Download Preclinical Drug Development

Preclinical Drug
Development
Miss Sirikan Nawapan
Objectives

To evaluate the anticancer properties of
this modified drug by studying
cell proliferation
apoptosis

tumor volumetric
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
Objectives
To 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
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
Introduce cDNA
into pcDNA3
pcDNA3
Transfect in to HepG2 cells line
Cytotoxicity test
stock solution
control conc1 conc2 conc3 conc4 conc5
HepG2 + ER ∆ 5
MTT assay
HepG2 + ER α
Dose dependent inhibition of HepG2 cells
by modified megestrol
120
% Cell survival
100
80
60
40
20
0
0
IC50
200
400
600
modified megestol
concentration
(µM) 800
1000
Time dependent inhibition of HepG2 cells
by modified megestrol
300
control
IC 50 of m-megestrol
% Original Cell No
250
200
150
100
50
0
0
10
20
30
Time (hr)
40
50
60
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
In vivo study
Objective
To study the efficacy and toxicity of the
modified drug in nude mice.
Components of in vivo study
1.
2.
3.
Pharmacokinetic study
Animal testing
Toxicity test
Pharmacokinetic study
Control
Female
Male
14C-Radiolabeled
Modified Megestrol
Blood, Urine, Feces samples
High Performance Liquid Chromatography
(HPLC)
Qualitative Distribution Study
Male
Female
Pregnant
Sacrificed and Sagittal Section (30m)
Whole Body Autoradiography
Animal testing
Vehicle
HepG2 + vER
control
Modified Megestrol
HepG2 + vER
male
HepG2 + vER
female
survivors
% ofVol
(% Orig
Tumor
(g)Vol)
Weight
Body
Animal Testing Endpoints
body weight (g) = total weight (g) – tumor volume(cm3)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Week
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
Phase I
staring dose
-Anticancer properties
of drug
IC50
-Toxicity testing
THE END