CHEMOPREVENTION: PRINCIPLES AND PROSPECTS

1. CHEMOPREVENTION: PRINCIPLES AND PROSPECTS Gary D. Stoner, Ph.D. Department of Internal Medicine College of Medicine and Public Health The Ohio State University

2. Strategies for Cancer Prevention • Reduce Exposure to Environmental Carcinogens • Identify Individuals at High Risk • Genetic factors • Biochemical factors • Chemoprevention • Dietary factors • Synthetic agents

3. Chemoprevention The prevention of cancer development by dietary or syntheticchemicals

4. Chemoprevention Drug Development Preclinical: Natural Products Synthetic Agents Clinical: In vitro Assays Anti-mutagenic Cell Trans- formation etc. In vivo assays Tumors Biomarkers Phase I Toxicity Metabolism Phase II Efficacy Biomarkers Phase III Efficacy Cancer Endpoint Toxicology

6. Chemopreventive Agents • Allyl sulfides • Calcium • Coumarins • Dithiolethiones • Indoles • Isoflavones • Isothiocyanates • Monoterpenes • NSAIDs • Polyphenols • Protease inhibitors • Retinoids • Selenium • Vitamins (A, C, D3,E)

7. Classes of Chemopreventive Agents • “Blocking” agents • Inhibitors of tumor initiation • “Suppressing” agents • Inhibitors of tumor promotion/ progression

8. Blocking (Anti-Initiating) Agents • Influence metabolic activation of carcinogens • Deactivate/detoxify carcinogens • Scavenge electrophiles and oxygen radicals • Increase level of fidelity of DNA repair

9. Carcinogen Metabolism Carcinogen Detoxification Activation Conjugates Genetic (DNA) Damage Excreted Cancer

10. Mechanism Inhibition of cytochrome P450 Induction of cytochrome P450 Examples dithiocarbamates ellagic acid diallyl sulfide isothiocyanates indole-3-carbinol -naphthoflavone Agents That Influence Carcinogen Activation M.A. Morse and G.D. Stoner, 1993

11. Isothiocyanates N-Nitrosomethylbenzylamine (NMBA) 3-Phenylpropyl isothiocyanate (PPITC) Benzyl isothiocyanate (BITC) 4-Phenylbutyl isothiocyanate (PBITC) Phenethyl isothiocyanate (PEITC) 6-Phenylhexyl isothiocyanate (PHITC)

12. NMBA Metabolism (NMBA) cytochrome p450 -hydroxynitrosamine [ CH3N = NOH ] methyldiazohydroxide benzaldehyde [ CH3+N  N ] methyldiazonium ion carbonium ion [ CH3+] O6-Methylguanine N7-Methylguanine

15. Protocol For Identification of Blocking Agents in Rat Esophagus NMBA (1x/wk/15 wks) 0 2 4 19 25 Inhibitor

16. Effect of Isothiocyanates on DNA Methylation and Tumorigenesis in Rat Esophagus ap < 0.05

17. Mechanism Introduction of phase II enzymes glutathione-S-transferases UDP-glucuronyl-transferases glutathione peroxidase Examples allyl sulfides, dithioethiones, isothiocyanates polyphenols selenium Agents That Deactivate/Detoxify Carcinogens

18. Sulforaphane Y. Zhang, P. Talalay, C.G. Cho, and G.H. Posner, A major inducer of anticarcinogenic protective enzymes from broccoli: isolation and elucidation of structure. Proc Natl Acad Sci USA 89 2399 (1992)

19. Induction of QR and GST in Mouse Tissues by Sulforaphane*(15 mol / mouse / day) QR = Quinone reductase GST = Glutathione-S-transferase

20. Protective Effects of Sulforaphane on DMBA-Induced Mammary Tumors in Rats* *Y.Zhang, PNAS 91: 3147, 1994

21. Suppressing Agents • Inhibit cell growth • Stimulate cell function • Stimulate apoptosis • Inhibit blood vessel formation (angiogenesis)

22. POLYAMINE SYNTHESIS ODC Ornithine Spermine Spermidine Putrescine Carbon dioxide • Chemopreventive Agents: • difluromethylornithine (DFMO) • curcumin

23. DFMO as an Anti-Tumor Agent • Azoxymethane-induced rat colon tumor model • inhibits tumor incidence and multiplicity • decreases cell proliferation • decreases activated ras expression • NMBA-induced rat esophagus model • inhibits tumorigenesis when given before,during and after NMBA • decreases cell proliferation and increases apoptosis • Human Neoplasias • Barrett’s esophagus; colonic polyps; oral leukoplakia; uterine cervix (CIN 3)

24. COOH Arachidonic Acid Cyclooxygenase Activity COX-1 COX-2 PGG2 Peroxidase Activity PGH2 Prostacyclin Prostaglandins Thromboxane PCI2 PGE2, PGF2, PGD2 TxA2 Action of COX Enzymes

25. Inhibitors of COX Activity • NSAIDs • Aspirin, Indomethacin, Ibuprofen, Naproxen • Effective chemoprevention agents in rodent skin, breast and colon tumor models • Aspirin may be effective as a prevention agent in human esophageal cancer • Selective COX-2 inhibitors • Celecoxib, Rofecoxib and others • Animal studies indicate that these agents are as effective and better tolerated than NSAIDs

26. COX Activity and Human Cancers Human Breast • Elevated COX-2 activity is seen in tumors • NSAID use is associated with reduction in susceptibility to breast cancer (Robertson, Harris) Human Colon • Colon polyps have elevated COX-2 activity • Treatment of FAP patients with Celecoxib leads to polyp regression

