Edward A. Sausville, M.D., Ph.D., Associate Director

The Developmental Therapeutics Program (DTP) provides cancer and AIDS drug discovery and development resources to the academic and industrial research communities. DTP offers, at little or no cost, screening of individual compounds, compound samples for research, large sets of compounds for high-throughput screening, and biopharmaceutics such as monoclonal antibodies and cytokines, tumor cell lines, and animals. DTP has contract resources for Good Manufacturing Practice (GMP) compound synthesis, formulation, pharmacology, and toxicology testing for therapeutic agents being developed for human clinical trials. DTP's Web-based databases and data-mining tools are expanding to better serve investigators. Those who use DTP products include the NCI, through the extramural clinical trials program with the Cancer Therapy Evaluation Program-held Investigational New Drug (IND) applications; the NCI intramural program; and extramural physician-held INDs for compounds developed through the Rapid Access to Intervention Development (RAID) initiative. DTP has maximized access to resources through new programs, services, and an expanded website.

DTP accomplishments involve two distinct spheres of activities: 1) the Grants portfolio managed by the Grants Contracts and Operations Branch (GCOB), which are similar to grants operations in other DCTD programs, and 2) our Contract Research Resources, which are used to Discover, Develop, and Deliver new anticancer therapies to patients in a very directed sense.

GRANTS

Method to Extend Research in Time (MERIT)
In 2002 (FY02), the Grants and Contracts Operations Branch (GCOB) supported five Method to Extend Research in Time (MERIT) awardees, a distinguished group of investigators who have maintained excellent track records in peer review and are likely to maintain productive research programs. Awardees included Dr. Rodney Croteau for a project on taxol biosynthesis, Dr. Samuel Danishefsky for the synthesis of antitumor natural products and carbohydrate-based vaccines, Dr. Yoshito Kishi for the synthesis of antitumor natural products, Dr. Stephen Lippard, for studies on the chemistry and biology of platinum anticancer drugs, and Dr. Andrew Myers for a project on natural and nonnatural DNA cleaving agents.

Biology-Chemistry Centers
The Biology-Chemistry Centers were created with an initiative, Cancer Drug Discovery: Diversity Generation and Smart Assays, to encourage the formation of teams of biologists and chemists to work together to develop and adapt new technology for more efficient drug discovery. Six grants were funded. The group headed by Dr. Stuart Schreiber, Harvard University, made progress in the emerging field of chemical genetics, in which compounds are used in place of genetic techniques to develop the equivalent of gene knockouts. Dr. Peter Vogt, Scripps Research Institute, La Jolla, CA made major strides in creating regulators of protein-protein interactions and protein-DNA interactions. Drs. Dale Boger and David Cheresh used combinatorial chemistry to uncover a small molecule which inhibits the binding of matrix metalloproteinase (MMP2) to integrin alpha 3 beta 1, that may be a new class of anti-angiogenic agent. Dr. John Lazo's laboratory at the University of Pittsburgh identified new leads for inhibiting dual-specificity phosphatases and to perturb tubulin/motor proteins. A major advance was the application of fluorous synthesis techniques for generating and purifying diversity libraries. This group also tested the NCI Diversity Set of 1990 structurally diverse compounds. They identified several leads that inhibited CDC25A with nanomolar potency.

National Cooperative Drug Discovery Group (NCDDG) Program
This program, created in 1982, is a cornerstone for GCOB drug discovery efforts. During FY02, five NCDDG products were in some phase of development through NCI assistance mechanisms, including RAID and Rapid Access to NCI Discovery Resources (R*A*N*D). Through RAID, the Biological Resources Branch has helped produce vaccines especially for NCDDGs that lack major industrial partners. Since the program's inception, the FDA has approved three agents (topotecan, gliadel, and DAB389IL-2) for marketing. Twelve agents have entered clinical trials, including two during FY02. The first, HTI-286, is a peptide-like molecule that binds to the vinca-binding domain on tubulin. The industrial collaborator, Wyeth-Ayerst, synthesized clinical material, completed toxicology, and filed the IND application. The second, LAQ824, is a histone deacetylase inhibitor designed by industrial partner Novartis.

