Preclinical Drug Development Miss Sirikan Nawapan.
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Transcript of Preclinical Drug Development Miss Sirikan Nawapan.
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