CHEMOPREVENTION: PRINCIPLES AND PROSPECTSGary D. Stoner, Ph.D.Department of Internal MedicineCollege of Medicine and Public Health The Ohio State University

Strategies for Cancer PreventionReduce Exposure to Environmental CarcinogensIdentify Individuals at High RiskGenetic factorsBiochemical factorsChemopreventionDietary factorsSynthetic agents

ChemopreventionThe prevention of cancer development by dietary or synthetic chemicals

Chemoprevention Drug DevelopmentPreclinical: Natural Products Synthetic AgentsClinical: In vitro AssaysAnti-mutagenic Cell Trans-formationetc.In vivo assays

TumorsBiomarkersToxicologyPhase I

ToxicityMetabolismPhase II

EfficacyBiomarkersPhase III

EfficacyCancerEndpoint

Qualities of an Ideal Chemopreventive Agent

Allyl sulfidesCalciumCoumarinsDithiolethionesIndolesIsoflavonesIsothiocyanatesMonoterpenesNSAIDsPolyphenolsProtease inhibitorsRetinoidsSeleniumVitamins (A, C, D3,E)Chemopreventive Agents

Classes of Chemopreventive AgentsBlocking agentsInhibitors of tumor initiationSuppressing agentsInhibitors of tumor promotion/ progression

Blocking (Anti-Initiating) AgentsInfluence metabolic activation of carcinogensDeactivate/detoxify carcinogensScavenge electrophiles and oxygen radicalsIncrease level of fidelity of DNA repair

Carcinogen MetabolismCarcinogenGenetic (DNA)DamageCancerConjugatesExcretedDetoxificationActivation

Agents That Influence Carcinogen ActivationMechanismInhibition of cytochromeP450

Induction of cytochrome P450Examplesdithiocarbamatesellagic aciddiallyl sulfideisothiocyanates

indole-3-carbinol-naphthoflavoneM.A. Morse and G.D. Stoner, 1993

IsothiocyanatesPhenethyl isothiocyanate (PEITC)Benzyl isothiocyanate (BITC)3-Phenylpropyl isothiocyanate (PPITC)4-Phenylbutyl isothiocyanate (PBITC)6-Phenylhexyl isothiocyanate (PHITC)N-Nitrosomethylbenzylamine (NMBA)

[ CH3N = NOH ][ CH3+N N ][ CH3+]benzaldehyde(NMBA)cytochrome p450-hydroxynitrosaminemethyldiazohydroxidemethyldiazonium ioncarbonium ionN7-MethylguanineO6-MethylguanineNMBA Metabolism

0225NMBA (1x/wk/15 wks)Inhibitor419Protocol For Identification of Blocking Agents in Rat Esophagus

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

TreatmentO6-Methylguanine(mol/mol guanine)Tumors / RatNMBA19.6 0.67.2 0.7NMBA + BITC16.0 0.4 6.5 0.6NMBA + PEITC 2.1 0.2a0.1 0.1aNMBA + PPITC 3.0 0.3a0.0 0.0aNMBA + PBITC 7.0 0.6a4.0 0.4NMBA + PHITC 20.7 1.011.9 0.7a

Agents That Deactivate/Detoxify CarcinogensMechanismIntroduction of phase II enzymesglutathione-S-transferasesUDP-glucuronyl-transferasesglutathione peroxidaseExamples

allyl sulfides, dithioethiones, isothiocyanatespolyphenolsselenium

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

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

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

Suppressing AgentsInhibit cell growthStimulate cell functionStimulate apoptosisInhibit blood vessel formation (angiogenesis)

Ornithine PutrescineSpermidineSpermineCarbon dioxideODCChemopreventive Agents:difluromethylornithine (DFMO)curcuminPOLYAMINE SYNTHESIS

DFMO as an Anti-Tumor AgentAzoxymethane-induced rat colon tumor modelinhibits tumor incidence and multiplicitydecreases cell proliferationdecreases activated ras expression NMBA-induced rat esophagus modelinhibits tumorigenesis when given before, during and after NMBAdecreases cell proliferation and increases apoptosis Human NeoplasiasBarretts esophagus; colonic polyps; oral leukoplakia; uterine cervix (CIN 3)

Action of COX Enzymes

Inhibitors of COX ActivityNSAIDsAspirin, Indomethacin, Ibuprofen, NaproxenEffective chemoprevention agents in rodent skin, breast and colon tumor modelsAspirin may be effective as a prevention agent in human esophageal cancer Selective COX-2 inhibitorsCelecoxib, Rofecoxib and othersAnimal studies indicate that these agents are as effective and better tolerated than NSAIDs

