screening of drugs and models for obesity

1. SCREENING OF ANTI-
OBESITY DRUGS
Feb 2011

2. Objectives
 To understand the need for new
anti obesity drugs
 To understand the
pathophysiology
 To understand how to manipulate
in vivo models
 To know basic in vitro tests

3. Overview
 Introduction
 Pathophysiology
 Problems in animal models
 Parameters assessed
 In vitro methods
 In vivo methods

4. Obesity
 Energy intake> Energy expenditure
 BMI>30 kg/m2
 Multifactorial
 Orexigenic peptide- NPY, AgRP, Orexin A &B,
galanin, endorphin, NE, GH-RH
 Anorectic peptide- Corticotrophin-RH, MSH,
CCK, GLP1,CGRP, bombesin

6. Need for anti obesity
drugs?
 One billion adults are
overweight
 >300 million are obese
 Prevalent in low income
countries.
 Obesity are linked to more
deaths worldwide than
underweight.

7. Need for anti obesity drugs?
Cont’d…
 In late 2009, $1.1 billion
market anti-obesity drugs
could nearly triple to reach
$3.1 billion by 2016
 No new anti-obesity drug
FDA approved since 1999

8. Animal models

9. Why mouse models ?
 Represenative for human disease
 Genome sequenced
 Acceptable reproduction time
 Large numbers can be handled
 Identification of disease genes
easier than in humans

10. Lack of Ideal model
 Obesity – a complex disorder
 Exact pathology - unknown
 Humans tend to enjoy eating
and are not forced to eat high fat
diet
 Humans do not have induced
gene mutations
 No single animal model can
display interplay of behaviour,
environment and genetic factors.

11. Animal models-
Parameters
 Food intake- intake and spillage
 Body weight
 Adipose tissue cell size and number- osmium
fixation method
 Body composition
 Locomotor /physical activity
 Plasma lipids, insulin and glucose levels

12. Animal
models
In vivo In vitro
Diet Virus
Hypothala
induce induce Genetic
mic
d d
Monoge Polyge
nic nic

13. 1. Diet Induced Obesity
 Rationale: calorie foods
 Animal: Adult female rat
230g
 Procedure: Animals
given cafeteria diet.
 Body wt, food intake,
locomotor activity and
serum insulin measured.
 After 3months, rats
sacrificed and adipose
tissue cell size, body
composition and lipid
content is determined

14. 2. Hypothalamic Obesity
 Rationale: Hypothalamus regulates food intake.
Venteromedial hypothalamic lesions food intake-
obesity in 3-4 months.
Surgical
Chemical

15. a).Surgically induced
hypothalamic
obesity
 Animal: female Sprague Dawley
rats 190g
 Procedure: high fat diet for 5-9
days. The cuts are made 1mm
lateral to the midline, extended
from 8.5-5.5mm anterior to ear
bars and from 3mm dorsally
from the base of the brain.

16. b). Chemically induced
hypothalamic obesity
 Animals: Mice/Rat (2-40 d old)
parameters
Observation of
x 3months
 Procedure:
Daily inj Monosodium-L-glutamate 2g/Kg ,
S/C x 5d
OR
Single Inj of Gold thioglucose 30-40mg/Kg ,
I/P
OR
Single Inj Bipiperidyl mustard 5-50mg/Kg,
I/P
OR

17. 3. Virus induced obesity
 Rationale: Some specific viruses target
hypothalamus leading to virus induced
disruption of critical brain CA pathways, leading
to obesity
 Animals: Mice
 Procedure: Mice infected with canine
distemper virus, develops obesity in 8-10
weeks.
 Other viruses: Rous-associated virus-7
Avian adenovirus SMAM-I
Ad-36

18. 4. Genetic models of obesity
Monogenic Polygenic

19. Yellow obese mouse (Aya)
 Rationale: Obesity inherited through
dominant gene, on Chromosome 2 at linkage
group 5, agouti locus.

