced protein-associated strand break 
responses and which contain Chinese hamster DNA. Our objectives now are to 
verify by repeated DNA transfer experiments that a dominant resistance gene has 
been identified and to determine its relationship to topoisomerase II. 

We have found that the activity of topoisomerase II measured by the protein- 
associated strand break response is dependent on the proliferation state of the 
cells. In non-proliferating 3T3 cells, this activity was much less than in 
proliferating cells. When quiescent cells were stimulated to proliferate, the 
topoisomerase II response increased at about the time of the first wave of DNA 
synthesis, but did not then oscillate in phase with the cell cycle. Thus the 
observed alteration in topoisomerase II activity appears to be related to the 
proliferation state of the cells and not to phase of the cell cycle. 

The epipodophyllotoxins (etoposide and teniposide) were studied. The formation 
of protein-associated strand breaks was verified in several cell lines and this 
was the only DNA effect detected in the pharmacologically reasonable dose range. 
The actions of these drugs on topooisomerase II were confirmed using enzyme 
purified from L1210 cells. 

Regulation of Chromosomal Protein Synthesis and Control of Cell Proliferation 

Previous work in this Laboratory utilized original methods for the electropho- 
retic separation and analysis of histone proteins to study the regulation of the 
synthesis of these proteins as a function of cell proliferation state. Although 
the synthesis of the major nucleosomal histones exhibited the expected reg- 
ulation in phase with DNA synthesis, this was not the case for certain variant 
histone species, notably H2A.Z, a species that had been characterized in this 
Laboratory. This variant species, which replaces histone H2A in a fraction 
of the nucleosomes, was found to be synthesized at a slow rate even in the 

340 



absence of DNA synthesis. The basal histone syntheses that occur in the 
absence of DNA synthesis in GO cells relative to Gl cells were distinguishable 
on the basis of the relative synthesis rates of the different histones. The 
effects of some anti-neoplastic drugs, including DNA and protein synthesis 
inhibitors and DNA crosslinkers, on the pattern of histone variant synthesis 
was studied. A model was proposed to explain observed interactions between 
protein and DNA synthesis inhibitors and histone synthesis pattern. 

A major component of this model involves changes in cytoplasmic histone pool 
sizes. However there was no reported adequate method to measure histone pool 
sizes. A new method was successfully developed to carry out these measurements 
with the required sensitivity. Utilizing this method, the state of newly 
synthesized histone species in the cytoplasm was observed. The possibility of 
contamination by histones from the nucleus was tested and was found not to be a 
problem. 

This now opens the way to answering some key questions about the mechanisms 
which regulate histone synthesis. Another aspect of this problem that we are 
investigating concerns changes in the level of cytoplasmic histone mRNA which 
may be related to histone pools in a feed-back regulation loop. 

In addition to the nucleosomal histones, the synthesis of histone HI variants 
was studied. Again changes in the variant synthesis patterns were observed in 
relation to cell proliferation state and in response to DNA synthesis in- 
hibitors. 

Further insight was gained on the mechanism of the regulation of DNA and histone 
synthesis during S-phase. Studies were carried out of the effects of protein 
synthesis inhibitors applied to cells at relatively low doses such that protein 
synthesis was reduced by only 50%. Although DNA and histone syntheses were 
initially inhibited proportionately, these syntheses then recovered despite 
the continued inhibition of overall protein synthesis. This was found to be 
brought about by a compensatory elevation of histone m-RNA. A mechanism thus 
exists which tends to preserve the duration of S-phase when Gl is lengthened 
because of nutritional or metabolic deficiencies. The mechanism will be studied 
studied further by using other means to lengthen Gl phase. The relevance to 
cancer chemotherapy is that this metabolic regulatory mechanism may be defective 
in some neoplastic cell lines and could lead to a strategem by which such cells 
could be selectively killed. 

In order to determine the histone variant regulatory mechanism at the gene 
level, we are attempting to isolate the human H2A.Z gene. Polynucleotide 
probes were obtained on the basis of 2 different regions of the known amino 
acid sequence of the protein. The probes were used as primers on mRNA derived 
from cells synthesizing H2A.Z. Radioactive cDNA's were then isolated and 
used to select clones from a human-lambda genomic library. Several clones 
were selected which hybridize equally with the 2 probes, and these clones 
are now being studied to determine whether any of them code for H2A.Z 



341 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06140-09 LMPH 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) 

Regulation of Hi stone Biosynthesis 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: William Bonner Head, Chromosome Structure LMPH NCI 



Others: Roy S. Wu 

Maurizio D' Incalci 
Henryk Panusz 



Head, Chromosome Structure 
and Function Section 

Cancer Expert 
Visiting Associate 
Visiting Scientist 



LMPH NCI 
LMPH NCI 
LMPH NCI 



COOPERATING UNITS (if any) Department or Biological Chemistry, School of Medicine, Univ. 
of California, Davis; Department of Biochemistry, 6WU Medical School and the 
Department of Biology, Georgetown University, Biotech Res. Labs., Inc. 



LAB/BRANCH 

Laboratory of Molecular Pharmacology, DTP, DCT, NCI 



SECTION 

Chromosome Structure and Function 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



2.3 



PROFESSIONAL: 



1.9 



0.4 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



n (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Our objective is to elucidate the mechanisms regulating histone biosynthesis in 
Gl, S and GO. 

Using methodology developed in our group over the last several years to resolve 
and characterize histone variants, we have been analyzing the patterns of 
histone synthesis during different cell behavioral states. Histones were 
found to be synthesized not only during S-phase, but also during Gl and the 
quiescent state (also termed extended Gl or GO). The qualitative pattern of 
histone synthesis differs between S-phase, Gl and quiescent cells, a finding 
which shows that the synthesis in Gl or quiescent cells is not due to contami- 
nation by S-phase cells. The histone synthesis in both Gl and quiescent cells 
Is not linked to DNA synthesis. Histones synthesized in quiescent cells are 
stable and seem to be incorporated into chromatin. The results suggest that 
the quiescent state Is not an extended Gl phase but a discrete state. This 
year, we published a comprehensive model concerning the regulation of histone 
and DNA synthesis. The cytoplasmic histone pool Is a crucial factor in this 
model and methods for analyzing the histone pool are being worked out. 

There is some evidence in the literature which suggests that there may be 
significant differences in the regulation of chromatin biosynthesis between 
normal and tumorigenic cells. Understanding these differences may lead to 
the development of protocols to protect normal cells or make tumorgenic cells 
more vulnerable to various treatments. 



342 



PHS 6040 (Rev. 1/84) 



GPO S14-S1S 



ZOl CM 06140-09 LMPH 
Project Description 

Introduction : 

We have found that when dividing cells cease division and become quiescent, 
they continue to synthesize histones at a reduced but significant rate. The 
pattern of synthesis of variant histones in quiescent cells differs from that 
in S-phase; therefore, this synthesis cannot be attributed to the presence of 
S-phase cells in the culture. 

In CHO cells where the pattern of histone synthesis has been examined during 
the cell cycle, reduced but significant synthesis has been found in both G2 and 
Gl (Wu and Bonner, Cell 27: 321-330, 1981). This basal pattern differs from 
both the S-phase and the quiescent patterns. These results strongly suggest 
that the quiescent state is not merely an extended Gl , but is a discrete state 
or cycle. 

Objectives : 

1) The development of greater understanding of the molecular mechanisms 
regulating chromatin biosynthesis and metabolism during the cellular states 
of proliferation and nonproliferation. 

2) The characterization of chromatin or cellular components that may be 
involved in these regulatory mechanisms. 

Methods : 

(1 ) Discontinuous electrophoretic separation of histones Including direct 
loading of histone extracts and two dimensional eletrophoresis. (Methods 
developed in this laboratory). 

(2) Peptide analyses on acrylamide gels to determine the relationship of 
proteins to each other. (Method developed in this laboratory). 

(3) Synchronization of cell lines, particularly human Hela cells and Chinese 
hamster ovary cells for studies on cell cycle. 

(4) Maintenance of cells and nuclei in viable non dividing states using 
modified and defined media. 

(5) Isolation and analysis of mRNA from different parts of the cell cycle or 
from quiescent cells. Cell free translation of mRNA. 

Major Findings and Accomplishments : 

1 . Effects of Inhibitors of DNA and Protein Synthesis on Gl , Quiescent, and 
S-Phase Histone Synthesis to DNA Synthesis 

Many studies have shown that inhibition of DNA synthesis immediately leads to a 
similar inhibition of histone synthesis even though total protein synthesis is 
not significantly inhibited. Our studies with hydroxyurea, a classical 
inhibitor of DNA synthesis, show that basal and S phase histone synthesis are 

343 



ZOl CM 06140-09 LMPH 
inmbited to different extents when DNA synthesis is inhibited. We have 
continued these studies with a variety of inhibitors of DNA synthesis Those 
agents which inhibit DNA synthesis only, give the same results as hydroxyurea 
However other agents which also inhibit protein syntheses do not selectively * 
inhibit the synthesis of particular histone variants but change the histone 
synthesis pattern in other ways. The crosslinking antineoplastic agents are 
in this class. It has previously been reported that inhibition of protein 
synthesis blocks the specific effect of DNA synthesis inhibitors on histone 
synthesis. We have recently published a model that offers an explanation 
for these interactions. ^ 

2. Development of Methodology to Study Histone Pools 

The amount of histone in the cytoplasm is thought to be a central control 
element in the regulation of histone and DNA synthesis. However, the histone 
pool has not been studied in any systematic way because of several technical 
difficulties. These include the small size of the pool, the problems of pur- 
ifying histone proteins from the cytoplasm, and the problem of distinguishing 
cytoplasmic histone from contaminating chromatin histone. Those few studies 
of histone pools have examined the labeling kinetics of some of the histone 
proteins rather than their mass. 

We have adapted our methodology for the analysis of histone variants in chro- 
matin to the analysis of histones in the cytoplasm and have overcome most of 
these problems. Newly synthesized H4 in the cytoplasm is doubly modified by 
an acetate and a phosphate. When cytoplasm is prepared X)y gentle lysis of 
cells with a nonionic detergent, then extracted with HCl and the extract freeze 
dried for electrophoresis on AUT-AUC gels, the pattern of cytoplasmic histone 
shows doubly modifed H4 as well as the absence of ubiquitin adducts of the 
H^A s. Thus cytoplasmic histone can be analyzed with little or no contami- 
nation from nuclear histone. 

We plan to use this newly developed method to study several aspects of the 
histone pool. The first set of questions concern the relationship of the pool 
histone to chromatin histone during normal S-phase. Do histones flux in one 
direction only from polyribosomes, through the pool to chromatin, or do 
histones flux out of chromatin into the pool? Are histones in the pool 
degraded? The second set of questions concern how the histones in the pool 
react to the inhibition of DNA and/or protein synthesis. Do the levels of 
histones in the pool change in such a way as to be consistent with auto- 
regu ation. The third set of questions concern the level and flux of histone 
in bl and GO as well as in S, and the transition between these states. 

3- In Vitro Assay for Aspects of Histone mRNA Regulation 

Our specific aim on this project is to develop an assay for the selective 
degradation of S-phase histone mRNA. There are no reports in the literature 
of such an assay. When whole cells are treated with hydroxyurea to inhibit DNA 
synthesis, histone mRNA disappears with a half-life of about 10 minutes. Other 
mRNA s, such as actin mRNA, are not affected. Using lysolecithin to make cells 
leaky, we can prepare an extract which continues translation. By using dot 
blotting, we can easily measure mRNA levels in the extracts and measure rates 
of degradation. 



344 



ZOl CM 06140-09 LMPH 

The specific degradation of histone mRNA Is presumably catalyzed by a nuclease, 
the activity of which can be rapidly modulated by a signal generated by the 
rate of DNA synthesis. This signal might be the level of the cytoplasmic 
histone pool. The significance of such a study is that if such an in vitro 
assay could be developed, this regulatory mechanism could be separated into its 
component parts. 

4. Histone HI Biosynthesis in Various Cell States 

The pattern of HI variant synthesis is very different between growing and 
quiescent cells. The synthesis of the variants Hl.l and HI. 2 was negligible 
in quiescent IMR-90, but significant when these cells were proliferating. In 
resting lymphocytes only 3 variants appear to be synthesized, namely HI. 3, 
HI, 4 and HI. 5, but after 50 hours of stimulation with PHA another subtype, 
likely to be HI. 2 is synthesized in significant amounts. In IMR-90 cells 
treated with hydroxyurea (HU) and 10 yM cytosine arabinoside (Ara-C), the HI 
synthesis pattern was found to be similar to that observed in quiescent cells. 

We conducted some other studies to evaluate whether anticancer agents which 
arrest cells in premitotic phase interfere with HI phosphorylation. We inves- 
tigated this point using two semisynthetic epipodophyllotoxin derivatives, VP16 
and VM26. Neither of them appear to inhibit the phosphorylation of HI as 
assessed by 33p_-jncorporation. The results of this study dre being prepared 
for publication. 

Significance to Biomedical Research and Program of the Institute : 

Cancer at one level is the inappropriate multiplication of cells. Our findings 
during the last year have suggested that analysis of histone variant synthesis 
and the histone variant genes may yield some insight into the relationship of 
different cell states in normal and neoplastic cells. 

Proposed Course ; 

1. To characterize the histone pool and to test the predictions of the 
published model as to the behavior of the pool. 

2. To develop methodology to study the selective sensitivity of histone mRNA. 
Publications ; 

1. Wu, R.S., West, M.H.P,, and Bonner, W.M.; Histone protein synthesis in 
human and other mammalian cells. In Stein, G. and Stein, J. (Eds.): 
Histone Genes . New York, John Wiley & Sons, 1984, pp. 457-483. 

2. Wu, R.S,, and Bonner, W,M,: Pattern of histone variant synthesis and 
implications for gene regulation. In Kumar, A. (Ed,): Eukaroytic Gene 
Expression , New York, Plenum, 1984, pp, 37-67, 

3. Seiler-Tuyns, A., Pantazis, P., Bonner, W., Hamer, D., and Kumar, A,: A 
mouse histone H4 gene carried by an SV40 vector is accurately expressed 
in infected monkey cells, DNA 3: 215-222, 1984 



345 



ZOl CM 06140-09 LMPH 

4. Pantazis, P., West, M.H.P., and Bonner, W.M.: Phosphorylation of 
histones in cells treated with hypertonic and acidic media. Mol . Cell. 
Biol . 4: 1186-1188, 1984. 

5. Pataryas, T.A., Sekeri-Pataryas, K.T., Bonner, W.M., and Aleporou- 
Marinou, V.: Histone variants of the insect Plodia interpunctella during 
meta morphosis. Comp. Biochem. Physiol . 77B : 749-753, 1984. 

6. West, M.H.P. , Wu, R.S., and Bonner, W.M.: Polyacryl amide gel electro- 
phoresis of small peptides. Electrophoresis 5: 133-138, 1984. 

7. Mueller, R.D., Yasuda, H., Bonner, W.M., and Bradbury, E.M.: Identifi- 
cation of uDiquitinated H2A and H2B in Physarum Polycephalum : 
Disappearance of these proteins at metaphase and reapperance at anaphase. 
J. Biol. Chem . 260: 5147-5153, 1985. 

8. Pantazis, P., and Bonner, W.M.: Specific alterations in the pattern 
of histone 3 synthesis during conversion of human leukemic cells to 
terminally differentiated cells in culture. Differentiation 28: 186- 
190, 1984. 

9. West, M.H.P., Pantazis, P., and Bonner, W.M.: Studies on nuclease 
digestion of chromatin phosphorylated in vivo . J. Biol. Chem . 260: 
4558-4560, 1985. 

10. Sariban, E., Wu, R.S., Erickson, L.C., and Bonner, W.M.: Interrelation- 
ship of protein and DNA synthesis during replication in mammalian 
cells. Mol. Cell Biol ., in press. 

11. Wu, R.S., Panusz, H., Hatch, C.L., and Bonner, W.M.: Histones. CRC Crit. 
Rev., in press. 



346 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06150-04 LMPH 



PERIOD COVERED 

October 1. 1984 to September 30. 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Protein-associated DNA Strand Breaks as Indicator of Topoisomerase II Inhibition 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: Yves Pommier Visiting Associate LMPH NCI 



Others: Jacqueline Wang-Peng 
Judith Markovits 
Donna Kerrigan 
Ronald E. Schwartz 
Kurt W. Kohn 



Sr. Investigator 
Guest Researcher 
Chemist 
Biologist 
Lab Chief 



MB NCI 
LMPH NCI 
LMPH NCI 
LMPH NCI 
LMPH NCI 



COOPERATING UNITS (if any) 

Merck Research Institute, West Point, PA (Dr. M. 0. Bradley); LCP, DTP, NCI; 
MCPB, COP, NCI; Institut Gustave Roussy (Dr. A. Jacquemin-Sablon) Villejuif, 
France 



LAB/BRANCH 

Laboratory of Molecular Pharmacology, DTP, OCT, NCI 



SECTION 

DNA Damage and Repair Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



PROFESSIONAL: 







CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues E (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The protein-associated DNA single and double-strand breaks induced in mammalian 
cells by DNA intercalating agents and epipodophyllotoxins have been shown to 
represent an effect on topoisomerase II. The aim of this project is to in- 
vestigate the relationship between the drug-induced protein-associated strand 
breaks and the physiological state and pharmacological sensitivity of cells. A 
correlation was found in V79 cells between protein-associated double-strand 
breaks and sister-chromatid exchange, mutation and cytotoxocity. A multi-drug 
resistant line of Chinese hamster cells exhibited reduced strand break responses 
to the intercalators, amsacrine and ellipticine, and to the epipodophyl lotoxin, 
etoposide; the uptake of these drugs into the resistant cells was not altered. 
Protein-associated strand breakage in response to amsacrine and etoposide was 
found to be dependent upon the proliferation state of NIH 3T3 cells; quiescent 
cells showed low sensitivity to this response, as well as to cell killing; when 
cells were stimulated to proliferate, the responses increased during the onset 
of DNA synthesis. Further work aims to elucidate the changes in topoisomerase(s) 
in resistant cells. 



347 



PHS 6040 (Rev. 1/84) 



GPO 9l4-9te 



ZOl CM 06150-04 LMPH 
Objectives : 

1. Determine the role of topoisomerase II in the production of sister 
chromatid exchanges, mutations and cell killing in mammalian cells. 

2. Determine the cytotoxic role of topoisomerase II trapping by intercalators 
and epipodophyllotoxins by comparing sensitive and resistant cell lines. 

3. Study the mechanism of cell resistance to topoisomerase II inhibitors. 

4. Determine the cell cycle-dependence of the protein-associated DNA strand 
breaks and cytotoxicity produced by intercalators and epipodophyllotoxins. 

Methods ; 

- Standard cell culture and synchronization methods. 

- Measurement of protein-associated ONA strand breaks by alkaline elution. 

- Measurement of sister chromatid exchanges, mutations and cytotoxicity by 
standard procedures. 

- Topoisomerase II (and nuclear protein) isolation by gel filtration, DNA 
affinity chromatography and glycerol gradient centrifugation. 

