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In- vitro and in-vivo screening of Hypoglycemics.

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In- vitro and in-vivo screening of Hypoglycemics.

  1. 1. In-vivo and In-vitro Screening of Hypoglycemics 29-01-2017 1 Presented by Kamlesh V. Warokar M.Pharm (Semister-I) (Dept. of Pharmacology) S.I.O.P., Pune.
  2. 2. • Introduction & Type Of D.M. • Symptoms of D.M. • Classification of Hypoglycemic drugs. • In-vivo screening of Hypoglycemics. • In-vitro screening of Hypoglycemics. • Evaluation Parameter. • References. 29-01-2017 2 Contents
  3. 3. INTRODUCTION • Diabetes Mellitus(DM) is a metabolic disorder characterized by hyperglycemia, glycosuria, hyperlipidaemia, & ketonaemia. • Hyperglycemia is a metabolic disorder characterized by high blood sugar level which may leads to diabetes mellitus. • High blood glucose happen when the body has too little insulin or when body can not use insulin properly. • On this basis diabetes mellitus classified as : Type I- Insulin Dependent Diabetes Mellitus(IDDM), Type II-Noninsulin Dependent Diabetes Mellitus (NIDDM). 29-01-2017 3 (K.D.Tripathi et al.)
  4. 4. 29-01-2017 4 1. Juvenile onset of DM. 2. Beta cells destruction in pancreatic islets. 3. Less common. 4. Have low degree of genetic predisposition. 1. Maturity onset of DM. 2. Low insulin circulation & moderate reduction in beta cell mass. 3. Most common. 4. High degree of genetic predisposition. TYPE 1 TYPE 2 (K.D.Tripathi et al.)
  5. 5. 29-01-2017 5 SYMPTOMS OF HYPERGLYCEMIA
  6. 6. 29-01-2017 6 Major Symptoms
  7. 7. 1. SULFONYLUREAS A. First generation- Tolbutamide, Chloropropamide. B. Second generation- Glipizide, Gliclazide. 2. BIGUANIDES- Metformin. 3. MEGLITINIDE- Repaglinide, Nateglinide 4. THIAZOLIDINEDOINES- Pioglitazone. 29-01-2017 7 CLASSIFICATION OF HYPOGLYCEMIC DRUGS (K.D.Tripathi, et al.)
  8. 8. 1. Streptozotocin induced diabetes 2. Alloxan induced diabetes 3. Pancreatectomy in dogs 4. Other diabetogenic compounds 5. Hormone induced diabetes 6. Insulin deficiency due to insulin antibodies 7. Virus induced diabetes 29-01-2017 8 METHODS FOR SCREENING OF HYPOGLYCEMICS 1. In vivo models for IDDM (Vogel,G.H.,et al.)
  9. 9. A. Spontaneous or genetically derived diabetic animal. B. Diet / Nutrition induced diabetic animal. C. Neonatal STZ induced diabetic animal. D. Transgenic diabetic animal. 29-01-2017 9 2. In vivo models for NIDDM (K. Srinivasan & P. Ramarao)
  10. 10. INVITRO SCREENING OF HYPOGLYCEMICS 1. Effect on Liver 2. Effect on Muscles 3. Effect on Pancreas 4. Effect on Adipose Tissue 29-01-201710 (Vogel,G.H.,et al.)
  11. 11. 29-01-2017 11
  12. 12. 29-01-2017 12 1. STREPTOZOTOCIN INDUCED DIABETES Male Wister Rats(150-220gm) are injected i.p. with 60mg/kg STZ prepared in citrated buffer. Three phases of changed blood glucose level are observed. Principle and Rationale Procedure IN VIVO MODELS FOR IDDM Rakieten et al.(1963) discovered diabetogenic activity of the antibiotic Streptozotocin. Basic principle is compound is found to be cytotoxic to beta-cells of the pancreas.
  13. 13. Initially after 3 hrs glucose level increased up to 150- 200mg/dl . After 8 hrs, serum insuline values are incresed up to 4 times. Hypoglycemic phase followed by hyperglycemia. After 24-48 hours hyperglycemia already occur reaching values 800mg/dl with glycosuria & ketonemia. Histologically beta cells are degranulated. 29-01-2017 13 After 14 days animal used for pharmacological test. (Vogel, al.)
