Approval of Natural Chemopreventive Product

1. Approval of Natural Chemopreventive Product

2. Scope of The Study • Preclinical evaluation (In vivo) • Toxicity testing • Acute toxicity • Subchronic toxicity • Chronic toxicity • Chemoprevention potency testing • Clinical evaluation • Phase I clinical trials • Phase II clinical trials • Phase III clinical trials • Post-clinical evaluation • Phase IV marketing post-marketing surveillance

3. Preclinical Evaluation • Crude extract from broccoli sprout. • Crude extract from curcumin. • The chemopreventive product (combination of curcumin and broccoli, lecithin use as a vehicle)

4. Toxicity Testing • Route of administration : Oral • Test 1: Range finding for LD50 • 5 mice/Sex/Dose plus control group. • 5 doses • Test 2: The LD50 determination • 5 mice/Sex/Dose plus control group. • 5 doses base on Test 1.

5. Toxicity Testing (Cont.) • Test 3: The LD50 determination (finely tuned) • 5 mice/Sex/Dose plus control group. • 5 doses base on Test 2. • Histopathological examination for dose-related lesions. • Test 4: Subchronic toxicity test • 5 mice/Sex plus control group. • Dose 10 times lower than LD50 (from Test 3) • Duration of treatment 90 days. • Observe animals for 360 days. • Histopathological examination for dose-related lesions.

6. Toxicity Testing (Cont.) • Test 5: Chronic toxicity test • 10 mice/Sex plus control group. • Dose : Lowest no effect dose in Test 4. • Duration of treatment 1 year. • Observe animals life span. • Histophathology.

7. Chemoprevention Potency Test • To evaluate the chemopreventive protency of the product. • 2 tests • Test I : No background of AFB1 exposed. • Test II : Exposed to AFB1 before the product administration. • HBV gene transgenic mice use in the studies to reduce number of mice. • Number of mice use • 10/sex/group

8. 3 weeks of age Positive control (Oltipraz) Chemopreventive agent Control Developed tumor Non tumor Non tumor Chemoprevention Potency Test I Daily administration of the product 1 week Expose to dietary AFB1 52 weeks

9. 3 weeks of age 1 weeks after AFB1 Positive control (Oltipraz) Chemopreventive agent Control Heavy tumor burden Reduce tumor burden Reduce tumor burden Chemoprevention Potency Test II AFB1 single dose (6 mg/kg by i.p.) 52 weeks

10. Clinical Evaluation : Phase I • Objectives • To obtain pharmacokinetics of the product following single p.o. dose of the product. • To investigate the induction of GST in lymphocytes. • To evaluate toxicity associated with a single p.o. dose of the product. • Subjects • 60 healthy normal volunteers (30 males + 30 females).

11. Clinical Evaluation : Phase I (cont.) • Treatment • 30 volunteers (15 males + 15 females) at dose X • 30 volunteers (15 males + 15 females) at dose Y • Pharmacokinetics study : Blood sample will be collect before administration and at 1, 2, 3, 4, 5, 6, 8, 16 and 24h after dosing to measure curcuminoid and sulforaphane levels. • Investigation of GST study : Blood sample will be collect before administration and at 6, 10 and 24h after dosing to measure GST levels. • Sample analysis • The samples will be measure by HPLC. • For toxicity grading, subjects will be evaluate for acute toxicity using standard U.S. National Cancer Institute toxicity criteria.

12. Clinical Evaluation : Phase II • Objective • To preliminary assess the efficacy of the product by examining modulation in the levels of several biomarkers of aflatoxin in urine. • To characterize in more detail the rang of dose-limiting toxicities in a potential target population including individuals infected with HBV. • Study : placebo control, double blind study

13. Clinical Evaluation : Phase II(cont.) • Subjects • Normal people (100 males + 100 females) • Product recipient (50 males + 50 females) • Placebo concurrent control (50 males + 50 females) • HBV carrier (50 males + 50 females) • Screening of subjects • Individual screening. • Determine base line level of aflatoxin–N-acetylcysteine in urine by HPLC.

14. Clinical Evaluation : Phase II(cont.) • Treatment • Daily administration for 8 weeks. • Placebo • Define dose 1 • Define dose 2 • Urine samples collect once a week for 9 weeks after that once in 2 weeks till week 17.

15. Clinical Evaluation : Phase III • Objective • The design of this phase is based on the finding in phase II to validate instruction for use and for imaging in the population. • Subjects • Normal people (200 males + 200 females) • Product recipient (100 males + 100 females) • Placebo concurrent control (100 males + 100 females) • HBV carrier (100 males + 100 females) • Liver resected (100 males + 100 females)

16. Clinical Evaluation : Phase III(cont.) • Screening of subjects • Individual screening. • Determine base line level of aflatoxin–N-acetylcysteine in urine by HPLC. • Treatment • Administration dose base on phase II. • Daily administration for 24 months. • Urine samples collect once a month for 12 months after that once in 2 months till month 24.

17. Post-clinical Evaluation : Phase IV • Objective • Designed to detect any rare or long-term adverse effects over a much larger population and timescale than was possible during the initial clinical trials. • Subjects • Same individual in phase III study. • Customers using the product. • Observation • Observe the side effects for 10 years after product on to the market.

18. Conclusion • The product is safe for the population and high chemoprevention potency. • The product will on to the market as dietary supplement and above 10 years old children milk.