Major Findings ; 

1. Correlations Between Intercalator-induced DNA Strand Breaks and Sister 
Chromatid Exchanges, Mutations and Cytotoxicity in Chinese Hamster Cells' 

Intercalator-induced DNA strand breaks in mammalian cells represent topoiso- 
merase II-DNA complexes trapped by intercalators. These complexes are 
detected as protein-associated DNA single-strand breaks (SSB) and DNA double- 
strand breaks (DSB) by filter elution. Using Chinese hamster lung fibroblasts 
(V79 cells) that were treated for 30 min with various concentrations of 
4'-(9-acridinylamino) methanesulfon-m-anisidide (amsacrine) or 5-imino- 
daunorubicin (5-ID), we measured DNA strand breaks (SSB & DSB), sister 
chromatid exchanges (SCEs), mutations at the hypoxanthine-guanine phosphoribo- 
syltransferase (HGPRT) locus and cell killing. Further, we correlated DNA 
strand breakage with the three other parameters. Both drugs induced SCEs, 
mutations and cell killing at concentrations which also produced reversible 
DNA strand breaks. While the quantity of DSB correlated with SCEs, mutations 
and cytotoxicity for both drugs, we found more SCEs, mutations and cytotoxicity 
per SSB in cells treated with 5-in than in those treated with amsacrine. These 
data show that the DSB (but not the SSB) induced by amsacrine and 5-ID at DNA 
topoisomerase II binding sites correlated closely with SCEs, mutations and 
cell killing, and could therefore be responsible for their production. The 
mechanism by which intercalators induce sister chromatid exchanges (SCEs) is 
not known. We propose that these drugs stimulate a subunit exchange of topo- 
isomerase II molecules within cleavable complexes at DNA replication forks. 
The subunit exchange would lead to a SCE after an additional round of DNA 
replication. Details of this model have been discussed in our publication 
(Pommier et al.. Cancer Res., 1985 , in press). We also propose that the 

348 



ZOl CM 06150-04 LMPH 

trapping of topoisomerase cleavable complexes (formation of DNA double-strand 
breaks) could lead to frame shift mutations. 

2. Role of Topoisomerase II Inhibition (Formation of Cleavable Complexes) by 
Intercalators and Epipodophyllotoxins in the Cell Killing (Antitumor) Effect 
of these Drugs 

Our approach has been to use cells resistant to topoisomerase II inhibitors. A 
Chinese hamster cell line (0C3F/9-0H) had been made resistant to 9-hydroxy- 
ellipticine by Dr. A. Jacquemin-Sablon (Institut Gustave Roussy, Villejuif, 
France). We selected these cells because 1) they had been made resistant to a 
drug which is a potential topoisomerase II inhibitor; 2) the parent cell line 
(DC3F) could be used as a control; and 3) the resistance to ellipticines was not 
due to a reduced cellular uptake of the drug. Resistant cells (DC3F/9-0H) were 
first tested for their cross-resistance to ellipticinium, amsacrine and etopo- 
side (VP-16). DC3F/9-0H cells appeared to be highly resistant not only to 
the two intercalators (amsacrine and ellipticinium) but also to etoposide 
which does not bind DNA. Resistance to amsacrine was not due to a modified 
uptake of the drug. Additional drug transport studies showed that resistance 
to adriamycin was, on the other hand, associated with a reduced uptake of 
the drug. 

We then measured the formation of protein-associated DNA strand breaks in both 
cell lines. Resistant DC3F/9-0H cells produced markedly less protein-associated 
DNA single- and double-strand breaks than sensitive DC3F cells after a 30 min 
exposure to amsacrine, ellipticinium or etoposide. Similar results were found 
by treating isolated nuclei from each cell line. 

These results show that resistance to intercalators and etoposide can occur 
without drug uptake change in cells which, however, may have plasma membrane 
modifications (reduced uptake of adriamycin). In addition, the results 
suggest that 1) resistance to amsacrine, ellipticinium and etoposide could be 
due to the reduced trapping of topoisomerase II within cleavable complexes, 
and 2) topoisomerase II modifications are present in resistant (DC3F/9-0H) 
relative to sensitive (DC3F) cells. 

This last point has been further investigated. DNA from DC3F/9-0H (resistant) 
cells has been isolated and transfected to mouse NIH 3T3 cells. 7 resistant 
transfected cell lines have been obtained. The integration of Chinese hamster 
(DC3F/9-0H) DNA into mouse cells (3T3) was confirmed by dot-blot hybridization. 
The resistant transfected cells (NAD) appeared to be as resistant to amsacrine 
as were the donor resistant cells. NAD cell nuclei, produced also much less 
protein-associated DNA breaks than did 3T3 cell nuclei NAD cells had also 
lost their reduced adriamycin uptake. These results suggest that resistance 
to intercalators and etoposide in DC3F/9-0H cells is due to the presence of 
a factor that reduces the ability of topoisomerase II to be trapped by inter- 
calators and etoposide. 

Additional data support this hypothesis. Purified topoisomerase II from 
DC3F/9-0H cells was as sensitive to amsacrine as that from DC3F or L1210 
cells. Nuclear extracts from DC3F/9-0H cells, however, did not produce 
amsacrine-dependent topoisomerase II cleavable complexes. It appears therefore 
that a modulating factor that reduced the effect of amsacrine maybe present 

349 



ZOl CM 06150-04 LMPH 

in the nuclear extract from resistant cells. The nature of this modulating 
factor is yet unknown. The fact that crude nuclear extracts from DC3F/9-0H 
cells and from resistant transfected cells (NAD) had an unusually high level 
of DNA linking activity suggests that this HNA linking activity might play a 
role as a modulating factor. 

3. Protein-associated DNA Breaks in Relation to Cell Cycle 

Previous experiments (Minford et al., Cancer Res., 1984) suggested that 
intercalator-induced protein-associated DNA breaks may be increased in repli- 
cating cells. 

Mouse 3T3 cells stop proliferation when they reach a certain density on the 
plate. The cells can be initiated to proliferate by replating at a lower 
density and feeding with fresh medium containing calf serum. We found that 
amsacrine-dependent DNA breakage in proliferating cells was much greater 
than in arrested cells. We also studied the DNA breakage as a function of 
time following replating of the cells and found peaks of DNA synthesis at 5-6 
h and about 10 h after replating the cells into fresh medium. Assays were 
done by taking cells at various times, and incubating them for 30 min with 
10 mM amsacrine. Filter elution assays for DNA double-strand breaks were 
then carried out, and showed peaks of DNA breakage occurring at about the 
times of the peaks of [•^H]-thymidine incorporation. 

Proposed Course : 

1. Study the formation of sister chromatid exchanges (and mutations) in 0C3F/ 
9-OH resistant cells in order to further correlate the formation of sister 
chromatid exchanges to that of protein-associated DNA breaks and the possible 
involvement of topoisomerase II in sister chromatid exchange formation and cell 
killing (antitumor activity). 

2. Isolate purified topoisomerase II from sensitive (DC3F) and resistant 
(DC3F/9-0H) cells and compare their cleavage pattern in the absence and presence 
of amsacrine, ellipticinium and etoposide. This could be done in sequencing 
gels. 

3. Re-transfect the DNA from the first generation of transfected cells (NAD) 
into NIH 3T3 cells in order to isolate the gene(s) involved in resistance to 
topoisomerase II inhibitors and the modulating activity of topoisomerase II, 
which seem to be associated with drug resistance. 

4. Further study the cell cycle-dependence of amsacrine-induced protein- 
associated DNA breaks by performing experiments in isolated nuclei. This 
procedure should limit drug uptake differences during cell cycle and allow a 
better analysis of the relationship between topoisomerase Il-induced DNA breaks 
and DNA synthesis. We plan also to study the cell cycle-dependence of amsacrine 
cytotoxicity and etoposi de-induced protein associated DNA breaks and cyto 
toxicity. 

Publications : 

1. Minford, J., Kerrigan, D,, Nichols, M. , Shackney, S., and Zwelling, L.A.: 

350 



ZOl CM 06150-04 LMPH 

Enhancement of the DNA breakage and cytotoxic effects of intercalating 
agents by treatment with sublethal doses of l-g-D-Arabinofuranosylcytosine ■ 
or hydroxyurea in L1210 cells. Cancer Res . 44: 5583-5593, 1984. 

2. Pommier, Y., Zwelling, L.A., Kao-Shan, C.S., Wang-Peng, J., and Bradley, 
M.O.: Correlations between intercalator-induced DNA strand breaks and 
sister chromatid exchanges, mutations, and cytotoxicity in Chinese hamster 
cells. Cancer Res ., in press. 

3. Pommier, Y., Jacquemin-Sablon, A., Schwartz, R.E., and Kohn, K.W.: 
Alterations of DNA topoisomerases in Chinese hamster cells resistant to 
DNA intercalators and VP-16. Proc. Am. Cancer Res. 26: 344, 1985. 



351 



DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06160-02 LMPH 



PERIOD COVERED 

October 1. 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or lass. Title must fit on orte line between the borders.) 

Mechanism of Action of DNA Cross! inking Agents 



PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 



PI: 



Others: 



Kurt W. Kohn 

Neil Gibson 
John Hartley 
Daniel Yarosh 
Roy S. Wu 



Lab Chief 

Visting Fel low 
Visiting Fel low 
Cancer Expert 
Cancer Expert 



LMPH NCI 

LMPH NCI 
LMPH NCI 
LMPH NCI 
LMPH NCI 



COOPERATING UNITS (If any) 

Laboratory of Medicinal Chemistry and Biology, OCT, NCI 
Surgical Neurology Branch, NINCDS 



LAB/BRANCH 

Laboratory of Molecular Pharmacology, DTP, DCT, NCI 



SECTION 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



3.4 



PROFESSIONAL: 



2.4 



1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
D (a2) Interviews 



[3 (b) Human tissues D (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Chi oroethyl nitrosoureas (CIEtNUs) kill susceptible human tumor cells by chloro- 
ethylation of guanine-06 positions in DNA, leading to the production of inter- 
strand crosslinks. Chi oroethyl ating agents potentially superior to CIEtNUs are 
being studied. 2-chl oroethyl (methyl sulfonyl )methanesulfonate ("ClEtSoSo") 
(NSC 338947) was found to have the desirable feature of lacking a prominent 
chemical side-reaction, hydroxyethylation of DNA oases, produced by the CIEtNUs. 
In reactions with purified DNA, ClEtSoSo produced only chloroethyl products 
whereas a ClEtNU produced more hydroxyethylation than chloroethyl products. 
Despite the lack of hydroxyethylation reaction, ClEtSoSo is at least as effective 
as any ClEtNU against experimental tumors. 

Another chloroethylating agent, mitozolomide, produced a greater diversity of 
products than did the ClEtNU; this was considered to be an undesirable feature 
of this drug. 

The susceptibility of certain human tumor cell lines to CIEtNUs depends on a 
deficiency of guanine-06-alkyltransferase. New methods are being developed 
for isolating this enzyme from human liver and for assaying the enzyme activity 
in tumor tissues. 

Studies are in progress to test for relationships between clinical response of 
malignant glioma patients and DNA crosslinking responses of cells from these 
patients' tumors to aziridinylbenzoquinone. 



352 



PHS 6040 (Rev. 1/84) 



GPO gi4-ate 



ZOl CM 06160-02 LMPH 
Project Description 

Objectives : 

1. Define the mechanism of action of new anticancer drugs that kill sus- 
ceptible human tumor cells by chloroethylation of DNA guanine-06 positions; 
find compounds that produce predominantly this reaction with a minimum of 
side-reactions. 

2. Develop improved methods of isolation and assay of guanine-06-alkyltrans- 
ferase, the key enzyme that determines the susceptibility of human tumor cells 
to chloroethylating agents; investigate the properties of this enzyme and clone 
genes in order to determine why the enzyme is deficient in certain cells. 

3. Test for correlations between clinical responses to DNA crossl inking drugs 
and crossl inking responses in cell cultures derived from the tumors. 

Methods : 

1. Reaction of radioactive drugs with DNA. 

2. Separation of reaction products by high pressure liquid chromatography 
(HPLC). 

3. Organic synthesis of predicted purine adducts. 

4. Mass spectrometry identification of purine adducts. 

5. Alkaline elution methods developed in this Laboratory for the determination 
of DNA lesions in mammalian cells. 

6. Chromatographic methods of enzyme isolation. 

7. Restriction enzyme assay of synthetic oligonucleotides containing an ab- 
normal base. 

8. Gene transfer by DNA transfection and detection of transfered DNA by molec- 
ular hybridization. 

Major Findings : 

2-Chloroethyl(methanesulfony1)methanesulfonate ('ClEtSoSo') (NSC 338947) 

This compound (colloquially 'ClEtSoSo') is being considered for clinical de- 
velopment by NCI as a new type of alkylating agent. We had found ClEtSoSo 
to produce DNA interstrand crosslinking and DNA-protein crosslinking in Mer" 
(guanine-06-alkyltransferase deficient) human tumor cell lines, the 
effects being indistinguishable from those produced by 2-chloroethylnitroso- 
ureas (ClEtNUs) of the non-carbamoylating type. ClEtSoSo selectively killed 
Mer- relative to Mer"*" human tumor cells, as did ClEtNUs, and the degree of 
selectivity was even greater than that produced by any ClEtNU tested. 



353 



ZOl CM 06160-02 LMPH 
The major question addressed during the current year was whether, as was 
expected on chemical grounds, ClEtSoSo would produce a more restricted range of 
of reaction products with DNA than do the ClEtNUs. ClEtNUs were known to 
produce hydroxyethyl adducts of DNA bases as major products, and it appeared 
unlikely that these reactions would contribute to antitumor activity. 

Purified DNA was therefore reacted with [^^C-ethyl ]-labelled ClEtSoSo and 
ClEtNU (the non-carbamoylating cis-2-OH-CCNU derivative was used). The DNA 
was hydrolysed and the reaction products were separated by HPLC. Whereas 
cis-2-OH-CCNU yielded 5 HPLC peaks, ClEtSoSo produced only 3 peaks. Two 
of the peaks were identified by chemical synthesis and mass spectrometry: 
N7-chloroethylguanine, and N7-hydroxyethyl guanine. Whereas cis-2-OH-CCNU 
produced all both of these products (and in fact more N7-hydroxyethyl 
than N7-chloroethylguanine), ClEtSoSo by contrast produced N7-chloroethyl- 
guanine and no detectable hydroxyethyl product. 

The results support our premise that ClEtSoSo is free of certain side reactions, 
namely hydroxyethyl ati on reactions, which are major reactions of all ClEtNUs. 
The fact that ClEtSoSo at least equalled any ClEtNU in its effectiveness against i 
a broad spectrum murine tumor systems in special NCI tests gives additional 
support to the proposition that hydroxyethyl ati on reactions are unnecessary for 
antitumor activity. 

Mitozolomide (M&B 39565) 

This compound was developed in England as a possible pro-drug for chloroethyl 
triazenoimidazolecarboxamide (ClEtTIC) and is undergoing clinical trial in 
Europe. We found that mitozolomide and ClEtTIC produce interstrand crosslinks 
and DNA-protein crosslinks and exhibit selective actions on Mer" cells in a 
manner similar to the ClEtNUs. l^C-chloroethyl -labeled mitozolomide was studied 
along with ClEtSoSo and ClEtNU in the studies described above. Mitozolomide 
generated DNA reaction products which on HPLC separation yielded 9 peaks, an 
even greater number than was produced by the ClEtNU. Among these peaks were 
all 3 hydroxyethyl and chloroethyl guanine species mentioned above, with the 
N7-hydroxyethyl predominating over the N7-chloroethyl guanine. 

These results indicate that mitozolomide generates an even greater diversity 
of chemical reactions than do (non-carbamoylating) ClEtNUs. From the point of 
view of the strategy to reduce reaction diversity and to develop compounds 
which produce only the necessary reactions, the clinical development of mitozo- 
lomide would not be supported. 

Guanine-06-alkyl transferase (G06AT) 

A deficiency of G06AT activity makes some human tumor cells (Mer- phenotype) 
susceptible to killing by chloroethyl ati ng agents. When G06AT activity is 
normal (Mer+ phenotype), chloroethyl adducts are rapidly removed from 
guanine-06 positions. When the enzyme is deficient (Mer" cells), the removal 
of these adducts is reduced or delayed, and the adducts react further to 
produce potentially lethal interstrand crosslinks. These conclusions, which 
are now generally accepted on the basis of work from this and other 
laboratories, show the importance of understanding the properties of G06AT 
and the origin of the deficiency in Mer* cells. 

354 



ZOl CM 06160-02 LMPH 

A new procedure was developed for purification of G06AT from human liver. The 
steps are: (1) preparation of extract by Waring blender; (2) ammonium sulfate 
precipitation and storage; (3) Affigel Blue chromatography; (4) mercury-agarose 
chromatography; (5) single-strand DNA agarose chromatography; (6) phenylagarose 
chromatography. The aim is to purify enough human enzyme for antibody pro- 
duction and amino acid sequencing. 

As an approach to cloning of the gene, DNA transfer of G06AT from human Mer"*" to 
Men" cells was attempted. Co-transfer of the pSVneo gene was used to aid the 
selection. A clone was isolated which contained G06AT activity associated with 
a 22 kilodalton protein, in addition to pSV2neo sequences. Encouraged by this 
result, transfer was attempted into CHO cells so that the transfer of human DNA 
In the hamster cells could be monitored. The transfer of GOeAT activity into 
CHO cells however was unsuccessful. 

The development of a simple and sensitive assay for G06AT was undertaken. A 
deoxypolynucleotide was obtained containing 06-methyl guanine at a defined 
position in the sequence. The sequence was designed so that the 06-methyl - 
guanine is at a restriction site which is blocked by the presence of this 
altered base. The assay for G06AT activity depends on the removal of the 
methyl group from the 06 position which would then allow restriction endo- 
nuclease cutting at this site. 

Test of Correlation Between DNA Crosslinking and Clinical Response of Malignant 
Gliomas 

In a collaborative study with the Surgical Neurology Branch, NINCDS, we are 
testing for a possible correlation between clinical response to DNA crosslinking 
drugs and the crosslinking response of cell lines derived from the tumors. The 
Surgical Neurology Branch routinely establishes cultures from most of their 
patients operated for malignant gliomas, and the resulting cell strains are 
stored frozen. In the current study, the clinicians select from their records 
patients who they felt responded to aziridinylbenzoquinone (AZQ) and patients 
who failed to respond. They prepare the corresponding cell cultures from frozen 
stocks and forward them to us as coded samples. In the first set of cultures, 
2 cell strains from responders showed greater interstrand crosslinking responses 
in vitro than did 2 cell strains from non-responders. 

Significance ; 

1. It was shown that 2 major classes of chemical side-reactions can be eli- 
minated without impairment of the experimental antitumor activity of an 
extraordinarily effective class of drugs. Elimination of unnecessary side- 
reactions may improve the therapeutic index of these drugs. 

2. The studies of human guanine-06-alkyltransferase (G06AT) aim to provide an 
assay by which tumor samples can be tested for potential sensitivity to treat- 
ment with chloroethylating drugs. In addition, an understanding of the origin 
of the G06AT deficit in some human tumor cells could lead to improved strategies 
for selective killing of potentially susceptible tumors. 

3. A demonstrated positive correlation between DNA interstrand crosslinking 
and clinical response of individual tumors to particular drugs would be a key 

355 



ZOl CM 06160-02 LMPH 

step in efforts to develop more potent and specific crossl inking agents. It 
could also lead to predictive drug sensitivity tests. 

Proposed Course : 

1. Develop assays for guanine-06-alkyltransferase (G06AT) in tumor tissues. 
An immunochemical assay for G06AT protein and a restriction enzyme assay for 
G06AT activity will be developed. 

2. Study possible correlations between G06AT activity, HNA crosslinking and 
the sensitivities of various human tumor cell types and of patients bearing 
the tumors to DNA crosslinking drugs. 

3. Study possible synergism between DNA crosslinking agents and epipodophyllo- 
toxins. 

Publications : 

1. Szmigiero, L., Erickson, L.C., Ewig, R.A.G., and Kohn, K.W.: DNA strand 
scission and cross-linking by diaziridinylbenzoquinone (diaziquone) in 
human cells and relation to cell killing. Cancer Res . 44: 4447-4452, 
1984. 