Molecular Targets for Drug Discovery Program
A new Molecular Targets for Drug Discovery program was initiated in 2000 as a joint effort of the Divison of Cancer Treatment and Diagnosis and Division of Cancer Prevention. In addition to characterization studies on targets, investigators were encouraged to develop assays for targets that could be used to develop interventions for cancer treatment and prevention. By the end of FY02, awards had been made for 13 cooperative agreements, 51 exploratory grants, six small business awards, and six supplements to existing awards. In one example, a study with apoptosis as the target process and antimycins as tools for downregulating the pro-life Bcl-xL survival protein, David Hockenbery of the Fred Hutchinson Cancer Research Center, Seattle, WA, reported mapping the drug-binding pocket on Bcl-xL by information-guided mutational analysis, and developing a computational model for antimycin docking studies. An exciting approach to identifying and validating new targets was reported by another grantee, Dr. Gregory Hannon of Cold Spring Harbor Laboratory in NY. From the original goal of a genome-wide search for cancer-relevant genes in a Drosophila model by synthetic lethality analysis, this PI perfected the methodology for large-scale gene silencing by small interfering RNA. He first validated it in Drosophila, where genome-wide expressed sequence tags have been created, and then applied it to mammalian cells. Another grantee, Dr. Pat Berg of George Washington University, discovered a breast cancer-relevant gene in which she noticed a disparity in the expression of the gene between white and African-American patients. NCI provided a one-year supplement to firm up the observation, anticipating a co-funding request to the National Center for Minority Health and Health Disparities for continued support. Because of the enthusiastic response and encouraging progress reports, the Molecular Targets for Drug Discovery initiatives were reissued and approved for release in early FY 2003.

Non-Mammalian Initiative
Two projects supported by this initiative use yeast to learn more about the importance of specific RNAs in the mechanism of action of 5-fluorouracil, and Xenopus oocytes to identify new signaling proteins activated in response to DNA damaging agents. Yeast is used to delineate mechanisms of the transcription factor PLZf, which is known to be important in acute myelocytic leukemia and acute promyelocytic leukemia and to identify peptidomimetics that interfere with its activity. Drosophila tracheal development is exploited as a model to identify new angiogenesis genes to better understand how hypoxia influences angiogenesis, and Dictyostelium is used as a model system to discover new signaling mechanisms important in sensitivity and resistance to DNA damaging agents. One success of the initiative was an investigation of the breast cancer gene BRCA-1 in yeast. Dr. Rong Li of the University of Virginia discovered new BRCA-1/Jun co-activator interactions.

Toxicology
With the Toxicology and Pharmacology Branch, GCOB supports two program announcements for research projects that take advantage of recent technology advances to develop toxicology models that predict specific organ toxicities. FY02 funded projects covered a range of topics, including the use of zebra fish to predict toxicities, an activity-based proteomic approach using various kinases, phosphatases, and p450s, and a novel tissue chip approach. Others are developing novel approaches to predicting neurotoxicity, hepatic toxicity, and pulmonary injury.

International Cooperative Biodiversity Groups
This program is funded by the NIH, the National Science Foundation, and the Department of Agriculture Foreign Agricultural Service. Program goals are biodiversity conservation, drug development, and economic growth. Since the first awards were made, funded groups have gained access to the biodiversity of countries in North, Central and South America, Southeast Asia, and Africa through multi-institutional collaborations. Five groups are now funded. In FY02, the NCI contribution was $400,000 to the total annual commitment of about $4.17 million. So far, the program has screened for cancer, HIV, malaria, tuberculosis, leishmaniasis, and Chagas diseases. Several novel compounds have been discovered and published.

Flexible System To Advance Innovative Research for Cancer Drug Discovery by Small Businesses (FLAIR)
Two main features distinguish FLAIR from regular Small Business Innovation Research/Small Business Technology Transfer (SBIR/STTR) grants. First, it allows larger phase I and phase II budgets that are required for successful discovery and development projects. Second, it provides support for developing innovative agents and therapeutic approaches from basic discovery through proof of principle in a clinical trial. The best measure of FLAIR success is the support of two agents that have entered clinical trials. ProLx Pharmaceutical is evaluating a small molecule that inhibits thioredoxin, a new molecular target important in cancer, in a phase I clinical trial. DEKK TEC, Inc. is evaluating a novel phenylhydrazone as an immune modulator for treating anogenital cancer associated with human papilloma virus infection. The interest and need for this program is exemplified by 52 applications submitted in the current grant round.

