Antidiabetic screening

Content-
1. Introduction
2. types of diabetes
3. screening methods of antidiabetic drugs
4. models for IDDM- chemically induced diabetes, hormones induced, virus induced, insulin antibodies induced and surgically induced diabetes.

1. SCREENING METHODS OF ANTIDIABETIC DRUGS PRESENTED BY: SHUBHA SHARMA M.SC. PHARMACOLOGY RUHS-CMS, JAIPUR

2. INTRODUCTION The pancreas is an organ composed of exocrine and endocrine cells. Islets of langerhans , which forms the endocrine part , consist of four types of cells alpha, beta, delta and PP cells. Glucose stimulates the beta cells to release insulin, which then promotes glucose uptake and storage. “ Diabetes mellitus is characterized by derangement in carbohydrate, protein and fat metabolism caused by the complete or relative insufficiency of insulin secretions and/ or insulin action.”

3. TYPES OF DIABETES There are 2 major types of diabetes: 1) Type 1 Diabetes (associated with insulin deficiency) Also called as Insulin- dependant diabetes mellitus (IDDM)/ juvenile onset Type 1 diabetes is believed to be an autoimmune condition. It happens when immune system mistakenly attacks and destroys the beta cells in your pancreas that produce insulin . The damage is permanent.

4. 2) Type 2 Diabetes (associated with insulin resistance) Also called as Non Insulin- dependant Diabetes mellitus (NIDDM) Most common form of diabetes. Usually occurs in adulthood but diagnosis is increasing in the younger generation. The exact cause is unknown. Contributing factors may include genetics, obesity, hyperinsulinemia and insulin resistance. Insulin resistance may be due to the defect at receptor level or at the postreceptor level . This means your body can’t use insulin efficiently.

5. SCREENING OF ANTIDIABETIC DRUGS

6. MODELS FOR IDDM (TYPE I) Chemically induced diabetes Alloxan Streptozotocin Hormones induced diabetes Virus induced diabetes Insulin antibodies- induced diabetes Surgically induced diabetes

7. MODELS FOR NIDDM (TYPE II ) 1. Chemically induce d Diabetes i . Neonatal STZ model for NIDDM 2. Genetic models Monogenic model for NIDDM a)  Yellow Mouse (The Agouty Mouse) b)   Obese and Diabetic Mouse Polygenic model for NIDDM a )  New Zealand Obese mouse (NZO) b)   Japanies KK Mouse

8. CHEMICALLY INDUCED DIABETES 1. Alloxan induced Diabetes: Alloxan have capacity to produce reversible diabetes. It is a toxic cyclic urea analogue which destroys β-cells of the islets of Langerhans in pancreas. This compound causes severe necrosis of pancreatic β-cells . It has been suggested that allloxan induces the production of H 2 O 2 and some free radicals such as O 2 and OH- that produce first damage and later the death of β-cells.

9. Mechanism Alloxan has two distinct pathological effects: Alloxan is highly reactive molecule that is readily reduced to dialuric acid, Dialuric acid is then auto-oxidized back to alloxan resulting in the production of free radicals. These free radicals damage the DNA of β -cells and cause cell death. Its ability to react with protein SH groups , especially the membrane proteins like Glucokinase on the β - cells , finally resulting in the cell necrosis ..

10. Procedure Animals: Rats of Wistar or Sprague–Dawley strain(150-200 g) Inducing agent: Alloxan (100-175 mg/kg s.c. ) Maintain rats at standard environment and laboratory chow. All the animals , which are given alloxan , receive glucose and insulin for one week and food ad libitum. Thereafter, single daily dose of 28 IU insulin is administered s.c. The blood glucose level shows triphasic change, first a rise at 2Hr , followed by hypoglycemic phase at 8Hr , finally an increase at 24Hr probably due to depletion of β -cells of insulin.

11. Evaluation: Any suitable method for estimation of  Glucose level  Insulin level  Hepatic glycogen level Compare results of standard with control group animals.

12. Drawbacks High mortality in Rats Causes ketosis in animals due to free fatty acid generation Diabetes induced is reversible Some species like guinea pigs are resistant to its diabetogenic action Alloxan has been almost completely replaced by streptozotocin (STZ) because of these drawbacks

13. 2. Streptozotocin -induced Diabetes STZ [2- deoxy-2-(3-methyl-3-nitrosourea)1-D-glucopyranose] It is a broad-spectrum antibiotic, which is produced from Streptomyces achromogens . It is used to induce both type 1 diabetes and type 2 diabetes at dose of Rat: 50-60 mg/kg i.p Mice: 175-200mg/kg i.p or i.v Dogs: 15mg/kg for 3 days

14. Mechanism of causing B-cell damage a)  By process of methylation b)  Free radical generation and c)  Nitric oxide production

15. Procedure STZ induces diabetes in almost all species of animals. Diabetic dose varies with species and the optimal doses required in various species are: a)  Rats(50-60mg/kg, i.p . or i.v. ) b)  Mice(175-200mg/kg, i.p . or i.v. ) c)  Dogs(15 mg/kg for 3 days) The blood glucose level shows the same triphasic response as seen in the alloxan treated animals, with hyperglycemia at 1Hr ,followed by hypoglycemia which lasts for 6 Hr , and stable hyperglycemia by 24-48Hr after STZ administration.

