Em antidiabetic drugs

EM antidiabetic drugs

1. Evaluation methods of antidiabetic drugs Dr Shinde Viraj Ashok Junior Resident – 3 Department of Pharmacology Guided by Dr Sonali Pimpalkhute Associate Professor Department of Pharmacology EM of antidiabetic drugs 1

2. overview EM of antidiabetic drugs 2

3. introduction Diabetes mellitus Characterized by hyperglycemia; altered metabolism of lipids, carbohydrates & proteins; & an ↑ risk of vascular & non vascular complications Also characterized by glycosuria, hyperlipidemia, negative nitrogen balance & sometimes ketonuria EM of antidiabetic drugs 3

4. Types of Diabetes Mellitus Type I IDDM Beta cell destruction (autoimmune/idiopathic) Low insulin levels Less common , less genetic predisposition Type II NIDDM No or moderate Beta cell reduction Low , normal , high insulin levels More common , more genetic predisposition Insulin resistance EM of antidiabetic drugs 4

5. Current treatment modalities Different insulin preparations and different insulin delivery systems Sulfonylureas Biguanides Meglitinides Thiazolidinediones α - glucosidase inhibitors EM of antidiabetic drugs 5

6. Evaluation of antidiabetic drugs EM of antidiabetic drugs 6

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8. Experimental animals EM of antidiabetic drugs 8

9. Methods to induce experimental IDDM EM of antidiabetic drugs 9

10. Alloxan induced Diabetes Alloxan is a cyclic urea (tetra- oxo - hexahydropyrimidine ) Mechanism of action - Free radical damage to beta cell DNA - Reacts with protein SH group causing cell necrosis Triphasic response: at 2hr, hyperglycemia at 8hr, hypoglycemia at 24 hr, hyperglycemia Alloxan standard solution is prepared at a strength of 5gm/100ml kept at pH 4.5 EM of antidiabetic drugs 10

11. procedure Rabbits - New Zealand strain weighing 2.0 to 3.5 kg - Infused via ear vein with 150mg/kg alloxan for 10 min - 70% - become hyperglycemic & uricosuric Rats - Sprague Dawley (SD) strain weighing 150-200 gms - A lloxan is injected by -Intravenous - 65mg/kg - Subcutaneous 100–175 mg/ kg - Intraperitoneal 100–175 mg/ kg EM of antidiabetic drugs 11

12. Contd.. Neonatal model [Modified] - Kodoma et al. - Male SD rats 2, 4, or 6 days of age - A fter a 16 h fast, injected IP with 200 mg/kg alloxan monohydrate - M ost severe diabetic symptoms occurred in rats injected on day 6 Male Beagle dogs - Weighing 15–20 kg - 50 mg/kg alloxan , IV - Subsequently, animals receive daily 1000 ml 5% glucose solution with 10 IU Regular insulin for one week & food ad libitum - Thereafter, a single daily dose of 28 IU insulin s.c . EM of antidiabetic drugs 12

13. Contd.. Drawbacks Risk of high mortality (up to 50%) Unstable at physiological pH, so care is taken to see that it is preserved at a pH of 4.5. Dosage variation with age & species Ketosis due to free fatty acid generation GUINEA PIGS are resistant to diabetogenic action EM of antidiabetic drugs 13 This process has been almost completely replaced by streptozocin for inducing diabetes because of these drawbacks

14. Streptozotocin induced Diabetes Streptozotocin : - 2-deoxy-2-[3-methyl-3-nitrosourea] 1-D glucopyranose - Initially used for antibacterial & anti-tumor activity - From fungus Streptomyces achromogenes - Rakieten et al : first described Diabetogenic property - Induces permanent DM : By methylation Free radical generation Nitric oxide production EM of antidiabetic drugs 14

15. Contd.. Procedure - Wistar rats weighing 150–220 g - 8 hrs fasting - 65 mg/kg streptozotocin in citrate buffer (pH 4.5) IP - 50 mg /kg IV - Triphasic response - 1 hr : Hyperglycemia - 6 hr : Hypoglycemia - 24-48 hr : Hyperglycemia EM of antidiabetic drugs 15

16. Modifications of method Multiple low doses of streptozotocin induce immune pancreatic insulitis in rats mimicking immune type 1 diabetes in humans Different animal models are tried like golden hamster, Yorkshier pig EM of antidiabetic drugs 16

17. Contd.. Advantages Almost replaced alloxan because of - Greater selectivity towards β cells - Lower mortality rate - Longer duration diabetes induction EM of antidiabetic drugs 17

18. Contd.. Disadvantages Highly unstable at room temperature (preserved at - 20 C) Single dose may not give results therefore streptozotocin might also be given 2 divided doses 4 hrs apart Necessary to maintain cold temperature GUINEA PIGS and RABBITS are resistant EM of antidiabetic drugs 18

