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SCREENING OF ANTI DIABETIC AGENTS BY G. ANIL KUMAR M. PHARMACY-1 st yr- PHARMACOLOGY, UCPSc, wgl.
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SCREENING OFANTI DIABETIC AGENTS

BY G. ANIL KUMAR

M. PHARMACY-1st yr-PHARMACOLOGY, UCPSc, wgl.

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INTRODUCTION• Dibetes mellitus is a chronic metabolic disorder

characterised by a high blood glucose concent r ation-hyperglycaemia due to insulin deficiency and/or insulin resistance.

• The pancras is an organ composed of 98% exocrine and 2% endocrine cells .Islets of langerhans, which forms the endocrine part of of pancreas, consists of 4 types of cells A, B and D cells.

• Glucose stimulates the beta cells to release insulin, which then promotes glucose uptake and storage in various tissues.

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• Two major types of diabetes, one associated with insulin deficiency called type-1 diabetes mellitus(IDDM), and other associated with insulin resistance called Tpe-2 diabetes mellitus(NIDDM).

• The consequences of diabetes are complex and are life threatening.

• Pre diabetes?

• What is insulin resistance?

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MODELS FOR IDDM

• CHEMICALLY INDUCED DIABETES

• Chemically induced Type-1 diabetes is most commonly used animal model of diabetes.

• Alloxan and Streptozotocin are the 2 chemicals which are widely used for this purpose.

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ALLOXAN INDUCED DIABETES

• Alloxan is a cyclic urea analog.

• Mechanism of action:

• -By generation of free radicals.

• -Iteracts with the proteins whish containing SH group.

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PROCEDURE:

• Rabbits weighing 2-3 kg are used for the purpose.

• Alloxan is infused via the ear marginal vein at a dose of 150mg/kg for 10 minutes.

• For wistar rats the dose is of 100-175mg/kg and the route of administration is s.c

• DRAWBACKS:• Mortality rate is high.• Diabetes induced is reversible.

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STREPTOZOTOCIN INDUCED DIABETES:

• STZ is a broad spectrum antibiotic, produced from Strptomyces achromogens.

• MECHANISM OF ACTION:

• -Free radical generation.• -By the process of methylation.• -Nitric oxide production.

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PROCEDURE:

• STZ induces diabetes in almost all species of animals.

• For rats the dose is 50-60mg/kg(i.p/ i.v).

• For mice the dose is 175-200mg/kg(i.p/i.v).

• For dogs 15mg/kg for 3 days.

• Streptazotocin damages the beta cells specifically.

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TRIPHASIC RESPONSE:

• Upon administration of these chemicals the blood glucose levels of animals varies from time to time.

• First raise at 1 hr.

• Followed by hypoglycaemic phase at 6 hrs.

• Followed by increse at 24 hrs

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• STZ has many advantages when compared to Alloxan like

• -less mortality

• -diabetsity is irreversible

• - Greater selectivity towards the beta cells

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HARMONE INDUCED DIABETES DIABETES MELLITUS:

• Dexamethasone a long acting glucocorticoid, is used to produce NIDDM, when administered at a dose of 2-5mg/kg i.p. twice daily over a number of days in rats.

• INSULIN ANTIBODIES- INDUCED DIABETES:

• Giving bovine insulin to guinea pigs produces anti-insulin antibodies. Intravenous injection of pig anti insulin serum to rats induces a dose dependent increse in blood glucose levels.

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DIABETES INDUCED BY VIRAL AGENTS:

• Viruses are thought to be one of the etiologic agents for IDDM. Viruses may produce diabetes mellitus by

• Infecting and destroying of bets cells in pancreas,

• A less infecting or cytologic variant producing a comparable damage by eliciting immune autoreactivity.

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SURGICALLY INDUCED DIABETES:

• Induction of diabetes mellitus can be achieved through the surgical removal of all or part of the pancreas.

• In partial pancreatectomy more than 90% of the organ must be removed to produce diabetes.

• Total removal of the pancreas results in an insulin dependent form of diabetes.

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DISADVANTAGES:

• Loss of pancreatic enzymes that are necessory for proper digestion.

• Surgical removal of the pancreas results in loss of alpha and delta cells in addition to the beta cells. This causes the loss of the counter regulatory harmones glucagon and somatostatin.