2. Szmigiero, L., and Kohn, K. W.: Mechanisms of DNA strand breakage and 
interstrand cross-linking by diaziridinylbenzoquine (diaziquone) in 
isolated nuclei from human cells. Cancer Res . 44: 453-4457, 1984. 

3. Sariban, E., Erickson, L. C, and Kohn, K. W.: Effects of carbamoyl ati on 
on cell survival and DNA repair in normal humane embryo cells (IMR-90) 
treated with various l-(2-chloroethyl )-l-nitrosoureas. Cancer Res . 44: 
1352-1357, 1984. 

4. Yarosh, D.8.: The role of 0^-methylguanine-DNA methyltransferase in cell 
survival, mutagenesis and carcinogenesis. Mutat. Res. 145 : 1-16, 1985. 

5. Zlotogorski, C, and Erickson, L.C.: Pretreatment of human colon tumor 
cells with DNA methylating agents inhibits their ability to repair 
chloroethyl monoadducts. Carcinogenesis 5: 83-87, 1984, 

6. Yarosh, D.B., Rice, M., Day, R. S, III, Foote, R.S., and Mitra, S.: 
0^-methylguanine-DNA methyltransferase in human cells. Mutat. Res . 
131: 27-36, 1984. 

7. Yarosh, D. B., Ziolkowski, C, and Day, R.S., III: Conversion of 
Human Cells to Carcinogen Resistance by DNA Transfection. In Bishop, 
J.M., Graves, M., and Rowley, J, (Eds.): Genes and Cancer . New York, 
Liss, in press. 



356 



ZOl CM 06161-02 LMPH 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Topoisomerase II as Target of Action of Anticancer Drugs 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

PI: Yves Pommier 
Others: Jon K, Minford 

Michael Mattern 

Judith Markovits 

Joseph Covey 

Donna Kerrigan 

William Mattes 

Kurt M. Kohn 



Visiting Associate LMPH NCI 

Medical Staff Fellow LMPH NCI 

Cancer Expert LMPH NCI 

Guest Researcher LMPH NCI 

Staff Fellow LMPH NCI 

Chemist LMPH NCI 

Staff Fellow LMPH NCI 

Lab Chief LMPH NCI 



COOPERATING UNITS (if any) 

Institut Gustave Roussy (J. 



B. LePecq) Villejuif, France 



LAB 



,B/BRANCH ^ , , 

aboratory of Molecular Pharmacology, DTP, DCT, NCI 



Damage and Repair Section 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



6.0 



PROFESSIONAL: 



5.0 



1.0 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



D (b) Human tissues E (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The effects of DNA intercalating agents and epipodophyllotoxins are being studied 
upon purified mammalian topoisomerase II. DNA intercalators and epipodophyllo- 
toxins inhibit DNA topoisomerase II while trapping the enzyme within topoisomerase 
II-DNA DNA cleavable complexes. The cleavable complexes consist of two enzyme 
subunits which are covalently bound to the 5'-termini of the DNA breal<. The DNA 
breal^s can be detected only after protein denaturation by sodium dodecyl sulfate 
and proteinase K digestion. The breaks also reverse upon salt addition in the 
absence of added nucleotide. The cleavable complexes trapped by intercalators 
and epipodophyllotoxins are analogous to intermediates in the DNA breaking-rejoin- 
ing reaction of topoisomerase II. It is by this reaction that the enzyme carries 
out DNA strand passage and DNA topoisomerization reaction. Another mode of inter- 
action between topoisomerase II-DNA complexes and intercalator is the forced 
reversal or unlocking of cleavable complexes. This effect has been observed 
with high concentrations of 2-methyl-9-hydroxyellipticinium and with the bisinter- 
calator, ditercalinium. The unlocking of cleavable complexes seems to be related 
to DNA binding and may not be accompanied by enzyme inhibition. 

The localization of intercalator-induced topoisomerase II action in the nuclear 
genome was investigated. It was found that certain repeated sequences are en- 
riched at a favored distance from sites of intercalator-induced topoisomerase 
II-DNA linkage. The results have implications regarding the localization of 
topoisomerase II action in the loop structure of nuclear chromatin. 



357 



PHS 6040 (Rev. 1/84) 



GPo ai4-eis 



ZOl CM 06161-02 LMPH 
Objectives : 

1. Determine the role of topoisomerase II in the formation of protein- 
associated DNA breaks by intercalators and epipodophyllotoxins in mammalian 
cells, 

2. Study the modes of interaction between DNA intercalators or epipodo- 
phyllotoxins and topoisomerase II-DNA complexes. 

3. Determine the mechanisms(s ) by which topoisomerase II recognizes potential 
DNA cleavage sites within isolated DNA. 

4. Investigate the localization of topoisomerase II action in the nuclear 
genome. 

Methods : 

1. Purification of topoisomerase II and I from mammalian cells by 1) Gel 
filtration of 0.35 M salt extract from isolated nuclei 2) DNA-cellulose 
chromatography 3) Glycerol gradient centrifugation. SDS-PAGE electropho- 
resis is used to test enzyme purity. 

2. Filter binding assay to detect the formation of covalent complexes between 
radioactively labeled DNA ([3h]-SV40) and topoisomerases II and I. 

3. Electrophoresis of radioactively [32p]-iabeled DNA (pBR 322, SV-40) in 
in agarose or in polyacrylamide-urea (sequencing methods) gels to detect 
the sequence specificity of the topoisomerase Il-induced DNA double- and 
single-strand breaks respectively. 

4. Controlled DNA scission, electrophoresis, blotting and DNA hybridization. 
Major Findings : 

Effects of the DNA Intercalators. Amsacrine (m-AMSA) and Ellipticinium 
(2-methyl-9-hydroxyellipticinium) Upon LI 210 DNA Topoisomerase ~lT 

Previous studies have indicated that the protein-associated DNA strand breaks 
detected in mammalian cells treated with intercalating drugs are produced by 
topoisomerase II and that the enzyme becomes covalently bound to DNA at the 
termini of the breaks. 

Using isolated nuclei preparations we have found an unexpected difference 
between amsacrine and ellipticinium. Amsacrine produced DNA double-strand 
breaks at all concentrations, and the yield of breaks tended towards sat- 
uration, leading to approximately one DNA double-strand break per 5 x 10^ 
base pairs. Ellipticinium, on the other hand, produced more complicated 
effects. At concentrations below 10 pM, DNA breaks were produced whereas 
at higher concentrations the breaks were not formed. The lack of break 
formation at high ellipticinium concentrations was due to an active inhi- 
bition of the DNA cleavage reaction because at these same concentations, 
the amsacrine-induced breaks were also inhibited by ellipticinium. 

358 



ZOl CM 06161-02 LMPH 

By performing experiments where we mixed amsacrine and el 1 ipticinium, it 
appeared that the production of breaks by low ellipticinium concentrations 
and the inhibition of the breaks by high ellipticinium concentrations, 
resulted from two opposite but competitive modes of interactions upon the 
same nuclear target enzymes. 

Similar types of experiments were performed using topoisomerase II purified 
from L1210 nuclei. Amsacrine produced both DNA single- and double-strand 
breaks in the presence of topoisomerase II. 

These breaks were inferred to be associated with enzyme covalently bound to 
the 5'-termini of the DNA breaks. The enzyme-associated DNA breaks were 
produced by amsacrine at concentrations ranging from 1 to 20 yM. At the 
same time that the breaks were formed, the DNA topoisomerization reactions 
of topoisomerase II (ATP-dependent DNA relaxation and catenation) were 
inhibited. As observed in the nucleus preparations, ellipticinium had more 
complicated effects upon topoisomerase II. At low concentrations (below 2 
yM), ellipticinium produced enzyme-associated DNA breaks and enzyme inhibition 
whereas at high concentrations (above 5 yM), it inhibited the protein- 
associated DNA breaks formed by the topoisomerase II alone or in the presence 
of amsacrine. This "unlocking" effect of high ellipticinium concentrations 
did not seem to be associated with enzyme inhibition. 

These results show that two modes of interactions exist between inter- 
calators and topoisomerase II-DNA complexes: 1) trapping of enzyme-DNA 
complexes leading to the formation of cleavable complexes and enzyme 
inhibition; 2) unlocking (forced reversal) of cleavable complexes 
leading to the inhibition of topoisomerase Il-induced DNA breaks in 
the absence of inhibition of enzyme activity. The similarities between 
the results obtained with purified topoisomerase II and those obtained 
in isolated nuclei, further supports the inference that the protein-associated 
DNA breaks produced by antitumor intercalating drugs are mediated by topo- 
isomerase II and associated with enzyme inhibition. 

Effects of the Bisintercalator, Ditercal inium Upon L1210 DNA Topoisomerase II 

Ditercalinium consists of 2 intercalative rings (7H pyridocarbazole) linked by 
2-N'*'(CH2)2-l-piperidyl rigid chain. The rigidity of the linking chain prevents 
the stacking of the 2 intercalative rings and the folding of the molecule. 

Treatment of L1210 cells with ditercalinium at concentrations lower than 2 yM 
for 24 hr did not produce any detectable DNA damage. At concentrations 
above 2 yM the DNA was extensively degraded and an immediate cytolysis 
was observed. In addition, nucleoids from ditercalinium treated cells were 
more compact than those from untreated cells at drug concentrations where 
no DNA damage could be detected. This nucleoid compaction was not produced 
in L1210 cells that had been made resistant to the drug. These complexes 
results suggest that ditercalinium does not trap topoisomerase II cleavable 
complexes as do monointercalators. In addition, the results of the nucleoid 
sedimentation experiments suggests that chromatin compaction is a major 
effect of ditercalinium. 



359 



ZOl CM 06161-02 LMPH 

The effects of ditercalinium were then studied upon purified L1210 topoiso- 
merase II. By contrast to monointercalators, ditercalinium did not stimulate 
the formation of topoisomerase II cleavable complexes. Ditercalinium, how- 
ever, altered topoisomerase II activity in three ways. 

1) Ditercalinium prevented the formation of cleavable complexes formed by 
the enzyme alone or in the presence of amsacrine and etoposide (VP-16). 
This effect was comparable to the "unlocking" effect of high elliptinium 
concentrations. 2) Ditercalinium inhibited the relaxation of SV-40 DNA by 
topoisomerase II in the presence of ATP. This inhibition seemed to be 
related to DNA unwinding rather than to a direct enzyme inhibition. 3) 
Ditercalinium stimulated DNA catenation (or aggregation) by topoisomerase 
II. This stimulation was maximum between 0.1 and 1 yM of ditercalinium 
and was ATP-dependent. Ditercalinium concentrations above 1 yM inhibited 
the catenation reaction. 

Effects of the Epipodophyllotoxins, Etoposide (VP-16) and Teniposide (VM-26) 
Upon LI 210 DNA Topoisomerase II 

Epipodophyllotoxins have been reported to induce protein-associated DNA breaks 
in mammalian cells and to stimulate the formation of cleavable complexes formed 
by calf thymus topoisomerase II. The possible interaction of epipodophyllo- 
toxins with topoisomerase II plus the fact that these drugs are active anti- 
tumor agents that do not bind DNA, prompted us to investigate their mechanism 
of action. 

The production of DNA breaks and DNA-protein crosslinks by etoposide and 
teniposide was first studied in intact cells. Both compounds produced 
DNA strand breaks and DNA protein crosslinks in mouse leukemia L1210 
cells, in human colon carcinoma HT-29 cells and in viral transformed 
human embryonic VA-13 cells. At all drug concentrations the frequency 
of DNA strand breaks was equal to the frequency of DNA-protein cross- 
links within experimental error. In addition, no DNA breaks were 
detected under nondeproteinizing conditions. Similar results were 
obtained by treating isolated nuclei with etoposide and teniposide. 
It appeared therefore that the DNA strand breaks produced by etoposide and 
teniposide were protein-associated and probably represent topoisomerase 
II cleavable complexes. Two additional observations were made from the 
studies performed in whole cells and isolated nuclei: 1) teniposide 
was several times more potent than etoposide at equimolar concentrations, 
and 2) the two human cell lines (HT-29 and VA-IS) produced approximately 
3 times less protein-associated DNA strand breaks than the mouse leukemia 
L1210 cells. The production of protein-associated DNA strand breaks thus 
varies with the compound and cell line used. 

The effects of epipodophyllotoxins were then studied on purified L1210 topoiso- 
merase II. Both etoposide and teniposide stimulated the formation of cleavable 
complexes and inhibited the DNA relaxing activity of topoisomerase II. The 
formation of cleavable complexes was stimulated by ATP and by its nonhydro- 
lyzable analog, ATP-y-S. In agreement with the results obtained in cells, we 
found that teniposide was approximately 10-fold more potent than etoposide. 
The trapping of cleavable complexes by epipodophyllotoxins appeared to be 
different from that produced by amsacrine: 1) It was resistant to the 



360 



ZOl CM 06161-02 LMPH 

reversing effect of 0,5 M^ NaCl , and 2) the location of the cleavable complexes 
within pBR 322 were different for epipodophyllotoxins and of amsacrine. 

The present data strongly suggest that the protein-associated DNA breaks 
produced by the epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), 
are due to the trapping of topoisomerase II cleavable complexes and are 
associated with enzyme inhibition. In addition, the cleavable complexes 
trapped by epipodophyllotoxins differ in some respects from those trapped 
by amsacrine. 

Genomic Localization of Intercalator-induced Protein-Associated Strand Breaks 
(PASBs) 

The possibility was investigated that intercalator-induced PASBs occur selec- 
tively at certain locations in the genome. Methods were devised for the 
isolation of DNA sequences close to the sites of intercalator-induced protein 
linkage, presumably linkages between DNA strand termini and topoisomerase II. 
Molecular hybridization of dot-blots and of Southern blots indicated that 
(TG)n (n>10-20) sequences in both mouse and human DNA were enriched at a 
distance of about 6 kilobases from the sites of protein linkage. A similar 
enrichment was observed for the Alu class of repeated sequences in human cells. 
The results were not entirely as expected and can be interpreted in terms 
of the loop structure of chromatin with special functions involving topo- 
isomerase II bound to the nuclear scaffold. 

Proposed Course ; 

1, Mao the cleavage sites of topoisomerase II using DNA sequencing techniques 
and [32p]-iabeled DNA (pBR 322 and SV-40). Various compounds will be used to 
stimulate the formation of cleavable complexes (amsacrine, elliptinium, 
epipodophyllotoxins). 

2, Determine whether there is any consensus among the DNA sequences at which 
topoisomerase II cleaves DNA. Determine the recognition sequences of topo- 
isomerase II. 

3, Use acridine derivatives having different DNA affinities and different DNA 
binding specificities (GC versus AT). If the trapping of cleavable complexes 

is determined by drug intercalation sites, the cleavage pattern should vary from 
one compound to another depending on the binding sites of the drugs. 

Publications ; 

1, Pommier, Y., Mattern, M.R., Schwartz, R., and Zwelling, L.A.: Absence of 
swivelling of sites of intercalator-induced protein-associated deoxyribo- 
nucleic acid strand breaks in mammalian cell nucleoids. Biochemistry 23: 
2922-2927, 1984. 

2. Pommier, Y., Mattern, M.R., Schwartz, R., Zwelling, L.A., and Kohn, K.W.: 
Changes in deoxyribonucleic acid linking number due to treatment of 
mammalian cells with the intercalating agents, 4'-(9-acridinylamino) 
methanesulfon-m-anisidide (m-AMSA). Biochemistry 23: 2927-2932, 1984. 



361 



ZOl CM 06161-02 LMPH 

3. Pommier, Y., Schwartz, R.E., Kohn, K.W., and Zwelling, L.A.: Formation 
and rejoining of deoxyribonucleic acid double-strand breaks induced in 
isolated cell nuclei by antineoplastic intercalating agents. Biochemistry 
23: 3194-3201, 1984. 

4. Pommier, Y., Zwelling, L.A., Schwartz, R. E., Mattern, M.R., and Kohn, 
K.W.: Absence of a requirement for long range DNA torsional strain in the 
the production of protein-associated DNA strand breaks in isolated 
mammalian cell nuclei by the DNA intercalating agent 4'-(9-acridinylamino) 
methanesulfon-m-aniside (m-AMSA). Biochem. Pharmacol . 33: 

3909-3912, 1984. 

5. Pommier, Y., Schwartz, R.E., Zwelling, L.A., and Kohn, K.W.: Effects of 
DNA intercalating agents on topoisomerase Il-induced DNA strand cleavage in 
isolated mammalian cell nuclei. Biochemistry , in press. 

6. Pommier, Y., Minford, J.K., Schwartz, R.E., Zwelling, L.A., and Kohn, K.W.: 
Effects of the DNA intercalators, 4'-{9-acridinylamino)methanesulfon-m- 
aniside (m-AMSA, amsacrine) and 2-methyl-9-hydroxye11ipticinium (2-Me-9-0H- 
E+) on topoisomerase Il-mediated DNA strand cleavage and strand passage. 
Biochemistry , in press. 

7. Minford, J.K., Pommier, Y. , Filipski, J., Kohn, K.W., Kerrigan, 0., Mattern, 
M.R., Michaels, S., Schwartz, R.E., and Zwelling, L.A.: Isolation of 
intercalator-dependent protein-linked DNA strand cleavage activity from 
cell nuclei and identification as DNA topoisomerase II. Biochemistry , in 
press. 

8. Minford, J.K., Pommier, Y., Uhlenhopp, E,, Mattern, M.R., Kerrigan, D., 
Mattes, W., and Kohn, K.W,: Topoisomerase II activity of cells, nuclei and 
in vitro is a target for the epipodophyllotoxins. Biochemistry , in press. 



362 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06170-01 LMPH 



PERIOD COVERED . ^^ , ^ 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Isolation of Human Variant Hi stone Genes 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) 

PI: William Bonner Head, Chromosome Structure LMPH NCI 



and Function Section 



Others: Christopher Hatch Staff Fellow 



LMPH NCI 



COOPERATING UNITS ftf any; Department of Biological Chemistry, School of Medicine, Univ. 
of California, Davis, Department of Biochemistry, GWU Medical School and the 
Department of Biology, Georgetown University, Biotech Res, Labs., Inc. 



LAB/BRANCH 

Laboratory of Molecular Pharmacology. DTP. DCT, NCI 



SECTION 

Chromosome Structure and Function 



INSTITUTE AND LOCATION 

NCI. NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



1.4 



PROFESSIONAL: 



1.2 



0.2 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
D (a1) Minors 
D (a2) Interview's 



D (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Our objective is to isolate the genes for the human histone H2A family, in 
particular the H2A.Z gene. 

A number of putative H2A.Z clones have been isolated and are being further 
screened. 



363 



PHS 6040 (Rev. 1/84) 



GPO ai4-Bia 



Z01 CM 06170-01 LMPH 
Project Description 

Introductio n: 

The regulation of the various histone variants differs greatly with respect 
to the rates of protein and DNA synthesis. Isolation of the genes for the 
variant histones will help elucidate these regulatory mechanisms. 

Objective : 

To isolate human histone genes particularly the gene for H2A,Z. 

Methods : 

1. Recombinant DNA techniques. 

2. Use of synthetic probes to prime reverse transcriptase and search genomic 
libraries. 

Major Findings and Accomplishments : 

In order to isolate the various histone genes, we have utilized the human 
genomic library originally established in Phil Leder's laboratory by the 
insertion of human placental DNA into the charon 28 lambda bacteriophage, A 
"complete" library of the human genome in lambda was grown with C600 E. coli 
bacteria and plated in 15 cm diameter plates. Each plate was replica plated 
in duplicate with sterile nitrocellulose filters. The filters were processed 
and incubated with radioactively labeled DNA probes. 