  14. 14. 29-01-2017 14 2. ALLOXAN INDUCED DIABETES Purpose and Rationale Frerichs(1968) & Creutzfeldt(1971)- Chemically induced diabetes in animals. Procedure Animal is injected with a single dose [100 mg/kg body weight] dissolved in normal saline by i.p. route Animals are kept for 48 hours during which food and water is allowed.
  15. 15. 29-01-2017 15 Blood glucose levels show triphasic response with hyperglycemia for 1 hour followed by hypoglycemia that lasts for 6 hours & stable hyperglycemia after 48 hours. Animals showing fasting blood glucose level above 140 mg/dl after 48 hour are considered diabetic For a period of six weeks, drug samples to be screened are administered orally
  16. 16. 29-01-2017 16 After six weeks of treatment, blood samples are collected from 8 hour fasting animals (can be collected via orbita sinus through a pipette) The serum glucose level is estimated by glucose oxidase- peroxidase method [GOD-POD] using autoanalyser. (Vogel,G.H.,et al.)
  17. 17. 29-01-2017 17 Animal used Beagle dogs 60 mg/kg(i.v) Wistar rat 100- 175 mg/kg(s.c) Rabbits 150mg/kg (via ear vein)
  18. 18. 29-01-2017 18 Mechanism Of Action (S. Lenzen.)
  19. 19. 29-01-2017 19 3. PANCREATECTOMY IN DOGS Principle or Rationale Procedure Dogs-(12-16kg) Anesthesia- Phenobarbital sodium (50mg/kg). Remove fur and disinfect. Incision- Xyphoid to Umbilicus. Self retaining retractor applied. Pancreas is brought in operating field. Pancreas separated from duodenum and dissected freely. Diabetes can be achieved by removal of pancreas. Bomskov(1910)- severe diabetic symptoms in dogs. Von Mehring & Minkowski(1890)- polyuria, polydipsia, after removal of pancreas. (Vogel,G.H.,et al.)
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  21. 21. 29-01-2017 21
  22. 22. 29-01-201722 • distal pancreatectomy surgery medicine surgical procedures.mp4
  23. 23. 29-01-2017 23 4. OTHER DIABETOGENIC AGENTS Purpose and Rationale Procedure Chleators such as dithizone injected in rabbit at dose of 40- 100mg/kg which leads to triphasic glycemic reaction. Phase 1- Increased blood glucose level after 2hr. Phase 2- Hypoglycemic phase after 8hr. Phase 3 – Permanent hyperglycemia after24-72hr. Diabetogenic agents are Dithizone, Goldthioglucose or monosodiun glutamate. Rabbit, Cats, Hamster rat and mice can be employed for this model. (Vogel,G.H.,et al.)
  24. 24. 29-01-2017 24 5. HORMONE INDUCED DIABETES Growth hormone Cotes et al., (1949) Animals used are generally adult Cats and Dogs. Repeated administration of GH induce diabetes with the symptoms of ketonuria & ketonemia. Rats does not shows diabetes but grow faster and shows striking hypertrophy of pancreatic islet. (Karthikeyan M.,et al.)
  25. 25. 29-01-2017 25 Ingle (1941) Forced fed rats, guinea pig and rabbits without forced feeding are used. In rats adrenal cortex is stimulated by corticotropin, which secret steroids and induce steroid diabetes. Dexamethasone is mostly used. Corticosteroid (Karthikeyan M.,et al.)
  26. 26. 29-01-2017 26 Adult rats 150-200gm dexamethasone 2-5 mg/kg i.p. Repeated injection of same dose level is carried out for a period of 20-30 days resulting in Diabetes. The sample to be screened is administered through a suitable route, blood glucose is measured. PROCEDURE (Vogel,G.H.,et al.)