19. Hormone induced Diabetes Growth hormone induced diabetes Dogs & cats after repeated administration of GH exhibit severe form of diabetes Rats are resistant Corticosteroid induced diabetes Combined with forced feeding rats produce symptoms of diabetes mellitus Also used in rabbits & guinea pigs EM of antidiabetic drugs 19

20. Insulin antibodies induced diabetes Giving bovine insulin with Complete Freud's adjuvant to guinea pigs produces anti-insulin ab IV injection of 0.25 – 1.0 ml guinea pig anti-insulin serum to rats induces hyperglycemia up to 300 mg% It persists as long as abs are capable of reacting with insulin remaining in circulation Higher doses produce ketonemia , ketonuria , acidosis may be fatal & in low doses, hyperglycemia is reversible EM of antidiabetic drugs 20

21. Other diabetogenic compound Chelator : Dithizone Single IV dose of 40–100 mg/kg To cats, rabbits, golden hamsters & mice Dithizone injection Causes a triphasic glycemic reaction in rabbits EM of antidiabetic drugs 21

22. Virus induced Diabetes mellitus Diabetes mellitus due to Infecting & destroying of β cells Damage by eliciting immune autoreactivity to β cells Produces systemic effect RNA picornavirus , CoxsackieB4, encephalomyocarditis (EMC-D & M variant), Mengo 2T EM of antidiabetic drugs 22

23. Spontaneous models BioBreeding (BB) Rat Spontaneous diabetes was initially diagnosed by Chapel brothers in 1974 Clinical presentation is similar to that of its human counterpart One of rodent model in which significant ketosis occurs in absence of obesity Within several days, diabetic animals are severely hyperglycemic , hypoinsulinemic & ketotic unless insulin treatment is instituted EM of antidiabetic drugs 23

24. NOD Mouse (non-obese diabetic) Similar to BB Rat Autoimmune mechanism attributed to CD 4 + & CD 8 + T cells dependent Feeding small amounts of human insulin conjugated with cholera toxin leads to suppression of disease EM of antidiabetic drugs 24

25. Surgical pancreatectomy in dogs Procedure (Classic model)- (1971) Male Beagle dogs 12–16 kg A nimal is anaesthetized with an IV 50mg/kg pentobarbital sodium Through a midline incision on abdomen - pancreas is carefully removed B leeding vessels & pancreatic duct - ligated EM of antidiabetic drugs 25

26. Contd.. Disadvantages of pancreatectomy Loss of α & δ cells– loss of counter regulatory hormones Loss of enzymes required for proper digestion Total resection in rats - very difficult Diabetes development appear to be strain specific EM of antidiabetic drugs 26

27. Contd.. Combined : Pancreatectomy + chemical agents Produce stable diabetes in cats & dogs Reduces organ damage associated with chemical induction EM of antidiabetic drugs 27

28. Transgenic animals Elastase I promoter fused to diptheria A gene produces mice without pancreas Lymphocytic choriomeningitis virus glycoprotein (LCMV GP) when expressed in mice causes CD8+ T cell mediated diabetes Other transgenic models where viral infection produces transgenic effect EM of antidiabetic drugs 28

29. Methods to induce experimental NIDDM EM of antidiabetic drugs 29

30. Streptozotocin induced diabetes Wistar neonates - Streptozotocin - 100 mg/kg I.P. as a single dose on day 2 or day 5 OR in divided doses on 2 nd & 4 th day Severe β cell destruction produces hyperglycemia β – cells partially regenerate – rats may become normoglycemic by 3 weeks age EM of antidiabetic drugs 30

31. Spontaneous animal models EM of antidiabetic drugs 31

32. Species Research suitability C57 BL/6J ob mice (Tubby mouse) Neuroendocrine factors; satiety & hypothalamic insulin responses; thermoregulation KK mice Muscle & liver insulin resistance; effects of antidiabetic drugs NZO mice Hyperphagic , obese, hyperglycemic, insulin resistance, mildly glucose intolerant C57BL/Ks db mice (AR) Effect of genomic & nutritional modifiers on ß-cell replication & survival; drugs mitigating insulin resistance & receptor function EM of antidiabetic drugs 32

33. Species Research suitability Jcr -LA- cp rats Genomic predisposition; role of hyperinsulinemia Sand rats Over nutrition induced insulin resistance; low insulin degradation; low islet secretion threshold; reversible insulin effects on receptor function GK rats Insulin secretion abnormalities; drug effects in moderate, insulin resistant DM SHR/N- cp rats Interrelation of hyperinsulinemia , hypertension & nutrition on kidney lesions; genomic predisposition to these lesions EM of antidiabetic drugs 33