Our primary effort has been towards the isolation of the genes encoding the 
minor variant form of H2A, H2A,Z, The isolation of non-S-phase histone genes 
has been difficult. This problem is most acute in the case of the gene for 
histone H2A.Z., since the amino acid sequence of this polypeptide has been 
found to be quite divergent from that of the other histone H2A sequences while 
it appears that the amino acid sequence of H2A.Z is conserved in a wide 
variety of species, the nucleotide sequences encoding this polypeptide may be 
more divergent between species. An H2A.Z related gene, called H2A.F, has been 
isolated from chicken, but cross-hybridization of this gene to homologous 
sequences in human, mouse, or sea urchin DNA was not found. This demonstrated 
that rather divergent nucleotide sequences could be coding for the same 
protein sequence and reinforced our belief that it would be difficult to 
utilize heterologous histone H2A gene probes to search for the human H2A,Z 
gene sequence, A more general approach not subject to these drawbacks 
involves the preparation of probes from known amino acid sequences. These 
specific probes for the detection of the desired histone mRNA's or genes can 
be obtained by the chemical synthesis of oligonucleotides which represent all 
possible codon combinations for a short run of amino acids in a given protein. 
H2A residues 24-28 were of particular interest to us since it has been de- 
termined that all H2A histone variants which had been sequenced in plants and 
animals have a common amino acid sequence from residues 21 to 29, We designed 
a 14-nucleotide length oligonucleotide sequence mixture that would contain 
sequences in this region. The probe designed contained a mixture of 14-base 
long synthetic oligonucleotides containg 16 possible combinations of sequence. 

364 



ZOl CM 06170-01 LMPH 

In order to both increase the specificity and stability of hybridization 
between the designed probe and the homologous sequences within the human 
genomic lambda library the synthetic oligonucleotide mixture was utilized as a 
primer for making partial cDNA copies of mRNA onto which it hybridized. In 
order to increase the proportion of H2A.Z mRNA relative to the other H2A mRNA s 
the mRNA treated with hydroxyurea for one hour prior to RNA purification from 
these cells. This treatment reduces the amount of major H2A variant mRNA and 
therefore would effectively increase the relative proportion of minor H2A 
variant mRNA. The oligonucleotide probe mixture was used to prime synthesis 
of radioactive cDNA copies of template mRNA. The -^^P-labeled cDNA's were 
then used to probe the human genomic lambda library. On the order of 
1/500-1/1000 of the human-lambda clones were observed to hybridize the 
radioactive probes. 

We decided to make a new synthetic probe mixture complementary to the mRNA 
encoding amino acid residues #4 through #8 of H2A.Z and H2A.F. A 14-base long 
synthetic probe mixture with 32 different possible sequence combinations was 
designed and commercially synthesized. Both this probe and the previously 
utilized 14-mer probe mixture were used to make -^-^P-labeled cONA probes and 
each of these probes were used to probe duplicate nitrocellulose replicas of 
the human genomic-lambda library. The autoradiographs of the filters from 
each plate were aligned to see which plaques showed positive hybridization 
to both of these independent probes. Although many plaques were doubly- 
positive, a smaller fraction appeared to match quite well in signal intensity. 
The plaques corresponding to the best matches were isolated, the phage was 
grown, the DNA was isolated, digested with restriction enzymes, run on 
agarose gels, and southern-transfered onto nitrocellulose. Nitrocellulose 
filters made from transfer of DNA from duplicate gels were probed separately 
with the two 32p_iat3eled cDNA's of forty lambda clones tested, five clones 
contained similar patterns of restriction fragments labeled by hybridization 
to each of the 32p_cDNA's. 

It therefore appears that human-lambda DNA clones have been found which 
hybridize selectively to two independent probes for the H2A.Z gene. 

This project is now at the crucial stage of determining whether or not the DNA 
of the chosen human-lambda clones can be used successfully to hybrid select and 
in vitro translate the mRNA encoding H2.Z. 

Significance : 

Isolation of histone genes of the H2A family would permit studies into the 
molecular basis of the different regulation of histone variants relative to 
DNA replication. 

Proposed Course : 

1. To test the doubly positive clones with an H2A.F gene (probably related to 
H2A.Z) from chicken (donated by Prof. Julian Wells.) 

2. To construct a synthetic probe using recently developed technology of 
inserting inosine into wobble positions. A longer probe could be constructed 
which could be used to search a genomic library directly. 

365 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06171-01 LMPH 



PERIOD COVERED 

October 1, 1984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) 

Chromatin Synthesis and the Control of Cell Proliferation 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) 

PI: William Bonner Head, Chromosome Structure LMPH NCI 

and Function Section 



Others: Roy S. Wu 



Cancer Expert 



LMPH NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Molecular Pharmacology, DTP, DCT. NCI 



SECTION 

Chromosome Structure and Function 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda. Maryland 20205 



TOTAL MAN-YEARS: 



PROFESSIONAL: 



1.3 



0.9 



0.4 



CHECK APPROPRIATE BOX(ES) 

n (a) Human subjects 
D (a1) Minors 
D (a2) Interviews 



n (b) Human tissues Q (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

Our Objective is to understand the relationship of various cell states and the 
transitions between them as reflected in the control of histone and chromatin 
Diosynthesis. One phenomenon which we have studied is the differential 
sensitivity of the growth and chromosome cycles to the rate of protein 
synthesis. The chromosome cycle seems to be relatively invariant even though 
other results show that DNA synthesis is inhibited in parallel to the 
inhibition of protein synthesis. To resolve this apparent paradox, we studied 
histone and DNA synthesis under different conditions of protein synthesis and 
cell growth. 

We have found and characterized a phenomenon which we have named chromosome 
cycle compensation. As protein synthesis is inhibited, the mRNA level of S- 
phase histones rises to compensate for the inefficiency of protein synthesis. 
There is evidence in the literature that normal and tumorigenic cells may differ 
significantly in their level of chromosome cycle compensation. Understanding 
this mechanism may lead to some insights into the growth characteristics of 
normal and tumorigenic cells. 



366 



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GPO 9t 4-9le 



ZOl CM 06171-01 LMPH 

Project Description 

Introduction : 

The biochemical mechanisms which regulate cell growth remain largely unknown. 
For example it has been widely documented that as cell growth is slowed, the 
chromosome related activities and cell cycle phases are relatively unaffected. 
The biochemical basis for the phenomenon was unknown. Our studies this year 
have resulted in an explanation for this phenomenon in CHO cells. 

Objective : 

1) The development of a greater understanding of the relationship of the 
mechanisms regulating chromatin biosynthesis to the molecular mechanisms 
regulating cell proliferation. 

2) A characterization of the responses of these regulatory mechanisms to the 
development of new regimens or compounds that might control inappropriate 
proliferation of transformed cells. 

Methods : 

(1) Discontinuous electrophoretic separation of histones including direct 
loading of histone extracts and two dimensional eletrophoresis. (Methods 
developed in this laboratory). 

(2) Synchronization of cell lines, particularly human Hela cells and Chinese 
hamster ovary cells for studies on cell cycle. 

(3) Maintenance of cells and nuclei in viable non dividing states using 
modified and defined media. 

(4) Isolation and analysis of mRNA from different parts of the cell cycle or 
from quiescent cells. 

(5) FACS analysis of ceil cycle distributions including the BrdU antibody 
technique for measuring S phase cells. 

Major Findings and Accomplishments : 

Mechanism for the Relative Invariance of the Chromosome Cycle 

The information reported in the literature concerning the regulatory mechanisms 
between DNA and protein synthesis has generally been obtained using high 
concentrations of inhibitors which slow either DNA or protein synthesis to 1-5% 
of their control level and which prevent cell growth. 

We decided to investigate the reaction of cells to lower concentrations of 
inhibitors of DNA and protein synthesis, concentrations which still allowed 
cell growth. We have found that when cells are treated with concentrations 
of cycloheximide that inhibit protein synthesis by about 50%, the rates of 
DNA and histone synthesis are immediately inhibited by approximately 50%, but 
then recover over the next two hours to rates which are close to their control 
rates. The recovery is selective for S-phase histone synthesis compared to the 

367 



ZOl CM 06171-01 LMPH 
synthesis of nonhistone proteins and certain oasal variants (i.e. H2A.X and 

These findings suggest that proliferating CHO cells favor replication related 
synthesis when total protein synthesis is inhibited. We have found several 
observations in the literature which support our findings. It is known that 
cells grow exponentially in low concentrations of cycloheximide, but with 
increasing doubling times. For example in some cell types one can inhibit 
protein synthesis by almost 50% without lengthening S-phase. Our findings 
of a recovery of histone and ONA synthesis after inhibition of protein 
synthesis provides a biochemical basis for this observation. 

This differential sensitivity of G1 and S-phase to the rate of protein syn- 
thesis has usually been interpreted in terms of a specific sensitivity of Gl . 
Our results show that this explanation is not completely correct, and that 
explanation is that the synthesis of proteins related to the chromosome cycle 
is increased to counteract the overall inhibition of protein synthesis. 

Cycloheximide slows the elongation step of translation and as such may not be 
a natural method of regulating protein synthesis. We are studying other more 
physiological methods of limiting protein synthesis. One method which we have 
preliminary data for is the isoleucine starvation of CHO cells. When put into 
isoleucine-lacking media, CHO cells finish S-phase and accumulate in a GO 
state, according to their histone synthesis pattern. The histone mRNA level 
elevates soon after introduction of the CHO cells into the isoleucine- 
minus media, then decreases. 

Significance : 

There are suggestions in the literature that normal and tumorgenic cells 
respond very differently in terms of cell cycle distribution when their 
growth is slowed. Our findings provide a molecular basis for this effect 
and thus provides experimental framework for studying this phenomenon. 

Proposed Course : 

1. To characterize the effect of slow growth on the chromosome cycle in 
several cell lines, particularly matched pairs of normal and transformed 
lines, such as mouse 3T3 and BP-3T3 or human IMR-90 and VA-13. 

2. To test whether chromosome cycle compensation exists in these cells or 
whether there are significant differences. 

Publications : 

None 



368 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06172-01 LMPH 



PERIOD COVERED 

October 1 , 1 984 to September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line between the tmrders.) 

Base Sequence Selective DNA Alkylation Reactions 



PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) 



PI: Kurt W. Kohn 

Others: William B. Mattes 
John A. Hartley 
Ronald Rahn 
Judith Markovits 



Lab. Chief 



LMPH NCI 



Staff Fellow LMPH NCI 

Visiting Fellow LMPH NCI 
Guest Researcher 

Guest Researcher LMPH NCI 



COOPERATING UNITS (if any) 



LAB/BRANCH 

Laboratory of Molecular Pharmacology, DTP, DCT, NCI 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 



3.2 



PROFESSIONAL: 



3.0 



0.2 



CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
n (a2) Interviews 



n (b) Human tissues 



(c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

A new project was undertaken to investigate the DNA base-sequence selectivity 
of alkylation reactions. The main objective is to determine whether base- 
sequence selectivities contribute to the anti-tumor activities of alkylating 
agents and whether the effectiveness of these drugs could be enhanced by 
structural modifications that would optimize the selectivity for certain 
sequences. A second objective is to utilize alkylation reactions to probe DNA 
structure and conformation in solution, especially with reference to the major 
groove. A DNA fragment being utilized in these studies contains the 5 -flanl< 
of the human c-H-ras-1 oncogene which contains a very GC-rich region, a possible 
hot-spot for alkylation reactions. DNA sequencing methodology was used to 
localize alkylations at guanine-N7 positions. Marked differences in reaction 
intensities were observed at different guanines in a DNA sequence, as well as 
between different nitrogen mustards. Some major sequence-dependent patterns 
were observed and are being investigated quantitatively. 



369 



PHS 6040 (Rev. 1/84) 



GPO SI4-Sie 



ZOl CM 06172-01 LMPH 
Project Description ; 

Objectives 

1. Determine the base sequence selectivities for the reactions of alkylating 
agents with DNA. 

2. Investigate the structural and conformational dependences of the selec- 
tivities. 

3. Devise new alkylating agent structures with enhanced base sequence 
selectivities, especially those targeted for certain oncogenes. 

Methods 

1. High-resolution polyacrylamide gel electrophoresis of end-labeled DNA re- 
striction fragments previously reacted with alkylating agents and chemically 
cleaved at sites of guan1ne-N7 alkylation. 

2. Quantitative densitometry and computer analysis of electrophoretic gel 
autoradiograms. 

3. Determination of interstrand crossl Inking of restriction fragments by DNA 
denaturation and selective enzymatic degradation of single-strands. 

4. Analysis of binding of [195mpt] cisplatin to DNA restriction fragments. 
Major Findings : 

Reaction of Nitrogen Mustards at Guan1ne-N7 Positions 

We began with the hypothesis that, since alkylating agents react predominantly 
with guanine in DNA, a significant aspect of the biological action of the drugs 
could be due to selective reaction at GC-rich regions of the genome. In order 
to determine where highly GC-rich regions may occur in the human genome, a 
computer search of the DNA sequence data base at NIH was carried out. The 
search was for segments in which at least 17 of 20 bases were G or C. Among the 
genes retrieved, several oncogenes were represented, most notably H-ras which 
has an extraordinarily high GC region in its 5'-flank. We therefore are 
focusing our study on the plasmid pbc-Nl which is pBR 322 with a human c-H- 
ras-1 Insert. We have prepared a restriction fragment of pbc-Nl containing 
pBR sequences and the adjacent very GC-rich (85% GC) 5'-flank of c-H-ras-1. 
The initial studies however were conducted using pBR 322 not containing the 
insert. 

The major findings to date are the following: (1) there are marked differences 
in reactivity among guanines in a DNA sequence; (2) the differences are not 
markedly dependent on the solvent conditions of reactions; (3) the guanine 
selectivity pattern is different for different nitrogen mustards; (4) the 
potential inter-strand crosslinking sites — e.g. 5'-GC-3' -- show relatively 
low reactivity for HN2, melphalan and spirohydantoin mustard; (5) these sites 
however show a markedly enhanced reaction with uracil mustard; (6) runs of G's 
(potential sites of intra-strand crosslinking) exhibited disproportionately high 

370 



ZOl CM 06172-01 LMPH 

reactivities; (7) quinacrine mustard exhibited a uniquely different pattern of 
reactions. 

Reaction of Chloroeth.ylatinq Agents at Guanine-N7 Positions 

Similar experiments were conducted using 2-chloroethyl (methyl sulfonyl )methane- 
sulfonate (ClEtSoSo), cis-2-OH-CCNU and mitozolomide. As described in another 
report, these compounds chloroethylate (and in some cases hydroxyethylate) DNA. 
We wanted to compare these compounds for selectivity for reaction. In these 
studies only reaction at guanine-N7 was observed, although it will be of 
interest to devise methods for observing reaction at guanine-06. 

The major findings were that cis-2-OH-CCNU exhibited marked differences in 
reaction intensities with different guanines. Especially striking were the 
disproportionately strong reactions at runs of 4 or more G's. ClEtSoSo differed 
from all other alkylating agents so far studied, in that the reaction inten- 
sities for all G's appeared to be the same. 

In view of this uniform reactivity and also of the bulky structure of the 
molecule, ClEtSoSo may be useful as a foot-printing reagent, especially for the 
major groove. 

The results indicate that ClEtSoSo differs significantly from the other 2 
chloroethylating compounds in the base sequence-selectivity of its reaction 
with DNA. 

Selective Binding of Cisplatin in DNA 

The relative binding of [195mpt]_cisplatin to restriction fragments of pbc-Nl 
was studied. Marked differences were observed in binding to the different 
restriction fragments, even after normalization for guanine content. The 
very GC-rich regions of c-H-ras-1 exhibited especially intense binding of 
cisplatin. This again suggests that highly GC-rich regions may be significant 
targets for DNA-reactive anti -cancer drugs. 

Significance : 

1. These studies may lead to the development of new alkylating agents with high 
base-sequence selectivities, especially for certain oncogene sequences. 

2. The studies may also yield new information about the structure and confor- 
mations of DNA in solution. 

Proposed Course : 

1. Carry out quantitative densitometry and computer analyses of the sequence- 
dependence of the reactions of a wide variety of nitrogen mustards. 

2. Carry out molecular graphics analyses in order to obtain structural hypo- 
theses to account for the observed selectivities. Test these hypotheses by 
synthesis of new nitrogen mustard structures that would be predicted to show 
strong selectivities. 



371 



ZOl CM 1)6172-01 LMPH 

3. Study congeners of CIEtSoSo currently being synthesized at Southern 
Research Institute in order to determine whether sequence selectivity 
can be restored to this class of compounds. 

4. Devise a method to measure reactions at guanine-06 positons. 

5. Extend these studies to DNA in chromatin and intact cells. 
Publications : 

None 



372 



SUMMARY REPORT 

ASSOCIATE DIRECTOR FOR THE CANCER THERAPY EVALUATION PROGRAM 

DIVISION OF CANCER TREATMENT 

NATIONAL CANCER INSTITUTE 

October 1, 1984 - September 30, 1985 

I. General Organization 

The Cancer Therapy Evaluation Program (CTEP) is responsible for the administra- 
tion and coordination of the majority of the extramural clinical trials supported 
by DCT. These programs include the activities of the Clinical Cooperative 
Groups, the Phase I and Phase II new drug development contractors, and the hold- 
ers of investigator-initiated grants (ROl and POl) relating to cancer treatment. 
Certain programs in developmental radiotherapy, such as high LET radiation, are 
administered in the Radiation Research Program. The Phase I development of bio- 
logic response modifiers is handled by the Biological Response Modifiers Program. 

The Investigational Drug Branch (IDB) is responsible for sponsoring trials of new 
investigational drugs and of evaluating them for efficacy and toxicity. It does 
this by: 1) Obtaining Investigational New Drug exemption (IND) authorization 
from the Food and Drug Administration (FDA); 2) Coordinating and monitoring the 
Phase I trials of new agents developed by the DCT; 3) Planning with members of 
the Clinical Investigations Branch (see below) overall strategies for activity 
(Phase II) studies in specific tumor types; 4) Monitoring the results of the 
clinical trials; 5) Ensuring that clinical investigators using investigational 
new drugs are in compliance with federal regulations regarding the use of such 
agents; 6) Regulating the distribution of investigational new drugs for which 
NCI is the sponsor; 7) Maintaining close contact and ongoing dialogue with the 
pharmaceutical industry in an attempt to ensure that new drug development 
proceeds in a coordinated way. 

The Clinical Investigations Branch (CIB) is responsible for clinical studies 
conducted under the Cooperative Agreement mechanism and those Phase II/III 
trials done under contract. It manages the clinical oncology and nutrition 
portfolios of ROl and POl grants. 

The Biometric Research Branch (BRB) provides statistical consultation to the 
other branches of CTEP, to the extramural and some intramural activities of 
other programs in DCT, and to the statistical centers of the clinical cooper- 
ative groups. It also carries on research in statistical methodology relating 
to cancer clinical trials. 

The Office of the Associate Director (OAD) integrates the efforts of the 
Branches. This goal is accomplished by weekly staff meetings, in which issues 
of concern to the program are discussed with the full staff, and by weekly 
branch chief meetings, where issues are further defined and decisions are made. 
The process of protocol review is administered within the OAD, by a central Pro- 
tocol and Information Office. The Protocol and Information Office is also the 



373 



receipt point at NCI for all protocols entered into the PDQ system. The OAD is 
responsible for overall program supervision and budgetary allocation. 

