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Screening of anti obesity drugs
 

Screening of anti obesity drugs

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screening of drugs and models for obesity

screening of drugs and models for obesity

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  • Leptin- leptos means thin.Leptin produced from adipocytes, released in pulsatile manner, inversely related to cortisol level. Leptin acts on hypothalamic nuclei, at specific leptin receptors2gps of nuclei in arcuate nucleus. AGRP agouti related peptide. POMC preproopiomelanocortin
  • Epidemic of obesity-globesityOverwt grading I BMI 25-30 , II 30-40, III >40
  • 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.

Screening of anti obesity drugs Screening of anti obesity drugs Presentation Transcript

  • SCREENING OF ANTI- OBESITY DRUGS Feb 2011
  • Objectives To 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
  • Obesity Energy 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 obesitydrugs? One billion adults are overweight >300 million are obese Prevalent in low income countries. Obesity are linked to more deaths worldwide than underweight.
  • 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 model Obesity – a complex disorder Exact pathology - unknown Humans tend to enjoy eating and are not forced to eat high fat diet Humans do not have induced gene mutations No 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 models In vivo In vitro Diet Virus Hypothalainduce induce Genetic mic d d Monoge Polyge nic nic
  • 1. Diet Induced Obesity  Rationale: calorie foods  Animal: Adult female rat 230g  Procedure: 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 Obesity Rationale: Hypothalamus regulates food intake. Venteromedial hypothalamic lesions food intake- obesity in 3-4 months. Surgical Chemical
  • a).Surgically inducedhypothalamic 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.
  • b). Chemically inducedhypothalamic obesity Animals: Mice/Rat (2-40 d old) parameters Observation of x 3months Procedure:Daily inj Monosodium-L-glutamate 2g/Kg , S/C x 5d ORSingle Inj of Gold thioglucose 30-40mg/Kg , I/P ORSingle Inj Bipiperidyl mustard 5-50mg/Kg, I/P OR
  • 3. Virus induced obesity Rationale: Some specific viruses target hypothalamus leading to virus induced disruption of critical brain CA 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
  • 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 Diabetesmouse mouse Autosomal recessive  Autosomal recessive mutation on mutation on chromosome 6 chromosome 4 Inbred stock of  Inbred stock of C57BL/6J strain C57BL/KsJ strain Obesity,  Obesity, hyperglycaemia, insulin hyperglycaemia, resistance
  • Fat mouse Tubby mouse Late onset obesity  Late onset Autosomal recessive  Autosomal recessive ‘Fat mutation’  Tub mutation Chromosome 8  C57BL/6J inbred strain coding for CPE  Additional: CPE involved in sensorineural insulin metabolism deafness,retinal Additional: infertility degradation
  • Fatty rat Obese SHR rat Zucker fatty rat  Mating SHR female Most widely used rat (kyoto wistar)with Autosomal recessive normotensive Fa/fa homozygous Sprague Dawley rat  Inbred strains after Obese by 3-5 weeks age 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 ModelsJapanese KK mouse NZO mouse Most suitable  New Zealand obese Large body size mouse mice inbred  6month age- renal Yellow obesity(AY) disease, autoimmune transferred to KK disorder mice KK-Ay mice Delayed onset obesity
  • Other polygenic models OLETF 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,
  • 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 agonist.
  • In vitro assaysContd’…..3. Assay for Neuropeptide Y It stimulates appetite. Six receptors Y 1-6 Y5,Y1 antagonist- new drug targets4. 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 linesFor leptin and leptin mRNA:1. Rat Preadipocytes- epididymal fat pad2. Rat primary cultured mature adipocytes3. 3T3-L1 adipocytes- mouse fibroblasts
  • Practical Implications Dietary models- represent behaviour and environmental factors Genetic models- for understanding genetics of human obesity Polygenic models- human obesity is also polygenic
  • References Drug 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