• The total resection pf pancreas in rats very difficult achieve.

• But the combination therapy reduces the risk of toxicity of the chemicals on other body parts

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GENETIC MODELS:

• These are the animal models in diabetes , which are diabetic by birth.

• Eg: -NOD mouse (a product of inbreeding for 80 generations)

• - BB rat

• What is the importance of in breeding???

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MODELS FOR NIDDM

• NEONATAL STZ MODEL OF NIDDM

• Neonatal rats of wistar or sprague-dawley strain are treated with STZ(80-100mg/kg),i.p at birth or with in the first 5 days following the birth

• This results in the development of Type-2 diabetes in their adult stage.

• But high doses of the STZ can induce Type-1 dibetes in adulr rats.

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OTHER CHEMICALLY INDUCED NIDDM MODELS

• These include:

• -Glucocorticoids• -Thiazide diuretics• -Diazoxide• -HIV protease inhibitors

• But this is however, temporary in nature..

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GENETIC MODELS OF NIDDM

• MONOGENIC MODELS OF OBESITY AND NIDDM:

• The phenotypes of the monogenic rodent models include obesity, hyper insulinaemia, transient or sustained hyperglycaemia and hyperlipidaemia.

• The animal models used are:• -Yellow Mouse - Obese and diabetic Mouse• -Tubby Mouse -Fat Mouse• -Zukker diabetic fatty rat

• What is phenotype and what is genotype?

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• POLYGENIC MODELS OF OBESITY AND NIDDM:

• These models simulate the human conditions more closely.

• -NZO Mouse -Japanese KK Mouse• -NSY Mouse -PBB/Ld Mouse• -Chinese Hamster -South African hamster

• What is monogenic disorder and what is polygenic disorder? MODY, NDM.

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TRANSGENIC ANIMALS:

• Transgene is randomly incorporated into the host genome and some offapring will therefore express the modified gene.

• Eg: The extra cellular ligand binding domain of the human insulin receptor has been expressed as a stable, soluble protein, which binds to the insulin with high affinity.

• Incorporation of this gene into the Mouse leads to accumulation of high affinity insulin binding protein in the plasma, which leads to chronic hyperglycaemia in that Mouse.

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KNOCK-OUT ANIMALS:

• Knock-out animals are produced by using a genetic construct that will disrupt normal gene.

• The genes that are manipulated to cause defective insulin secretion include:

• 1. GLT-2 2. Glucokinase• 3. Hepatic nuclear factors• 4. Islet amyloid polypeptide

• The genes that increase body fat include • -Knock-out of leptin gene -Knock-out of beta-3 rec.

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• Tha genes that are manipulated to cause insulin resistance coresponds to:

• 1. Insulin receptor 2. insulin receptor subsrate 1&2

• 3. Glucose transporters

• 4. Hexokinase-2

• 5.Tumor necrosis factor-alpha

• 6. Fatty acid binding protein etc..

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NORMOGLYCEMIC ANIMAL MODELS• RABBIT MODEL:

• Rabbit is the animal of choice for this model

• PROCEDURE:

• Mixed breed rabbits of either sex weighing 3 to 4.5 kg are chosen and divided into groups of 4 to 5.

• For the evaluation of insulin and insulin like compounds, food is stopped a day earlier to the experiment.

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• For the evaluation of hypoglycaemic agents, the animals have free access to normal diet till the experiment.

• The test drug is given orally through a gavage in the dose of 1ml/kg in 0.4% starch suspension.

• Control group receives only the vehicle, different doses are tried on other groups.

• Blood is withdrawn from the ear vein immediately before, and 1, 2, 3, 4, 5, 24, 48, and 72 h after treatment.

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• Blood glucose is determined in 10µl blood samples using the hexokinase enzyme method and blood sugar values are plotted against time.

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INVITRO METHODS ON ISOLATED ORGANS: • ISOLATED PANCREAS OF RAT:

• The invitro perfusion of isolated pancreas help us in studying the effect of the drug on insulin, glucagon and somatostatin secretion without interference from other organ changes.

• PROCEDURE:

• Animals used are male wistar rats weighing 200 to 250 g.

• The pancreas is removed under pentobarbital (50mg/kg i.p) anaesthetia.

