Approval of Natural Approval of Natural Chemopreventive ProductChemopreventive Product

Scope of The StudyScope of The Study

• Preclinical evaluation (In vivo)Preclinical evaluation (In vivo)– Toxicity testingToxicity testing

Acute toxicityAcute toxicity Subchronic toxicitySubchronic toxicity Chronic toxicityChronic toxicity

– Chemoprevention potency testingChemoprevention potency testing

• Clinical evaluationClinical evaluation– Phase I clinical trialsPhase I clinical trials– Phase II clinical trialsPhase II clinical trials– Phase III clinical trialsPhase III clinical trials

• Post-clinical evaluationPost-clinical evaluation– Phase IV marketing post-marketing surveillancePhase IV marketing post-marketing surveillance

Preclinical EvaluationPreclinical Evaluation

• Crude extract from broccoli sprout.Crude extract from broccoli sprout.

• Crude extract from curcumin.Crude extract from curcumin.

• The chemopreventive product The chemopreventive product (combination of curcumin and broccoli, (combination of curcumin and broccoli, lecithin use as a vehicle)lecithin use as a vehicle)

Toxicity TestingToxicity Testing

• Route of administration : OralRoute of administration : Oral

• Test 1: Range finding for LDTest 1: Range finding for LD5050

– 5 mice/Sex/Dose plus control group.5 mice/Sex/Dose plus control group.– 5 doses5 doses

• Test 2: The LDTest 2: The LD5050 determination determination– 5 mice/Sex/Dose plus control group.5 mice/Sex/Dose plus control group.– 5 doses base on Test 1.5 doses base on Test 1.

Toxicity Testing (Cont.)Toxicity Testing (Cont.)

• Test 3: The LDTest 3: The LD5050 determination (finely tuned) determination (finely tuned)– 5 mice/Sex/Dose plus control group.5 mice/Sex/Dose plus control group.– 5 doses base on Test 2.5 doses base on Test 2.– Histopathological examination for dose-related lesions.Histopathological examination for dose-related lesions.

• Test 4: Subchronic toxicity testTest 4: Subchronic toxicity test– 5 mice/Sex plus control group.5 mice/Sex plus control group.– Dose 10 times lower than LDDose 10 times lower than LD5050 (from Test 3) (from Test 3)– Duration of treatment 90 days.Duration of treatment 90 days.– Observe animals for 360 days.Observe animals for 360 days.– Histopathological examination for dose-related lesions.Histopathological examination for dose-related lesions.

Toxicity Testing (Cont.)Toxicity Testing (Cont.)

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

Chemoprevention Potency TestChemoprevention Potency Test

• To evaluate the chemopreventive protency of To evaluate the chemopreventive protency of the product.the product.

• 2 tests 2 tests – Test I : No background of AFB1 exposed.Test I : No background of AFB1 exposed.– Test II : Exposed to AFB1 before the product Test II : Exposed to AFB1 before the product

administration.administration.

• HBV gene transgenic mice use in the studies to HBV gene transgenic mice use in the studies to reduce number of mice.reduce number of mice.

• Number of mice useNumber of mice use– 10/sex/group10/sex/group

Chemoprevention Potency Test IChemoprevention Potency Test I

3 weeks of age3 weeks of age

Chemopreventive Chemopreventive agentagent ControlControl

Non tumorNon tumor

52 weeks52 weeks

Developed tumorDeveloped tumor

Expose to dietary Expose to dietary AFB1AFB1

Positive control Positive control (Oltipraz)(Oltipraz)

Non tumorNon tumor

Daily administration Daily administration of the productof the product1 week1 week

Chemoprevention Potency Test IIChemoprevention Potency Test II3 weeks of age3 weeks of age

1 weeks after AFB11 weeks after AFB1

Chemopreventive Chemopreventive agentagent ControlControl

Reduce tumor burdenReduce tumor burden

52 weeks52 weeks

Heavy tumor burdenHeavy tumor burden

AFB1 single dose AFB1 single dose (6 mg/kg by (6 mg/kg by

i.p.)i.p.)

Positive control Positive control (Oltipraz)(Oltipraz)

Reduce tumor burdenReduce tumor burden

Clinical Evaluation : Phase IClinical Evaluation : Phase I

• ObjectivesObjectives– To obtain pharmacokinetics of the product To obtain pharmacokinetics of the product

following single p.o. dose of the product.following single p.o. dose of the product.– To investigate the induction of GST in To investigate the induction of GST in

lymphocytes.lymphocytes.– To evaluate toxicity associated with a single To evaluate toxicity associated with a single

p.o. dose of the product.p.o. dose of the product.

• SubjectsSubjects– 60 healthy normal volunteers (30 males + 30 60 healthy normal volunteers (30 males + 30

females).females).

Clinical Evaluation : Phase I (cont.)Clinical Evaluation : Phase I (cont.)

