screening methodes of anti-diabetic drugs

Diabetes mellitus is chronic metabolic disease , occurs when the pancreas is not producing insulin or produced insulin cannot be used by the body, or combination of both.

1. A SEMINOR ON PRESENTED BY BORUDE SHAMAL R. M.PHARM UNDER GUIDANCE OF PROF. H. J. PAGAR DEPARTMENT OF PHARMACOLOGY DR.VITHALRAO VIKHE PATIL COLLEGE OF PHARMACY, VILAD GHAT, AHMEDNAGAR [2018-019] PRECLINICAL SCREENNING of ANTI-DIABETIC DRUG

2. CONTENT Introduction to Diabetes Type of Diabetes Signs & Symptoms Complication of DM Anti-diabetic drug [Oral Hypoglycemic Drugs] Screening methods Evaluation parameter References

3. diabetes mellitus [dm] Diabetes mellitus is a chronic metabolic disease, occurs when the pancreas is not producing insulin or produced insulin cannot be used by the body, or combination of both. When any of these things happens the body is unable to get sugar from blood into the cells. Hyperglycemia- high blood-sugar level . Glucose – form of sugar found in the blood, chief energy source. Lack of insulin or resistance to insulin – Increases sugar in your blood. This can leads to many health problem.

4. TYPES OF DIABETES mellitus 1.Type 1 Diabetes Mellitus Type 1 diabetes is an autoimmune condition. It's caused by the body attacking its own pancreas with antibodies . In people with type 1 diabetes, the damaged beta cell of pancreas doesn't make insulin. This type of diabetes may be caused by a genetic predisposition. It happens when immune system mystically attacks & destroy beta cells in the pancreas that normally produce insulin. This is also called insulin dependent DM.

5. TYPE OF DM [ CONTd …..] 2.Type 2 Diabetic mellitus Also called as non-insulin dependent DM. Most common form of Diabetes. Usually occurs in adulthood but generally diagnosis is increase in the younger generation. Type 2 DM starts as a insulin resistance. This means your body can't use insulin efficiently. Insulin production decreases which leads to high blood sugar. The exact cause is unknown, contributing factors may be genetic, lack of exercise, & being overweight.

6. TYPE OF DM [ CONTd …..] 3.Gestational DM Gestational DM is due to insulin blocking hormone during pregnancy. This type of diabetes occurs only during pregnancy. Blood glucose level are high during pregnancy in women. There is high risk of type 2 diabetes & cardiovascular diseases.

7. TYPE OF DM [ CONTd …..] 4.Pre-diabetes At least 79 million people are diagnosed with pre-diabetes each year. It is above average blood glucose level, not high enough to be classified under type 1 & type 2 diabetes. Causes -long-term damage to the body, including heart & circulatory system . Starts with unhealthy eating habbits & inadequate exercise.

8. SIGNS AND SYMPTOMS Frequent urination Excessive thirst Unexplained weight loss Extreme hunger Sudden vision changes Numbness in the hands or feet Feeling very tired much of the time Very dry skin Sores that are slow to heal More infections than usual

9. complications Nerve damage (neuropathy) Cardiovascular disease , High blood pressure Kidney damage (nephropathy) Eye damage (retinopathy) Poor wound healing Skin conditions - e.g. skin rashes

10. ORAL HYPOGLYCEMIC DRUG 1.Sulphonylureas a) First generation - e.g.Tolbutamide, chlorpromide b) Second generation – e.g. Glibenclamide, Glipizide 2.Biguanides --e.g. Metformine 3.Meglitinide –e.g. Repaglinide, nateglinide 4.Thiazolidinediones –e.g. Rosiglitazone, Pioglitazone 5.Alpha-glucosidase inhib . - e.g. Meglitol, Acarbose K. D. Tripathi

11. SCREENNING OF ANTI-DIABETIC DRUGS Methods of screening A. In-vivo Models 1. For IDDM Streptozotocin Induced diabetes Alloxan Induced diabetes Virus Induced diabetes Hormone Induced diabetes Insulin deficiency due to insulin antibodies Other diabetagenic compounds Vogel G. H.et.al

12. contd…… 2.For NIDDM Spontaneous or genetically derived diabetic animal Diet / Nutrition induced diabetic animal Neonatal STZ induced diabetic animal Transgenic diabetic animal K. Srinivasan and P. Ramrao B. In-Vitro Models Effect on Liver Effect on Muscles' Effect on Pancreas Effect on Adipose tissue

13. Animals used for screening Sand Rat Neonatal Mice Wister Rat Spiny mice NOD Mouse BB Mouse

14. IN-VIVO MODELS FOR IDDM 1) Streptozotocine induced diabetes Principle - Rakieten et.al.(1963) discovered diabetogenic activity of antibiotics streptozocin.Basic principle is the compound is found to be cytotoxic beta cells of pancreas. Wister rat :

15. [contd …..] Procedure – Male Wister rats(150-220gm)are injected i.p with 60mg/kg STZ prepared in bufferd solution Three phases of changes blood glucose level are observed

16. Initially after 3 hrs level of glucose increased up to 150-200mg/dl. After 8 hrs serum insulin values are increased up to 4 times. Hypoglycemic phase followed by hyperglycemia After 24-48 hrs hyperglycemia alredy occur reaching values 800mg/dl with glycosuria & ketonemia. Histologically beta cells are degranulated After 14 days animals are used for pharmacological test. Vogel G. H. et.al

17. IN-VIVO MODELS FOR IDDM [contd….] 2) Alloxan Induced diabetes Purpose and Rationale – Frerichs (1968) & Creutzfeldt (1972) – chemical induced diabetes in animal. Principle & Procedure - Alloxan have capacity to produce reverse diabetes It is a toxic cyclic urea analogue which destroys the beta cells of Langerhans in pancreas. This compound causes sever necrosis in pancreatic beta cells. It has been suggested that it produce hydrogen peroxide & some free radicals such as oxygen & OH that produce damage to the beta cells & then death of cell.

