In-vivo Screening Procedures For Anti-Diabetic Drugs
Preclinical testing of drugs in experimental animals or in vitro for their biological and toxic effects and potential clinical applications.
In-vivo Screening Procedures
1. Models For Insulin Dependent Diabetes Mellitus [IDDM]
2. Models For NIDDM
3 Models For Insulin Sensitivity and Insulin Like Activity
Animals Used For The Screening Of Anti-Diabetic Drug
Obese mouse
Diabetic mouse
Sand mouse [Psammomys obesus]
Spiny mouse [Acomys cahirinus]
BB rats
KK mouse
Yellow mouse
NOD mouse
Yellow KK mouse
New Zealand obese mouse
Tuco-tuco [clenomys talarum]- these are burrowing rodents from Argentina.
Chinese hamster [Cricetulus griseus]
Chemical Agents Capable Of Inducing Diabetes
A) Irreversible beta cytotoxic agents:
Alloxan
Streptozocin
Diphenyl thiocarbazine
Oxine-9- hydroxyquinolone
Vacor
B) Reversible beta cytotoxic agents
6- aminonicotinamide
l-asparginase
Cyanide
Cyproheptadine
C) Other agents
Anti insulin antibodies
Somatostatins
Catecholamines
1. Models For Insulin Dependent Diabetes Mellitus [IDDM]
Alloxan induced diabetes
Alloxan: is a cyclic urea compound, which induces permanent diabetes.
It is a highly reactive molecule, which produces free radical damage to beta islet cells & causes cell death.
Dose: – In rats Alloxan at dose of 100 mg/kg produces diabetes.
In rabbits dose of 150 mg/kg infused through marginal ear vein produces diabetes in 70% of the animals.
PPT Anti Diabetic In vivo screening procedure antididabetic drugs
Procedure: –
Albino rats of either sex [150-200g] are injected with a single dose of alloxan monohydrate [100 mg/kg body weight] dissolved in normal saline by i.p. route.
Blood glucose levels show triphasic response with hyperglycemia for one hour followed by hypoglycemia that lasts for six hours & stable hyperglycemia after 48 hours.
Animals showing fasting blood glucose level above 140 mg/dl after 48 hour of alloxan administration are considered diabetic
For a period of six weeks, drug samples to be screened are administered orally
After six weeks of treatment, blood samples are collected from 8 hour fasting animals through a caudal vein
Serum is separated by centrifuge (3000 rpm) under cooling (2-4 °C) for ten minutes
The serum glucose level is estimated by glucose oxidase-peroxidase method [GOD-POD kit] using autoanalyser.
1.2 Streptozotocin induced diabetes
Streptozotocin: is a broad-spectrum antibiotic, which causes beta islet cell damage by free radical generation.
It induces diabetes in almost all species of animals excluding rabbits and guinea pigs.
Dose: – Diabetogenic dose: In Mice: 200mg/kg i.p
Beagle dogs: 15 mg/ kg i.v for three days.
Procedure: –
Streptozotocin [60 mg/kg body weight] is prepared in citrated buffer [ph 4.5]
Albino rats of either sex weighing 150-200 g are injected i.p with above solution
Animals showing fasting blood glucose levels > 140mg/dl after 48 hours of streptozotocin administration are considered diabetic.
· After six weeks of treatment blood samples are collected from 6 hr fasted animals through caudal vein
·Serum is separated by centrifuge (3000 rpm) under cooling (2-4 °C) for ten minutes
· Serum glucose level is estimated by glucose- peroxidase method [GOD-POD kit] using autoanalyser.
1.3 Virus induced diabetes
Principle: –
Viruses are one of the etiological agents for IDDM. They produce diabetes mellitus by infecting and destroying beta cells of pancreas.
Various human viruses used for inducing diabetes include RNA picornovirus, encephalomyocarditis [EMC-D], coxsackie B4 [CB-4].
Procedure: –
6-8 week old mice are inoculated by 0.1 ml of 1:50 dilutions of D-variant encephalomyocarditis [EMC] through i.p.
0.1ml of above dilution contains 50 PFU [ plaque forming units] of EMC virus.(mortality due to this concentration of virus is approximately 10-20%)
Less infecting variant produces a comparable damage by eliciting autoimmune reactivity to the beta cells.
· Infected animals are considered hyperglycemic if there non fasting levels exceed by 250mg/dl the levels of uninfected animals of the same strain.
· Drug samples to be screened are administered orally for a period of 6 weeks
· After 6 weeks of drug treatment, blood glucose estimation is done to determine the anti diabetic activity.