Screening of antiobesity 23may2012
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screening of drugs/ agents for obesity. the models are described here
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Screening of antiobesity 23may2012
- 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
- 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
- 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
- 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
Notas do Editor
- Epidemic of obesity-globesityOverwt grading I BMI 25-30 , II 30-40, III >40
- Dexfenfluramine,sibutramine, orlistat and rimonabant.
- MultifactorialComplex Orexigenic peptide- NPY, AgRP, Orexin A &B, galanin, endorphin, NE, GH-RHAnorectic peptide-POMC,RH, MSH, CCK, GLP1,CGRP, bombesin
- Placebo subtracted weight loss >5% maintained for >1 year is the efficacy end point for approval.
- Neuroanatomy should be similar.
- Body composition is estimated: carcasses oven dried at 95*C for 6-9 days till constant wt is reached. Lipid content is measured in gonadal and retroperitoneal fat pads. For this, adipose tissue is homogenised with 2:1 chloroform-methanol mixture and washed with water. The resulting mixture separates into two phases, lower one has pure lipid extract.
- Make flow chart
- it could be argued that this approach is not physiological (e.g. an overnight fast would be a major stressor for a mouse)For a fast screening method the reduction of food intake can be an effective method, can provide info for relative potencies, and the duration of action of compounds.
- Venteromedial hypothalamic lesions food intake- obesity in 3-4 months.