  27. 27. 6. VIRUS INDUCED DIABETES Principle & Rationale Jevunile onset diabetes mellitus may be due to virus infection and beta-cell specific autoimmunity. (Craighead 1978). 29-01-2017 27 6-8 week old mice are injected with D- variant of encephalomyocarditis [EMC] i.p. Procedure
  28. 28. 29-01-2017 28 Pre treatment is given with cyclosporin A. Drug to be screened is administered orally for a period of 6 weeks. (Karthikeyan M.,et al.)
  29. 29. 29-01-2017 29 7. INSULIN DEFICIENCY DUE TO INSULIN ANTIBODIES Principle and Rationale Moloney and Coval(1995); Wright(1968)- A transient diabetic syndrome can be induced by injection of guinea pig anti-insulin serum. (Vogel,G.H.,et al.)
  30. 30. 29-01-2017 30 Bovine insulin dissolved in acidified water (pH 3.0) at dose of 1mg/ml Injected to male guinea pig (s.c). Anti-insulin Sera is collected after two weeks of antigenic challenge. PREPRATION OF ANTIBODY
  31. 31. 29-01-2017 31 Adult albino rats are injected with 0.25-1.0 ml guniea pig anti insulin serum. Increased of blood glucose level upto 300mg/dl. The drug sample to be screened is given and glucose level is analysed to determine the activity. PROCEDURE
  32. 32. 29-01-201732 A. SPONTANEOUSLY or GENETICALLY DERIVED DIABETIC ANIMAL INVIVO MODELS FOR NIDDM  Spontaneously diabetic animals of type 2 diabetes may be obtained from the animals with one or several genetic mutations transmitted from generation to generation (e.g., ob/ob, db/db mice) or by selected from non-diabetic outbred animals by repeated breeding over several generation [e.g., (GK) rat, Tsumara Suzuki Obese Diabetes (TSOD) mouse]. (K. Srinivasan & P. Ramarao)
  33. 33. 29-01-2017 33 1. BB rat 2. WBN/KOB rat 3.GOTO/KAKIZAKI rat 4. ZUKKAR fatty rat 5. WDF/ TA-FA rat 6. BHE rat 1. KK mice 2. KK-Ay mice 3. NOD mice 4. Obese hyperglycemic mice 5. New Zealand obese mice 6. Transgenic mice RATS MICE Other Rat and Mice
  34. 34. 29-01-2017 34 BB RAT • Bio Breeding Rat. • Insulin deficiency & insulitis due to beta-cells destruction. • Nakhooda et al. 1978.
  35. 35. 29-01-2017 35 • Classic model of hyperinsulinemic obesity. • Obesity due autosomal recessive gene which develops at an early age. • Diabetic phenotype develops due to lipotoxicity to beta cells. ZUKKER DIABETIC FATTY RAT
  36. 36. 29-01-2017 36 KK- Ay MICE • Derived from KK mice. • Carry yellow obese gene • Demonstrate the extra pancreatic action of glimepirid (Satoh et al. 1994). • Also evaluate ciglitazone (Sohda et al. 1990) . KK- MICE • Nakamura (1962), reported on a diabetic strain of the KK mouse. • It is obese animale & showed polyphagia and polyuria. • Mice at the age of seven months or older showed glucosuria and blood sugar levels up to 320mg%.
  37. 37. 29-01-201737 SAND RAT • Small rodent (gerbil). • Indigenous to desert region. • Access to food and water is limited. • Exhibit the genetic predisposition, if fed with high calorie laboratory diet. B. DIET / NUTRITION INDUCED DIABETIC ANIMAL  Some of the animal models exist in which diabetes is induced neither by chemicals nor by genetic defect. Sand rat, Tuco-Tuco and Spiny mouse are important models of nutritionally induced obesity and type 2 diabetes.