Grants Highlights

Dr. William Fenical, Scripps Institution of Oceanography, University of California-San Diego: Dr. Fenical's group discovered a new genus of actinomycete bacteria, a vast new source of potentially useful microorganisms from deep-sea sediments.

Drs. Ben Shen and Jon Thorson, University of Wisconsin-Madison: Enediynes are potent cytotoxic compounds biosynthesized by bacteria. These compounds have a 9- or 10-membered ring containing two triple bonds separated by a double bond. Dr. Shen showed that disrupting the single bacteria PKS gene stops the biosynthesis of a nine-membered ring enediyne.

Dr. Leonard Katz, Kosan Biosciences, Inc.: Epothilone D, being developed by Kosan Biosciences, is a complex polyketide that inhibits depolymerization of microtubules. It is in Phase I clinical trials as an anticancer agent.

CONTRACT RESEARCH RESOURCES ACCOMPLISHMENTS

For many years the NCI has conducted a vigorous program of discovery and development for new cancer treatment drugs. DTP uses a variety of award and support mechanisms to increase the rate of discovery of new molecular entities and approaches relevant to cancer, and to expedite the flow of these new discoveries to the clinic. This section describes the programs responsible for deciding which discoveries should receive support for preclinical development toward clinical trials, plus the DTP branches responsible for the oversight of DTP development contracts.

Drug Development Group (DDG)
The DDG approves anticancer agents for access to DTP drug development contract resources. Successful DDG drug candidates lead to an NCI-held IND application and NCI-sponsored clinical trials. Sixteen new agents were recently approved for stage 1B activities (in vivo testing, pre-range-finding toxicology, pre-formulation), four new agents were approved for stage 2A studies (additional in vivo testing, range-finding toxicology, formulation studies), and three agents were approved for stage 2B IND-directed toxicology studies: NSC 710464, an aminoflavone prodrug with activity against renal tumors in animal models, NSC 710305, a benzothiazole prodrug (Phortress) with activity against estrogen receptor (ER)+ and ER- breast xenograft models, and NSC 694501, a pyrrolobenzodiazepine (SJG136) with activity against breast, melanoma, and ovarian xenograft animal models.

Rapid Access to Intervention Development (RAID)
This program gives academic researchers access to DTP's development resources. Applications are reviewed by a panel of extramural experts. Resources provided to the originating investigator may include GMP synthesized material, formulation research, pharmacological methods, or IND-directed toxicology to support an investigator-held IND application and clinical trials. Following is a summary of recently approved projects:

1. Dr. Selwyn Vickers, University of Alabama, Birmingham will apply tissue-specific targeted CRAd in treating pancreatic adenocarcinoma. RAID will support GMP production for a Phase I clinical trial and safety testing.
2. Dr. Paul Shami, University of Utah will focus on glutathione S-transferase activated nitric oxide donors in cancer treatment. RAID will support non-GMP production, additional in vivo efficacy experiments, pharmacology, and preliminary toxicity studies.
3. Dr. A. Douglas Kinghorn, University of Illinois, Chicago will focus on pervilleine A and analogs, multi-drug resistance inhibitors. RAID will support collection and isolation of pervilleines A-F and further in vivo efficacy studies to identify one candidate suitable for further development.
4. Dr. Leisha Emens, Johns Hopkins University will focus on chemoimmunotherapy for metastatic breast cancer treatment. RAID will support formulation, as well as fill and release testing of peptides derived from HER-2/neu and mutated k-ras (as a negative control) for clinical administration.
5. Dr. William Read, Washington University will focus on the production of sodium phenylbutyrate. RAID will support GMP production of sodium phenylbutyrate for the phase I clinical trial.
6. Dr. Thomas Burke, University of Kentucky will focus on SN-38 liposomal prodrug featuring tumor-targeting and pH-mediated self activation. RAID will support synthesis of 8-10 grams of SN-38 to support the investigator's studies; the applicant will return to RAID for continued development once a lead candidate is identified.
7. Dr. Howard Bailey, University of Wisconsin will focus on the preclinical development of an anticancer agent, SL-11093. RAID will support GMP production for phase I clinical trials and a multicycle single-species toxicity study.
8. Dr. Nurulain Zaveri, SRI International will focus on SR 16158, a novel endocrine approach to breast cancer therapy. RAID will support synthesis of radiolabeled material, further in vivo efficacy testing, GMP synthesis, pharmacology, and preliminary toxicology.
9. Dr. Gary Schwartz, Memorial Sloan-Kettering Cancer Center will focus on the development of a combination of the pro-apoptotic drug safingol and standard chemotherapies in patients with advanced solid tumors. RAID will support formulation development, GMP synthesis, and required pharmacology and toxicology to allow conduct of a phase I trial of safingol in combination with cisplatin.
10. Dr. Dajun Yang, University of Michigan will focus on preclinical and clinical testing of (-) gossypol, a potent small-molecule inhibitor of Bcl-XL as a novel and targeted anticancer therapy for cancer. RAID will support non-GMP synthesis of (-)-gossypol to allow further in vitro and in vivo efficacy testing with correlative pharmacology.
11. Dr. Doris Benbrook, University of Oklahoma Health Sciences Center will focus on SHetA2 will inhibit cancer recurrence without retinoid-associated toxicities. RAID will support non-GMP synthesis of ShetA2, pharmacology, and preliminary toxicology.
12. Dr. Timothy Kinsella, Case Western Reserve University will focus on IPdR, an oral prodrug for tumor radiosensitization. RAID will support formulation development and GMP synthesis of IPdR to support a phase I clinical trial with 14-day drug exposure.
13. Dr. Andrew Raubitschek, City of Hope National Medical Center will focus on HEFi-1 antibody for clinical trials. RAID will support antibody production and ligand manufacture.
14. Dr. T-C Wu, Johns Hopkins University will focus on the production of DNA vaccine encoding calreticulin linked to E7 for treating patients with advanced cervical carcinoma. RAID will support GMP production of the vaccine and IND-directed toxicology.

