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By Goutami Perala

Screening Of Antidiabetic Drugs

A seminar presentation on

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Over view

Introduction Screening methods Animals used In vivo methods In vitro methods Genetic knock out models Summary References

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Introduction

Diabetes : group of metabolic diseases in which a person has high blood sugar

Normal range : -random : 82 to 110 mg/dL-Post lunch :140 mg/dL

Symptoms : -Polyuria-Polydipsia -Polyphagia

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Types

- Type 1 : by a lack of insulin output because of auto-immune damage to the pancreas gland

- Type 2 :insufficient production of insulin in the pancreas a resistance to the action of insulin in the body's cells - especially in muscle, fat and liver cells

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CURE IS DIFFICULT ….

Sulfonylureas Meglitinides Metformin

Thiazolidinediones α- glucosidase inhibitors

Peptide analogues Insulin

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SCREENING MODELS OF ANTIDIABETICS

IN VIVO

MODELS FOR IDDM

NORMOGLYCEMIC MODELS

MODELS FOR NIDDM

IN VITRO

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ANIMALS USED

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Models for IDDM

EXPERIMENTAL MODELS GENETIC MODELS

Chemical induced

Hormone induced

Viral induced

Surgically induced

Insulin antibodies

Genetic alteration

AlloxanStreptozotocin

Dexamethasone

-Encephalo-mylocarditis -Mengo -Picorna -Coxsackie -Reovirus

All or part of the pancreas

Diabetes like condition is induced

-NOD Mouse -BB Rat

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Alloxan induced Diabetes Induces permanent diabetes MOA

Directly toxic to beta cells Interacts with sulfhydril enzyme and

inhibits it 1. Hexokinase activity 2. Protein kinase activity3. Induces mitochondrial abnormalities4. Damages the DNA (fragmentation)

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Animal used

Wistar rat 100-175 mg/kg

Baboons 65-200 mg/kg

Rabbits 150mg/kg

Beagle dogs 60 mg/kg

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ALLOXAN INDUCED

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

Blood glucose levels show triphasic response with hyperglycemia for 1 hour followed by hypoglycemia that lasts for 6 hours & stable hyperglycemia after 48 hours.

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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

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 kit] using autoanalyser.

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Limitations

High mortality Ketosis Some species are resistant to alloxan

e.g. guinea pig Streptozocin has almost completely

replaced alloxan

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Streptozotocin induced DM

Broad spectrum antibiotic – 200 mg/kg i.p.

MOA

β cell damage by free radical injury Fragmentation of DNA ( alkylation ) Nitric oxide generation Induces diabetes in all species

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Streptozotocin [60 mg/kg body weight] is prepared in citrated buffer [ph 4.5]

Animals showing fasting blood glucose levels > 140mg/dl after 48 hours of streptozotocin administration are considered diabetic.

Albino rats of either sex weighing 150-200 g are injected i.p with above solution.

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After six weeks of treatment blood samples are collected from 6 hour fasting animal

Serum is separated by centrifuge (3000 rpm) under cooling (2-4 °C) for ten minutes

Serum glucose level is estimated by glucose- peroxidase method [GOD-POD kit] using autoanalyser

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Advantages

Greater selectivity towards beta cells Low mortality Longer and irreversible diabetes Guinea pigs and rabbits are resistant

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Hormone induced DM

PRINCIPLE: - Dexamethasone: is a long acting glucocorticoid possessing immunosuppressant action in the islets and produces type 1 diabetes.

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PROCEDURE

Adult rats 150-200gm dexamethasone2-5 mg/kg i.p

Repeated injection of same dose level is carried out for a period of 20-30 days resulting in IDDM

The sample to be screened is administered through a suitable route, blood glucose is measured

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Limitations

Long standing procedure

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VIRAL INDUCED DM

Rna picorna virusCoxsackie virusEncephalomyocarditisMengo-2tRenovirusLymphocytic choriomeningitis

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6-8 week old mice are

inoculated by 0.1 ml of 1:50

dilutions of encephalomyocarditis [EMC] i.p. Hyperglycemic:non fasting levels exceed

by 250mg/dl

Drug to be screened is 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

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Limitations

Must be cultured to affect rat/ mice beta cells

Time consuming

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SURGICALLY INDUCED METHODS

PRINCIPLE: Surgical removal of pancreas result in insulin dependant form of DM state

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Surgically induced animals

Partial pancreatectomized animals e.g. dog, primate, pig & ratsAdvantages Disadvantages Avoids cytotoxic effects of chemical diabetogens on other body organs

cumbersome technical andpost operative procedures

Resembles human type 2 diabetes due to reduced islet beta cell mass

•Digestive problems due to excision of exocrine portion of the pancreas•Loss of counter regulatory response to hypoglycaemia

High mortality

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Insulin antibody induced diabetes

Principle: - A transient diabetic syndrome can be induced by injecting guinea pigs with anti insulin serum.

