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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