Inter-Institute Program for AIDS-Related Therapeutics (IIP)
This joint effort of the National Institute of Allergy and Infectious Diseases and the NCI is designed to facilitate the preclinical development of microbicides against HIV and therapies to treat HIV, AIDS-associated malignancies, and AIDS-associated opportunistic infections. Services include high-throughput screening, animal model studies, formulation, pharmacology, and toxicology studies, and bulk substances acquisition. During this reporting period, the following projects were approved:

1. Dr. Stephen John Brown, AIDS ReSearch Alliance will focus on Prostratin, a potential adjuvant for HIV-infected individuals on highly active antiretroviral therapy. IIP support for this project will include synthesis of non-GMP material and preliminary pharmacology and toxicology studies.
2. Dr. John Donnelly, Chiron Corp. will focus on nonhuman primate evaluation of therapeutic immunization strategies for HIV. Proposed studies will evaluate immunogenicity of the prime-boost approach in macaques infected with SIV and treated with antiretroviral drugs, and the ability of immune responses to control viral replication after cessation of antiretroviral treatment.
3. Dr. Walter Chambliss, University of Mississippi will focus on 8-aminoquinoline derivative for treating Pneumocystis carnii pneumonia. This application seeks to provide sufficient quantities of GMP-quality NPC1161B to allow the conduct of preclinical and formulation development studies needed for an IND.

Rapid Access to NCI Discovery Resources (R*A*N*D)
To assist academic and nonprofit investigators in the discovery stage of anticancer drug research including the development of high-throughput screening assays, recombinant target protein production and characterization, and compound library generation. An extramural review panel advised by NCI staff reviews applications. Projects approved in the second and third cycles include:

1. Dr. Robert Shapiro, Harvard Medical School will focus on optimization of small-molecule lead compounds to inhibit angiogenin. R*A*N*D support will include the synthesis of small molecules capable of inhibiting angiogenin and suppressing the growth and metastasis of human tumors.
2. Dr. Glyn Dawson, University of Chicago will focus on palmitoyl:protein thioesterase inhibitors as antitumor drugs. R*A*N*D support will include the anticancer evaluation of existing inhibitors of PPT1 in vitro and in vivo.
3. Dr. Suzy Torti, Wake Forest University School of Medicine will focus on optimizing tach-family chelators for cancer therapy. R*A*N*D support will include the synthesis of analogs of tachpyr with improved pharmacological properties.
4. Dr. Adnan Elfarra, University of Wisconsin-Madison will focus on determination of the in vivo antitumor efficacy of novel glutathione-dependent thiopurine prodrugs. R*A*N*D will support the synthesis of 6-(2-acetylvinylthio)purine and its in vivo efficacy testing.
5. Dr. William Parker, Southern Research Institute, and Dr. Eric Sorscher, University of Alabama-Birmingham will focus on synthesis of prodrugs for mutant E. coli purine nucleoside phosphorylase (PNP). R*A*N*D will support the synthesis of sufficient quantities of purine nucleoside prodrugs for planned in vivo studies.
6. Dr. Anton Wellstein, Georgetown University will focus on therapeutic targeting of the receptor for pleiotrophin, the ALK kinase. DTP will develop and conduct a high-throughput screen and collaborate with Dr. Wellstein's group on characterizing active inhibitors.

