screening of antidiabetic agents

SCREENING OF ANTIDIABETIC : 

1 SCREENING OF ANTIDIABETIC PRESENTED BY:- Salunkhe Pankaj Shrikant Semester 1st M.Pharm {Pharmacology} Department of Pharmacology R.C.Patel college of pharmacy,Shirpur SHRI

Content :- : 

2 Content :- History Definition & Classification of Diabetes Insulin -Structural feature -Mode Of Action Oral hypoglycemic agent -Classification & Mode of Action Glucose Estimation Method Screening techniques -In Vivo -In Vitro References

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3 DM Known since ages as “Madhumeha” i.e. sweetness of disease urine in Ayurveda by ‘Sushruta’ During first century Greek physician ‘Aeretaus’ gives term Diabetes as “to flow through” In 1755 by ‘Dobson’ demonstrated Diabetes as by presence of sugar in urine. In 1909 by ‘De Mayer’ gives statement about insulin is ‘the non-digestive part of pancreas & islet cell was responsible for pancreatic diabetes’. History :-

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4 -It is chronic metabolic disorder, resulting from insulin deficiency characterized by hyperglycemia, altered metabolism of carbohydrate, protein , lipid & increases the risk of vascular complications. -Diabetes is not a single disease, it is heterogeneous group of syndrome, characterized by an evaluation of Blood Glucose caused by a relative deficiency of Insulin. Definition :- 7,5

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6 INSULIN LACK HYPERGLYCEMIA KETOSIS Glycosouria Osmotic Diuresis Loss of Electrolyte Na+, K+, Ca++ Intracellular K+ Depletion Hypotension, Shock Acidosis Loss of Fixed cation in Urine Dehydration Hypoosmolarity of Blood Loss of Water vomiting Ketonurea Hyperventilation Impairment of Glucose entry in Brain Impairment of consciousness Intracellular Dehydration

Classification :- : 

7 Classification :- 1) Type-I / Juvenile onset / Insulin Dependent Diabetes Mellitus :- It occurs in nonbiased or before age 30. Autoimmune Disease of β-cell. Autoimmune type 1A antibody destroy β-cell. Circulating insulin is low or very low so patient prove to ketosis. Patient must rely on Exogenous insulin to control the hyperglycemia. Mostly Subcutaneous rout is preferred. 6,9,5,9

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8 2)Type II /Maturity Onset /Non-Insulin dependent Diabetes Mellitus :- Usually occurs after age 40. Obesity, lack of exercise is major risk factor. Moderate reduction of β-cell. Insulin circulation is less / normal / high ; may occur hyperglycemia or hypoglycemia. Here, abnormalities in Glucoreceptor in β-cell so that they respond at higher glucose concentration. Oral hypoglycemic agent are as a part of treatment , exercise , dietary modification.

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9 3)Type III / MODY (Maturity Onset Diabetes in Young) :- In which dysregulation of glucose sensing or insulin secretion is due to mutation of particular gene. Due to Hormonal disorder. Treatment varies often response to OHA 4)Type IV / Gestational Diabetes Mellitus :- Glucose intolerance associated with Pregnancy. It Is important to tight glycemic control close to normal range during pregnancy ,because- hyperglycemia can lead to congenital abnormalities in fetus Treatment- controlled by diet,exersise

Insulin: : 

10 Insulin: Structural feature:- High mol. wt. polypeptide having wt. about 5808 Amino acid linkages 51 aa arranged in 2 chain. A chain -acidic – 21 aa B chain -basic -- 30 aa C –peptide linkage connect to A & B chain. 2 disulphide bond are linked in both aa. 5,6,7

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11 # MOA of Insulin:- α α β β

Oral hypoglycemic agent : 

12 Oral hypoglycemic agent Classification :- 1) Insulin secretagogus – they promote the insulin release from β-cell of pancreas. e.g.. Sulfonylurea :- Tolbutamide,Acetohexamide 2) Insulin sensitizer – (i) it increases insulin uptake & utilization by target tissue. e.g.Thiazolidinediones:-Rosiglitazone,Pioglitazone (ii) it reduces hepatic glucose output, largely by inhibiting hepatic gluconeogenesis e.g. Biguanides : - Metfomin,Phetformin 3) α-Glycosidase inhibitor – they act by delaying the digestion of carbohydrate thereby decreasing glucose absorption e.g. Acarbose, Miglitol 7,9

