Approval of Natural Chemopreventive Product

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

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

Toxicity Testing Route of administration : OralRoute of administration : Oral Test 1: Range finding for LD 50Test 1: Range finding for LD 50 –5 mice/Sex/Dose plus control group. –5 doses Test 2: The LD 50 determinationTest 2: The LD 50 determination –5 mice/Sex/Dose plus control group. –5 doses base on Test 1.

Toxicity Testing (Cont.) Test 3: The LD 50 determination (finely tuned)Test 3: The LD 50 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 testTest 4: Subchronic toxicity test –5 mice/Sex plus control group. –Dose 10 times lower than LD 50 (from Test 3) –Duration of treatment 90 days. –Observe animals for 360 days. –Histopathological examination for dose-related lesions.

Toxicity Testing (Cont.) Test 5: Chronic toxicity testTest 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.

Chemoprevention Potency Test To evaluate the chemopreventive protency of the product.To evaluate the chemopreventive protency of the product. 2 tests2 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.HBV gene transgenic mice use in the studies to reduce number of mice. Number of mice useNumber of mice use –10/sex/group

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

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

Clinical Evaluation : Phase I ObjectivesObjectives –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. SubjectsSubjects –60 healthy normal volunteers (30 males + 30 females).

Clinical Evaluation : Phase I (cont.) TreatmentTreatment –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 analysisSample 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.For toxicity grading, subjects will be evaluate for acute toxicity using standard U.S. National Cancer Institute toxicity criteria.U.S. National Cancer InstituteU.S. National Cancer Institute

Clinical Evaluation : Phase II ObjectiveObjective –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 studyStudy : placebo control, double blind study

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

Clinical Evaluation : Phase II (cont.) TreatmentTreatment –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.

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

Clinical Evaluation : Phase III (cont.) Screening of subjectsScreening of subjects –Individual screening. –Determine base line level of aflatoxin–N- acetylcysteine in urine by HPLC. TreatmentTreatment –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.

Post-clinical Evaluation : Phase IV ObjectiveObjective –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. SubjectsSubjects –Same individual in phase III study. –Customers using the product. ObservationObservation –Observe the side effects for 10 years after product on to the market.

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