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.
Screening of antiobesity 23may2012
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
Life styleEnvironment Multifactorial Genetics Diet
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
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
• 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 sacrificedAdipose 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
• Rationale: Hypothalamus regulates food intake. Surgical Chemical
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-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
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.
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 activityInduce 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 targetsRole of leptin Ob gene product. Receptor: lepr or OB-R - Northern blot analysis - RIA
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 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.