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Screening Technique : 

14 Screening Technique In vivo - Chemical Induced Diabetes i) Alloxan induced Diabetes ii) Streptozotocin induced Diabetes iii) 8-TSQ Induced diabetes - Viral Induced diabetes - Genetically diabetic animal - Assay of Insulin i) Blood sugar lowering effect in Rabbit ii) Hypoglycemic Seizures in mice iii) Blood Sugar determination in mice - Blood Glucose lowering activity of Antidiabetic Drugs i) In Rabbits ii) In Rats - Euglycemic Clamp technique - Inhibition of Glucose absorption in vivo 10,21

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15 In Vitro -Isolated Rat pancreas2 -Isolated Diaphragm of rat

Glucose Estimation Method : 

16 Glucose Estimation Method Glucose Oxidase method-12 Glucose is oxidized in Gluconic Acid by glucose oxidize. The hydrogen peroxide liberated is reduced by peroxides & Oxygen transferred to an accepter, which is colour less in the reduced form but coloured in oxidized form. Glucometer- Urine analysis- Qualitative,Cheap,Convenient. Benedict Reagent are used. but Diagnosis can’t based- may show false +ve, false-ve 3,4,5,

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17 Hexokinase enzyme method-25 The procedure is a fluorometric rate method measuring the formation of NADPH catalyzed by immobilized glucose-6-phosphate dehydrogenase and hexokinase held within a tiny stirrer. The enzyme stirrer is stable for at least two months and can be used over eight-hundred assays without any loss of activity. Glycosylated Haemoglobin (HbA1c)3 Measurement of blood glucose level in diabetic suffer from variation due to dietary intake of previous day. Long term objective assessment degree of diabetic control is better done by measurement of glycosylated haemoglobin HbA1c,a minor haemoglobin component present in normal person. This is because non-enzymatic glycosylation of haemoglobin takes place over 120 day’s. This gives an estimated of diabetic control for the Preceding 6-10 day’s.

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18 Glucose Tolerance Test (GTT)- 3,4,5 Fasting Blood glucose level is first determined & then blood glucose level are sampled minutes to hour after an oral dose of glucose(75gm in 300 ml water) . Blood and urine sample are collected at half hourly interval.

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19 In Vivo Screening (1) Alloxan induced diabetes:- Purpose – Induction of diabetes using chemical alloxan. In Most species Triphasic time course is observed. (Initial rise of glucose is followed by decrease,due to depletion of islets from insulin,again followed by sustain increase of blood glucose). Procedure- 3 Group are selected as (Control, Standard,Test )

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20 Evaluation Use any suitable method for collection of blood. and estimation of blood glucose level. Compare the result of test with standard and control.

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21 2) Streptozotacin induced diabetes:- Purpose – Induction of diabetogenic activity of antibiotic streptozotacin. The compound turned out cytotoxic to β-cell of pancreas. Initially blood glucose increases up to 150-200 mg %, 6-8 hour after insulin increase up to 4 times (hypoglycemic phase) after 24-48 hyperglycemia up to 800 mg%. Procedure- Male Wistar Rat – 150-200 gm Group – Test, Standard, Control STZ. Dose – 60 mg/kg intravenously Evaluation Use any suitable method for collection of blood. and estimation of blood glucose level. Compare the result of test with standard and control.

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22 3) Other Diabetogenic compound induced diabetes:- Purpose – Several other compound to induce symptoms of diabetes and/or obesity. These compound also shows triphasic glycemic reaction. Procedure- Animal – Cats ,Rabbits ,Golden Hamsters ,Mice Group – Test, Standard, Control Chemical – Dithizone 8-TSQ (8-p-toluene-sulfonylamino-quinoline) 8-BSQ (8-benzene-sulfanylamino-quinoline) Dose – 40-100 mg/kg intravenously Evaluation Use any suitable method for collection of blood. and estimation of blood glucose level. Compare the result of test with standard and control.

