screening of drugs/ agents for obesity. the models are described here

1. SCREENING OF ANTI-
OBESITY DRUGS
Dr. Akanksha William
23 May 2012

2. 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

3. • Introduction
• Burden of the disease
• Pathophysiology
• Ideal animal model
• Problems in animal models
• Parameters assessed
• In vitro methods

4. 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

6. Disease burden
• WHO -1.5 billion obese
• U.S. 68% (largest market)
• India-60% affected
Children- 14.3% boys
- 9.3% girls

7. 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

8. Pathophysiology

9. Life
style
Environment Multifactorial Genetics
Diet

11. 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.

12. 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.

13. 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

14. Hypothalamic
Diet induced Virus induced Genetic models
obesity
• Normal vs. • Surgical • Canine • Spontaneously
high fat diet • Chemical distemper obese rat
• Modification virus(antigenic • WBN/KOB
ally related to • Zukar fatty rat
• Gold
measles)
thioglucose • WDF/TA-FA
induced • Borna disease RAT
• Monosodium- • Rous • OLETF RAT
associated
glutamate • Obese SHR
induced virus 7
• JCR:LA-
obesity • Avian
Corpulent
adenovirus
• Spontaneously
• Ad 36 human
obese mouse
adenovirus
• Growth
hormone
deficient dwarf
rat

15. DIET INDUCED
OBESITY

16. • Rationale: calorie foods
• Animal: Adult female rat 230-250gms

17. 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

18. 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

19. Hypothalamic Obesity

20. • Rationale: Hypothalamus regulates food intake.
Surgical
Chemical

21. 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.

22. Chemically induced obesity
• Animals: Mice/Rat (2-40 d old)

23. 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

24. Virus induced obesity

25. • Rationale: Some specific viruses target
hypothalamus leading to virus induced disruption of
feedback pathways, leading to obesity
• Animals: Mice

26. 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

27. Genetic models of obesity
Monogenic Polygenic

28. Yellow obese mouse (Aya)
• Rationale: Obesity inherited through dominant gene,
on Ch- 2 at linkage group 5, agouti locus.

29. Obese mouse
• Autosomal recessive mutation on chromosome 6
• Inbred stock of C57BL/6J strain
• Obesity, hyperglycaemia, insulin resistance

30. Diabetes mouse
• Autosomal recessive mutation on chromosome 4
• Inbred stock of C57BL/KsJ strain
• Obesity, hyperglycaemia, insulin resistance

31. Fat mouse
• Late onset obesity
• Autosomal recessive
• ‘Fat mutation’
• Chromosome 8
• Additional: infertility

32. Tubby Mouse
• Autosomal recessive
• Late onset
• Tub mutation
• C57BL/6J inbred strain
• Additional: sensorineural deafness, retinal degeneration

33. Fatty rat
• Zucker fatty rat
• Most widely used
• Autosomal recessive
• Fa/fa homozygous
• Obese by 3-5 weeks age

34. 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

35. WDF/ta-fa rat
• Wistar fatty rat
• Tranfer of fatty gene (fa) from Zucker rat to
Wistar Kyoto rat

36. Polygenic Models
Japanese KK mouse
• Most suitable
• Large body size mice inbred
• Yellow obesity(AY) - KK mice
• KK-Ay mice
• Delayed onset obesity

37. NZO mouse
• New Zealand obese mouse
• 6month age- renal disease, autoimmune disorder

38. 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

39. Transgenic models
Rationale: 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)

40. • 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.

42. IN VITRO ASSAYS

43. 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

44. To study 3 agonist activity
Induce weight loss by increased thermogenesis,
suppression of leptin gene expression

45. 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

46. 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

47. 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

48. 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

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

50. THANK YOU