Pharmaceutical Resources Branch (PRB)
PRB activities include acquisition of bulk materials, formulation development, analytical testing, clinical dosage-form production, and stability testing of clinical dosage forms.

PRB Compound Highlights

17-allylamino geldanamycin: This prodrug of geldanamycin is undergoing Phase I/II clinical trials. The bulk drug is being synthesized from geldanamycin, analyzed, and converted to finished dosage form.

Peptide vaccines: Several peptide vaccines were manufactured to support ongoing Phase I/II clinical trials.

E7389: This potent halichondrin B analog was received from Eisai Pharmaceuticals.

SJG-136: This pyrrolobenzodiazepine was received from CRC, UK. A co-solvent formulation containing dimethylacetamide was developed and two vial sizes, one for U.K. clinical trials and one for U.S. clinical trials, were manufactured by the production contractor.

Safingol: A counterion formulation containing lactate was developed along with an emulsion formulation. Based on toxicology consideration, the emulsion formulation will be pursued and a clinical batch will be produced this year.

Toxicology and Pharmacology Branch (T&PB)
The development of new agents in DTP is typically carried out in two stages, DDG IIA and IIB. In the preliminary IIA stage, the kinetics of the drug in question are determined in various animal species after plasma analysis methodology is developed and validated. The maximum tolerated dose (MTD) of the drug is determined. In stage IIB, full scale IND-directed toxicology studies are performed using the MTD from the earlier stage as the pivotal dose. Scheduling is established from the preclinical efficacy and pharmacology studies, the stage IIA toxicology studies and the projected clinical regimen. T&PB is responsible for conducting schedule optimization studies, pharmacological methods development, dose/range-finding studies, and IND-directed toxicity testing with correlative pharmacology and histopathology. In FY 2002 T&PB conducted pharmacology and toxicology studies on 38 approved projects, leading to eight published papers and 25 abstracts or presentations made at national scientific meetings, seven DDG presentations, and four educational presentations.

T&PB Compound Highlights

Aminoflavone Prodrug (NSC 710464): The preclinical pharmacology and toxicology data was summarized and presented to the DDG for approval at the 2B level. The toxicology study protocol to evaluate pulmonary toxicity in dogs following a 3 hr continuous infusion was developed. Study will be conducted in FY03.

5-F-Benzothiazole Lysyl Prodrug (NSC 710305): Initiated a comprehensive study to measure CYP1A1, initially limited to PBMCs, and liver and lung tissues from dogs. Conducted a pharmacokinetics/range-finding study in NHP by a 1 hr civ to determine if effective concentrations could be administered without pulmonary toxicity. Conducted an IND-directed study in dogs as a 1 hr civ on a q7dx3 schedule.

BNP-7787 (NSC 716976) +/- Oxaliplatin (NSC 266046): Conducted a combination neurotoxicity study in rats to determine if BNP7787 modulated oxaliplatin toxicity. BNP was administered intravenously prior to oxaliplatin which was administered by the ip route. The study design was a collaboration between T&PB and Bionumerik.

CDDO (NSC 711193) & CDDO-Me (NSC 713200): In vitro stability studies with both CDDO and CDDO-Methyl Ester were conducted in aqueous buffers and in plasma. This was followed by pharmacokinetic studies with CDDO-Me in mice and rats by the i.v. and oral routes of administration.

Betulinic Acid (NSC 113090): Initiated and completed cell permeation evaluation in caco-2 cells to determine potential for bioavailability. Completed 28-day IND-directed dermal studies in rats and rabbits to complete evaluation of this agent.

Liposomal DB67 (NSC 708298): Conducted range-finding toxicity studies in rats and dogs, on a dx5 schedule as an iv bolus. This completed the evaluation of DB67 since this agent was licensed to Novartis.