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23 4) Virus induced Diabetes :- Principle- -It causes type-I diabetes mellitus. -The D-variant of Encephalomyocarditis virus (EMC-D) selectively infects and destroys pancreatic β-cell . -Islet cell destruction was associated with chronic islet cell inflammation, elevation of islet cell antibody, and prolonged presence of viral RNA in the pancreas. 5) Genetically diabetic animal :- # BB Rat- Bio Breeding rat is a model of spontaneous diabetes associated with insulin deficiency due to autoimmune destruction of pancreatic beta cell. # EBN/KOB Rat- Spontaneous hyperglycemia, glycocosourea have been observed in aged male of Wistar Strain.

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24 # WDF /TA-FA Rat- Commonly referred to as the fatty rat, is a genetically obese, hyperglycemic rat established by transfer of fatty gene from Zucker rat to wistar Kyoto rat. # NOD Mice- NOD mice is a model of Type-I diabetes NOD mice Develop diabetes between 100 and 200 days of age. As well as rapid weight loss, polyuria, severe Glucosuria. NOD mice caused by specific T-cell mediated, destruction of insulin producing beta cell. # Chinese Hamster – (Crecetulus griseus) The no. of pancreatic islet was decreased , and the cell of the remaining islet was decreased and other cell were abnormal. BSL of diabetic hamster were elevated from normal of 110mg% up to 600 mg%.

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25 NOD Mice Bio Breeding rat Chinese Hamster – (Crecetulus griseous)

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26 Assay of Insulin # Blood sugar lowering effect in Rabbits- Purpose- A biological assay of insulin preparation in comparison with a suitable standard using the blood sugar lowering effect in rabbits. Procedure- 6 rabbit in each group. wt.at least 1.8kg 24 h before test rabbit is provided with food ad libidum. During test food & water withheld until blood sample taken.

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27 Evaluation – Calculate the response of each rabbit to each injection. From the sum of two BSL values substrate it’s response to standard solution 1 & from standard solution 2. USP

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28 # Hypoglycemic seizure in mice10,23 Purpose- Twin crossover method for bioassay of Insulin using blood glucose level in mice. Procedure- Animal - Swiss albino (wt. 20 _ 2 gm) Group - 4 Group (each >10 animal ) Solution - Standard ( 2 diluting with 0.9% Nacl, PH-2.4) Test ( 2 diluting with 0.9% Nacl, PH-2.4) Non fasting mice are used. ( s.c.rout) Evaluation- The mice are observed for 1.5 hour. The no. of mice are recorded that are dead, convulse. +

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29 # Blood Sugar determination in mice Purpose- Biological assay of Insulin using hypoglycemic seizures in mice. Procedure- Animal - 96 mice of either sex (wt. 20 _ 5 gm) Group - 4 Group Solution - Standard ( diluting with 0.9% Nacl, PH-2.4) Test ( diluting with 0.9% Nacl, PH-2.4) Mice deprived food 2-20 hour immediately preceding test Each of prepared sol.0.1 ml/10gm injected s.c. Not less than 2.5 h later, each sol. Administered to second group. Evaluation- Exactly 2.5 h later, a sample of 50 µl of blood is taken from orbital venous sinus of each mice. BSL is determine. Potency is calculated by twin-cross-over assay

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30 Blood glucose lowering activity of antidiabetic drug # Blood glucose lowering activity in Rabbit Purpose- Rabbit has been used since many year for standardization of insulin. Procedure- Groups (std., test, control) rabbits of either sex (3.0-4.5 kg) Animal kept on Normal diet. Oral glucose lowering agent are applied by gavages in 1 ml/kg of 0.4% starch suspension or i.v. in solution. several dose are given to different group’s. control group receive vehicle only. Evaluation- By puncturing of ear vein, blood 10 µl is withdrawn immediately and 1, 2, 3, 4, 5, 24, 48, & 72 hours. BSL is determine. Average blood sugar values are plotted verse time for each dosage

