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Screening of antiobesity 23may2012

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

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


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  • 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.
  • Transcript

    • 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
    • 5. Disease burden• WHO -1.5 billion obese• U.S. 68% (largest market)• India-60% affected Children- 14.3% boys - 9.3% girls
    • 6. 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
    • 7. Pathophysiology
    • 8. Life styleEnvironment Multifactorial Genetics Diet
    • 9. 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.
    • 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• No single animal model can display interplay of behavior, environment and genetic factors.
    • 11. 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
    • 12. HypothalamicDiet 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
    • 13. DIET INDUCED OBESITY
    • 14. • Rationale: calorie foods• Animal: Adult female rat 230-250gms
    • 15. Animals given cafeteria diet.Body wt, food intake, locomotor activity and serum insulin measured. After 3months, rats sacrificedAdipose tissue cell size, body composition and lipid content is determined
    • 16. 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
    • 17. Hypothalamic Obesity
    • 18. • Rationale: Hypothalamus regulates food intake. Surgical Chemical
    • 19. 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.
    • 20. Chemically induced obesity• Animals: Mice/Rat (2-40 d old)
    • 21. Inj Monosodium-L-glutamate2g/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
    • 22. Virus induced obesity
    • 23. • Rationale: Some specific viruses target hypothalamus leading to virus induced disruption of feedback pathways, leading to obesity• Animals: Mice
    • 24. 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
    • 25. Genetic models of obesity Monogenic Polygenic
    • 26. Yellow obese mouse (Aya)• Rationale: Obesity inherited through dominant gene, on Ch- 2 at linkage group 5, agouti locus.
    • 27. Obese mouse• Autosomal recessive mutation on chromosome 6• Inbred stock of C57BL/6J strain• Obesity, hyperglycaemia, insulin resistance
    • 28. Diabetes mouse• Autosomal recessive mutation on chromosome 4• Inbred stock of C57BL/KsJ strain• Obesity, hyperglycaemia, insulin resistance
    • 29. Fat mouse• Late onset obesity• Autosomal recessive• ‘Fat mutation’• Chromosome 8• Additional: infertility
    • 30. Tubby Mouse• Autosomal recessive• Late onset• Tub mutation• C57BL/6J inbred strain• Additional: sensorineural deafness, retinal degeneration
    • 31. Fatty rat• Zucker fatty rat• Most widely used• Autosomal recessive• Fa/fa homozygous• Obese by 3-5 weeks age
    • 32. 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
    • 33. WDF/ta-fa rat• Wistar fatty rat• Tranfer of fatty gene (fa) from Zucker rat to Wistar Kyoto rat
    • 34. Polygenic Models Japanese KK mouse• Most suitable• Large body size mice inbred• Yellow obesity(AY) - KK mice• KK-Ay mice• Delayed onset obesity
    • 35. NZO mouse• New Zealand obese mouse• 6month age- renal disease, autoimmune disorder
    • 36. 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
    • 37. 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)
    • 38. • 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.
    • 39. IN VITRO ASSAYS
    • 40. 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
    • 41. To study 3 agonist activityInduce weight loss by increased thermogenesis,suppression of leptin gene expression
    • 42. Assay for Neuropeptide Y It stimulates appetite. Six receptors Y 1-6 Y5,Y1 antagonist- new drug targetsRole of leptin Ob gene product. Receptor: lepr or OB-R - Northern blot analysis - RIA
    • 43. Isolated adipocyte cell linesFor leptin and leptin mRNA:1. Rat Preadipocytes- epididymal fat pad2. Rat primary cultured mature adipocytes3. 3T3-L1 adipocytes- mouse fibroblasts
    • 44. 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
    • 45. 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
    • 46. • Animal models and their value in predicting drug efficacy and toxicity. 2011; 15 - 16.
    • 47. THANK YOU