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Preclinical Drug Development

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  1. Preclinical Drug Development Miss Sirikan Nawapan

  2. Objectives • To evaluate the anticancer properties of this modified drug by studying cell proliferation tumor volumetric apoptosis DNA fragment • To provide the evidence for supporting the safety of use in patient treatment

  3. Components of Preclinical Drug Development 1. In vitro study : Cytotoxicity assay 2. In vivo study : Pharmacokinetic study : Animal testing : Toxicity test

  4. In vitro study Objectives To study the effectiveness of the drug on the growth of HepG2 cells and to provide the appropriate concentration using in vivo study.

  5. Components of in vitro study 1. Preparation of HepG2 cells 2. Cytotoxicity test 2.1 Dose dependent inhibition 2.2 Time dependent inhibition 3. Determination of cell cycle and apoptosis

  6. Preparation of HepG2 cells line cDNAs contain the full-length ER and ER∆5 Introduce cDNA into pcDNA3 pcDNA3 Transfect in to HepG2 cells line

  7. stock solution conc4 conc2 control conc1 conc3 conc5 Cytotoxicity test HepG2 + ER ∆ 5 MTT assay HepG2 + ER α

  8. Dose dependent inhibition of HepG2 cells by modified megestrol IC50

  9. Time dependent inhibition of HepG2 cells by modified megestrol

  10. Determination of Cell Cycle and Apoptosis Cell suspensions and Propidium Iodide Staining Analyzed by Flow cytometry HepG2 cell lines + ApopAlert LM-PCR Ladder Assay kit

  11. In vivo study Objective To study the efficacy and toxicity of the modified drug in nude mice. Components of in vivo study • Pharmacokinetic study • Animal testing • Toxicity test

  12. Control Male Female Blood, Urine, Feces samples 14C-Radiolabeled Modified Megestrol High Performance Liquid Chromatography (HPLC) Pharmacokinetic study

  13. Male Female Pregnant Qualitative Distribution Study Sacrificed and Sagittal Section (30m) Whole Body Autoradiography

  14. Animal testing Vehicle Modified Megestrol HepG2 + vER HepG2 + vER HepG2 + vER control male female

  15. Animal Testing Endpoints body weight (g)= total weight (g) – tumorvolume(cm3) Tumor Vol (% Orig Vol) Body Weight (g) % of survivors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Week

  16. Toxicity Test Test 1:The LD50 determination 5 doses and 6 mices/sex/dose Test 2:The LD50 determination (finely tune) 5 doses and 6 mices/sex/dose Test 3:Acute toxicity test 3 dose of test 2 and 6 mices/sex/dose Test 4:Subchronic toxicity test 1/20 dose of test 2 ,10 mices/sex/dose Test 5: Chronic toxicity test No-effect dose in test 4,10 mices/sex/dose

  17. -Anticancer properties of drug -Toxicity testing IC50 Summary Tested drug In vitro In vivo Clinical trials Phase I staring dose

  18. THE END