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31 # Blood glucose lowering activity in Rats- Purpose- Rats are used for screening as well as for quantitative evaluation of blood glucose lowering agent. Procedure- Male Wistar rats (180-240 gm) are kept on standard diet . Group’s of 4-7 non-fasted animal are treated orally or intraperitoneally with various doses. Control group receive only vehicle. Evaluation- Blood is withdrawn 10 µl from tip of tail immediately before and 1, 2, 3, 5, 24, 48 hours. BSL is determine. Average blood sugar values are plotted verse time for each dosage

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32 # Euglycemic Clamp Technique:- Purpose- Useful method of quantifying in vivo insulin sensitivity in human. In this technique a variable glucose is delivered to maintain euglycemia during insulin infusion. Procedure- -Male Wistar rats (150-200 gm ) are fasted overnight. -Anaesthetized with Pentobarbital ( 40 mg/kg, i.p. ) -Catheter’s are inserted into jugular vein for blood collection and a femoral vein for insulin and glucose infusion. -Insulin infusion rates, 6 and 30 mU/kg/min. -Glucose infusion rate is adjusted so as to maintain BSL at basal level

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33 Evaluation- Blood glucose conc. are determined from sample collection at 5-min interval during the 90 min clamp technique. The glucose metabolic clearance rate is obtained by dividing glucose infusing rate by steady state blood glucose infusion rate Euglycemic clamp Technique

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34 # Inhibition of Glucose absorption in Vivo- Purpose- The inhibition of glucose absorption determined by blood glucose after administration of starch or disaccharides. Procedure- - Male Wistar rat (150-200mg) kept on standard diet, free access to tap water. -16 hour prior to experiment food is withdrawn but not water -2.5 gm/kg Starch receive by stomach tube for control group & with various dose -amylase inhibitor in test group -After, 10, 20, 30, 60, 120 & 240 min, blood is withdrawn and determined blood glucose. -The animal are sacrificed after these interval & residual starch in stomach , intestine is determined.

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35 Evaluation- The values of starch content in stomach & intestine as well as blood glucose , serum insulin values are compared bet control and treated animal.

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36 # Isolated Rat Pancreas:- 2 Purpose- Used for studying the effect of drug on Insulin, glucagons, somatostatin secretion. Procedure- Adult Wistar rat (150-200 gm) are fed ad libitum . Pancreas are removed under Pentobarbital (50 mg/kg i.p.) Through a portal vein canula Krebs-ringer bicarbonate buffer with 2% bovine albumin & 5.5 mmol./l glucose is perfuse at rate of 1.75 ml/min. at pressure 100mmHg. Perfusate is collected every min. for 30 min. after first 5 min. test drug added till 15 min. next 16-30 min glucose is perfuse In Vitro Screening

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37 Evaluation Insulin, Glycogen, Somatostatin are estimated using Radioimmunoassay. The effect of test drug on hormone secretion of pancreas in response to elevated glucose level is compared with the control.

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38 # Isolated diaphragm of rat- Purpose- Determination of Insulin based on the stimulation of glucose uptake by the isolated diaphragm from rat . Procedure – -Animal - Male Sprague Dawley (70-100 gm) -Animal sacrificed during anesthesia and diaphragm are carefully removed, spred out and divided into two equal pieces -Hemi diaphragm are incubated in Krebs buffer solution with carbogen with 5µM glucose, insulin or compound to be tested. -After 30 min. hemidiphragm are blotted on tissue, grounded on porcelin mortar pestle chilled with liquid nitrogen -After 4 hour at -200C Sample centrifuged 2000g for 10 min.

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39 29 Preparation of Diaphragm

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41 Evaluation- The concentration dependent of [ U-14 C] glucose uptake and conversion into glycogen and concentration by insulin or insulin mimetic compound are determined.