II. Organizational and Personnel Changes 

During the past year Dr. Edwin T. Jacobs retired from Government service; 
Drs. Silvia Marsoni , Mercedes Lassus, Elihu Estey, and Elizabeth Read left the 
program for positions elsewhere, to be replaced by Drs. Bruce Cheson, Hoo Chun, 
Gisele Sarosy, and F. Andrew Dorr. Drs. Michael Stewart and J. Michael Hamilton 
also joined the staff of CIB. Dr. Michael Hawkins was recruited from the 
University of Wisconsin as Head, Biologies Section in IDB. Dr. Maryann Roper 
also joined the staff of the Biologies Section. Dr. Lawrence V. Rubinstein 
joined the staff of the BRB, where Dr. Frances J. Mather of Tulane University 
spent a sabbatical year and Dr. Sate Songorabadi served as a Visiting Fellow. 
Dr. Brigid Leventhal, on sabbatical from the Johns Hopkins University, served 
as Special Assistant to the Associate Director. 

III. Highlights in Program Development 

A) Immunodeficiency Syndrome (AIDS) 

Over the past year, staff members of the CTEP have been heavily involved in ac- 
tivities related to AIDS. Dr. Killen has chaired the NCI committee coordinating 
extramural support for AIDS research. This group works closely with other com- 
ponents of the NIH actively involved in supporting AIDS research. The committee 
is composed of program staff from the NCI's Division of Cancer Biology and Diag- 
nosis, Division of Cancer Cause and Prevention, and Division of Cancer Treatment. 
The projects funded span a spectrum of disciplines and involve widely dispersed 
geographic locations including major patient centers on both coasts. Major 
areas under study include the following: 

Virology - Investigators at several institutions are involved in attempts 
to characterize further the nature of the viral infection with the HTLV- 
III family. Efforts along these lines have been aided immeasurably by the 
extensive network of collaboration set up between extramural investigators 
and the Laboratory of Tumor Cell Biology, DTP, DCT, NCI (see the Report of 
the Associate Director for the Developmental Therapeutics Program). 

Immunology - Research in this area involves further characterization of 
the nature of the immunologic defect in AIDS patients as well as patients 
with possible prodromes or in high-risk groups. Activity includes evalu- 
ation of all components of the immune response both in viv o and in vitro 
and at all levels, down to individual cell function. Also, under study are 
various antibodies, including one directed at sperm and T cells, and immune 
complexes or other evidence implicating a possible autoimmune etiology. 
Several investigators are also involved in immunogenetic profile studies, 
with preliminary evidence from several centers pointing toward a predom- 
inance of certain HLA haplotypes in affected patients. 

Epidemiology - A number of affected AIDS or high-risk groups are under ac- 
tive prospective observation, including cohorts of male homosexuals, 
heterosexual drug addicts, native Haitians, children, and individuals with 



374 



several possible prodromes, including the syndrome of diffuse lymphadeno- 
pathy. Plans for other studies are under development, and include multi- 
institutional investigations of health care workers, prisoners, etc. 

Therapy - During the past year the leading hypothesis in therapy has been 
generated by Broder's observation that Suramin, an inhibitor of retroviral 
reverse transcriptase, also inhibits HTLV-III replication in vitro. Broder 
has also shown that Suramin treatment of AIDS patients results in a fall of 
lymphocyte-associated reverse transcriptase levels. In response to this ex- 
citing development, CTEP has coordinated a multicenter working group for the 
further testing of Suramin in patients with AIDS and ARC. Participating 
investigators are affiliated with most of the premier institutions involved 
in the AIDS epidemic. The clinical trials will be accompanied by clinical 
pharmacologic studies of Suramin and correlative viral studies. 

B) Development of New Agents 

Highlights of the year include: 

1. Introduction of 15 New Agents into Phase I Trial : Of particular interest will 
be results with flavone acetic acid, an agent with broad solid tumor activity 
in preclinical testing, and Suramin, the first agent introduced into clinical 
trial as an antiviral agent for the treatment of AIDS. Among the biologies 
three monoclonal antibodies have begun clinical testing. 

2. Drugs Entering Phase II : The following drugs entered Phase II trials during 
the year: pibenzimol, trimetrexate, taxol, caracemide, acodazole, and the 
radiosensitizer SR-2508. 

3. Creation of a Biologies Evaluation Section : During this past year IDB began 
a systematic approach to the development of a Phase II and III program with 
biologic agents. The BES undertook a comprehensive review of the current 
status of the interferons, and as a result has targeted chronic myelogenous 
leukemia, hairy cell leukemia, and non-Hodgkin's lymphoma as specific areas 
for large-scale comparative trials. In addition the staff is organizing 

a definitive trial of the role of BCG in the treatment of superficial bladder 
cancer. 

4. Important Clinical Trials Initiated ; During the past year several clinical 
trials are worthy of special note. The Phase I program with Suramin is the 
first full-scale test of an agent specifically directed against the HTLV-III 
virus. 

HMBA is a polar planar compound that selectively differentiates Friend eryth- 
roleukemia and HL60 cells in vi tro at non-cytotoxic doses. This drug is now 
well into Phase I trials; closes of 19.2 g/m'^ given as a continuous infusion 
over 5 days yield plasma levels over 1 mmolar, the concentration necessary 
to induce differentiation in the in vitro models. Since this effect is 
dose-dependent in vitro. Phase I trials are continuing in order to escalate 
the dose further. 

Extensive plans have been drawn up for two active platinum analogs, CBDCA and 
CHIP in ovary cancer, small cell carcinoma of the lung, and head and neck 



375 



cancer, in order to define the roles of these drugs in initial therapy. 
Randomized scheduling trials with trimetrexate in head and neck cancer and 
possibly breast cancer are being arranged at the present time. Ifosphamide 
has been shown to have significant activity in adult soft tissue sarcoma, 
and three randomized Phase III trials (intergroup sarcoma, NCI-C, and ECOG) 
are currently being developed to define its role in combination with doxoru- 
bicin with or without DTIC in initial therapy. The role of homoharringtonine 
in combination therapy (especially with VP-16) is still be determined in 
acute myelocytic leukemia. Similarly the role of deoxycoformycin +_ inter- 
feron is being determined in hairy cell leukemia. A randomized Phase III 
trial of non-cross resistant regimens including the M.D. Anderson MIME regi- 
men is now being negotiated with M.D. Anderson and Memorial Hospitals. 

Phase II clinical trials are continuing on 35 cytotoxic drugs. 

5. Identification of drug activity : Plans for the platinum analogs CBDCA and 
CHIP have been noted above. Homoharringtonine is active in acute leukemia; 
hypotension, previously the dose-limiting toxicity, is manageable if the 
drug is given as as a slow infusion. The activities of AZQ in brain tumors, 
AMSA in acute leukemia, and mitoxantrone in breast cancer are being further 
defined in randomized controlled trials against BCNU as a single agent, 
daunorubicin in combination, and doxorubicin, respectively. Phase II data 
have suggested that PCNU may be superior to BCNU in the treatment of brain 
tumors; Phase III comparative trials are just beginning. 

6. Closing of IND's : IND's for maytansine and trifluorothymidine were closed 
because of a lack of clinical activity. 

7. Areas of Special Emphasis : A Request for Application (RFA) to study in 
depth certain areas in biochemical modulation will shortly be issued. An 
RFA for the study of differentiation agents is in preparation. 

8. Liaison with European Clinical Drug Development : During the past year major 
efforts were continued to develop collaborative liaisons with the components 
of the EORTC and the Cancer Research Campaign in Great Britain. The goal is 
to create a two-way flow of drugs across the Atlantic with minimum time 
delay. This effort also seeks to reduce unnecessary duplication of trials 
which had occurred in the past because investigators often wished to (or 
needed to) reproduce the results of a reported study in their own country. 

This renewed effort is a response to the increased efforts devoted to cancer 
drug development in Europe. NCI is interested in the possibility of bringing 
promising drugs into the U.S. after early clinical trials in Europe. In 
addition to promoting increasing attendance of American and European investi- 
gators at meetings of mutual interest, the DCT is supporting an increased 
level of monitoring for certain high priority Phase I trials in Europe. 

9. Reduction of drug cost : Emphasis on improved management practices and 
tighter protocol review during the past year continue to yield savings in the 
cost of clinical drugs. We estimate a decrease of about $1.4 M in FY '84 
compared with FY '83. 



376 



10. Development of Case Report Forms and Study Summaries : To enhance the 
value of CTEP's data base on the clinical activity of experimental agents, 
standardized case report forms have been developed for use by the drug 
development contractors. In addition, standardized study summaries will 
be required for all trials using experimental drugs performed in cancer 
centers. These summaries will greatly increase the reliability and com- 
pleteness with which trials results are reported to CTEP and will improve 
the management of the clinical drug development program. 

11. Site Visit Monitoring : All 19 cooperative groups supported by the Cancer 
Therapy Evaluation Program now have active monitoring programs administered 
by the groups themselves under CTEP oversight. Since 1982 a total of 475 
member institutions and 465 affiliate institutions have been audited; during 
the current year 114 members, 66 affiliates, and 45 CCOPs were site-visited. 

The Quality Assurance and Compliance Section directly oversees the moni- 
toring of Phase I and cancer centers studies. Phase I studies are monitored 
three times per year. Since 1982, 43 cancer centers site visits (involving 
33 institutions) have been accomplished. Sixteen were carried out in the 
current year. 

C) Group Studies 

Noteworthy contributions are being made by the clinical cooperative groups in 
several areas, and interesting new initiatives by the groups are in progress. 
Early results from the NSABP's clinical trial comparing modified radical mastec- 
tomy to breast-preserving surgery demonstrates that the lesser surgery plus 
radiotherapy is as effective as conventional primary treatment in preventing 
recurrence. Additional followup is necessary. The GITSG has completed a con- 
trolled trial indicating benefit for the use of adjuvant radiotherapy plus chemo- 
therapy compared to surgery alone for patients with resectable colorectal cancer. 
P06 has recently conducted a randomized trial in osteosarcoma which conclusively 
demonstrated the benefit of postoperative adjuvant chemotherapy over a non- 
treatment control. This trial addressed a controversial issue whose clarifica- 
tion was essential for optimal patient care, as well as for future studies in 
osteosarcoma. CALGB has active, group-wide studies of the lymphoproliferative 
disorders including sophisticated immunological and cytogenetic evaluations and 
therapy including promising new agents such as interferon and deoxycofori^ycin. 
SWOG is planning a large Phase III comparison of two of the newer, more aggres- 
sive chemotherapy regimens for non-Hodgkin's Lymphoma with CHOP, long considered 
"standard" therapy by many practicing oncologists. In a study of women with 
advanced breast cancer, MAOP has initiated the first groupwide trial involving 
response-surface methodology; successful use of these statistical and mathe- 
matical techniques may permit the development of a much more rational approach 
to the construction of optimal drug combinations. CCSG has demonstrated that 
routine histopathologic classification of non-Hodgkin's lymphoma is the best 
criterion for choice of therapy for patients with generalized involvement. The 
National Wilms' Study #3 suggests that tumors designated "favorable histology" 
can be successfully treated with less intensive regimens. Long-term survival 
rates of 90% have been demonstrated for patients with non-metastatic favorable 
histology Wilms' Tumor. CCSG has demonstrated that elevation of serum ferritin 
levels in neuroblastoma is associated with a significantly poorer prognosis than 
was normal ferritin and that this correlation is independent of stage and age 



377 



at diagnosis. Breast adjuvant chemotherapy studies ongoing In three cooperative 
groups (NSABP, SEG, CALGB) are exploring regimens that are substantially more 
aggressive In dose intensity than therapies tried over the past decade. 

The increasing participation of two or more cooperative groups in a single trial 
is a laudable trend that we have been actively promoting. Examples of such 
intergroup studies organized during the past year include: 

1. Intergroup Prostate Study: This trial will attempt to confirm impressive 
preliminary results suggesting that the combination of an LHRH agonist (leu- 
prolide) with an antiandrogen (flutamide) yields much better results than 
the LHRH agonist alone. 

2. Intergroup Colon Adjuvant Trial: This study is designed to replicate an 
earlier, smaller trial conducted by the NCCTG. Preliminary analysis suggests 
that patients receiving 5FU plus levamisole following curative surgery for 
colon cancer may have fewer recurrences than those treated with surgery 
alone. 

3. Head and Neck Intergroup Study: Patients with localized, advanced disease 
will be treated in a multimodality study, testing the hypothesis that three 
cycles of cisplatin plus 5FU adds significantly to the efficacy of standard 
local therapy. 

Several ongoing Intergroup efforts continue, Including: 

1. The Intergroup Rhabdomyosarcoma Study Group has developed a prognostic 
classification system based on cytohistologic subtypes that identifies those 
patients who require more Intensive therapy. 

2. Intergroup Soft Tissue Sarcoma Study: Patients with primary soft tissue 
sarcoma (STS) after definitive surgery and/or radiation therapy are rando- 
mized to either high-dose intermittent doxorubicin adjuvant therapy or a 
control arm of no further therapy. Most major national and regional groups 
participate. 

3. Intergroup Mesothelioma Study: Patients with limited unilateral thoracic 
mesothelioma receive combined surgery and radiation therapy and are random- 
ized to receive adjuvant doxorubicin or a control arm. Several major 
national groups participate. 

4. Intergroup Melanoma Study: Patients with intermediate thickness primary 
melanoma are randomized to have an excision with a 2 cm versus 4 cm margin 
and to have elective lymph node dissection or not. All cooperative groups 
participate as well as the NCI of Canada and the Melanoma Clinic, Australia. 

E) Cancer and Nutrition Program 

Current studies are exploring various aspects of the relationship between 
nutrition and malignancy. 

1. Heber and associates are continuing to examine the efficacy of enteral 
nutritional supplements in the prevention and treatment of protein-calorie 



378 



malnutrition in the patient with lung cancer. Hormonal and metabolic aberra- 
tions will be examined in these patients to determine patterns of nutritional 
disease which may be amenable to specific nutritional intervention. 

2. Bernstein et al are examining the development of food aversions and taste 
alterations in children receiving cancer chemotherapies. The temporal rela- 
tionship between the food items and the therapy, as well as the novelty of 
the food item, appear to be important in the development of food aversions. 

3. Ota et al are studying the effect of intravenous nutritional support on the 
cellular kinetics of rectal carcinoma. Patients are being randomized either 
to receive immediate surgery or to receive one week of preoperative hyper- 
alimentation. Biopsy specimens collected prior to and during surgery will 
be analyzed for cellular growth parameters. This study will help to define 
the role of nutritional support in the cancer patient. 

F) NCI -Pan American Health Organization; Collaborative Cancer Treatment 
Research Program (PAHOrCCTRPl" 

The collaboration between U.S. and Latin American investigators in the develop- 
ment of clinical studies continues to be the major goal of this program. An 
extensive re-evaluation of goals has resulted in: 1) concentration primarily in 
Phase II trials in diseases prevalent in the area such as penile, vulvar, esopha- 
geal, head and neck, gastric, lung, and cervical cancers; 2) design of master 
Phase II protocols in the diseases listed above, of which two have been approved 
and activated, and the remainder have been completed and will be submitted for 
activation; 3) the establishment of a scientific advisory committee to coordinate 
scientific directions, to review critically protocols and publications, and to 
evaluate membership status; 4) assess feasibility and scientific merit of pro- 
posed Phase III studies prior to submission to NCI. These will preferably be 
performed by more than one center to ensure adequate accrual. More effort will 
be made to assure proper patient entry, interim evaluation, reporting procedures, 
and final analyses. These trials will be performed in diseases prevalent in this 
area of the world, which should enable the group to make unique contributions. 

G) Clinical Trials Review 

CTEP has enbarked upon an ambitious attempt to review the effectiveness of the 
clinical trials mechanism sponsored by the NCI. Initially the review will focus 
upon the performance of the cooperative groups during the time interval 1979- 
1984. The reviews will be disease-oriented and will be conducted by small com- 
mittees of experts selected by CTEP. For each disease category the reviews will 
attempt to answer the following questions: 1) What were the most pressing clini- 
cal research issues in cancer treatment during the specified time period? 2) 
Were these issues dealt with adequately by the cooperative groups? 3) Was there 
an undesirable degree of duplication of activity in the cooperative group system? 
4) What was the quality of the clinical trials methodology employed by the 
groups, with specific reference to the design, execution, analysis, and reporting 
of trials over the five-year period of interest? 

Thus far the committees for testis cancer and non-small lung cancer have met, 
concluded deliberations, and are in the process of preparing reports. The review 
will continue with many other disease sites and with certain areas of categorical 



379 



concern, such as the drug development program. We anticipate that the results of 
the review will be extremely important in modifying our approach to the organi- 
zation and coordination of clinical trials in the coming years. 

H) Organization and Coordination of Clinical Trials 

With increasing opportunities for clinical implementation of exciting new devel- 
opments in the laboratory, the cooperative groups face a future full of bright 
possibilities. Since the likelihood of a relatively flat budget is fairly strong 
for the near future, the size of the clinical trials mechanism cannot be expected 
to increase significantly. For these reasons selection and prioritization of 
questions suitable for clinical trials are perhaps more important than ever 
before. In addition, experience over the past few years strongly suggests that 
the size of certain kinds of trials, particularly comparative trials where survi- 
val is a major endpoint, should probably be much larger than has been customary 
in the past. All these observations point to the need for an increasing level 
of coordination among clinical trials groups, probably at the national level. 

CTEP has begun discussion with the cooperative group chairmen about ways to 
achieve these ends. During the next few months we plan to develop a system for 
identification, prioritization, and implementation of key questions in cancer 
therapy at the national level. Doing so on a broad scale may involve a substan- 
tial alteration in the way many cooperative groups do their business. The goal 
of achieving a maximally effective clinical trials program clearly justifies 
explorations along these lines. 

I. Modification of the Guidelines for Peer Review of the Clinical Trials Groups 

In connection with our anticipated increased emphasis on large trials involving 
the cooperation of several groups, it will be necessary to define carefully the 
terms according to which the cooperative groups are to be reviewed. Tradition- 
ally the peer review process rewards accrual and the generation of studies by a 
group. If the number of trials is to decrease (with the advent of larger 
trials), groups will have to be evaluated, at least in part, on the effective- 
ness of their participation in high-priority studies, whether or not the stucly 
was generated within the group. We also wish to replace the overemphasis on raw 
accrual figures with a more balanced assessment that will include strong atten- 
tion to the quality of the ideas submitted to clinical trial by a group. Accor- 
dingly we have begun to rewrite the guidelines for cooperative group review with 
these and the other similar issues in mind. 

J. Cost of Clinical Trials 

As part of our attempts to rationalize the process of budgeting for clinical 
trials, we have determined that a systematic examination at the resource needs of 
the various types of clinical trials is necessary. A working group within CTEP, 
chaired by Ms. Kim Morgan (Program Analyst, OAD), is attempting to develop 
methodology to define these resource requirements. We expect that methodology 
will be set by the end of FY *85. The project will then enter a phase of data 
gathering. Having a more precise knowledge of the costs of clinical trials will 
make budgeting for them more rational and therefore more consistent across the 
clinical trials groups. 



380 



i^'sSlfc. 



K. Representation in International Activities 

CTEP is responsible for providing clinical input into various international 
agreements adminstered by DCT. The PAHO Treatment Research Programs have al- 
ready been mentioned, as have the increasingly close links with the European 
Organization for Research on the Treatment of Cancer (EORTC) which has had for 
several years a strong link with NCI. A member of the EORTC attends the Phase I 
Working Group meetings in Bethesda; CTEP staff is represented on the Protocol 
Review Committee of the EORTC. CTEP staff also provides clinical input into the 
bilateral agreements between the U.S. and France, Italy, Germany, and Japan. 
Major emphasis in these agreements thus far has been on new drug testing. 

L. Analyses in Progress 

In its role as coordinator and sponsor of most NCI-supported clinical trials of 
cancer therapies, CTEP has the important responsibility of assessing the suita- 
bility of current approaches and methodologies. Among the many questions 
currently under investigation by CTEP staff are: 

1. How accurately do the findings in preclinical toxicology predict (a) a safe 
starting dose in Phase I trials, and (b) the pattern of organ toxicities 
seen in the clinic? 