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Preparation of antibody

Bovine insulin, dissolved in acidified water [ph 3.0] at a dose of 1mg /ml

Anti insulin sera is collected after two weeks of antigenic challenge

Injected into guinea pigs

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Procedure Adult albino rats are injected with 0.25-1.0 ml of guinea pig anti- insulin serum.

increase of blood glucose level up to 300 mg/ dl.

The drug sample to be screened is given and blood glucose level is analyzed to determine the activity.

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Limitations

Effect persist as long as antibodies remain in the circulation

Large doses and prolonged administration- ketonemia, ketonuria, glycosuria and acidosis are fatal to animals

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GENETIC MODELS

PRINCIPLE: Autoimmune destruction of pancreatic β cells in association with autoantibody production.

Shares many characteristics with human IDDM

Spontaneously DM on hereditary basis

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NOD MOUSE

BB RAT

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PROCEDURE

MICE ARE BRED IN LAB BY SIB

MATINGS OVER 20-80

GENERATIONS

SPONTANEOUS DEVELOPMENT OF IDDM IS OBTAINED• DEVELOPS

ABRUPTLY AT 100-200 DAYS OF AGE

ANIMALS ARE TREATED WITH DRUG SAMPLE TO BE SCREENED• BLOOD

SAMPLE IS ANALYSED FOR GLUCOSE

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LIMITATIONS

Ketosis Glycosuria Weight loss High mortality

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MODELS FOR NIDDM

Neonatal STZ model Genetic models

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NEONATAL STZ MODEL FOR NIDDM

Principle: Pancreatic beta cells destructionDecrease in pancreatic insulin

stores

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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

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Genetic models for NIDDM

Monogenic models of obesity

and NIDDM

Polygenic models of obesity and

NIDDM

Animal models of NIDDM with

unknown hereditary and

environmental component

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MONOGENIC MODELS FOR OBESITY AND NIDDM

PROTOTYPE:ObesityHyperinsulinemiaHyperglycemiaHyperlipidemia

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TUBBY MOUSE

YELLOW MOUSE

ZUCKER DIABETIC FAT RAT

OBESE ZUCKER RAT MODEL OFSYNDROME X

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Animal Characteristics Age at which NIDDM symptoms

develop

Yellow mouse(The Agouti mouse)

HyperglycemiaHyperinsulinemiaInsulin resistance

4-5 wks of age

Obese and diabetic mouse

Obesity; hyperglycemia

Insulin resistance

5-8 months

Tubby mouse Slowly developing obesity

Hypoglycemia , hyperinsulinemia

12wks

Fat mouse Obesity hyperglycemia

7-8wks

Zucker diabetic fatty rat

Obesity, insulin resistance

7-8wks

Koltesky and JCR :LA-Corpulent

Rats

HypertensiveProteinuria with kidney disease

5-6wks

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POLYGENIC MODELS OF OBESITY AND NIDDM

No single gene implicated

Interaction between environment and several genetic defects

Polygenic animal model represents human condition more closely

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NIDDM ANIMAL MODELS

NEW ZEALAND OBESE MOUSE

SOUTH AFRICAN HAMSTER

CHINESE HAMSTER

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Animal Characteristics Age - glucose intolerance

New Zealand obese(NZO) Mouse

Hyperphagic, hyperinsulinemic, insulin resistant,-

4wks-glucose tolerance decrease continuously with age

Japanese kk mouse Hyperinsulinemia, gluc intolerance, insulin resistance due to defect in receptor and post receptor signal transduction

Nagoya –Shibata- Yasuda (NSY) Mouse

Spontaneous diabetes develops in98%

48 weeks

Goto – Kakisaki rat best models of spontaneous non obese NIDDM, similar metabolic, vascular and hormonal disorders to humans