34. Transgenic & knock out animals Genes manipulated to cause Insulin Resistance - Insulin receptor - Insulin receptor substrate 1 & 2 - Glucose transporters - Hexokinase II - TNF α & fatty acid binding protein 2 Genes manipulated to cause Defective Insulin Secretion - GLUT - 2 - Glucokinase - Hepatic nuclear factor - Islets amyloid peptide EM of antidiabetic drugs 34

35. Diazoxide induced Diazoxide - T hiazide diuretic - Produces hyperglycemia by blocking potassium gated ATP channels in beta cells - Hyperglycemia is till effect of dose lasts (up to 2 hrs maximum being in initial 30 minutes) - Can be used for short glucose lowering ability experiments EM of antidiabetic drugs 35

36. Contd.. Procedure : - Normal Wistar fasted rats (180 ± 220 g) - Initial dose of 30 mg/kg, I.P. 30 min before administration of test drug - Supplementary doses (15 mg/kg, I.P.) during experiment every 15 min - Blood samples taken at intervals up to 3 h after administration of test substances EM of antidiabetic drugs 36

37. Models for evaluation EM of antidiabetic drugs 37

38. Models for evaluation of insulin and insulin-like activity In vivo models Blood sugar lowering effect in rabbits Hypoglycemic seizures in mice In vitro models Rat adipocytes Total uptake of glucose - in rat adipocytes - in isolated diaphragm of rats or mice EM of antidiabetic drugs 38

39. Blood sugar lowering effects in rabbits Biological assay of insulin preparations in comparison with a stable standard using blood sugar lowering effect in rabbits Procedure : - 4 groups of at least 6 randomly distributed rabbits (New Zealand strain) weighing at least 1.8 kg are selected - 24 hr before test : Each rabbit is provided with an amount of food that will be consumed within 6 hr EM of antidiabetic drugs 39

40. Contd.. Food & water is withheld until final blood sample has been taken Two solutions of standard preparation are made, containing 1 unit & 2 units of insulin per ml, respectively Solutions is injected S.C. to one group of rabbits, 0.3 & 0.5 ml EM of antidiabetic drugs 40

41. Contd.. One hour & 2.5 h after each injection, blood sample is taken from ear vein of each rabbit Blood sugar is determined by a suitable method (glucose oxidase ) Same procedure can also be carried out in rats. D oses are given intraperitoneally . EM of antidiabetic drugs 41

42. Hypoglycemic seizures in mice To evaluate for deleterious effects of hypoglycemia produced by test compound Procedure: - Ninety-six mice of either sex weighing 20 ± 5 g are randomly distributed into 4 groups - Mice are deprived of food 20 hr immediately preceding test EM of antidiabetic drugs 42

43. Contd.. Insulin solutions standard & of test preparation containing 30 & 60 milli Units/ml are prepared 0.5 ml/20 g subcutaneously Observation of mice for 1.5 h & number of mice is recorded that are dead, convulse or lie still for more than 2 or 3 s when placed on their backs EM of antidiabetic drugs 43

44. In-Vitro studies for insulin and insulin like activity Rat adipocytes Used in 1. Lipogenesis assay Measures insulin stimulated glucose transport & conversion into lipid soluble products 2. Glucose transport Measures total glucose uptake & its conversion into different products  EM of antidiabetic drugs 44

45. Contd.. Rat adipocyte membrane Assay of glucose transporter translocation Isolated membrane of rats & mice Glucose uptake by isolated diaphragm from mice & rats has been used to study effects of insulin & insulin-mimetic substances on muscle tissue EM of antidiabetic drugs 45

46. Models for evaluation of drugs which act as secretagogues A] Blood glucose lowering activity in 1. Rabbits 2. Rats 3. Dogs B] Euglycemic clamp technique C] Receptor binding of sulfonylureas EM of antidiabetic drugs 46

47. A] Blood glucose lowering activity in rabbits Insulin secretagogues will ↑ release of insulin & hence level of hyperglycemia achieved will be less in animals receiving these drugs Procedure : - Groups of 4–5 mixed breed rabbits (New Zealand strain) of either sex weighing 3.0–4.5 kg - Animals are on a normal diet till prior to experiment - Test drug administered by gavage in 1 ml/kg in 0.4% starch suspension or intravenously in solution EM of antidiabetic drugs 47

48. Contd.. Several doses are given to different groups One control group receives vehicle only Blood is withdrawn immediately before & 1, 2, 3, 4, 5, 24, 48 & 72 hr after treatment through ear veins For time-response curves values are also measured after 8, 12, 16, and 20 h EM of antidiabetic drugs 48

49. B] In vivo- Euglycemic clamp technique Useful method of quantifying in vivo insulin sensitivity in male vistar rats In this technique, a variable glucose infusion is delivered to maintain euglycemia during insulin infusion Whole-body tissue sensitivity to insulin, as determined by net glucose uptake, can be quantitated under conditions of near steady state glucose & insulin levels EM of antidiabetic drugs 49