18. Blood glucose level shows triphasic response hyperglycemia for 1 hr followed by hypoglycemia that lasts for 6 hrs & stable hyperglycemia after 48 hrs Animals showing fasting blood glucose level above 140mg/dl after 48 hrs are considered diabetic Animals are kept for 48 hrs during which food & water allowed Animal is injected with single dose [100mg/kg body wt] dissolved in normal saline by I. p. route

19. Serum glucose level is estimated by glucose – oxidase – peroxidase method [GOD-POD] using Autoanalyser Vogel G. H. et.al After six weeks of treatment blood samples are collected from 8 hr fasting animals [can be collected via orbita sinus through a pipette ] For a period of six weeks, drug sample to be screened are administered orally

20. IN-VITRO MODELS FOR NIDDM 1.Diet /nutrition Induced diabetes – Some of the animal model exist in which diabetes is induced neither by genetic defects nor by chemicals. Sand rat, Tuco-tuco & spiny mouse are important model for nutritionally induced obesity & type 2 diabetes.

21. contd……. 1) Sand rat- Small rodent Indigenous to desert region Access to food and water is limited Exhibit genetic predisposition, if fed with high calorie laboratory diet

22. Contd…….. 2) Spiny mice- Two species of spiny mouse acomys russates And cahirinus Have congenital hyperplasia of pancreatic islets K. Srinivasan and P. Ramrao

23. Contd……. 2. Genetically diabetic animal 1) The NOD mouse Non obese diabetic mouse These are the inbred strain of albino mice developed by Makino & co-workers in Japan. Develop diabetes abruptly at the 100-200 days of the age

24. Contd….. 2) The BB Rats [Bio Breeding Rat] Insulin deficiency is due to the autoimmune destruction of pancreatic beta cells. Male & female have equal severity & frequency of diabetes. Onset of clinical diabetes is sudden & occurs at about 60-120 days at age

25. IN-VITRO MODELS FOR NIDDM [CONTD..] 2.Neonatal STZ model – Neonatal STZ Rats – STZ (90mg/kg) i.p . At birth or within the first five days Rats showing fasting blood glucose level above 140mg/dl are considered as diabetic Drug sample to be screened is administered by suitable route & blood glucose level is analyzed K. Srinivasan and P. Ramrao

26. b. In-vitro models 1.Effect on Liver Isolated Hepatocytes Purpose & Rationale - Isolated Hepatocytes can be used to study the effect of drug on hepatic gluconeogenisis & other hepatic metabolic reactions such as ketone bodies formation & tricarboxylic formation. Animal – Male wister rat (200-250gm) anasthesized with hexabarbitol ( 150ml/kg)

27. [contd…..] Evaluation – The sample for analysis is withdrawn by catheter & are evaluated for net glucose production Procedure - Male Wister rat taken Hepatocytes isolated by collagenase method Cell suspension is preincubated Substrats are added Test drug added

28. [contd…..] 2. Effect on muscle cell Use of isolated diaphragm from mice & rat Glucose is evaluated by glucose oxidase method and pyruvate, lactate & acetoacetate are evaluated by enzymatic method. Isolated diaphragm from rats & divide it in equal part. Incubate with kreb –henselit buffer with 5mM glucose, insulin or compound to be tested.

29. After 30 min hemi diaphragm are blotted on the tissue & frozen I liquid nitrogen. Powdered tissue is dissolved in the 30 % KOH . After freezing sample are centrifuged . Glycogen pallets are wash with 70 % ethanol & labeled C14 glycogen is determined. Total glycogen is determined after hydrolysis to glucose. The total conc. dependence of glucose uptake & conversion into glycogen by insulin is determined. K. Srinivasan and P. Ramrao

30. [Contd…..] Evaluation Body wt. Lipid profile Serum insulin level Histopathology of pancreas & liver

31. REFERANCES 1. K.D.Tripathi, “Essentials of medical Pharmacology” Jaypee Publication 2006(7) 235-236 2. G. H. Vogel Springer“Drug Discovery and evaluation assay”, 2002, 999-1044 3. K. Srinivasan and P. Ramrao“Animal model in type 2 diabetes Reasearch and overview " Indian J Med Res 125 March 2007 , 451-472 4. Lenzen S “ The mechanisms of alloxan- and streptozotocin- induced diabetes”. (2008) Diabetologia 51: 216-226.

32. Contd…….. 5. Animal models for biological screening of anti- diabetic drugs: An overview Manish Pal Singh and Kamla Pathak Dept. of PIV- Manish Pal Singh and Kamla Pathak Euro. J. Exp. Bio., 2015, 5(5):37-48 Pelagia Research Library 6. Karthikeyan M, Balasubramanian T, Kumar P (2016) In-vivo Animal Models and In-vitro Techniques for Screening Antidiabetic Activity. J Develop Drugs 5: 153. doi:10.4172/2329-6631.1000153 7. Ramanathan et al. and N. Venkatesan World Journal of Pharmacy and Pharmaceutical Sciences