16. Advantages It has completely replaced Alloxan for inducing diabetes because of : Greater selectivity towards B-cells Lower mortality rate Irreversible diabetes induction

17. MODIFICATIONS Multiple low dose of STZ also induces diabetes by causing immune mediated pancreatic insulitis in rats. Cyclosporin -A when given with STZ enhances its diabetogenic efficacy. STZ combined with complete Freund’s adjuvant, administered 24h prior to STZ (25mg/kg)and then repeated in the three subsequent weeks, all produces hyperglycemia. Neither four administration of CFA nor of STZ alone result in persistent hyperglycemia.

18. Hormones Induced Diabetes Animal: Rats, Guinea pigs and Rabbits Inducing hormone: Dexamethasone. Eg . NIDDM form of diabetes is induced when dexamethasone,a long-acting glucocorticoid is administered at a dose of 2-5mg/kg i.p . twice a day in rats In other animal models(like guinea pigs and rabbits), corticotrophin is used to stimulate adrenal cortex that results in hormonal imbalance causing steroidal diabetes.

19. VIRUS INDUCED DIABETES  Principle: - various human viruses used for inducing diabetes include RNA picorno virus encephalomyocarditis [EMC-D] coxsackie B4 [CB-4]. Renovirus Mengo-2t

20. 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%) A less infecting variant produces a comparable damage by eliciting autoimmune reactivity to the beta cells. Infected animals are considered hyperglycaemic if there non fasting levels exceed by 250mg/dl 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.

21. Insuline Antibodies-induced Diabetes Giving Bovine insulin along with CFA to guinea pigs produces anti-insulin antibodies. Large doses and prolonged administration are accompanied by ketonemia , ketonuria , glycosuria and acidosis are fatal to animals Lower doses produce reversible type of diabetes after a few hours.

22. Surgically -induced Diabetes Surgical removal of all or a part of pancreas . Total removal of pancreas result in the insulin dependant type of diabetes. Experimental animals are required to maintain isulin therapy. P ancreatectomy with chemical agents can produce a stable form of diabetes in animlas such as cats and dogs. The combination therapy reduces the organ damage associated with chemical induction and minimizes the interventions.

23. Genetically diabetic animals The NOD Mouse Non Obese Diabetic Mouse These are the inbred strain of albino mice developed by Makino and coworkers in Japan . Autoimmune destruction of β-cell in association with Insulitis and auto-antibody production. Develop diabetes abruptly at the 100-200 days of age. Insulin treatment required for survival.

24. Genetically diabetic animals … . WBN/ KOB rat: InbreedWistarstrain . Decrease in number and size of islets at 12 weeks of age . Aged male Wistar strain shows diabetic symptoms. Impairedglucosetoleranceandglycosuriaat21week of age.

25. MODELS FOR NIDDM NEONATAL STZ MODEL Streptozotocin causes severe pancreatic beta cells destruction, accompanied by decrease in pancreatic insulin stores and rise in plasma insulin levels. Procedure : - • Neonatal rats of Sprague-Dawley strain are taken • Treated with STZ 80-100 mg/kg IP at birth or within 5 days of birth which leads to severe β cell destruction, deficiency in pancreatic insulin levels and rise in plasma glucose. • In contrast to adults, the β cells in neonates partially regenerate. • Following an initial spike in plasma glucose, these rats become normoglycemic by 3 weeks.

26. Other chemical methods • For NIDDM in rabbits - adrenaline (0.1 mg/kg SC). • The hyperglycemia is seen at 1 hour and lasts for 4 hours. Oral hypoglycemic agents can be screened by this method. • Other chemicals are 8-hydroxy quinoline , biphenyl thio carbazine , EDTA (partially depancreatized rats), thiazides, chlorthiazide , hydrochlorthiazide , diazoxide and furosemide.

27. Genetic models for NIDDM Monogenic models of obesity and NIDDM Yellow mouse (the Agouti mouse) Obese and diabetic mouse Tubby mouse Fat mouse Zucker diabetic fatty rat Koletsky and JCR: LA-Corpulent rats Polygenic models of obesity and NIDDM New Zealand obese mouse Japanese KK mouse Nagoya – Shibata – Yasuda mouse PBB/ Ld mouse Goto-Kakisaki rat Chinese Hamster South African Hamster

28. THANKS …