Feb 2011

SCREENING OF ANTI-OBESITY DRUGS

ObjectivesTo 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

Overview Introduction

Pathophysiology

Problems in animal

models

Parameters assessed

In vitro methods

In vivo methods

ObesityEnergy 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

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.

Globally, > 42 million

children

<5y age are overweight

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

Animal models

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

Lack of Ideal modelObesity – a complex disorderExact pathology - unknownHumans tend to enjoy eating and

are not forced to eat high fat dietHumans do not have induced

gene mutationsNo single animal model can

display interplay of behaviour, environment and genetic factors.

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

Animal model

s

In vivo

Diet induce

d

Hypothalamic

Virus induce

d

Genetic

Monogenic

Polygenic

In vitro

1.Diet Induced ObesityRationale: calorie foodsAnimal: Adult female rat

230gProcedure: 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

2.Hypothalamic ObesityRationale: Hypothalamus regulates food

intake. Venteromedial hypothalamic lesions food intake- obesity in 3-4 months.

SurgicalChemical

a).Surgically induced hypothalamic obesityAnimal: 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.

b). Chemically induced hypothalamic obesityAnimals: Mice/Rat (2-40 d old)Procedure: Daily inj Monosodium-L-glutamate

2g/Kg , S/C x 5dOR

Single Inj of Gold thioglucose 30-40mg/Kg , I/P

ORSingle Inj Bipiperidyl mustard 5-

50mg/Kg, I/POR

Single Inj 4-nitroquinoline l-oxide intracerebral

Obse

rvatio

n o

f para

mete

rs x

3m

onth

s

3. Virus induced obesityRationale: Some specific viruses target

hypothalamus leading to virus induced disruption of critical brain CA pathways, leading to obesity

Animals: MiceProcedure: Mice infected with canine

distemper virus, develops obesity in 8-10 weeks.

Other viruses: Rous-associated virus-7Avian adenovirus SMAM-IAd-36Borna disease virusAvian retrovirus

4. Genetic models of obesity

Monogenic

Polygenic

Yellow obese mouse (Aya)Rationale: Obesity inherited through

dominant gene, on Chromosome 2 at linkage group 5, agouti locus.

Obese mouseAutosomal recessive

mutation on

chromosome 6

Inbred stock of C57BL/6J

strain

Obesity,

hyperglycaemia, insulin

resistance

Autosomal recessive

mutation on

chromosome 4

Inbred stock of

C57BL/KsJ strain

Obesity,

hyperglycaemia, insulin

resistance

Diabetes mouse

Fat mouseLate onset obesityAutosomal

recessive‘Fat mutation’Chromosome 8

coding for CPECPE involved in

insulin metabolismAdditional:

infertility

Late onsetAutosomal recessiveTub mutationC57BL/6J inbred

strainAdditional:

sensorineural deafness,retinal degradation

Tubby mouse

Fatty ratZucker fatty ratMost widely usedAutosomal

recessiveFa/fa homozygous Obese by 3-5

weeks age

Mating SHR female rat (kyoto wistar)with normotensive Sprague Dawley rat

Inbred strains after several generation

SubstrainJCR: LA Corpulent rat

Vascular complications

Obese SHR rat

WDF/TA-FA rat

Wistar fatty rat

Tranfer of fatty gene (fa) from Zucker rat

to Wistar Kyoto rat

Polygenic ModelsJapanese KK mouse NZO mouse

Most suitableLarge body size

mice inbredYellow obesity(AY)

transferred to KK mice

KK-Ay miceDelayed onset

obesity

New Zealand obese mouse

6month age- renal disease, autoimmune disorder

Other polygenic modelsOLETF rat

Otsuka-Long evans-Tokushima-Fatty rat

nephropathy model

BSB model

AKR/J x SWR/J model

Transgenic modelsRationale: genes regulating energy

homeostasis are manipulatedProcedure – 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, α-MSH, AgRP, Melonocortin-4 receptor, melanocortin- 3 receptor.

In vitro assays1. 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 3 agonist.

In vitro assays Contd’…..3. Assay for Neuropeptide Y

It stimulates appetite. Six receptors Y 1-6Y5,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

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

Practical ImplicationsDietary models- represent

behaviour and environmental

factors

Genetic models- for understanding

genetics of human obesity

Polygenic models- human obesity

is also polygenic

New therapeutic targets

ReferencesDrug 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