Biological Resources Branch (BRB) Biopharmaceutical Production
The BRB oversees a government-owned, contractor-operated facility producing clinical-grade biological agents from bacterial, yeast, and mammalian cells, natural products from different organisms, as well as DNA, RNA, and viral materials. The Biopharmaceutical Development Program (BDP) provides biological drug development expertise and production capability to NIH-supported investigators.

Biologics Production Highlights

Monoclonal antibodies released for clinical use: MuB3, an anti-Lewis Y antibody for intramural NCI use; Ch 14.18, an anti-GD2 antibody for an ongoing cooperative group trial; and 7G7, an anti-IL2R antibody for intramural NCI use.

Other Mammalian Cell Products for NCI trials: multiple clinical lots of a pancreatic cell vaccine consisting of Panc 6.03 and Panc 10.3 cell lines for a RAID-sponsored phase I/II trial, and 23 GMP lots of patient-specific idiotype vaccine material were released for intramural lymphoma trials.

Recombinant proteins for NCI clinical trials: a clinical lot of LMB-2 immunotoxin, MR1-1 immunotoxin for a RAID-sponsored brain cancer study, and two toxicology lots of IL-7 cytokine; assay and process development or production studies were performed in five other RAID-sponsored projects.

PUBLICLY AVAILABLE RESOURCES FOR DRUG DISCOVERY

Materials Repositories
DTP maintains several materials repositories that are available to the extramural community and include the following:

Synthetics Repository: The synthetics repository receives, inventories, and distributes synthetic and pure natural products to screening contract laboratories. Additionally, more than 140,000 non-proprietary compound samples are available to the extramural investigational community for research purposes. In FY02, 7028 individual samples were sent to DTP screening labs for in vitro and in vivo evaluation, and 3838 compounds were shipped to investigators around the world.

Natural Products Repository: More than 61,200 plant and 10,000 marine samples have been collected and extracted since the beginning of the collection projects in 1986. Nearly 5500 vials and 195,272 plated extracts were distributed to researchers. More than 10,500 crude extracts were processed and 28,000 plates produced.

Radiolabeled Materials: About 90 radiolabeled compounds are available for distribution to licensed researchers. The radiolabeled inventory is maintained under contract by Research Triangle Institute, NC. Radiolabeled materials are used primarily to support preclinical pharmacology, toxicology, and mechanism of action studies.

Biologics Reference and Standards Reagents: The BRB distributes reference human and mouse cytokines, and monoclonal antibodies to peer-reviewed investigators for research purposes. Cytokines for human clinical trials are made available through a mechanism similar to that of the RAID program; projects are reviewed and approved by an ad hoc committee. The clinical trial is conducted at the applicant's institution under an investigator-held IND. The cytokines, including stem cell factor, FLT-3 ligand, thrombopoietin, IL-4, IL-6, and IL-7, are available to investigators from other NIH institutions following review by that institute.

Tumor/Cell Line Repository: This is a low-temperature repository of transplantable in vivo-derived tumors and in vitro-established tumor cell lines from various species. The materials are available to qualified investigators as a service to the research community under Material Transfer Agreements. In FY02, 269 shipments of 1856 ampules were shipped.

Angiogenesis Resources Center: This center, established in response to a directive from a special NCI Director subcommittee, makes human umbilical vein endothelial cells (HUVEC) available to qualified investigators who provide evidence of peer-reviewed support. Additionally, both in vitro and in vivo testing services are offered at no cost to investigators. In FY02, 70 compounds were tested in vitro for anti-angiogenic activity to inhibit cell growth, migration, and HUVEC tube formation.

Animal Production
The animal production contract facilities produce many inbred and hybrids of inbred strains of rats, mice, and guinea pigs. The DTP oversees this unique program, which furnishes genetically defined, pathogen-free laboratory animals and services to investigators at NIH, through NIH/NCI-funded research contracts, to NIH/NCI funded grantees, and to other government agencies. In FY02, the animal production program shipped 644,443 animals representing 19,512 orders.

