A seminar presentation on

Screening Of Antidiabetic Drugs

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By
Goutami Perala

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Over view
Introduction Screening Animals In In

methods

used

vivo methods vitro methods knock out models

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Genetic

Summary
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References

Introduction
Diabetes

: group of metabolic diseases in which a person has highblood sugar range : -random : 82 to 110mg/dL -Post lunch :140mg/dL

Normal

Symptoms

:
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-Polyuria
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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 -2/6/13 especially in muscle, fat and liver 44

CURE IS DIFFICULT .
Sulfonylureas Meglitinides Metformin

Thiazolidinediones - glucosidase inhibitors Peptide analogues Insulin

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SCREENING MODELS OF ANTIDIABETICS

IN VIVO IN VITRO

MODELS FOR IDDM MODELS FOR NIDDM NORMOGLYCE MIC MODELS

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ANIMAL S USED

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Models for IDDM

EXPERIMENTAL MODELS GENETIC MODELS

Chemic Hormone Viral Surgicall Insulin Genetic al induced induced y antibodi alteratio induced induced es n Alloxan Dexamet All or Diabetes -NOD Streptoz hasone Encephalpart of like Mouse otocin othe condition -BB Rat mylocar pancreas is ditis induced 2/6/13 88 -Mengo

Alloxan induced Diabetes

Induces MOA
Directly toxic to beta cells Interacts with sulfhydril enzyme and

permanent diabetes

inhibits it
1. Hexokinase activity 2. Protein kinase activity 3. Induces mitochondrial abnormalities 4. 2/6/13

Damages the DNA (fragmentation)

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Animal used

Baboons 65-200 mg/kg

Wistar rat 100-175 mg/kg

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Beagle dogs 60 mg/kg

Rabbits 150mg/k g

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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. 2/6/13 11

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 has almost completely replaced alloxan

Streptozocin

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Streptozotocin induced DM
Broad spectrum antibiotic 200 mg/kg i.p. MOA

cell damage by free radical injury of DNA ( alkylation )

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Fragmentation
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Nitric

oxide generation

Streptozotocin [60 mg/kg body weight] is prepared in citrated buffer [ph 4.5] Albino rats of either sex weighing 150-200 g are injected i.p with above solution.

Animals showing fasting blood glucose levels > 140mg/dl after 48 hours of streptozotocin administration are considered 2/6/13 diabetic.

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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 2/6/13

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Advantages
Greater Low

selectivity towards beta cells

mortality and irreversible diabetes pigs and rabbits are resistant

Longer Guinea

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Hormone induced DM
v

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

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PROCEDU RE
Adult rats 150-200gm dexamethasone25 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
v v v v v v

Rna picorna virus Coxsackie virus Encephalomyocarditis Mengo-2t Renovirus Lymphocytic choriomeningitis
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6-8 week old mice are inoculated by 0.1 ml of 1:50 dilutions of encephalomyoca Hyperglycemic:non fasting levels exceed rditis [EMC] i.p. 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

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Limitations
Must

be cultured to affect rat/ mice beta cells consuming

Time

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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 e.g.

pancreatectomized animals
Disadvantages
cumbersome technical and post operative procedures

Advantages

dog, primate, pig & rats

Avoids cytotoxic effects of chemical diabetogens on other body organs 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
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Insulin antibody induced diabetes

v

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

Injected into guinea pigs

Anti insulin sera is collected after two weeks of antigenic challenge

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Procedure

Adult albino rats are injected with 0.25-1.0 ml of guinea pig antiinsulin 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 28

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Limitations
v

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
v

PRINCIPLE: Autoimmune destruction of pancreatic cells in association with autoantibody production. Shares many characteristics with human IDDM Spontaneously DM on hereditary basis
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v v

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NOD MOUSE

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BB RAT

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PROCEDURE

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LIMITATIONS
Ketosis Glycosuria Weight High

loss

mortality

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MODELS FOR NIDDM

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NEONATAL STZ MODEL FOR NIDDM

Principle:
Pancreatic Decrease

beta cells destruction

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

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environmental component

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MONOGENIC MODELS FOR OBESITY AND NIDDM

v

PROTOTYPE:
Obesity Hyperinsulinemia Hyperglycemia Hyperlipidemia

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TUBBY MOUSE

ZUCKER DIABETIC FAT RAT

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OBESE ZUCKER RAT

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Animal

Characteristics

Yellow Hyperglycemia mouse(The Hyperinsulinemi Agouti mouse) a Insulin resistance Obese and Obesity; diabetic mouse hyperglycemia Insulin resistance
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Age at which NIDDM symptoms develop 4-5 wks of age

5-8 months

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POLYGENIC MODELS OF OBESITY AND NIDDM

v

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

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CHINESE

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Animal

Characteristics

New Zealand obese(NZO) Mouse

Hyperphagic, hyperinsulinemic, insulin resistant,-

Age glucose intolerance 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- Spontaneous diabetes 48 weeks Yasuda (NSY) develops in98% 2/6/13 43 Mouse

Animal model of NIDDM with unknown hereditary & v Principle: Animals taken from natural environment component

environment developed DM when fed normal laboratory diet

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SAND RAT

SPINY MOUSE
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TUCO TUCO
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Diet and nutrition induced

Advantages Best model for diabesity syndrome Toxicity of other chemical can be avoided
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Disadvantages Long period of dietary requirement 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

Genes

defective insulin secretion

GLUT-2
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TRANSGENIC TECHNIQUES

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Salient features
Advantages
Single gene mutations can easily be investigated in vivo

Disadvantages
Highly sophisticated and costly procedure

Dissection of complex genetics Expensive for regular screening become easier experiments

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Normoglycemic animal models

Rabbit Rat

Model

Model Model

Dog

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Rabbit model

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RAT MODEL
Male Wistar rats -250 gms

Divided into 4 groups of 7 each

Test dose is given as per dose in starch suspension Blood is drawn from the tip of the tail at 1,2,3,5,24 hours
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DOG MODEL
Beagle

dog 15-20kg

Food is stopped 18 hours prior to administration of test compound is collected up to 48 hours MODIFICATIONS

Blood

Dogs
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are pancreatectomized 2-3 years prior

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INSULIN ASSA YS

Four groups of six rabbits weighing at least 1.8 kg

Two standard solutions of insulin containing 2/6/13 unit and two units one

two dilutions of sample whose potency is being

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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
v v v v v

Assays of insulin &of insulin like activity Isolated organs, cell and membranes Insulin receptor Binding assay Assays of other glucose regulating peptide hormones
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Inhibition of polysaccharide degrading enzyme 2/6/13

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 2/6/13Glucose

uptake by the isolated

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Summary
IDDM

In vivo NIDDM

Chemical

Neonatal STZ Genetics

Non Diabetic Models Rat

In vitro Insulin assays Effect on secondary symptoms Isolated organs, cell and membranes Inhibition of polysacchari de degrading enzyme
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Hormone

Rabbit

Virus

Dog

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References
Vogel H.

screening methods 2nd edition

Gerhard Vogel- Drug Discovery and Evaluation models in type 2 diabetes research: An overview K. Srinivasan & P. Ramarao. Indian J Med Res 125, March 2007, pp 451-472

Animal

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Websites
http://en.wikipedia.org/wiki/Alloxan

http://www.springerlink.com/content/e24

http://www.pharmainfo.net/reviews/biolo

http://www.netdoctor.co.uk/diseases/fact

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HYDERABAD

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Thank you

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