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. of in vitro study 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 ER5 pcDNA3 Transfect in to HepG2 cells line Introduce cDNA into pcDNA3

7. HepG2 + ER 5 HepG2 + ER α MTT assay stock solution con c1 con c4 con c3 con c2 con c5 con trol Cytotoxicity test

8. IC 50 Dose dependent inhibition of HepG2 cells by modified megestrol

9. Time dependent inhibition of HepG2 cells by modified megestrol

10. Cell suspensions and Propidium Iodide Staining Determination of Cell Cycle and Apoptosis HepG2 cell lines Analyzed by Flow cytometry 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 1. Pharmacokinetic study 2. Animal testing 3. Toxicity test

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

13. Qualitative Distribution Study MaleFemalePregnant Sacrificed and Sagittal Section (30 m) Whole Body Autoradiography

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

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

16. Toxicity Test Test 1:The LD 50 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. Summary Tested drugIn vitroIn vivoClinical trials IC 50 Phase I staring dose -Anticancer properties of drug -Toxicity testing

18. THE END