COX Activity and Human CancersHuman BreastElevated COX-2 activity is seen in tumorsNSAID use is associated with reduction in susceptibility to breast cancer (Robertson, Harris)Human ColonColon polyps have elevated COX-2 activityTreatment of FAP patients with Celecoxib leads to polyp regression

Transcription factors

Transcription Factors and TumorigenicityAP-1Regulates 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 modelAgents that inhibit AP-1 activity, such as retinoids, are effective chemoprevention agents in this modelThe role of altered AP-1 activity in other epithelial tumors is unknown at present

Transcription Factors and TumorigenicityNFkBElevated activity seen in mouse skin modelElevated in human Barretts esophagus and esophageal cancersIn cell culture models, elevated NFkB activity correlates with increased resistance to apoptosisSelective inhibitors of NFkB activity are unavailable at present

Tumor AngiogenesisNeovascularization 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.

Human Clinical TrialsPhase IToxicity, metabolismPhase IIBiomarkersPhase IIILong term prospective studies

Target Populations for ChemopreventionHigh Risk IndividualsHereditary predispositionHigh exposure to carcinogensprecancerous lesionsprevious treatment for cancerGeneral Population ?

Chemoprevention Trials EvaluationModulation of Intermediate Endpoints (Biomarkers)Prevention and/or Reversal of Precancerous LesionsExtension of Latency Period for Cancer DevelopmentReduction of Cancer Incidence and Mortality

PopulationUsually high risk18-24 subjectsEscalating DoseObjectivesDetermine pharmacokinetic parametersMinimum and maximum tolerated doseTime course of the reversibility of side effectsDose selection for Phase II trialsPhase I Trials

Phase I Clinical Trial of Freeze-Dried Black Raspberries10 normal volunteers, > 18 years of agePhenol-free diet90 g BRB / day, 14 daysBlood and urineClinical signs of toxicity

Results of Phase I Clinical Trial of Freeze-Dried Black RaspberriesWell tolerated

blood levels of ellagic acid and several anthocyanins

Reduced levels of 8-OH-dG in urine

Phase II TrialsRandomized placebo controlledPopulationHigh risk100-200 subjectsObjectivesFurther evaluation of toxicityDose response vs. biomarker modulationDose selection for Phase III trials

BIOMARKERSAnti-InitiationPhase I enzyme activities Phase II enzyme activitiesDNA repair enzyme activitiesDNA adducts (carcinogen and free radicals)Hemoglobin adductsUrinary metabolitesDetoxification productsMutagenicity assaysMicronuclei, binucleated cellsChromosome damage (FISH)

BIOMARKERSAnti-Promotion/ProgressionProliferation Index: PCNA, Ki67, BrdU, 3H-thymidineODC activity; polyamine levelsGrowth factor and receptor expressionProstaglandin levelsCell differentiationBlood group antigensKeratinsRetinoid receptorsSquamous metaplasia mucociliary differentiationApoptosisMorphometricDNA ploidyNuclear/nucleolar-morphology

Barretts EsophagusNormalBarretts Esophagus

Esophageal Cancer

Multi-Center Clinical Trial Study SchemaBarretts EsophagusIntestinal Type MetaplasiaLow Grade DysplasiaBiopsies for SurrogateEndpoint for BiomarkersRandomizeDFMO 900mg, Once Dailyx 26 WeeksEndscopy with Biopsies forEndpoint Biomarkers26 Weeks, No TreatmentEndoscopy with Biopsies forEndpoint BiomarkersPlacebo, Once Dailyx 26 WeeksEndoscopy with Biopsies forEndpoint Biomarkers26 Weeks, No TreatmentEndoscopy with Biopsies forEndpoint Biomarkers

Determine if oral DFMO given in a randomized, placebo-controlled,double-blinded study will significantly alter:

Primary Endpoint:Ki-67 labeling indexSecondary Endpoints: p53 protein accumulation Levels of Cyclin D1, EGFR, or TGF-alpha DNA ploidy, nuclear and nucleolar morphometry Cellular apoptosis Pathology & morphology of Barretts

Overall Study Objectives

Phase III StrategiesTarget Populations

Length of Study

Conclusiveness of Study

Statistical Methods

Combination StrategiesSynergistic ActivityLower Doses of Each Lower Toxicity + Fewer Adverse EffectsMechanistic Combination Strategy (Anti- Initiator/ Anti-Proliferator)

CollaboratorsOSURobeena AzizPeter CarltonTong ChenAshok GuptaKeith HarrisTamaro HudsonBeth ListonMark MorseRon NinesHaiyan Qin

OSU - Former StudentsRajaram GopalakrishnanLeena KhareLaura KrestyDian WangQian-Shu WangAmerican Health FoundationFung-Lung ChungUniv. of MinnesotaStephen HechtSharon Murphy