  38. 38. 29-01-2017 38 • Two species of spiny mouse Acomys russates & cahirinus. • Have congenital hyperplasia of pancreatic islets. • When laboratory bred, A.cahirinus- weight gain on fat diet with hyperhlycemia. SPINY MICE (K. Srinivasan & P. Ramarao)
  39. 39. 29-01-201739 C. NEONATAL STZ MODEL FOR NIDDM Neonatal rats : STZ(90 mg /kg) i.p at birth or within the first five days Rats showing fasting blood glucose level above 140 mg/ dl are considered diabetic. Drug sample to be screened is administered by a suitable route and blood glucose level is analyzed (K. Srinivasan & P. Ramarao)
  40. 40. 29-01-2017 40 D. TRANSGENIC DIABETIC ANIMAL  Schaefer et al. 1994, described a transgenic mouse model of chronic hyperglycemia.  Effect of single gene or mutation on diabetes can be investigated in vivo.  Dissection of complex genetics of type 2 diabetes become easier.  Highly sophisticated and costly procedure for the production and maintenance.  Expensive for regular screening experiments.
  41. 41. Purpose & Rationale: Was Described extensively by Ross (1927), the perfusion of rat liver from the Portal vein is the most widely used technique. 29-01-2017 41 EFFECT ON LIVER 1. Isolated Rat Liver INVITRO SCREENING OF HYPOGLYCEMICS
  42. 42. 29-01-2017 42 2. Isolated Hepatocytes Purpose & Rationale: Isolated Hepatocytes can be used to study the effect of drugs on hepatic gluconeogenisis & other hepatic metabolic reactions such as ketone bodies formation and tricarboxylic acid cycle. (Vogel,G.H.,et al.)
  43. 43. 29-01-2017 43 • Male Wister Rats (200-250gm). • Anasthasized with hexobarbitol 150ml/kg.Animal • Isolate & washed with 100ml heparinized saline solution. • Oxygenated air tube connected to portal vein. • Perfusion is done & perfusate is collected. Isolation & Treatment • The sample for analysis are withdrawn by catheter & are evaluated for net glucose production. Evaluation
  44. 44. 29-01-2017 44 Glucose is evaluated by glucose-oxidase method. And pyruvate, acetoacetate & lactate are evaluated by enzymatic method. 1. The cell suspension is preincubated. 2.Substrats are added. 3. Test drug added. 1. Male Wister rat taken. 2. Hepatocytes isolated by collagenase method Procedure
  45. 45. 29-01-2017 45 EFFECT ON MUSCLE CELL Use of Isolated Diaphragm From Mice And Rats Isolate diaphragm from rats and divide it in equal parts. Incubate it in kreb- henselit buffer with 5mM glucose, insulin or compound to be tested. After 30 min, hemi diaphragm are blotted on tissue and frozen in liquid nitrogen. Powdered tissue is dissolved in 30% KOH. After freezing samples are centrifuged. Glycogen Pallets are washed with 70% ethnol & lebelled C14 glycogen determined. Total glycogen is determined after hydrolysis to glucose The total conc. dependence of glucose uptake & conversion into glycogen by insulin is determined.
  46. 46. • Body Weight. • HbA1c • Lipid Profile. • Serum Insulin Level. • Histopathology of Pancreas & Liver. 29-01-2017 46
  47. 47. 29-01-2017 47 References 1. Tripathi,K.D., “Essentials of medical pharmacology”, Jaypee publications, 2006 (7): 235-236. 2. Vogel,G.H., “Drug Discovery And Evaluation (Pharmacological Assays)”, Springer, 2002: 999-1044. 3. Gupta,S.K., “Drug Screening Method (Preclinical Evaluation of New Drugs)”, Jaypee Brothers Medical Publishers Pvt Ltd., 2009: 602-610. 4. K. Srinivasan & P. Ramarao, “Animal models in type 2 diabetes research: An overview”, Indian J Med Res 125, March 2007: 451-472. 5. S. Lenzen, “The mechanisms of alloxan- and streptozotocin-induced diabetes”, Diabetologia, Springer-Verlag, 2008 (51): 216–226.
  48. 48. 29-01-2017 48 5. Manish Pal Singh and Kamla Pathak, “Animal models for biological screening of anti-diabetic drugs: An overview”, Pelagia Research Library, European Journal of Experimental Biology, 2015, 5(5): 37-48. 6. Karthikeyan M, Balasubramanian T and Pawan Kumar, “In-vivo Animal Models and In-vitro Techniques for Screening Antidiabetic Activity”, J Develop Drugs 5, 2016 (2): 153-159.
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