2. To what extent can therapeutic responses seen in Phase I trials predict 
subsequent activity in Phase II? 

3. How accurately has the NCI's preclinical tumor panel predicted activity of 
new agents in human cancer? 

4. Have clinical adjuvant chemotherapy trials performed over the past ten years 
provided adequate tests of the hypotheses generated in laboratory models? To 
what extent can deviations from the laboratory models explain the borderline 
or negative results of many clinical trials? 

5. What are maximally efficient study designs for assuring that promising new 
therapies are not overlooked, and therapies which do not offer significant 
advantages over standard treatment are discarded after entry of the 
minimum number of patients? 

6. How may the concepts of biochemical modulation be most effectively applied 
and tested clinically? 

7. How may the concepts and techniques of statistical inference be extended or 
adapted to deal with such problems as the early termination of clinical 
trials, crossover interactions between treatment efforts and patient charac- 
teristics, and estimating the proportion of total variability in outcome 
explained by prognostic factors with censored survival data? 

M. Sponsorship of Meetings and Workshops 

In addition to the usual meetings of the new drug development contractors and the 
Biochemical Modulation Advisory Group and the numerous working sessions with 
extramural investigators necessary to carry out the programs mentioned above, 
several other meetings were of particular interest: 



[ \ 



1. National Symposium on Methodology and Quality Assurance in Cancer Clinical 
Trials 

This major national two-day symposium was organized and sponsored by the 
Biometric Research Branch to stimulate improvement in the methodology of 
cancer clinical trials. The symposium was attended by almost 300 investi- 
gators from cancer centers, cooperative groups and the pharmaceutical indus- 
try. The full proceedings will be published in Cancer Treatment Reports . 
Planning for a 1986 symposium on the same topic is underway. 

2. In connection with CTEP's effort to promote clinical research activity in 
surgical oncology, a workshop on adjuvant therapy after resection of 
isolated hepatic metastasis from colorectal cancer has been held and a 
protocol is being developed. A second workshop dealing with surgical 
quality control in the cooperative groups is to be held in July 1985 and 
two other workshops looking at adjuvant therapy to surgery in the treatment 
of hepatomas and rectal cancers are in the planning stage. 

3. As part of IDB's efforts to develop uniform toxicity criteria, cooperative 
group representatives met to discuss a draft set of criteria. We plan to 
push hard to establish common criteria, which would do much to enhance the 
interpretability and comparability of trials conducted by different groups. 

IV. Publications 

1. Chabner, B.A., Wittes, R.E., Hoth, D.F., and Hubbard, S.: Investigational 
Trials of Anticancer Drugs: Establishing Safeguards for Experimentation. 
Public Health Rep. 99:355-360, 1984. 

2. Chapman, R.A., Natale, R.B., Young, C.W., and Wittes, R.E.: Phase II trial 
of l-{2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-l-nitrosourea in small cell 
cancer of the lung. Am. J. Clin. Oncol. 7:413-414, 1984. 

3. Eisenberger, M.A., Simon, R., O'Dwyer, P.J., Wittes, R.E., and Friedman, 
M.A.: A reevaluation of nonhormonal cytotoxic chemotherapy in the treatment 
of prostatic carcinoma. J. Clin. Oncol. 3:827-841, 1985. 

4. Friedman, L., Simon, R., Verter, J., Wittes, J., and Wittes, R. (Eds.): 
Proceedings of the Workshop on Evaluation of Therapy, September 1983. 
Statistics in Medicine 3:305-486, 1984. 

5. Magee, M.J., Howard, J., Bosl, G.J., and Wittes, R.E.: Phase II trial of 
4 '-epi doxorubicin in advanced carcinoma of head and neck origin. Cancer 
Treatment Reports 69:125-126, 1985. 

6. Million, R.R., Cassisi, N.J., and Wittes, R.E.: Cancer of the Head and 
Neck. In DeVita, V., Hellman, S., and Rosenberg, S. (Eds.): Principles 
and Practice of Oncology , 2nd Edition. Philadelphia, J.B. Lippincott 
Company, 1985, pp. 407-506. 



382 



7. Natale, R.B., Shank, B., Hllaris, B.S., and Wittes, R.E.: Combination 
cyclophosphamide, adriamycin, and vincristine (CAV) rapidly alternating 
with combination cisplatinum and VP-16 (PVP) in the treatment of small 
cell lung cancer. Am. J. Med, (in press). 

8. Natale, R.B., and Wittes, R.E.: Alternating combination chemotherapy 
regimens in small cell lung cancer. Sem. Oncol . (in press). 

9. O'Dwyer, P. J., Leyland-Jones, B., Alonso, M.T., Marsoni , S., and 

Wittes, R.E.: Etoposide (VP-16-213): Current status of an active anticancer 
drug. New Eng. J. Med 312:692-699, 1985. 

10. Simon, R. and Wittes, R.E.: Methodologic guidelines reports of clinical 
trials (Editorial). Cancer Treat. Rep. 69:1-3, 1985. 

11. Simon, R.S., Wittes, R.E., and Ellenberg, S.S.: Randomized Phase II Clinical 
Trials. Cancer Treat. Rep, (in press). 

12. Stanton, G.F., Raymond, V., Wittes, R.E., Schulman, P., Budman, D., 
Baratz, R., Williams, L., Petroni , G., Geller, N., Hancock, C, Kreis, W. 
and Young, C.W.: Phase I and clinical pharmacologic evaluation of 4'-deoxy- 
doxorubicin in patients with advanced cancer. Cancer Research 45:1862-1868, 
1985. 

13. Sugarhaker, P.H., Gunderson, L.L., and Wittes, R.E.: Cancer of the Anal 
Region. In DeVita, V., Hellman, S., and Rosenberg, S. (Eds.): Principles 
and Practice of Oncology , 2nd Edition. Philadelphia, J.B. Lippincott 
1985, pp. 885-894. 

14. Sugarbaker, P.H., Gunderson, L.L., and Wittes, R.E.: Colorectal Cancer. 
In DeVita, V., Hellman, S., and Rosenberg, S. (Eds.): Principles and 
Practice of Oncology , 2nd Edition. Philadelphia, J.B. Lippincott Company, 
198b, pp. /9b-884. 

15. Thongprasert, S., Bosl, G.J., Geller, N.L., and Wittes, R.E.: Phase II 
trial of Methyl -GAG in patients with advanced head and neck cancer. 
Cancer Treat. Rep. 68:1301-1302, 1984. 

16. Wittes, R.E.: Bleomycin: Future prospects. In Sikic,B.I., Carter, S.K., 
Rozencweig, M.: Bleomycin Chemotherapy: Current Status and New Develop- 
ments . San Diego, Academic Press, Inc. (in press). 

17. Wittes, R.E.: Commentary: Detection and prevention of anthracycline- 
induced heart damage. Cancer Treat. Symposia 3:55-57, 1984. 

18. Wittes, R.E.: Moderator's Introduction: Single agent versus combination 
chemotherapy. Proceedings of the International Conference on head and 
neck cancer, July 22-27, 1984, Burlington, Ontario, Brian Decker, Inc. 
(in press). 



383 



19. Wittes, R.E.: Obstacles to improved end results in head and neck cancer. 
In Harrap, K.R., Davis, W., and Calvert A.H. (Eds.): Cancer Chemotherapy 
and Select ive Drug Development, Boston, Martinus Nijhoff Publishing, 1984, 
pp. 111-113. 

20. Wittes, R.E.: The role of induction chemotherapy in combined modality 
treatment programs. In Sikic, B.I., Carter, S.K., Rozencweig, M.: 
Bleomycin Chemotherapy: Current Status and New Developments . San Diego, 
Academic Press, Inc. {in press). 

21. Wittes, R.E.: Vitamin C and Cancer (Editorial). N. Eng. J. Med. 
312:178-179, 1985. 

22. Wittes, R.E. and Killen, J.Y. Jr.: Problems in the therapy of AIDS- 
associated malignancies. In Selikoff, I. J., Teirstein, A.S., and 
Hirschman, S.Z. (Eds.): Acquired Immune Deficiency Syndrome , New York, 
New York, The New York Academy of Sciences, 1984, pp. 454-460. 

23. Young, C.W., Stanton, G.F., Budman, D.R., and Wittes, R.E.: Initial 
clinical evaluation of a new anthracycline analogue: 4 '-Deoxy doxorubicin. 

In Spitzy, K.H. and Karrer, K. (Eds.): Proceedings of the 13th International 
Congress of Chemotherapy: Cytostat. Symposia II, Vienna, Austria, Verlag H. 
Egermann, 1983, pp. 215/13-215/16. 

24. Young, C.W., Wittes, R.E., Jain, K., and Casper, E.S.: Clinical evaluation 
of 4 'epi- doxorubicin. In Spitzy, K.H. and Karrer, K. (Eds.): Proceedings 
of the 13th International Congress of Chemotherapy: Cytostat. Symposia II, 
Vienna, Austria, Verlag H. Egermann, 1983, pp. 215/13-215/16. 



384 



BIOMETRIC RESEARCH BRANCH 

1. Statistical Planning and Scientific Monitoring of NCI Sponsored 
Extramural Clinical Trials 

A statistical review is performed of all NCI sponsored extramural clinical 
trials. The BRB attempts to ensure that such trials are planned, conducted 
and reported in a sound statistical manner. The BRB is particularly active 
in the planning and monitoring of inter-group trials. Trials which have re- 
ceived particular attention during the past year include: National Prostatic 
Cancer Project trials for the adjuvant treatment of prostatic cancer patients; 
planning of national prospective trial for limb perfusion of stage I-II mela- 
noma patients; planning of multi-institution retrospective analysis of limb 
perfusion for stage III melanoma patients; planning of national prospective 
trial for chemotherapy of patients who have had resection of isolated hepatic 
metastases; Pediatric Oncology Group trial of adjuvant chemotherapy for pa- 
tients with osteosarcoma; Intergroup trial of adjuvant 5-FU plus levamisole 
for colorectal cancer; Jamaica studies of the chemotherapy of HTLV associated 
T-cell lymphomas; Intergroup study of pediatric Hodgkin's disease; Intergroup 
studies of the treatment and histologic classification of rhabdomyosarcoma; 
Cancer and Leukemia Group B studies of relapse treatment for adult leukemia; 
M.D. Anderson studies of first line treatment for adult leukemia; Piedmont 
Oncology Association trial of tamoxifen versus megace for the treatment of 
metastatic breast cancer; planning multi-center trials of high dose chem- 
otherapy with autologous bone marrow rescue in a variety of solid tumors. 

The BRB serves as liaison to extramural statistical centers and makes recom- 
mendations concerning the funding of statistical activities. The branch 
conducted a programmatic site visit of the Southwest Oncology Group Statistical 
Center in Seattle in order to provide a basis for a critical funding decision. 
The BRB participates in the organization and funding of meetings and workshops 
of statisticians involved in major extramural cancer clinical trials in order 
to improve statistical and data management procedures for the conduct of these 
studies. A meeting of cooperative group statisticians was held in New Orleans 
in May 1985 and a meeting of statisticians, data managers and executive offi- 
cers is currently being planned for late 1985 to develop revised guidelines 
for the conduct of Intergroup studies. 

2. Collaborative Clinical Research 

The Biometric Research Branch staff provide statistical collaboration on the 
following clinical studies. Principal clinical collaborators are listed in 
parentheses. 

a. National clinical trials of the staging and treatment of early ovarian 
cancer (Dr. Robert Young). 

b. Multi-institution randomized clinical trials of nutritional support in the 
treatment of patients with solid tumors (Dr. Daniel Nixon). 

c. Multi-institution clinical trials of chemotherapy in advanced unresectable 
head and neck cancers (Dr. Mario Eisenberger) . 

d. Multi-institution clinical trials of surgery and adjuvant chemotherapy 
in resectable lung cancer (Dr. Carmack Holmes). 

e. Multi-institution clinical trials of magnetic resonance imaging 
(Dr. Ruzicka). 



385 



f . Intramural clinical trials of the Biological Response Modifier Program. 

g. Intramural antiemetic clinical trials of the Surgery Branch. 

h. National Registry of colorectal cancer patients who have had resection of 
hepatic metastases. This national registry currently contains about 700 
cases and Is used to distinguish the kinds of patients who do well or poor- 
ly after hepatic resections (Dr. Sugarbaker). 

1. Review of results In the chemotherapy of advanced gastric cancer 
(Dr. Klllen). 

j. Review of survival results In the chemotherapy of advanced prostatic 
cancer. 

k. Review of results with hexamethylmelamine In advanced ovarian cancer 
(Dr. Foster). 

1. Comparative analysis of functional outcomes In women undergoing mastectomy 
or excision plus radiotherapy for primary breast cancer (Dr. Gerber). 

m. Analysis of prospectively collected psychosocial data from a randomized 
trial of mastectomy versus excision plus radiotherapy for primary breast 
cancer (Dr. Schain). 

n. Planning the comparison of histologic classification systems for rhabdomyo- 
sarcoma (Dr. Ungerlelder) . 

o. Evaluation of results in the treatment of 1900 patients in phase I trials 
to determine the extent to which phase II drug activity can be predicted 
(Dr. Marsonl) . 

p. Comparison of children and adults with regard to toxicities and maximum 
tolerated doses of chemotherapeutic agents (Dr. Marsoni). 

q. Evaluation of prognostic significance of pathologic and clinical hetero- 
geneity of lymphocyte depleted Hodgkin's disease (Dr. Elaine Jaffe). 

r. Analysis of sperm counts in men taking 13-clsretinoic acid for skin dis- 
orders (Dr. DiGiovanna). 

s. Analysis of prognostic factors for patients with colorectal cancer. The 
St. Mary's Hospital (England) data base of over 20,000 cases is being 
used for these analyses (Dr. Fielding). 

3. Preclinical Drug Discovery 

a. The BRB is actively collaborating with the Developmental Therapeutics Pro- 
gram in the statistical design of screening procedures using a non-clono- 
genic assay with human tumor cell lines. We are also developing methods 
for the analysis of histologic specificity of results from the colony 
forming assay or the new assay. Computer programs have been written by 
the BRB that implement two methods for assessing histologic specificity 
and research on a third Improved method is underway. These methods have 
been used to evaluate histologic specificity for over 60 compounds that 
have undergone stage 2 testing in the colony forming assay against fresh 
human tumors. Blesults were reported at AACR. Methods under development 
for the design and analysis of the new non-clonogenlc assay emphasize 
estimation of log-cell-kill and determination of activity and dose-response 
using procedures with sound statistical bases. 

b. Evaluation of the 1975 "tumor panel experiment" in collaboration with Drs. 
Sylvia Marsoni and Daniel Hoth and investigators of the Developmental 
Therapeutics Program. 

c. Development of methodology for analysis of tumor growth curves for use in 
analysis of biological response modifier pre-clinical screens 

(Dr . Talmadge) . 



386 



d. Development of improved statistical designs for screening compounds using 
the P388 in-vivo system. This research was conducted in collaboration with 
Drs. Paull and Hodes. 

4. National Symposium on Methodology and Quality Assurance in Cancer 
Clinical Trials 

This major national two day symposium was organized and sponsored by the 
Biometric Research Branch. It was very successful in stimulating improvement 
in the methodology of cancer clinical trials. The topics were very broad 
ranging and the sjnnposium was attended by almost 300 investigators from 
cancer centers, cooperative groups and the pharmaceutical industry. The 
full proceedings are being edited by the Biometric Research Branch and will 
be published in Cancer Treatment Reports . Planning for a 1986 symposium on 
the same topic is underway. 

5. Development of Methodological Guidelines for the Publication of Reports 
of Cancer Clinical Trials 

These guidelines were developed in collaboration with Dr. Robert Wittes and 
adopted by the editorial board of Cancer Treatment Reports . They constitute 
nine specific recommendations that appear in every issue of CTR which can 
substantially improve the quality of clinical trial reports. It is hoped 
that other journals will adopt these guidelines. 

6. Evaluation of Cooperative Group Cancer Clinical Trials 

This important project utilizes small teams of extramural experts on a disease 
oriented basis to evaluate the past 5 years of cooperative group cancer clini- 
cal trials. The teams address whether the most important questions are 
being asked and whether trials are being planned and conducted in a manner 
that yields reliable answers. The BRB is involved in the planning and conduct 
of this project which is well underway. 

7. Investigator's Handbook 

The BRB participated in writing parts of this important document dealing with 
the statistical design, monitoring and analysis of clinical trials. 

8. CTEP - Information System 

The BRB provided extensive consultation on the hardware and software alterna- 
tives appropriate for the effective development and use of this system. The 
BRB secured an outside consultant to participate in this process of developing 
an RFP for the procurement of appropriate hardware and software. 

9. Development of methodology to evaluate the cost of various kinds of clini- 
cal trials and to evaluate the impact of funding patterns. This project 

is led by Kim Horgan. 

10. Comparative Studies to Evaluate Magnetic Resonance Imaging 

These multi-institution studies are being sponsored by the Radiation Research 
Program. BRB staff participated extensively in the design of these studies 



387 



in developing an estimate of requirements for coordinating center funding, 
and in providing statistical collaboration during their conduct. 

11. Computer Software Development 

a. Development of a microcomputer data base for the National Hepatic 
Metastases Registry. DBASE 3 on a Compaq Plus computer was used. 

b. Development of an interactive computer program for the generation of 
survival curves on a graphics terminal. This is a very powerful 
improvement of a previous program. It is written in the SAS language 
and has been requested by several major statistical centers. A 
description of this program will be published. 

c. Initiation of projects for the Small Business Innovative Research 
Program that would result in the development of computer systems 
for interactive analysis of data that would bring state-of-the-art 
methodology and interactive graphics to the disposal of statisticians, 
clinical investigators and bench scientists. 

d. Development of interactive computer programs for use by BRB and Devel- 
opmental Therapeutics personnel for assessing histologic specificity 
of new compounds in in-vitro assay against human tumors and cell 
lines. 

e. Development of an interactive computer program to calculate exact con- 
fidence limits for response rates and response rate differences for use 
by CTEP staff. 

12. New Methodology for Evaluating Chronic Toxicity as a Function of 
Cumulative Dose 

Assessment of the relationship of acute toxicity to dose of an administered 
drug is usually straightforward. This is not the case for chronic toxicity 
such as irreversible cardiac or lung damage. Because some patients receive 
low doses due to early death and because extended survival may be necessary 
to observe clinical evidence of toxicity, the usual analyses are biased. We 
have developed new statistical methodology for such problems. 

We are using these new methods to re-analyze the large data base of the South- 
west Oncology Group concerning the relation between cardiotoxicity and cumtila- 
tlve dose of mitoxantrone. Unfortunately, the original data base used by 
Von Hoff for analysis of adriamycin cardiotoxicity is no longer available. 
The new methodology also has broad applicability to studies of second malig- 
nancies and to epidemiologic investigations of occupational health. 

13. Testing for Qualitative Treatment by Subset Interactions 

A qualitative interaction is said to exist involving two treatments (A and B) 
when treatment A is best for one subset of patients but treatment B is best 
for another subset. Many major clinical trials in cancer, cardiovascular 
diseases and other areas conclude with a report of qualitative Interactions. 
Such claims are very important because of their implications on treatment 
selection for individual patients and because of their biological implications. 
We have, during the past year, developed the first valid statistical significant 
test of qualitative treatment by subset interactions. This test was used 
to re-analyze the National Surgical Adjuvant Breast Project's Protocol for 
the evaluation of tamoxifen. 



388 



These results will be published in the June 1985 issue of Biometrics . These 
results and other results concerning new methodology for subset analysis 
have been presented in invited talks before the Washington Statistical 
Society, Society for Clinical Trials and the National Heart, Lung, and Blood 
Institute's September 1984 Workshop on Subset Analysis. 

