CHEMOPREVENTION: PRINCIPLES AND PROSPECTS
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CHEMOPREVENTION: PRINCIPLES AND PROSPECTS. Gary D. Stoner, Ph.D. Department of Internal Medicine College of Medicine and Public Health The Ohio State University. Strategies for Cancer Prevention. Reduce Exposure to Environmental Carcinogens Identify Individuals at High Risk

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CHEMOPREVENTION: PRINCIPLES AND PROSPECTS

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Chemoprevention principles and prospects

CHEMOPREVENTION: PRINCIPLES AND PROSPECTS

Gary D. Stoner, Ph.D.

Department of Internal Medicine

College of Medicine and Public Health

The Ohio State University


Chemoprevention principles and prospects

Strategies for Cancer Prevention

  • Reduce Exposure to Environmental Carcinogens

  • Identify Individuals at High Risk

    • Genetic factors

    • Biochemical factors

  • Chemoprevention

    • Dietary factors

    • Synthetic agents


  • Chemoprevention principles and prospects

    Chemoprevention

    The prevention of cancer development by dietary or syntheticchemicals


    Chemoprevention drug development

    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


    Chemoprevention principles and prospects

    Chemopreventive Agents

    • Allyl sulfides

    • Calcium

    • Coumarins

    • Dithiolethiones

    • Indoles

    • Isoflavones

    • Isothiocyanates

    • Monoterpenes

    • NSAIDs

    • Polyphenols

    • Protease inhibitors

    • Retinoids

    • Selenium

    • Vitamins (A, C, D3,E)


    Chemoprevention principles and prospects

    Classes of Chemopreventive Agents

    • “Blocking” agents

      • Inhibitors of tumor initiation

  • “Suppressing” agents

    • Inhibitors of tumor promotion/ progression


  • Blocking anti initiating agents

    Blocking (Anti-Initiating) Agents

    • Influence metabolic activation of carcinogens

    • Deactivate/detoxify carcinogens

    • Scavenge electrophiles and oxygen radicals

    • Increase level of fidelity of DNA repair


    Chemoprevention principles and prospects

    Carcinogen Metabolism

    Carcinogen

    Detoxification

    Activation

    Conjugates

    Genetic (DNA)

    Damage

    Excreted

    Cancer


    Agents that influence carcinogen activation

    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


    Chemoprevention principles and prospects

    Isothiocyanates

    N-Nitrosomethylbenzylamine (NMBA)

    3-Phenylpropyl isothiocyanate (PPITC)

    Benzyl isothiocyanate (BITC)

    4-Phenylbutyl isothiocyanate (PBITC)

    Phenethyl isothiocyanate (PEITC)

    6-Phenylhexyl isothiocyanate (PHITC)


    Chemoprevention principles and prospects

    NMBA Metabolism

    (NMBA)

    cytochrome p450

    -hydroxynitrosamine

    [ CH3N = NOH ]

    methyldiazohydroxide

    benzaldehyde

    [ CH3+N  N ]

    methyldiazonium ion

    carbonium ion

    [ CH3+]

    O6-Methylguanine

    N7-Methylguanine


    Chemoprevention principles and prospects

    Protocol For Identification of Blocking Agents in Rat Esophagus

    NMBA (1x/wk/15 wks)

    0

    2

    4

    19

    25

    Inhibitor


    Chemoprevention principles and prospects

    Effect of Isothiocyanates on DNA Methylation and Tumorigenesis in Rat Esophagus

    ap < 0.05


    Agents that deactivate detoxify carcinogens

    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


    Chemoprevention principles and prospects

    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)


    Induction of qr and gst in mouse tissues by sulforaphane 15 mol mouse day

    Induction of QR and GST in Mouse Tissues by Sulforaphane*(15 mol / mouse / day)

    QR = Quinone reductase

    GST = Glutathione-S-transferase


    Protective effects of sulforaphane on dmba induced mammary tumors in rats

    Protective Effects of Sulforaphane on DMBA-Induced Mammary Tumors in Rats*

    *Y.Zhang, PNAS 91: 3147, 1994


    Suppressing agents

    Suppressing Agents

    • Inhibit cell growth

    • Stimulate cell function

    • Stimulate apoptosis

    • Inhibit blood vessel formation (angiogenesis)


    Chemoprevention principles and prospects

    POLYAMINE SYNTHESIS

    ODC

    Ornithine

    Spermine

    Spermidine

    Putrescine

    Carbon dioxide

    • Chemopreventive Agents:

    • difluromethylornithine (DFMO)

    • curcumin


    Chemoprevention principles and prospects

    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)


    Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    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


    Cox activity and human cancers

    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


    Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    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.


    Chemoprevention principles and prospects

    Human Clinical Trials

    • Phase I

      • Toxicity, metabolism

    • Phase II

      • Biomarkers

    • Phase III

      • Long term prospective studies


    Target populations for chemoprevention

    Target Populations for Chemoprevention

    High Risk Individuals

    • Hereditary predisposition

    • High exposure to carcinogens

    • precancerous lesions

    • previous treatment for cancer

      General Population ?


    Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    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


  • Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    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


  • Chemoprevention principles and prospects

    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)


  • Chemoprevention principles and prospects

    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


  • Barrett s esophagus

    Barrett’s Esophagus

    Normal

    Barrett’s Esophagus


    Esophageal cancer

    Esophageal Cancer


    Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    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


    Chemoprevention principles and prospects

    Phase III Strategies

    • Target Populations

    • Length of Study

    • Conclusiveness of Study

    • Statistical Methods


    Chemoprevention principles and prospects

    Combination Strategies

    • Synergistic Activity

    • Lower Doses of Each  Lower Toxicity + Fewer Adverse Effects

    • Mechanistic Combination Strategy (Anti- Initiator/ Anti-Proliferator)


    Chemoprevention principles and prospects

    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


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