Screening
The DTP maintains an in vitro and in vivo screening service for NIH and extramural anticancer and anti-HIV researchers. This no-cost service is available to investigators from academia as well as industry with the proviso that the materials to be screened must pass DTP criteria for acceptance. Details of the screening program include the following:

Routine in vitro testing: The in vitro anticancer drug screen includes 60 human tumor cell lines representing various tumor types. A pre-screen model comprised of three tumor cell lines is used for initial testing of most compounds and is amenable to high-throughput screening of chemical libraries. A cell-based anti-HIV drug screen created by DTP is offered as a routine screen to the research community. Input to the routine in vitro screens is via an online submission form. In FY02, 3274 compounds were submitted for in vitro anticancer testing.

High-throughput molecular targeted drug-screening models: A major emphasis in FY02 has been the development of an infrastructure for high-throughput, molecular targeted drug screening by the Screening Technologies Branch (STB). For four of these models, results of screening the NCI Structural Diversity Set of 1990 compounds are publicly available on the DTP Web site. Two screening models, the hypoxia-inducible factor-1a screen, developed as a DTP initiative, and the CCAAT/enhancer binding protein screen, developed in collaboration with Dr. Daniel Tenen of the Harvard University School of Medicine, have been used to screen the DTP plated-compound library of 140,000 samples. Featured on the STB homepage is a chemical structure-based self-organizing map analysis of these 140,000 compounds, developed by STB staff.

In vivo testing: Hollow fiber and xenograft models conducted at NCI-Frederick Cancer Research and Development Center. The hollow-fiber assay is the gating mechanism for compounds identified through the in vitro screening program. Sixty compounds were evaluated in the standard hollow-fiber assay comprising 12 cell types. Of these, 14 compounds met the standard for activity. Five were evaluated against the leukemia hollow-fiber model and two of these were chosen for more studies. Three hundred twenty compounds or compound combinations were evaluated in 162 separate in vivo model tumor experiments. The testing used xenograft and syngeneic tumor models. Efforts to develop new mouse models for evaluating anticancer agents are also ongoing. A method was developed to provide continuous infusion therapy in tumor-bearing mice so that compounds with short half-lives have a chance to demonstrate activity. Also developed were intra-renal implant models for renal cancer that demonstrated activity for dimethane sulfonates following IP and IV drug administration. Recent acquisition of the Xenogen Ivis in vivo imaging system provides a new approach to mouse tumor model development. Current efforts to develop a panel of luciferase transfected tumor models is underway that will quantify the tumors by showing photons per second calibrated compared among the experimental animals. Other in vivo model resources include a contract for the bulk of in vivo testing of compounds active in the hollow fiber or identified through another mechanism to be worthy of further exploration. During FY02, approximately 100 agents were shipped for assessment of an MTD using, at a minimum, one route of administration. In excess of 25 agents were shipped for preliminary evaluation in human tumor xenograft models based on activity in the hollow-fiber assay. Also included is the BTB Pharmacokinetic Center, which provides pharmacokinetic and metabolism studies in support of intramural and extramural investigators. Studies in this center include NSC 652287, a novel dithiophene compound with potent and selective activity against human renal cancer cell lines. The present investigation identified the major metabolite. These observations were key to understanding the compound's mechanism of action and toxicity. Investigators have also studied NSC 710305, a benzothiazole derivative. The current studies defined in vivo disposition of new analogs and prodrugs, helping select the best clinical candidate.

Collaborative Research and Development Agreements (CRADA)
STB developed a portfolio of CRADAs related to detailed mechanistic study and development of novel agents and discovery of new agents. A mature effort is exemplified by the CRADA with SAIC that has involved high-throughput screening of the entire NCI Natural Products Repository for new agents capable of affecting the growth of drug-resistant fungal and bacterial organisms important as opportunistic infections. This CRADA effort has resulted in isolation and characterization of more than 50 antimicrobial compounds.

Support For Molecular Target Characterization
Samples of protein, DNA, or RNA derived from the 60 tumor cell lines used for in vivo drug screening are distributed to extramural investigators as part of the DTP Molecular Targets Program. During this reporting period more than 4,000 samples were prepared and shipped to 68 investigators. Molecular target characterization information generated by these investigators is curated and made available on the DTP website, together with tools for relating the target information to tumor cell line responses to drug treatment. Information Technology Branch (ITB) staff members participate in and chair the DTP Molecular Targets Team, a committee that organizes DTP interaction with outside investigators who wish to measure molecular targets in the panel of human tumor cell lines. For approved proposals, DTP provides 60-cell-line materials at no charge and assists with data analysis. Researchers agree to return the data to DTP and allow it to be posted on the DTP website after allowing sufficient time for the researcher to publish. In collaboration with the DTP Molecular Targets Team, Dr. Steve Hewitt (Laboratory of Pathology, CCR) produced a set of cell-based microarrays of the 60-cell-line panel suitable for immunohistochemistry.