• TreatmentTreatment– 30 volunteers (15 males + 15 females) at dose X30 volunteers (15 males + 15 females) at dose X– 30 volunteers (15 males + 15 females) at dose Y30 volunteers (15 males + 15 females) at dose Y– Pharmacokinetics study : Blood sample will be collect before Pharmacokinetics study : Blood sample will be collect before

administration and at 1, 2, 3, 4, 5, 6, 8, 16 and 24h after dosing administration and at 1, 2, 3, 4, 5, 6, 8, 16 and 24h after dosing to measure curcuminoid and sulforaphane levels.to measure curcuminoid and sulforaphane levels.

– Investigation of GST study : Blood sample will be collect before Investigation of GST study : Blood sample will be collect before administration and at 6, 10 and 24h after dosing to measure administration and at 6, 10 and 24h after dosing to measure GST levels.GST levels.

• Sample analysisSample analysis– The samples will be measure by HPLC.The samples will be measure by HPLC.

• For toxicity grading, subjects will be evaluate for acute For toxicity grading, subjects will be evaluate for acute toxicity using standard toxicity using standard U.S. National Cancer Institute U.S. National Cancer Institute toxicity criteria.toxicity criteria.

Clinical Evaluation : Phase IIClinical Evaluation : Phase II

• ObjectiveObjective– To preliminary assess the efficacy of the To preliminary assess the efficacy of the

product by examining modulation in the levels product by examining modulation in the levels of several biomarkers of aflatoxin in urine.of several biomarkers of aflatoxin in urine.

– To characterize in more detail the rang of To characterize in more detail the rang of dose-limiting toxicities in a potential target dose-limiting toxicities in a potential target population including individuals infected with population including individuals infected with HBV.HBV.

• Study : placebo control, double blind studyStudy : placebo control, double blind study

Clinical Evaluation : Phase IIClinical Evaluation : Phase II (cont.)(cont.)

• SubjectsSubjects– Normal people (100 males + 100 females)Normal people (100 males + 100 females)

Product recipient (50 males + 50 females)Product recipient (50 males + 50 females) Placebo concurrent control (50 males + 50 Placebo concurrent control (50 males + 50

females)females)

– HBV carrier (50 males + 50 females)HBV carrier (50 males + 50 females)

• Screening of subjectsScreening of subjects– Individual screening.Individual screening.– Determine base line level of aflatoxin–Determine base line level of aflatoxin–NN--

acetylcysteine in urine by HPLC.acetylcysteine in urine by HPLC.

Clinical Evaluation : Phase IIClinical Evaluation : Phase II (cont.)(cont.)

• TreatmentTreatment– Daily administration for 8 weeks.Daily administration for 8 weeks.

PlaceboPlacebo Define dose 1Define dose 1 Define dose 2Define dose 2

– Urine samples collect once a week for 9 Urine samples collect once a week for 9 weeks after that once in 2 weeks till week 17.weeks after that once in 2 weeks till week 17.

Clinical Evaluation : Phase IIIClinical Evaluation : Phase III

• ObjectiveObjective– The design of this phase is based on the finding in The design of this phase is based on the finding in

phase II to validate instruction for use and for imaging phase II to validate instruction for use and for imaging in the population.in the population.

• SubjectsSubjects– Normal people (200 males + 200 females)Normal people (200 males + 200 females)

Product recipient (100 males + 100 females)Product recipient (100 males + 100 females) Placebo concurrent control (100 males + 100 Placebo concurrent control (100 males + 100

females)females)– HBV carrier (100 males + 100 females)HBV carrier (100 males + 100 females)– Liver resected (100 males + 100 females)Liver resected (100 males + 100 females)

Clinical Evaluation : Phase IIIClinical Evaluation : Phase III (cont.)(cont.)

• Screening of subjectsScreening of subjects– Individual screening.Individual screening.– Determine base line level of aflatoxin–Determine base line level of aflatoxin–NN--

acetylcysteine in urine by HPLC.acetylcysteine in urine by HPLC.

• TreatmentTreatment– Administration dose base on phase II.Administration dose base on phase II.– Daily administration for 24 months.Daily administration for 24 months.– Urine samples collect once a month for 12 months Urine samples collect once a month for 12 months

after that once in 2 months till month 24. after that once in 2 months till month 24.

Post-clinical Evaluation : Phase IVPost-clinical Evaluation : Phase IV

• ObjectiveObjective– Designed to detect any rare or longDesigned to detect any rare or long--term adverse term adverse

effects over a much larger population and timescale effects over a much larger population and timescale than was possible during the initial clinical trials.than was possible during the initial clinical trials.

• SubjectsSubjects– Same individual in phase III study.Same individual in phase III study.– Customers using the product.Customers using the product.

• ObservationObservation– Observe the side effects for 10 years after product on to Observe the side effects for 10 years after product on to

the market. the market.

ConclusionConclusion

• The product is safe for the population and The product is safe for the population and high chemoprevention potency.high chemoprevention potency.

• The product will on to the market as The product will on to the market as dietary supplement and above 10 years dietary supplement and above 10 years old children milk.old children milk.