South African hamster Non obese , ketonuria is common

Chinese hamster Non obese

Otsuka Long Evans Tokushima Fatty Rat

Polyuria, polydipsia, hyperinsulinemia, persistant hyperglycemia, hypertriglyceridemia and obesity

2 weeks – obesity18 weeks –hyperglycemia

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Animal model of NIDDM with unknown hereditary & environment componentPrinciple: Animals taken from natural

environment developed DM when fed normal laboratory diet

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SAND RAT

SPINY MOUSETUCO TUCO

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Diet and nutrition induced

Advantages Disadvantages Best model for diabesity syndrome

Long period of dietary requirement

Toxicity of other chemical can be avoided

No frank hyperglycaemia upon simple dietary treatment

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Transgenic or knockout animals Genes – insulin resistance

Insulin receptor Glucose tranporters Hexokinase II Tumour necrosis factor α

Genes –defective insulin secretion GLUT-2 Glucokinase Islet amyloid polypeptide

Genes- increases body fat β3 receptors knockout mouse Uncoupling protein knockout mouse

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TRANSGENIC TECHNIQUES

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Salient features…

Advantages Disadvantages

Single gene mutations can easily be investigated in vivo

Highly sophisticated and costly procedure

Dissection of complex genetics become easier

Expensive for regular screening experiments

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Normoglycemic animal models

Rabbit Model Rat Model Dog Model

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Rabbit model

Rabbits 3-4.5kg div into groups of 4-5• Insulin and

insulin like compound-

• Food stopped a day earlier

• Hypoglycemic agents-diet till experiment

Test drug –orally 1ml/kg in0.4%starch suspension• Control receives

only the vehicle• Different doses

are tried on other groups

Blood is withdrawn before &1,2,3,4,5,24,48 and 72 hr • Blood glucose is

determined • Blood sugar

values are plotted against time

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RAT MODEL

Blood is drawn from the tip of the tail at 1,2,3,5,24 hours

Test dose is given as per dose in starch suspension

Divided into 4 groups of 7 each

Male Wistar rats -250 gms

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DOG MODEL

Beagle dog 15-20kg Food is stopped 18 hours prior to

administration of test compound Blood is collected up to 48 hours

MODIFICATIONS Dogs are pancreatectomized 2-3

years prior Given pancreatic enzymes orally Test compound is given with an oral

suspension

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INSULIN ASSA YS

Four groups of six rabbits weighing at least 1.8 kg

Two standard solutions of insulin containing one unit and

two units respectively

two dilutions of sample whose

potency is being examined are

prepared

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After one hour and 2.5 h of each injection, a suitable blood sample is taken from the ear vein of each rabbit.

blood sugar determined by glucose oxidase method

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IN VITRO METHODS ON ISOLATED ORGANS AND CELLS

PRINCIPLE:To study the effect of the drug on insulin, glucagon and somatostatin secretion without interference from other organs

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In vitro methods

Assays of insulin &of insulin like activity

Isolated organs, cell and membranes

Insulin receptor Binding assayAssays of other glucose regulating

peptide hormonesInhibition of polysaccharide

degrading enzymeEffect on secondary diabetes

symptoms

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Isolated pancreas of rat Isolated pancreatic islets of rat Isolated rat liver Isolated hepatocytes of rat Assays for insulin or insulin like

substances on adipocytes Assays for lipid synthesis Assays for glucose transport Glucose uptake by the isolated diaphragm

from mice and rats

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Summary In vivo In vitro

IDDM NIDDM Non Diabetic Models

Insulin assays

Chemical Neonatal STZ Rat Effect on secondary symptoms

Hormone Genetics Rabbit Isolated organs, cell and

membranes

Virus Dog Inhibition of polysaccharide

degrading enzyme

Surgical Assays of other glucose

regulating peptide

hormones

Anti-bodies

Genetic

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References

Vogel screening methods 2nd edition H. Gerhard Vogel- Drug Discovery and

Evaluation Animal models in type 2 diabetes

research: An overview K. Srinivasan & P. Ramarao. Indian J Med Res 125, March 2007, pp 451-472

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Websites

http://en.wikipedia.org/wiki/Alloxan http://www.springerlink.com/content/

e2450r1101642j8w/ http://www.pharmainfo.net/reviews/b

iological-screening-procedures-anti-diabetic-drugs

http://www.netdoctor.co.uk/diseases/facts/diabetesnoninsulindependent.htm

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HYDERABAD

Thank you