50. C] Receptor binding of sulfonylureas Sulfonylureas block ATP - Dependent K+ channels in β-cell plasma membrane Binding to receptor depolarization of membrane chain of events leading to release of insulin Binding studies of sulfonylureas & other drugs can be performed on - Isolated pancreatic islets - Isolated insulinoma cells - Isolated intact membranes or solubilized membranes EM of antidiabetic drugs 50

51. Model for evaluation of insulin sensitizer drugs Activity in isolated organs & cells Effects on PANCREAS : Perfusion of isolated rat pancreas - To study influence of carbohydrates, hormones & drugs such as sulfonylureas on insulin, glucagon & somatostatin secretion EM of antidiabetic drugs 51

52. Models for evaluation of α- glucosidase inhibitors In vitro models- using everted sac technique in rat - To study effects of intestinal enzymes on substrates in an incubation vial In vivo models- Inhibition of glucose absorption in rat intestine - By measuring blood glucose after administration of starch or disaccharides with & without inhibitor - In addition, non-absorbed starch or disaccharides can be determined in intestine EM of antidiabetic drugs 52

53. Clinical evaluation EM of antidiabetic drugs 53

54. Phase i Safety study in normal volunteer Objectives of these studies is to provide metabolic, kinetic data as base for phase 2 studies First human exposure especially crucial if biosynthetic drugs are used EM of antidiabetic drugs 54

55. Contd.. Food & drug interaction is to be observed meticulously Special care is taken to evaluate patients general condition & change in regular habbits along with biochemical & electrophysiological monitoring EM of antidiabetic drugs 55

56. Phase ii and iii End points Primary end point Change in HbA1c Secondary end points Change in fasting blood glucose levels Lipid profile Clinical safety & toleration & lab safety Quality of life assessment EM of antidiabetic drugs 56

57. Phase iv Postmarketing study Any undesired progression of disease complication Care to find out beneficial effects in different complications which were not evaluated before To delineate additional information including drug's risks, benefits & optimal use EM of antidiabetic drugs 57

58. Clinical implications for antidiabetic evaluation Type 1 Diabetes Mellitus - Insulin is essential glucose-lowering therapy - All treatments for type 1 diabetes that are not insulin analogues or other insulin receptor ligands should be studied as add-on therapies to insulin - For study design interpretation & inference of new drug’s efficacy, pharmacodynamic interactions with insulin & need to adjust insulin doses to prevent hypoglycemia EM of antidiabetic drugs 58

59. Contd.. Type 2 Diabetes Mellitus Efficacy & safety of new products evaluated in - Placebo-controlled monotherapy trials - Placebo-controlled add-on therapy trials - Active-controlled trials EM of antidiabetic drugs 59

60. Studies of a test agent as monotherapy Participants : - Drug-naïve - Diabetes reasonably well controlled with diet & exercise Duration : up to 24 weeks - For phase 2 or phase 3 studies - If hyperglycemia persists or worsens beyond prespecified thresholds - Monitored & treated throughout study EM of antidiabetic drugs 60

61. conclusion An ideal antidiabetic agent is one which normalises all of metabolic disturbances Induced experimentally by removal of source or action of insulin Care must be taken in interpretation & extrapolation of results obtained from these animal models to humans Most useful screening method is based on depression of blood sugar in intact animals EM of antidiabetic drugs 61

62. references Drug Discovery and Evaluation, Pharmacological Assays Wolfgang H.Vogel , Bernward A. Schölkens , Jürgen Sandow Günter Müller , Second Edition Goodman & Gilman's The Pharmacological Basis of Therapeutics - 12th ed. Drug Screening Methods, SK Gupta. http://clinicaltrials.gov/ct2/show/NCT00684528 EM of antidiabetic drugs 62

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64. Limitations of current treatment ?? Does not prevent or reverse complications of diabetes None of the treatment regimen mimic normal physiological secretion rhythm of insulin Not much acceptable by patients (daily insulin injection) and Not cost effective Immunological reactions are common EM of antidiabetic drugs 64

65. C57BL/6J mice Not described Genomic characteristics leading to hypertension on high energy nutrition; hyperinsulinemia & hypertension EM of antidiabetic drugs 65 Zucker fa & ZDRt-fa rats Renal changes; possible hypertension; infertility Intrinsis islet mechanisms causing hypersecretion ; antidiabetic drugs

66. In-Vitro studies for insulin and insulin like activity 3. Insulin signaling cascade Differentiates between metabolic & mitogenic signaling in insulin responsive cells EM of antidiabetic drugs 66

67. Contd.. Effects on LIVER : Perfusion of isolated rat liver - To study dynamic response & transitions between various metabolic states Isolated hepatocytes - To study effect of drugs on hepatic gluconeogenesis & metabolic reactions such as ketone body formation & TCA cycle EM of antidiabetic drugs 67