We have recently generalized this method to test the hypothesis that some 
subsets of patients benefit from one treatment whereas for others the treat- 
ments are equivalent. This generalization was recently presented in conjunc- 
tion with other results in an invited presentation to the Society of Clinical 
Trials on the topic of combining evidence across clinical trials. The new 
methodology is applicable to determining whether results from different insti- 
tutions and different studies are "poolable." 

This research was conducted jointly with Dr. Mitchell Gail. The research is 
continuing on other new methodology for subset analyses. 

14. Modeling the Covariate Dependence of Binary Sequences 

There is an increasing interest in studying quality of life of cancer patients 
on clinical studies. Psychological factors may be studied by treating them as 
dichotomous variables which vary over time between the states of "good" and 
"poor." We have explored modelling these transitions by means of a Markov 
chain, incorporating covariate dependence by means of a logistic model for 
the transition probabilities. We have applied these methods to a large 
study of the psychological effects of mastectomy. This research was conducted 
with Dr. Larry Muenz and has been published in Biometrics . 

15. The Size of Cancer Clinical Trials 

Many phase III trials are too small to identify therapeutic effects of the 
magnitude that can be realistically expected. The implications of having 
numerous phase III trials of inadequate size is also not generally appreciated. 
These results were presented at the November 1984 Sjnnposium on Methodology 
and Quality Assurance in Cancer Clinical Trials and will be published in 
Cancer Treatment Reports . 

An evaluation of the appropriate size of phase II trials is also being con- 
ducted. Preliminary results were given in invited presentations before the 
Washington Statistical Society, NYU Medical Center and at the Memorial Sloan 
Kettering Cancer Center. 

16. Randomized Phase II Clinical Trials 

An evaluation of several kinds of randomized designs for phase II clinical 
trials has been conducted. Although some proposed randomized designs seem 
of little value for phase II trials, the concept of randomizing among new 
agents for cooperative group trials in common diseases can be useful. The 
objectives of randomized phase II trials have been carefully assessed and 
distinguished from the objectives of phase III trials, and the use of ranking 
and selection theory for planning the size of such trials is developed. 
These results have been accepted for publication in Cancer Treatment Reports . 



389 



17. Methodology For Antiemetic Studies 

A critical review of methodology used for the design, conduct and analysis 
of antiemetic studies in cancer patients was made in collaboration with 
Drs. Joseph Aisner and Ian Oliver. This manuscript has been submitted for 
publication. 

18. Designs For The Early Termination Of Clinical Trials 

We have continued research on methodology for early termination of randomized 
clinical trials when interim results for the experimental treatment are not 
promising. An easily utilized and effective two-stage design has been devel- 
oped and presented at the International meeting of the Biometric Society and 
the November 1984 Symposium on Methodology and Quality Assurance in Cancer 
Clinical Trials. A manuscript dealing with the use of this design as an 
alternative to nonrandomized pilot studies in the treatment of head and 
neck cancers is in preparation. 

19. Development of Methods For Treatment Planning Utilizing Mathematical 
Models 

Clinical trials of the past 5 years were evaluated relative to the predictions 
of the "Norton-Simon model." These results provide support for the concept 
of intensification of active therapy. This review will appear in a special 
issue of Cancer Treatment Reports dealing with the interface between the 
laboratory and clinic. 

20. Confidence Intervals For Differences In Response Rates 

Research is being conducted to develop improved confidence intervals for the 
difference in proportions that are easy to calculate and have appropriate 
coverage probabilities. The increased use of confidence intervals in published 
reports could substantially reduce misinterpretation of results by both 
authors and readers. Two manuscripts on the new methods we have developed 
are in preparation. 

21. Planning of Clinical Trial Strategies For Developing Effective 
Combinations 

We have embarked upon a multi-faceted research project dealing with the develop- 
ment of new designs for sequences of clinical trials to identify effective 
combination regimens. We are pursuing the first portion of this exciting 
new area and the results are very encouraging. These initial results were 
described in invited presentations to the Cancer and Leukemia Group B, 
April 1985 meeting and the Southeastern Oncology Group's June 1985 meeting. 
The results are being prepared for publication. 

22. Randomized Consent Designs 

An evaluation of the randomized consent (pre-randomization) design has been 
completed. These designs were being used with increasing frequency though 
their properties are not well understood. The results of this analysis were 



390 



published in the N Engl J^ Med and presented to the Oncologic Drug Advisory 
Committee of the FDA as well as at plenary sessions of the Society of Clinical 
Trials and the Tucson Adjuvant Clinical Trials meetings. It appears that 
this study has had important impact on limiting the use of this problematic 
design. 

23. Cost-benefit analysis of CEA monitoring in surgically "cured" colorectal 
cancer patients. 

24. Evaluation of methodology for determining the required sample size for a 
clinical trial when time-to-event is the primary endpoint. 

Publications : 

1. Eisenberger, M.A. , Simon, R. , O'Dwyer, P.J., Wittes, R.E. and Friedman, 
M.A. A re-evaluation of nonhormonal cytotoxic chemotherapy in the 
treatment of prostatic carcinoma. Journal of Clinical Oncology 

(in press). 

2. Ellenberg, S.S., Eisenberger, M.A. : An efficient design for phase III 
studies of combination chemotherapies . Cancer Treatment Reports 

(in press) . 

3. Ellenberg, S.S.: Randomization designs in comparative clinical trials. 
New England Journal of Medicine : 310: 1404-1408, 1984. 

4. Feld, R. , Rubinstein, L.V. and Weisenberger, T.H.: Sites of recurrence 
in resected stage I non-small cell lung cancer (NSCL): A guide for future 
studies. Journal of Clinical Oncology 2: 1352-1358, 1984. 

5. Gail, M. and Simon, R. Testing for qualitative interactions between 
treatment effects and patient subsets. Biometrics (in press). 

6. Gastrointestinal Tumor Study Group, The: Prolongation of D=disease free 
interval in surgically cured rectal carcinoma. New England Journal of 
Medicine : 312: 1465-1472, 1985. 

7. Gerber, L.H., Helfgott, R.K. , Gross, E.G., Hicks, J.E., Ellenberg, S.S., 
Peck, G.L.: Vertebral abnormalities associated with synthetic retinoid 
use. Journal of American Academy of Dermatology ; 10: 817-823, 1984. 

8. Lessner, H.E., Mayer, R.J., Ellenberg, S.S., Stablein, D.M. : Adjuvant 
therapy for colon cancer (Letter to the Editor) . New England Journal 
of Medicine 311: 410, 1984. 

9. Lokich, J., Ellenberg, S., Gerson, B., Knox, W.E., Zamcheck, N.: 
Plasma clearance of carcinoembryonic antigen (CEA) following hepatic 
metastectomy . Journal of Clinical Oncology : 2: 462-465, 1984. 

10. Miller, D.L., Vermess, M. , Doppman, J.L., Simon, R.M., Sugarbaker, P.H., 
O'Leary, T.J., Grimes, G. , Chatterju, D.G. and Willis, M. : CT of the liver 
and spleen with EOE-13: Review of 225 examinations. Am. J . Roent . 143: 
235-243, 1984. 



391 



11. Muenz, L.R. and Rubinstein, L.V.: Modelling the covariate dependence of 
binary sequences. Biometrics 41: 91-102, 1985. 

12. Norton, L. and Simon, R. The Norton-Simon Hypothesis Revisited. Cancer 
Treatment Reports (in press). 

13. Patronas, N.J., DiChiro, G. , Kufta, C. , Bairamian, D. , Kornblith, P.L., 
Simon, R.M. and Larson, S.M. : Prediction of Survival in Glioma Patients 
by Means of Positron Emission Tomography (PET). Journal of Neurosurgery 
62: 816-822, 1985. 

14. Shackney, S.E., Levine, A.M., Fisher, R.J., Nichols, P., Jaffe, E., 
Schuette, W.H., Simon, R., Smith, C.A., Occhipinti, S.J., Parker, J.W. , 
Cossman, J., Young, R.C. and Lukes, R.J. The biology of tumor growth 
in the non-Hodgkin' s Ijrmphomas . In 1985 Year Book of Cancer (in press). 

15. Shoemaker, R.H., Wolpert-DeFilippes, M.K. , Kern, D.H., Lieber, M.M. , 
Kamuch, R.W. , Melnick, N.R. , Miller, W.T., Salmon, S.E., Simon, R.M., 
Venditti, J.M. and Von Hoff, J.D. : Application of a human tumor colony 
forming assay to new drug screening. Cancer Research 45: 2145-2153, 1985. 

16. Simon, R. The size of phase HI cancer clinical trials. Cancer Treatment 
Reports (in press). 

17. Simon, R. , Wittes, R.E. and Ellenberg, S.S. Randomized phase II clinical 
trials. Cancer Treatment Reports (in press). 

18. Simon, R. and Wittes, R.E. : Editorial: Methodologic Guidelines for 
Clinical Trial Reports. Cancer Treatment Reports 69: 1-3, 1985. 

19. Smith, F.P., Ellenberg, S.S., Mayer, R.J., Lessner, H.E., Horton, J.B.: 
Phase II study of MOF-Streptozotocin (methyl-CCNU, vincristine, 5- 
fluorouracil and streptozotocin) in advanced colorectal cancer: a GITSG 
study. Journal of Clinical Oncology : 23: 770-773, 1984. 

20. Sugarbaker, P.H. , Vermess, M. , Doppman, J.L. and Simon, R. Improved 
detection of focal lesions with computerized tomographic examination 
of the liver using ethiodized oil emulsion (EOE-13) liver contrast. 
Cancer 54: 1489-1495, 1984. 



392 



DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE 
NOTICE OF INTRAMURAL RESEARCH PROJECT 



PROJECT NUMBER 



ZOl CM 06308-14 BRB 



PERIOD COVERED 

October 1, 1984 through September 30, 1985 



TITLE OF PROJECT (80 characters or less. Title must fit on one line Ixtween the borders.) 

Blometrlc Research Branch 



PRINCIPAL INVESTIGATOR (Ust other professional personnel betoiv the Principal Investigator) (Name, title, laboratory, and institute affiliation) 

Richard M. Simon, Chief, Biometric Research Branch, CTEP, DCT, NCI 

Others : 

Susan S. Ellenberg, Statistician, BRB, CTEP, DCT, NCI 

Lawrence V. Rubinstein, Statistician, BRB, CTEP, DCT, NCI 

Frances J. Mather, IPA, BRB, CTEP, DCT, NCI 

Sate Songhorabadi , Visiting Fellow, BRB, CTEP, DCT, NCI 



COOPERATING UNITS fffanw Developmental Therapeutics Program, DCT, NCI; Radiation 
Research Program, DCT, NCI; Biological Response Modifiers Program, DCT, NCI; 
Clinical Oncology Program, DCT, NCI; Environmental Epidemiology Branch, DCE, NCI. 



UB/BRANCH 

Biometric Research Branch 



INSTITUTE AND LOCATION 

NCI, NIH, Bethesda, Maryland 20205 



TOTAL MAN-YEARS: 

5.0 


PROFESSIONAL: 

4.0 




OTHER: 

1.0 


CHECK APPROPRIATE BOX(ES) 

D (a) Human subjects 
n (a1) Minors 
n (a2) Interviews 


D (b) Human tissues 


5 (c) Neither 



SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 

The Biometric Research Branch (BRB) is the statistical component for scientific 
planning and monitoring of the national and international research program of 
the Division of Cancer Treatment. The branch provides statistical leadership 
for all extramural activities of the division. The branch is also responsible 
for statistical consultation and collaboration with the intramural activities 
of the Biological Response Modifier Program, Developmental Therapeutics Program, 
and Radiation Research Program and performs collaborative research with compo- 
nents of the Clinical Oncology Program. 

The Biometric Research Branch performs statistical planning and evaluation of 
all NCI supported therapeutic clinical trials. The branch performs scientific 
monitoring for the statistical aspects of the conduct and analysis of trials 
performed via cooperative agreement or contract. Primary statistical direction 
is provided by the branch for the conduct of selected national and international 
studies of therapeutic interventions, prognostic factors, pre-clinical screening 
and diagnostic imaging. The branch performs evaluations of therapeutic inter- 
ventions based upon syntheses of results from multiple studies. 

The Biometric Research Branch conducts research on experimental designs, bio- 
metric methods and biomathematical approaches for the development and efficient 
evaluation of improved cancer treatments. 



393 



PHS 6040 (Rev. 1/84) 



CLINICAL INVESTIGATIONS BRANCH (CIB) 
CONTENTS 



1.0 Personnel 

2.0 Cooperative Agreement Programs 

2.1 Cooperative Clinical Trials Groups 

2.1.1 Listing of Groups 

2.1.2 Description of Programs 

2.1.3 Summary of Accoraplishments 

3.0 Grant Programs 

3.1 Program Project Grants (POl) 

3.2 ROl Grant Programs 

3.2.1 Clinical Oncology 

3.2.2 Cancer and Nutrition Program 

3.2.3 Supportive Care (Under XOl Grant Program) 

3.2.4 Surgical Oncology 

3.3 Cooperative Agreements 

3.3.1 Conversion of Cooperative Agreements 

3.3.2 Immunodeficiency Syndrome (AIDS) 

4.0 Contract Programs 

4.1 Medicine Section 

4.1.1 Istituto Nazionale 

4.1.2 Phase II-III Drug Evaluation 

4.1.3 Statistical Support for the GITSG 

4.2 Nutrition Section 

4.3 Pediatric Section 

4.4 ECTO - EMMES 

4.5 PAHO - CCTRP 

5.0 Miscellaneous 

5.1 Clinical Trials Review 

5.2 Suramin Programs for HTLV Patients 

6.0 Staff Publications 

7.0 Staff Presentations 

8.0 Conferences - Workshops - Seminars 



394 



The Clinical Investigations Branch (CIB) is responsible for the scientific admin- 
istration of the national cooperative clinical trials groups (the Cooperative 
Group Program); for scientific monitoring of the disease-oriented contracts, an 
individual investigator-initiated clinical oncology grant program, a nutrition 
grant program, the pediatric Phase I contracts, the surgical oncology grant 
program, a series of Intergroup studies; and scientific administration of the 
Program Projects grants in clinical cancer treatment. 

1.0 Personnel 

1. Michael A. Friedman, M.D. — Chief, CIB; Acting Head, Nutrition Section 

2. John Y. Killen, Jr., M.D. — Head, Medicine Section, Associate Chief, CIB 

3. Richard S. Ungerleider, M.D. — Head, Pediatric Section 

4. Frederick Avis, M.D. — Head, Surgery Section 

5. Bruce Cheson, M.D. — Senior Investigator, Medicine Section 

6. Ann Fields — Secretary to the Chief 

7. Carol Tippet — Secretary 

8. Jill Johnston — Secretary 

9. Helen Bradley — Stay-in-School, Secretary 

Dr. Friedman is responsible for the overall administration of the Branch 
and coordination of its activities with the Cancer Therapy Evaluation Pro- 
gram, the Grants Administration Branch, and the Division of Extramural 
Activities. Regional Studies Review Committee (CRSRC). He also supervises 
the Project Officers on clinical contracts, and the Program Directors on 
grants and cooperative agreements. 

Dr. Killen is Associate Chief, CIS and Head of the Medicine Section. He is 
program director for the Clinical Cooperative Group Program. He is also 
the Project Officer for the two EMMSS Corporation contracts ("Supportive 
Services for Extramural Clinical Trials" and "Statistical Support for the 
Gastrointestinal Tumor Study Group"), the Milan, Italy contract "Multimodal 
Treatment of Primary Breast Carcinoma" and the International Bone Marrow 
Transplant Registry Contract. He serves as liaison to the EORTC Protocol 
Review Committee. 

Dr. Ungerleider is Head of the Pediatric Section, CIB, and is the Program 
Director of the POl and ROl grants for pediatric cancers. He is Program 
Director for the cooperative groups which are conducting studies of pediat- 
ric cancers, and is the Project Officer for the pediatric Phase I contracts. 
He is program director for the pediatric Program Project Grants. 

Dr. Avis is the Head of the Surgery Section. His main responsibility is to 
promote surgical oncology research on a national level. This is accomplished 
by heading the program to encourage surgeons to submit ROl and POl grants 
under a program announcement, to hold workshops on various timely subjects 
and promote surgical oncology training programs. He is also the DCT repre- 
sentative to the NCI Nutrition Working Group. 

Dr. Cheson is Cancer Expert for the Medicine Section, CIB. He is program 
director for the POl Clinical Treatment Grants involving the hematologic 
malignancies and bone marrow transplantation. He is scientific liaison to 

395 



SWOG, CALGB, SECSG, MAOP, Polycythemia Vera Study Group, Leukemia Intergroup, 
the Lymphoma Pathology Reference Center, as well as the ATLL Project at 
University of the West Indies, and the RFP for the evaluation of Differentia- 
tion Agents in Human Malignancies. Or. Cheson also functions as CTEP coor- 
dinator for extramural activities in AIDS, including the multi-Institutional 
Suramin studies, autologous bone marrow transplantation. Interferon therapy 
in non-IIodgkin's Lymphomas, and hairy cell leukemia. Dr. Cheson served on 
the planning committee for the Feasibility Study for a National Bone Marrow 
Transplant Registry. 

2.0 Cooperative Agreement Programs 

2.1 Cooperative Clinical Trials Group 

The present Cooperative Group program was initiated in 1955 by the 
Cancer Chemotherapy National Service Center, to test new agents from 
the NCI drug development program. In the early 197 O's this grant pro- 
gram was transferred to the Division of Cancer Treatment. During the 
raid-197 0's, NCI initiatives established other disease-specific coopera- 
tive clinical trials groups on the advice of advisory bodies which per- 
ceived a need unmet at the time by the existing grant-funded program. 
In 1979 the DCT Board of Scientific Counselors conducted an in-depth 
review of the progress of clinical research supported through grants 
and contracts by the DCT. As a result of this review, and the past 
history of DCT staff involvement with the cooperative groups, funding 
for the entire clinical trials program of the Division was converted 
to the cooperative agreeiaent mechanism in 1982-83. Consolidation of 
the NCI's cooperative clinical trials programs was completed in 1983 
with the transfer of the National Bladder Cancer Treatment Group and 
the National Prostatic Cancer Treatment group to the DCT. 

2.1.1 Listing of the Cooperative Clinical Trials Groups 

Multlmodality Multidisease Groups 

Cancer and Leukemia Group B (CALGB) 
Eastern Cooperative Oncology Group (ECOG) 
Mid-Atlantic Oncology Program (MAOP) 
North Central Cancer Treatment Group (NCCTG) 
Northern California Oncology Group (NCOG) 
Piedmont Oncology Association (POA) 
Southeastern Cancer Study Group (SEG) 
Southwest Oncology Group (SWOG) 

Multlmodality Groups Devoted to a Major Oncologic Area 

Children's Cancer Study Group (CCSG) 
Gynecologic Oncology Group (GOG) 
Pediatric Oncology Group (POG) 
Lung Cancer Study Group (LCSG) 



396 



Single Modality Group 

Radiation Therapy Oncology Group (RTOG) 

Single Disease Groups 

Intergroup Rhabdomyosarcoaia Study (IRS) 

National Surgical Adjuvant Breast and Bowel Project (NSABP) 

National Wilms' Tumor Study Group (NWTSG) 

Polycythemia Vera Study Group (PVSG) 

Radiotherapy riodgkin's Disease Group (RHDG) 

Leuke:nia Intergroup (LIG) 

National Prostatic Cancer Project (NPCP) 

National Collaborative Group A (Bladder Cancer) 

Brain Tumor Cooperative Group (BTCG) 

GI Tumor Study Group (GITSG) 

Lung Cancer Study Group (LCSG) 

Special Activities Groups 

European Organization for Research on Treatment for Cancer 

(EORTC) Operations and Statistical Office 
Lymphoma Pathology Reference Center (LPRC) 

Radiologic Physics Center (RPC) (see Radiation Research Program) 
Quality Assurance Review Center (QUARC) 
Nutrition Oncology Research Cooperative Agreement (NORCA) 

2.1.2 Description of the Clinical Cooperative Group Program 

The Cancer and Leukemia Group 8 (CALGB), founded in 1955, studied 
primarily hematologic malignancies until the 197 O's, when it also 
developed multimodal studies in solid tumors. In 1982 the group 
elected a new chairman and focussed its scientific scope to the 
treatment of leukemia, lymphoma, lung and breast cancer. 