27. Growth factor receptor Other stimuli ras ceramide Elevated ROS levels raf Elevated NFkB activity MEK-1 Erks Elevated AP-1 activity Altered gene expression Increased cell proliferation, resistance to apoptosis Transcription factors

28. Transcription Factors and Tumorigenicity • AP-1 • Regulates transcription of genes involved in cell proliferation, differentiation and modulation of extracellular matrix • Elevated AP-1 activity is causally related to tumor promotion and progression in the mouse skin model • Agents that inhibit AP-1 activity, such as retinoids, are effective chemoprevention agents in this model • The role of altered AP-1 activity in other epithelial tumors is unknown at present

29. Transcription Factors and Tumorigenicity • NFkB • Elevated activity seen in mouse skin model • Elevated in human Barrett’s esophagus and esophageal cancers • In cell culture models, elevated NFkB activity correlates with increased resistance to apoptosis • Selective inhibitors of NFkB activity are unavailable at present

30. Tumor Angiogenesis • Neovascularization is essential for sustained tumor growth as well as establishment of metastases. • Anti-angiogenesis factors include endostatin, a product of plasminogen, that inhibits new vessel growth in tumor xenografts in mice. Endostatin is presently being evaluated in human clinical trials for its efficacy against metastatic disease.

31. Human Clinical Trials • Phase I • Toxicity, metabolism • Phase II • Biomarkers • Phase III • Long term prospective studies

32. Target Populations for Chemoprevention High Risk Individuals • Hereditary predisposition • High exposure to carcinogens • precancerous lesions • previous treatment for cancer General Population ?

33. Chemoprevention Trials Evaluation • Modulation of Intermediate Endpoints (Biomarkers) • Prevention and/or Reversal of Precancerous Lesions • Extension of Latency Period for Cancer Development • Reduction of Cancer Incidence and Mortality

34. Phase I Trials • Population • Usually high risk • 18-24 subjects • Escalating Dose • Objectives • Determine pharmacokinetic parameters • Minimum and maximum tolerated dose • Time course of the reversibility of side effects • Dose selection for Phase II trials

35. Phase I Clinical Trial of Freeze-Dried Black Raspberries • 10 normal volunteers, > 18 years of age • “Phenol-free” diet • 90 g BRB / day, 14 days • Blood and urine • Clinical signs of toxicity

36. Results of Phase I Clinical Trial of Freeze-Dried Black Raspberries • Well tolerated •  blood levels of ellagic acid and several anthocyanins • Reduced levels of 8-OH-dG in urine

37. Phase II Trials • Randomized placebo controlled • Population • High risk • 100-200 subjects • Objectives • Further evaluation of toxicity • Dose response vs. biomarker modulation • Dose selection for Phase III trials

38. BIOMARKERS • Anti-Initiation • Phase I enzyme activities • Phase II enzyme activities • DNA repair enzyme activities • DNA adducts (carcinogen and free radicals) • Hemoglobin adducts • Urinary metabolites • Detoxification products • Mutagenicity assays • Micronuclei, binucleated cells • Chromosome damage (FISH)

39. BIOMARKERS • Anti-Promotion/Progression • Proliferation Index: PCNA, Ki67, BrdU, 3H-thymidine • ODC activity; polyamine levels • Growth factor and receptor expression • Prostaglandin levels • Cell differentiation • Blood group antigens • Keratins • Retinoid receptors • Squamous metaplasia mucociliary differentiation • Apoptosis • Morphometric • DNA ploidy • Nuclear/nucleolar-morphology

40. Barrett’s Esophagus Normal Barrett’s Esophagus

41. Esophageal Cancer

42. Multi-Center Clinical Trial Study Schema Barrett’s Esophagus Intestinal Type Metaplasia Low Grade Dysplasia Biopsies for Surrogate Endpoint for Biomarkers Randomize DFMO 900mg, Once Daily x 26 Weeks Placebo, Once Daily x 26 Weeks Endoscopy with Biopsies for Endpoint Biomarkers Endscopy with Biopsies for Endpoint Biomarkers 26 Weeks, No Treatment 26 Weeks, No Treatment Endoscopy with Biopsies for Endpoint Biomarkers Endoscopy with Biopsies for Endpoint Biomarkers

43. Overall Study Objectives • Determine if oral DFMO given in a randomized, placebo-controlled, • double-blinded study will significantly alter: • Primary Endpoint: Ki-67 labeling index • Secondary Endpoints: • p53 protein accumulation • Levels of Cyclin D1, EGFR, or TGF-alpha • DNA ploidy, nuclear and nucleolar morphometry • Cellular apoptosis • Pathology & morphology of Barrett’s

44. Phase III Strategies • Target Populations • Length of Study • Conclusiveness of Study • Statistical Methods

45. Combination Strategies • Synergistic Activity • Lower Doses of Each  Lower Toxicity + Fewer Adverse Effects • Mechanistic Combination Strategy (Anti- Initiator/ Anti-Proliferator)

46. Collaborators OSU Robeena Aziz Peter Carlton Tong Chen Ashok Gupta Keith Harris Tamaro Hudson Beth Liston Mark Morse Ron Nines Haiyan Qin OSU - Former Students Rajaram Gopalakrishnan Leena Khare Laura Kresty Dian Wang Qian-Shu Wang American Health Foundation Fung-Lung Chung Univ. of Minnesota Stephen Hecht Sharon Murphy