Informatics
Oversight of DTP informatics is carried out by ITB. ITB informatics capabilities are subdivided into: 1) the development of web-based analysis tools and data dissemination, and 2) analysis tools applications.

Web-based Data and Tools
The DTP website provides a single access point for the intramural and extramural research community to learn what services and data are available from DTP, and to request services and data. Available at this site are data for downloading from the in vitro cancer screening of more than 37,000 compounds and 15,000 natural product extracts, AIDS data for 43,000 compounds, and yeast screening results for more than 60,000 compounds, and data from molecular target screens. Researchers will find ordering instructions for obtaining pure compounds, tumor cell lines, natural products extracts, and biological materials. Tools for searching and analyzing data include searches by chemical name, NSC number, substructure, and COMPARE searches.

ITB was able to complete a number of projects designed to improve usability of the website and to substantially increase data availability. Highlights of these projects include:

Completion of the Data Warehouse: A set of relational database tables were constructed that enabled all data to be held in one set of tables that are accessed by a library of software codes written in Java. This software library provides a consistent, unified way to access all DTP data.

New data from molecular targeted assays: One of the first tests of functionality of the new architecture was making data from the STB Molecular Targeted Assays available through the DTP website. Only three weeks elapsed from the first delivery of data for three high-throughput assays from STB to its online availability. Use of Structured Vector Graphics allowed for feature-rich presentation of data in histograms and scatter plots. Users can search for data about compounds of interest and visualize the data from each assay and across assays.

COMPARE software: COMPARE is a pattern recognition algorithm developed in the mid-1980's to analyze screening assay results, and remains a vital part of DTP work and one of the most important tools available on the DTP web site. In 2002, a re-design was completed to make COMPARE more flexible and easily expandable to include new tuypes of data. The new design allows suppliers to run COMPARE on the data for their own compounds in a secure manner.

Analysis Tools Applications For Drug Discovery
Highlights of compound-directed collaborations include the paullone series of compounds, which are small-molecule inhibitors of cyclin-dependent kinases. ITB staff members participated in the discovery and development of paullones. In early 2002, a CRADA with Enzon was signed that involved the design of paullone prodrugs. In 2001, the ITB received funding from the U.S. Army Medical Research Institute of Infectious Diseases to develop therapeutics against attacks with bioterror materials such as anthrax, botulinum neurotoxins, Staphylococcus aureus enterotoxins, and filoviruses such as Marburg and ebola. The ITB has made major strides in developing potential therapeutics for anthrax antitoxin infection since the project's inception, including setting up and standardizing a high-throughput assay to screen for inhibitors of anthrax Lethal Factor (LF), and identifying 19 lead compounds as inhibitors of anthrax LF. Based on experimental data obtained from the Lazo group at the University of Pittsburgh on cdc25 phosphatases, ITB developed a common binding mode and a mechanism of binding for a series of cdc25 phosphatase inhibitors. The binding site model has been supported by protein digestion studies and kinetics studies. ITB has optimized, compared and utilized several phosphatase crystallographic structures for purposes of evolving a structural hypothesis for cdc25b inhibitor mechanism of action.

WORKSHOPS/MEETINGS/PUBLICATIONS

Drug Development Workshops and Presentations
Drug development workshops acquaint the research community with requirements and issues that arise in bringing a preclinical discovery to the clinic, and with the drug development resources available through DTP. The intended audience includes cancer researchers from academia and industry who have preclinical discoveries and are unfamiliar with the drug development process or who may not be aware of the new DTP mission. The most recent workshop was held at the Burnham Institute, La Jolla, CA, in September 2002. T&PB toxicologists codeveloped a continuing education short course, Challenges in Development of Anticancer Drugs, for the 2002 Society of Toxicology Meeting and gave a talk, Preclinical Development Philosophy Between Different Classes of Anticancer Drugs.

Publications
In FY 2002, DTP staff counted more than 125 publications in scientific journals plus numerous abstracts and poster presentations. A complete list of these publications can be found at the DTP website.