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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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slide1

CHEMOPREVENTION: PRINCIPLES AND PROSPECTS

Gary D. Stoner, Ph.D.

Department of Internal Medicine

College of Medicine and Public Health

The Ohio State University

slide2

Strategies for Cancer Prevention

  • Reduce Exposure to Environmental Carcinogens
  • Identify Individuals at High Risk
      • Genetic factors
      • Biochemical factors
  • Chemoprevention
      • Dietary factors
      • Synthetic agents
slide3

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

slide6

Chemopreventive Agents

  • Allyl sulfides
  • Calcium
  • Coumarins
  • Dithiolethiones
  • Indoles
  • Isoflavones
  • Isothiocyanates
  • Monoterpenes
  • NSAIDs
  • Polyphenols
  • Protease inhibitors
  • Retinoids
  • Selenium
  • Vitamins (A, C, D3,E)
slide7

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
slide9

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

slide11

Isothiocyanates

N-Nitrosomethylbenzylamine (NMBA)

3-Phenylpropyl isothiocyanate (PPITC)

Benzyl isothiocyanate (BITC)

4-Phenylbutyl isothiocyanate (PBITC)

Phenethyl isothiocyanate (PEITC)

6-Phenylhexyl isothiocyanate (PHITC)

slide12

NMBA Metabolism

(NMBA)

cytochrome p450

-hydroxynitrosamine

[ CH3N = NOH ]

methyldiazohydroxide

benzaldehyde

[ CH3+N  N ]

methyldiazonium ion

carbonium ion

[ CH3+]

O6-Methylguanine

N7-Methylguanine

slide15

Protocol For Identification of Blocking Agents in Rat Esophagus

NMBA (1x/wk/15 wks)

0

2

4

19

25

Inhibitor

slide16

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
slide18

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)
slide22

POLYAMINE SYNTHESIS

ODC

Ornithine

Spermine

Spermidine

Putrescine

Carbon dioxide

  • Chemopreventive Agents:
  • difluromethylornithine (DFMO)
  • curcumin
slide23

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)
slide24

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

slide25

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
slide27

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

slide28

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
slide29

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
slide30

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.
slide31

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 ?

slide33

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
slide34

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
slide35

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
slide36

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
slide37

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
slide38

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)
slide39

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

slide42

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

slide43

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
slide44

Phase III Strategies

  • Target Populations
  • Length of Study
  • Conclusiveness of Study
  • Statistical Methods
slide45

Combination Strategies

  • Synergistic Activity
  • Lower Doses of Each  Lower Toxicity + Fewer Adverse Effects
  • Mechanistic Combination Strategy (Anti- Initiator/ Anti-Proliferator)
slide46

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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