References:- : 

42 References:- Dr .Shivaji P. Gawade. How and What’s Of Pharmacology. Nirali Publication,1995 ; 1st :231-232 Dr.Mohd. Aslam,Dr.Surender Singh. Research Methodology and Statistics in Pharmacology. Academe publication,2006;1st :168-172 Cortan,Kumar,collins.Robbin’s Pathology Basic of Disease .Harcourt Asia Ltd. 2000;6th:915 Harsh Mohan. Textbook of Pathology . Jaypee Brother’s ,2006;5th 843,83 K.D.Tripathi. Essential of medical Pharmacology. Jaypee Brother’s .,2003;5th :380-387 H.P. Rang, M.M.Dale, J.M.Ritter, P.K.Moore .Pharmacology. Churchill Livingstone.2003;5th:236-251.

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43 F.S.K.Barar. Essential of Pharmacotherapeutics. S.Chand,2005;9th :340-349 Arthur C. Guyton,john E.hall, Textbook of Medical Physiology .Harcourt Brace & Company,1998;9th :982 Richard D.Howland, Mary J. Mycek. Lippincott’s Illustrated reviews, Pharmacology, Lippincot william’s & Wilkins,2002;3rd edition:281-289 H. Gerhard Vogel . Drug Discovery and Evaluation Pharmacological assay.springer.2002;2nd:Chapter-k.:947-1022 The United states Pharmacopeias , Asian edition, USP-28,2005;(121):2287 N.Raghuramulu, K.Madhavan Nair & S. Kalynasundaram . A Manual of Laboratory Techniques, national institute of Nutrition,ICMR,Hydrabad,2003;2nd 103-104 Anne Waugh, Allison Grant, Ross & Willison’s,Anatomy And physiology in Health & Illness. Churchill, 2001; 9th: 225 , 235,237

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44 S.Bhardwaj et.al, Antihyperglycemic activity of aq. Extract of leaves of cocculus hirsutus(L.) in alloxan induced diabetic mice, Indian journal of Pharmacology, feb.2006;38:49-53. P.D .Djomeni et.al.,Hypoglycemic effect of methylene chloride root extract of ceiba pentandra in normal and Diabetic rat, Indian journal of Pharmacology, June 2006 ; 38 : 194-197 Shweta Guptaet.al, Antidiabetic ,antihypercholesterolae- -mic and antioxidant effect of occium santum (L) seed oil, Indian journal of Experimental Biology,april-2006;44:302-304. Venugopal P. Menon et.al. Antidiabetic & Antihyper lipidaemic effect of alcoholic syzium cumini seed in alloxan induced diabetic rat , Journal of Ethno pharmacology ,2004,91;209-213

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45 P.R.Ortiz Andrade et.al. Antidiabetic effect on alloxianiszed and normoglycemic rat of Tournefortia harrtwegiana.Journal of Ethno pharmacology , 2005, 101 :37-42 S.Hemlata et.al. Effect of aq.extract of fruit of Withania caagulens on glucose utilization by rat Hemidiaphragm.Indian Journal of Natural Product;21(2): 20-22 Rajanarayana K, et.al.,Antidiabetic,antihyperlipidemic and Free radical scavenging activities of Ayurvedic Medicine, Indian Drug, March 2002;37(3):130-131 21) Alan G. Baxter*, Rowena C. Duckworth, Models of type1 (autoimmune) diabetes; Comparative Genomics Centre, James Cook University, Australia; Drug Discovery Today: Disease Models Vol. 1, No. 4 2004;451-453.

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46 Romel Somwar1, Xian ping Fang2, Gary Sweeney2,*Drug Discovery Today: Disease Models Vol. 2, No. 3 2005:181-188 Dr. Libor Velisek .,Hypoglycemic Seizures Institution: Albert Einstein College of Medicine at Yeshiva University Mentor : By Ka Lai Poon www.diabetescare.com www.americanassociationofdiabetis.com www.hopkinsmedicine.org www.diabetis.diabetisjournal.com/ www.tubules.com/.../mice_8nod_sept.2004.jpg W.L.M.Perry. Pharmacological Experiment’s on Isolated preparation, E.&.S. Livingstone, 1970;2nd ;32

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47 ‘?’ ANY QUESTIONS

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48 Thank you