SCREENING OF ANTI-OBESITY DRUGS

Dr. Akanksha William

23 May 2012

Objectives

• To review the patho physiology of obesity

• Need for new anti obesity drugs

• To understand the basis of using animal models

• To know in vitro tests

• Introduction

• Burden of the disease

• Pathophysiology

• Ideal animal model

• Problems in animal models

• Parameters assessed

• In vitro methods

Obesity• Energy intake> Energy expenditure

BMI(wt/m2) CLASSIFICATION

18.5-24.9 NORMAL

25-29.9 Over weight/PRE OBESE

30-34.9 OBESE class I

35-39.9 Obese class II

>40 Obese class III

Disease burden

• WHO -1.5 billion obese

• U.S. 68% (largest market)

• India-60% affected

Children- 14.3% boys

- 9.3% girls

Need for anti obesity drugs

• 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

Pathophysiology

Multifactorial

Life style

Genetics

Diet

Environment

Ideal animal model

• Representative for human disease

• Genome sequenced

• Acceptable reproduction time

• Large numbers can be handled

• Placebo subtracted weight loss >5% maintained for

>1 year is the efficacy end point for approval.

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

• No single animal model can display interplay of

behavior, environment and genetic factors.

Parameters assessed

• Food intake- intake and spillage

• Body weight

• Adipose tissue cell size and number

• Body composition

• Locomotor /physical activity

• Plasma lipids, insulin and glucose levels

Diet induced

• Normal vs. high fat diet

Hypothalamic obesity

• Surgical • Chemical• Modification• Gold

thioglucose induced

• Monosodium-glutamate induced obesity

Virus induced • Canine distemper virus(antigenically related to measles)

• Borna disease• Rous

associated virus 7

• Avian adenovirus

• Ad 36 human adenovirus

Genetic models • Spontaneously

obese rat • WBN/KOB• Zukar fatty rat• WDF/TA-FA

RAT• OLETF RAT• Obese SHR• JCR:LA-

Corpulent • Spontaneously

obese mouse• Growth

hormone deficient dwarf rat

DIET INDUCED OBESITY

• Rationale: calorie foods

• Animal: Adult female rat 230-250gms

Animals given cafeteria diet.

Body wt, food intake, locomotor activity and serum insulin measured.

After 3months, rats sacrificed

Adipose tissue cell size, body composition and lipid content is determined

Disadvantages-acute food intake model

• Stimulating food intake by fasting• Insensitive to drugs that have delayed onset of

action• Drugs that increase energy expenditure• Lipase inhibitors

Hypothalamic Obesity

• Rationale: Hypothalamus regulates food intake.

SurgicalChemical

Surgically induced 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.

Chemically induced obesity

• Animals: Mice/Rat (2-40 d old)

Inj Monosodium-L-glutamate 2g/kg , s/c x 5 days

Inj of Gold thioglucose 30-40mg/kg , i/p

Inj Bipiperidyl mustard 5-50mg/kg, i/p

Inj 4-nitroquinoline l-oxide intracerebral

Virus induced obesity

• Rationale: Some specific viruses target

hypothalamus leading to virus induced disruption of

feedback 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

Borna disease virus

Avian retrovirus

Genetic models of obesity

Monogen

ic

Polygeni

c

Yellow obese mouse (Aya)

• Rationale: Obesity inherited through dominant gene, on Ch- 2 at linkage group 5, agouti locus.

Obese mouse

• Autosomal recessive mutation on chromosome 6

• Inbred stock of C57BL/6J strain

• Obesity, hyperglycaemia, insulin resistance

Diabetes mouse

• Autosomal recessive mutation on chromosome 4

• Inbred stock of C57BL/KsJ strain

• Obesity, hyperglycaemia, insulin resistance

Fat mouse

• Late onset obesity

• Autosomal recessive

• ‘Fat mutation’

• Chromosome 8

• Additional: infertility

Tubby Mouse

• Autosomal recessive

• Late onset

• Tub mutation

• C57BL/6J inbred strain

• Additional: sensorineural deafness, retinal degeneration

Fatty rat

• Zucker fatty rat

• Most widely used

• Autosomal recessive

• Fa/fa homozygous

• Obese by 3-5 weeks age

Obese SHR rat

• Mating SHR female rat (kyoto wistar)with

normotensive Sprague Dawley rat

• Inbred strains after several generation

• Substrain- JCR: LA Corpulent rat

• Vascular complications

WDF/ta-fa rat

• Wistar fatty rat

• Tranfer of fatty gene (fa) from Zucker rat to

Wistar Kyoto rat

Polygenic Models

Japanese KK mouse

• Most suitable

• Large body size mice inbred

• Yellow obesity(AY) - KK mice

• KK-Ay mice

• Delayed onset obesity

NZO mouse

• New Zealand obese mouse

• 6month age- renal disease, autoimmune disorder

Other polygenic models

• OLETF rat -Otsuka-Long Evans-Tokushima-Fatty rat

nephropathy model

• BSB model

• AKR/J x SWR/J model

• M 16- to study genetics of growth and obesity

Transgenic modelsRationale: genes regulating energy homeostasis are

manipulated

• KO 3 gene – in white and brown adipose tissue

• KO Uncoupling protein -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 ASSAYS

To study metabolic activity in brown adipose tissue

Male fatty rat, 10 weeks age are given test drug od s/c

Rats sacrificed after 14 weeks. Brown and white fat removed

UCP and GLUT4 determined with western blot analysis

To study 3 agonist activity

Induce weight loss by increased thermogenesis,

suppression of leptin gene expression

Assay for Neuropeptide Y

It stimulates appetite. Six receptors Y 1-6

Y5,Y1 antagonist- new drug targets

Role of leptin

Ob gene product. Receptor: lepr or OB-R

- Northern blot analysis

- RIA

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 Implications

• Dietary models- represent behavior and environmental

factors

• Genetic models- for understanding genetics of human

obesity

• Polygenic models- human obesity is also polygenic

• New therapeutic targets

References

Drug screening methods - S K Gupta

Drug Discovery and Evaluation - Vogel

Pharmacology- Rang and Dale

Steven P Vickers.The utility of animal modelsto evaluate

novelanti-obesity agents. British Journal of

Pharmacology.2011; 164: 1248–1262.

Biology of Obesity: Lessons from Animal Models of

Obesity. Journal of Biomedicine and Biotechnology

doi:10.1155/2011/197636

• Animal models and their value in predicting drug efficacy and toxicity. 2011; 15 - 16.

THANK YOU