The Children's Cancer Study Group (CCSG) is a multiraodality or- 
ganization concerned exclusively with pediatric malignancies. 
They conduct major Phase II and III studies in hematologic and 
solid tumors, and collect information about the long-term ef- 
fects of cancer therapy. 

The Eastern Cooperative Oncology Group (ECOG), founded in the 
1950' s, developed and remains committed to multimodal solid 
tamor studies as well as studies in the hematologic malignancies. 
This large national group has made major contributions in hema- 
tologic breast and gastrointestinal malignancies. It was a 
pioneer group in the development of a quality control monitoring 
program and the implementation of biologic response modifiers 
(BRM) studies. 

The Gastrointestinal Tumor Study Group (GITSG) began activity in 
1974 as a disease-specific contract-supported research group. 
Its primary mission has been and continues to be the evaluation 



397 



of aew surgical adjuvaat and corabiaed modality therapies for 
gastroiatestinal malignancy. 

The Gynecoloj^ic Oncology Group (GOG) coordinates the specialties 
of gynecology, radiation, medical oncology, and pathology for 
research in gynecologic cancers. They have done a systeiaatic 
analysis of Phase II drug activity in several gynecologic malig- 
nancies as well as Phase III studies in early ovarian and uterine 
cancers. 

The Mid-Atlantic Oncology Program (MAOP) oegan activity in 1982 
as one of two Regional Clinical Trials Groups funded in response 
to an NCI 8.FA, The objectives of the group are to perform re- 
search through joint participation of physicians from private 
practice and academic centers in cooperative clinical effort. 

The National Surgical Adjuvant Breast and Bowel Project (NSABP) 
is a focussed multimodality group. In the past, it concentrated 
exclusively on primary and adjuvant treatment of breast cancer, 
but now it is also involved in studies of primary colorectal 
cancer. 

The National Wilms' Tumor Study Group (NiVTSG) is an intergroup 
organization incorporating the pediatric cooperative clinical 
groups. Their third study (;'^WTS-3) is primarily concerned with 
refinement of therapy. Nl-JTS-l and NWTS-2 conclusively demon- 
strated that most children with this tamor c-in now be expected 
to survive if they are managed by corabiaed modality therapy 
from the outset, and that prognosis is closely related to his- 
topathologic findings. They are closely monitoring the late 
effects of cancer therapy in young children. They are in the 
process of developing a successor (N'v;TS-4) study. 

The Worth Central Cancer Treatment Group (NCCi'G), organized in 
1980, consists of the Mayo Comprehensive Cancer Center and 10 
clinics in the North Central region. The objectives of this 
Regional Clinical Trials Group is to make promising cancer re- 
search accessible to patients in their region, and to conduct 
clinical research of high quality in a community setting. 

The Northern California Oncology Group (NCOG), a regional group 
organized in 1976, has developed programs in brain tumors and 
high LET radiation and radiosensitizer studies. They have par- 
ticipated in the direction of the head and neck Intergroup stu- 
dies and have expertise in biological response modifiers and 
hyperthermia. 

The Pediatric Oncology Group (POG) is a multimodality organiza- 
tion formed in 1980 from pediatric members of SWOG and CALG3. 
They have initiated new Phase 11 and III studies with a major 
interest in the classification of childhood leukemias through 
the use of cell markers. They have recently completed a crucial 
randomized trial of adjuvant therapy in osteogenic sarcoma. 



398 



The Piedmont Oncology Association (POA) was the first of two 
new Regional Cooperative Groups to be funded in 1982 as a result 
of an RFA issued by the NCI in 1981. Its center of operation is 
located at the Bowman Gray School of Medicine, and its member- 
ship is composed largely of trained oncologists in private 
practice in the Piedmont area. 

The Polycythemia Vera Study Group (PVSG), founded in 1967, had 
protocols to determine the natural history, course, and optimum 
therapy of polycythemia vera. Currently it is funded for fol- 
lowup and final analyses of their primary protocol, which has 
shown an increase in incidence of leukemia in patients with 
chlorambucil as compared to radioactive phosphorus or phlebotomy. 

The Radiation Therapy Oncology Group was formed in 1971 follow- 
ing a multi-institutional methotrexate study in head and neck 
cancer. Their protocols explore the methodology and technique 
of radiation therapy as applied in various tumor types, and 
diseaseoriented studies exploring more than one modality of 
treatment using radiation therapy as primary focus for study. 
Studies include time-dose relationships, the use of radiosen- 
sitizers, high LET radiation, hyperthermia, and a study of the 
late effects of radiation therapy. 

The Southeastern Cancer Study Group (SEG) is a multimodality 
group involved in studies in leukeiaia, lymphoma, lung cancer, 
breast cancer and genitourinary cancers. 

The Southwest Oncology Group (3W0G) is a large national group, 
with a major focus on Phase II and III studies. They have made 
significant contributions in AilL, myeloma, lymphoma, and breast 
cancer. 

The Intergroup Rhabdomyosarcoma Study Group (IRS) is composed of 
members af POG and CCSG. Their first study developed a staging 
system, demonstrated varied prognoses depending on site, and 
evaluated the effect of multimodal therapy. Their second study 
has incorporated special treatment considerations relating to 
primary site of disease, and prospectively evaluated a new histo- 
pathologic grading system. IRS-III has as its objectives im- 
proved survival and relapse-free survival rates with the fewest 
long-term complications. 

The Radiotherapy Hodgkin's Disease Group (RHDG) has studied 
whether survival in localized Hodgkin's disease was different 
when patients received involved field of radiation, or extended 
fields. The trial is presently in long-term followup. 

The Lung Cancer Study Group (LCSG) is composed of member insti- 
tutions and statistical centers studying surgical, radiation and 
chemotherapeutic treatments for patients with early lung cancer. 
Combination studies of chemotherapy with surgery or radiotherapy 
in patients with localized disease are ongoing. 



399 



The National Prostatic Cancer Project (NPCP), converted to a 
cooperative agreement, Is composed of several centers studying 
cheraotherapeutlc and hormonal treatments for patients with ad- 
vanced metastatic disease and evaluates the role of surgery 
versus radiotherapy in patients with localized disease. Addi- 
tionally, studies evaluating the role of chemotherapy or hor- 
mones as adjuvant for patients with loco-regional disease at 
high risk risk for dissemination are being conducted. The group 
is presently involved with a study evaluating flutamide and 
leuprolide and is responsible for the statistical evaluation of 
the data. 

The National Collaborative Group A (National Bladder Cancer 
Group) is evaluating the role of intravesical chemotherapy in 
patients with superficial bladder cancer. Combined modality 
studies for patients with invasive disease are currently in pro- 
gress, including one study of adjuvant chemotherapy for patients 
with Invasive disease resected for cure. Additionally, one 
trial of chemotherapy in patients with metastatic disease is in 
progress. A large data bank from patients with superficial dis- 
ease (stages A and 0) has been collected for the past several 
years and analysis is soon to be completed. 

The Brain Tumor Cooperative Group (BTGG) was formerly a contract 
supported group administered by the Radiation Research Program. 
It was converted to a cooperative agreement after successful 
peer review of a grant application in 1983. This group is per- 
forming one major Phase III study and one Phase II study in 
malignant brain tumors. Recent interest has been generated by 
this group in studying intra-arterial chemotherapy, and refining 
interstitial radiation approaches. 

The following special activities groups provide support services 
for groups: 

The Operations and Statistical Office of the EORTC is partially 
funded by DCT. A representative of CTEP serves on its protocol 
review committee. 

The Lymphoma Pathology Reference Center (LPRC) provides expert 
review of pathological material for the groups performing ther- 
apeutic research in malignant lymphoma. 

The Quality Assurance Review Center (QQARC) provides radiation 
therapy quality control for three national cooperative groups 
(CCSG, CALGB, POG) and two pediatric intergroup studies (IRS, 
NWTSG). 

The Radiologic Physics Center site visits and reviews the radi- 
ation treatment of cooperative groups. 



400 



2.1.3 Sununary of Accomplishuieacs 

Noteworthy accoinpllshmeats of the cooperative clinical trials 
groups are nuiaerous, bat selected exaiuples are: 

1. CALGB has active, ;jroup-«ide studies of the lympho prolifer- 
ative disorders lacluding sophisticated immunological and 
cytogenetic evaluations, and therapy including interesting 
new agents such as interferon and deoxycoformycin. 

2. 3W0G is planning a large Phase III comparison of two of 
the newer, more aggressive chemotherapy regimens for non- 
Hodgkin's Lymphoma with CHOP, which has been considered 
"standard" therapy. 

3. In a study of women with advanced breast cancer, MAOP has 
initiated the first group-wide trial involving response- 
surface methodology. 

4. FOG has recently conducted a randomized trial in osteosarcoma 
which conclusively demonstrated the benefit of postoperative 
adjuvant chemotherapy over a non-treatnent control. This 
trial addressed a controversial issue whose clarification 
was considered essential for future studies in osteosarcoma. 

5. CCSG has demonstrated that routine histopathologic classifi- 
cation of QOii-'Iodgkiii's lympiio^aa is the best criterion for 
choice of therapy with non-localized involvement. 

6. The National '//ilms' Study #3 suggests that tumors designated 
"favorable histology" can be successfully treated with less 
intensive regimens. Long-term survival rates of 90% have 
been demonstrated for patients with non-metastatic favorable 
histology Wilms' Tumor. 

7. CCSG has demonstrated that elevation of serum ferritin levels 
in neuroblastoma is associated with a significantly poorer 
prognosis tlian was normal ferritin and that this correlation 
is independent of stage and age at diagnosis. 

8. The NSABP reported early results from a clinical trial com- 
paring modified radical mastectomy to lesser breast-pre- 
serving surgery. The data demonstrate that the lesser 
surgery is as effective as conventional primary treatment 

in preventing recurrence. Additional followup is necessary. 

9. The GITSG has completed a controlled trial indicating bene- 
fit for the use of adjuvant radiotherapy plus chemotherapy 
compared to surgery alone for patients with resectable 
rectal cancer. 



401 



Intergroup Studies 

The importance of participation of multiple cooperative groups 
in single, high-priority clinical trials is becoming Increasingly 
apparent. Such intergroup studies permit more rapid completion 
of important trials than is possible by single cooperative 
groups. During the past year, with considerable involvement 
by program staff, three new intergroup studies have been initi- 
ated. These are: 

1. The Intergroup Rhabdomyosarcoma Study Group has developed a 
prognostic classification system based on cytohistologic sub- 
types that identifies those patients v/ho require more inten- 
sive therapy. 

2. Intergroup Prostate Study: This very high priority study 
will attempt to confirm the impressive, albeit preliminary, 
results showing that the LHRH agonist, leuprolide, when com- 
bined with the antiandrogen, flutamide, yields a very high 
percentage of prolonged remissions in men with advanced 
prostatic cancer. 

3. Intergroup Colon Adjuvant Trial: This study is designed to 
replicate an earlier, s.naller trial conducted by the NCGTG. 
Preliminary analysis suggests that patients receiving 5FU 
plus levamisole following curative surgery for colon cancer 
may have fewer recurrences than those treated with surgery 
alone. 

Additionally the following studies continue: 

1. Intergroup Testicular Study: This is a collaboration among 
seven cooperative groups and four large institutions having 
an interest in testicular cancer. The protocol is a random- 
ized controlled study of adjuvant chemotherapy of Stage II 
resectable testicular cancer and a monitoring of Stage I 
testicular cancer. 

For Stage II the study compares the disease-free and overall 
survival for surgery alone (with combination chemotherapy 
for relapse) versus surgery plus early adjuvant chemotherapy. 
Stage I patients are registered and monitored to identify 
prognostic variables which may predict recurrence in this 
group. The protocol also includes important biologic studies 
such as histologic typing, serum marker studies, and studies 
of the accuracy of lymphangiograms, GT scans, and ultrasono- 
graphy. Progress presentations have been made at various 
cooperative group meetings: GALGB, SEG, SvJOG, and NCOG. This 
study is nearing completion, and full analyses are forth- 
coming. 

2. Intergroup Soft Tissue Sarcoma Study: Patients with primary 
soft tissue sarcoma (STS) after definitive surgery and/or 



402 



radiation therapy are randomized to high dose intermittent 
adriamycin adjuvant therapy versus a control arm. Most 
major national and regional groups participate. 

3. Intergroup Mesothelioma Study: Patients with limited uni- 
lateral thoracic mesothelioma receive combined surgery and 
radiation therapy and are randoiaized to receive adjuvant 
adriamycin versus a control arm. Several major national 

'" grcJiips participate. 

4. Intergroup Melanoma Study: Patients with intermediate thick- 
ness primary melanoma are randomized to have an excision with 
a 2 cm versus 4 cm margin and to have elective lymph node 
dissection or not. All cooperative groups plan to partici- 
pate as well as the NCI of Canada and the Melanoma Clinic, 
Australia. 

5. Head and Neck Intergroup Study: Patients with localized, 
advanced disease will be treated in a multimodality study. 

6. Intergroup Pancreas: Patients with resectable pancreatic 
cancer will receive post-op therapy in a controlled trial. 

3.0 Grant Program 

3.1 Program Project Grants (POl) 

At the present time there are 31 active clinical program project grants. 
Program project grants provide research support for broadly based acti- 
vities that blend preclinical and clinical activities. 

Each grant involves a number of investigators, each of whom conducts a 
research project designed to elucidate one or more aspects of a common 
goal. These efforts are conducted in an organized fashion in order to 
facilitate the interactions of these participating investigators. This 
approach is designed to acquire knowledge more effectively than would 
a simple aggregate of research projects operating without organization 
and thematic integration. 

Historically, the program has supported highly successful research pro- 
jects that have made significant contributions. By bringing together 
basic and clinical investigators, the program has been able to provide 
excellent patient care and also explore basic elements in tumor biology. 

Although clinical research is the main thrust of all programs, substan- 
tial efforts in more basic elements are present. These activities in- 
clude drug development and pharmacology, cell kinetics, immunobiology, 
marrow transplantation, his topathology, and hematology. 

Among the many iateresting and important program project grants are: 

1. Frel et al in the Solid Tumor Autologous Marrow Program (STAMP) 
have presented some of the highest response rates in patients 

403 



2. 



3. 



with advanced cancer treated with high doses of combinations of 
drugs including multiple alkylating agents. 

Kersey et al have a program project in pediatric radiation oncology 
and bone marrow transplantation that is currently investigating the 
utility of autologous marrow that has been purged of residual leu- 
kemia cells by treatment with a "cocktail" of monoclonal antibodies 
against lymphoblasts. Success in the use of autologous transplanta- 
tion will widely extend the use of transplantation beyond that cur- 
rently possible with allogeneic transplant. 

Simone et al have made many contributions to the therapy of acute 
lymphoblastic leukemia in children, which include establishing the 
necessity for CNS therapy, first demonstrating a greater than 50 
percent cure rate, and first establishing the clinical relevance 
of T-cell markers on lymphoblasts. They are currently engaged in 
attempts to overcome resistance to therapy and to better understand 
the pathogenesis of childhood leukemias. 

O'Reilly et al have demonstrated the use of soybean lectin-based 
fractionation procedure for T-cell depletion in bone marrow trans- 
plantation for severe combined immunodeficiency disease. 

Morton et al are involved in new surgical concepts featuring ad- 
vanced pathological staging of primary malignancies and early iden- 
tification of metastatic disease by immunological techniques in- 
volving measurements of serum and urinary tumor associated antigens, 
tumor specific serum antibodies, and circulating immune complexes. 



P.I. 
Thomas, E. Donnall 
Clarkson, Bayard D. 
Dicke, Karel 
Herbst, Arthur L. 

Freireich, Emil J. 
Storb, Rainer 
Schlossman, Stuart 

Rosenberg, Saul A. 
Clarkson, Bayard D. 



CLINICAL TREATMENT - POls CATALOG 

Title 

Adult Leukemia Research Center 

Human Hematopoietic Tumors Program Project 

Studies in Autologous Bone Marrow Transplantation 

UCCRC: Clinical-Laboratory Studies in Gynecologic 
Cancer 

Human Leukemia Research Center Program 

Aplastic Anemia Center 

The Biology of Treatment of Human Leukemia and Lym- 
phoma 

Clinical and Laboratory Studies of Malignant Lymphomas 
Cancer Chemotherapy Program Project 



404 



P._I._ 

Laszlo, John 
McGaire, William L. 

Carbone, Paul 
O'Reilly, Richard J. 

Moertel, Charles 
Santos, George 
Gale, Robert P. 
Blunie, Karl G. 

Nathan, David G. 
Sinione, Joseph V. 
Krivit, Williau 
Siraone, Joseph V. 
Miller, Denis R. 
Morton, Donald 
Morton, Donald 
Seigler, Hilllard 

Salmon, Sydney 
Frei, Emil 
Henderson, Edward 
Bertino, Joseph R. 
Creaven, Patrick J. 
Wilson, Charles B. 



GLINICAL TREATMENT - POls CATALOG (continued) 

Title ; 

Clinical Cancer Research Prograai 

Medical Oncology Program Project - Therapeutic 
Research 

Experitaental Therapy of Solid Tumors 

Transplantation of Lethal Congenital Immunodeficien- 
cies 

New Approaches to Treatment of Gastrointestinal Cancer 

Sone Marrow Transplantation in Human Disease 

A Program in Bone Marrow Transplantation 

Bone Marrow Transplantation for Hematologic Malig- 
nancies 

A Program for Investigation of Childhood Tumors 

Leukemia Program Project Grant 

Pediatric Oncology /Marrow Transplantation Project 

Studies of Childhood Solid Tumors 

Hematopoietic Malignancies in Children 

Surgical Iinmunology and Immunotherapy of Human Cancer 

New Approaches to Surgical Oncology 

Diagnosis and New Therapeutic Modalities in Surgical 
Oncology 

Medical Oncology Program Project 

Solid Tumor Autologous Marrow Program 

Clinical Cancer Research 

Clinical Pharmacology and Cancer Chemotherapy 

Clinical Pharmacology in Cancer Therapeutics 

Program for Treatment of Malignant 3rain Tumors 



405 



3.2 ROl Grant Programs 

3.2.1 Clinical Oncology 
Description 



The purpose of this program is to support research aimed at im- 
proved treatment of cancer patients. The range of the projects 
supported includes basic and clinical studies which are directly 
or ultimately aimed at improving methods of cancer therapy, in- 
cluding chemotherapy, radiation therapy, immunotherapy, surgery 
and supportive care. While many of these studies concern basic' 
mechanisms of antineoplastic agents in cells, animals, and 
humans, there is an emphasis on clinical studies which relate 
directly to human cancer treatment. Improved methods of experi- 
mental design and statistical methods for clinical cancer studies 
are an integral part of the research fostered by this program. 
The program currently includes 93 grants. 

Accomplishments 

Many preclinical and clinical studies are worthy of note. A. 
few examples are: 

1. Plunkett et al and Capizzi and co-workers are exploring the 
cellular pharmacology of antitumor agents (especially Ara-C) 
in human leukemlas. 

2. . Baylin and co-workers are investigating the effects of DMFO 
in