20. Obese Diabetes
mouse mouse
 Autosomal recessive  Autosomal recessive
mutation on mutation on
chromosome 6 chromosome 4
 Inbred stock of
 Inbred stock of
C57BL/6J strain
C57BL/KsJ strain
 Obesity,
 Obesity,
hyperglycaemia, insulin
hyperglycaemia,
resistance

21. Fat mouse Tubby mouse
 Late onset obesity  Late onset
 Autosomal recessive  Autosomal recessive
 ‘Fat mutation’  Tub mutation
 Chromosome 8  C57BL/6J inbred strain
coding for CPE  Additional:
 CPE involved in sensorineural
insulin metabolism deafness,retinal
 Additional: infertility degradation

22. Fatty rat Obese SHR rat
 Zucker fatty rat  Mating SHR female
 Most widely used rat (kyoto wistar)with
 Autosomal recessive normotensive
 Fa/fa homozygous
Sprague Dawley rat
 Inbred strains after
 Obese by 3-5 weeks
age several generation
 Substrain
JCR: LA Corpulent
rat
Vascular
complications

23. WDF/TA-FA rat
 Wistar fatty rat
 Tranfer of fatty gene (fa) from Zucker rat to
Wistar Kyoto rat

24. Polygenic Models
Japanese KK mouse NZO mouse
 Most suitable  New Zealand obese
 Large body size mouse
mice inbred  6month age- renal
 Yellow obesity(AY) disease, autoimmune
transferred to KK disorder
mice
 KK-Ay mice
 Delayed onset
obesity

25. Other polygenic models
 OLETF rat
Otsuka-Long evans-Tokushima-Fatty rat
nephropathy model
 BSB model
 AKR/J x SWR/J model

26. Transgenic models
Rationale: genes regulating energy homeostasis
are manipulated
Procedure – gene for diphtheria toxin A chain is
used to link to the gene chosen to be knocked out
 KO 3 gene – in white and brown adipose tissue
 KO Uncoupling protein in brown fat
(thermogenesis)
 KO mice lacking Steriodogenic factor I (SF-I)
 Overexpression of corticotropin releasing factor
gene, GLUT-4 gene, human agouti-related protein
complementary DNA
 Genes for leptin, leptin receptor, growth hormone,

27. In vitro assays
1. To study metabolic activity in brown adipose
tissue
Assay for uncoupling protein and GLUT4
Procedure: OLETF rat, 10 weeks age are given
test drug OD S/C . Rats sacrificed at 14 weeks
age. Brown and white fat removed. UCP and
GLUT4 determined with western blot analysis.
2. To study 3 agonist activity
they induce wt. loss by increased
thermogenesis,
suppression of leptin gene expression
c-AMP response element-luciferase receptor gene assay for
agonist.

28. In vitro assays
Contd’…..
3. Assay for Neuropeptide Y
It stimulates appetite. Six receptors Y 1-6
Y5,Y1 antagonist- new drug targets
4. Role of leptin
Ob gene product. Receptor: lepr or OB-R
a)Assay for leptin mRNA level in adipose tissue
- Northern blot analysis
b) RIA for measurement of plasma leptin

29. Isolated adipocyte cell lines
For leptin and leptin mRNA:
1. Rat Preadipocytes- epididymal fat pad
2. Rat primary cultured mature adipocytes
3. 3T3-L1 adipocytes- mouse fibroblasts

30. Practical Implications
 Dietary models- represent
behaviour and environmental
factors
 Genetic models- for
understanding genetics of
human obesity
 Polygenic models- human
obesity is also polygenic

31. References
 Drug screening methods - S K Gupta
 Drug Discovery and Evaluation - Vogel
 Pharmacology- Rang and Dale
 Biology of Obesity: Lessons from Animal
Models of Obesity. Journal of Biomedicine
and Biotechnology doi:10.1155/2011/197636