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• Once the pancreas is removed , through a portal vein canula , Krebs-Ringer bicarbonate buffer with 2% bovine albumin and 5.5 mmol/l glucose is perfused at a rate of 1.75 ml/min.

• The temperature of the perfusion fluid is kept at 37.5c and the pressure at which it is perfused is about 100 mmHg.

• The perfusate is collected every minute for 30 min.

• After the first 5 min. of perfusion, test compound is added till the 15 min.

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• From 16th min. till 30 min, glucose of 5.5 mM and 16.6mM is perfused.

• The samples collected are stored at -20c.

• Hormones insulin, glucagon and somatostatin are estimated radioimmunologically.

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ISOLATED RAT LIVER:

• PROCEDURE:

• Male wistar rats weighing 200 to 250 g are anaesthetized with 150mg/kg hexobarbital i.p. after which the liver is removed from the animal.

• It is washed with 100 ml heparinized (5 IU/ml) physiological saline solution at 37c for 3 min. through the portal vein.

• The preparation is then transferred to perfusion apparatus, where portal vein canula is attached to tubing containing the oxygenated medium.

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• Perfusion is don with Krebs-Ringer bicarbonate buffer with 25% bovine erythrocytes, 1.6% bovine serum albumin, and 22.5 mmol/l Na- L- lactate at a rate of 30ml/min.

• Seventy ml are used for recirculation over 2 hrs.

• The test compound is added to the perfusate medium in a concentration of 40-100 µmol/l.

• The perfusate is bubbled with70 ml gas mixture/min.

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Glucose uptake by the isolated diaphragm from mice and rats

• This model is useful for the study of the effects of insulin and insulin like substances on muscle tissue.

• TO STUDY GLYCOGEN SYNTHESIS:• Diaphragms are obtained from male sprague

dawley rats weighing 70 to 100gm

• The hemidiaphragms are incubated in Kreb s-Hensleit buffer which has continuous supply of gas and also has the 5mM of c14 labeled glucose.

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• After 30 min. the hemidiphragms are frozen in liquid nitrogen.

• The powdered tissue is dissolved by heating for 45 min. at 100c in 30% KOH, and then 70%ethanol is added to it.

• After freezing at -20c , the samples are centrifuged at 2000g for 10 min.

• Then the c14 labeled glycogen is determined by liquid scintillation counting.

• Total glycogen is determined after hydrolysis to glucose. (1 N HCL at 100c for 3 hrs.)

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TO STUDY GLUCOSE TRANSPORT:

• Freshly disected rat diaphragms are incubated for 30 min at 37c in HEPES-buffured saline , under constant bubbling with gas.

• The diaphragms are then washed 2 times with the same buffer without glucose, and incubated further for 30 min. in 5 ml of the above buffer with test compound or insulin.

• Glucose transport is initiated by addition of 50 ml of 10mM of tritium labeled deoxy glucose in the absence or presence of 25 mM cytochalasin B(control).

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• After 15 min. the diaphragms arte rinsed four times with ice-cold buffer containing 10mM glucose, 25 mM cytochalasin B, then blotted with the filter paper and homogenized.

• Samples of the supernatant in a volume of 1ml are centrifuged at 1000g for 15 min. and mixed with scintillation cocktail and counted for radioactivity.

• Glucose transport is calculated as difference between diaphragm associated radioactivity measured in the absence and presence of cytochalasin.

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CONCLUSION:

• Many of the animal models described apparently share similar characteristic features of type 2 diabetes and have allowed experimentation that would be impossible in humans.

• None of the known single species is exactly equivalent to human diabetes, but each model act as a essential tool for investigating genetic, endocrine, metabolic, morphologic changes that could also operate during the evolution of type 2 diabetes in humans.

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• Though there are some limitations like expensiveness, practical difficulties, extreme care and ethical considerations associated with the use of non rodent animal models, the detailed investigations in these diabetic animal species are urgently required for better understanding of the disease mechanisms.

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• REFERENCES:• 1. S.K Guptha. Screening of anti diabetic agents. In:

Drug screening methods(Pre clinical evaluation of new drugs), Jaypee brother medical publishers, New Delhi, 2009, pp. 589-610.

• 2. N.S Parmar, Shiva Prakash. Screening of anti diabetic activity. In: Screening methods in pharmacology, Narosa publishing house, New Delhi, 2006, pp. 289-293.

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• THANK YOU