Preclinical Drug Development

Miss Sirikan Nawapan

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

Components of Preclinical Drug Development

1. In vitro study

: Cytotoxicity assay

2. In vivo study

: Pharmacokinetic study

: Animal testing

: Toxicity test

In vitro study

ObjectivesTo study the effectiveness of the drug

on the growth of HepG2 cells and to provide the appropriate concentration using in vivo study.

Components of in vitro study 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

Preparation of HepG2 cells line

cDNAs contain the full-length ER and ER∆5

pcDNA3

Transfect in to HepG2 cells line

Introduce cDNA into pcDNA3

2HepG + ER ∆ 5

2 + ERα

MTT assay

stock solution

conc1

conc4

conc3

conc2

conc5

control

Cytotoxicity test

0

20

40

60

80

100

120

0 200 400 600 800 1000modified megestol concentration (µM)

% Cell su

rviva

l

IC50

Dose dependent inhibition of HepG2 cells by modified megestrol

Time dependent inhibition of HepG2 cells by modified megestrol

0

50

100

150

200

250

300

0 10 20 30 40 50 60Time (hr)

% Orig

inal C

ell No

controlIC 50 of m-megestrol

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+

In vivo study

ObjectiveTo 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

Pharmacokinetic study

Control

Male

Female

Blood, Urine, Feces samples

High Performance Liquid Chromatography (HPLC)

14C-Radiolabeled Modified Megestrol

Qualitative Distribution Study

Male Female Pregnant

Sacrificed and Sagittal Section (30m)

Whole Body Autoradiography

Animal testing

HepG2 + vERHepG2 + vERHepG2 + vER

Modified Megestrol

male femalecontrol

Vehicle

Animal Testing Endpoints

Week

Tu

mo

r V

ol (

% O

rig

Vo

l)B

od

y W

eig

ht

(g)

% o

f su

rviv

ors body weight (g) = total weight (g) – tumor volume(cm3)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

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

Summary

Tested drug In vitro In vivo Clinical trials

IC50

Phase I staring dose

-Anticancer properties of drug

-Toxicity testing

THE END