Antidiabetic screening

Points to be discussed…
 Introduction
 Screening methods
 IDDM Screening methods
 Chemically induced diabetes
 Surgically induced diabetes
 NIDDM Screening methods
 Chemically induced diabetes
 Normoglycemic animal model
 Isolated pancreas of rat [in vitro]
 In vitro assay of insulin on adipocyte
 Glucose uptake by isolated diaphragm from mice/rat
 Insulin receptor binding assay

Introduction
 DM is a metabolic disorder characterized by abnormal
carbohydrate, protein, fat metabolism caused by the
complete or relative insufficiency of insulin secretion
and/or insulin action.
 Type-I: IDDM
 Specific/complete loss of β-cell
 Type-II: NIDDM
 Associated with insulin resistance/obesity/hyperinsulinaemia

β-Cell mass
β-cell growth
Neogenesis
/Replication/Hypertroph
y
β-cell loss
Necrosis/Apoptosi
s/Hypotrophy

Type-I: IDDM
Type-1 Diabetes Mellitus
• Absolute lack of insulin
• β cell mass reduction
Mechanisms for
destruction of islet cells
• Genetic susceptibility
• Acute autoimmunity
• Environmental insult

Type-II: NIDDM
Type 2 Diabetes Mellitus
• Insulin resistance
• Relative impairment in insulin
secretion
Mechanisms for
development of NIDDM
• Defect at Insulin receptor level
• Defect at β-cell function
• Combination of both

Screening methods
IDDM
Chemically Induced DM
Surgically Induced DM
Miscellaneous
Genetic model
Hormone induced DM
Insulin antibody induced DM
DM induced by viral agent
NIDDM
Normoglycemic Animal model
Miscellaneous
Genetic model
Isolated pancreas of Rat [in vitro]
In vitro assay of insulin on
adipocyte
Glucose uptake by isolated
diaphragm from mice/rat
Insulin receptor binding assay

Most common animal model of
DM
3 types of chemical
• Specifically damage β-cell
• Cause temporary inhibition of
insulin production/secretion
• Diminish the metabolic efficacy
of insulin in target tissue

Alloxan Induced DM
Mechanism of
action
Free oxygen
radical
Free radical β-
cell death
Reaction with -
SH group of
protein
Cell necrosis

Alloxan induced DM
Triphasic response: Rise at 2 hr – hypoglycemic at 8hr – hyperglycaemic at
24hr
30% either die/temporary hyperglycaemic
70% become hyperglycaemic/Uricosuric
Animal are given glucose + Food ad libitum and regular insulin [28 IU per day-
single dose] for one week
Alloxan infusion [150mg/kg] via ear marginal vein for 10 minute
Rabbit 2-3kg are used

Alloxan induced DM
Disadvantage
• High mortality
• Ketosis in animals
• Diabetes induced- reversible
• Guinea pig are resistant to its effect
Newer method – STZ induced DM

Streptozotocin (STZ) induced DM
Broad
spectrum
antibiotic
produced
from
streptomyces
acromogens
Induce DM in
almost all
species
Cyclosporine-
A enhance
STZ-
diabetogenic
efficacy

STZ induced DM
Mechanism
of action
Methylation
Free radical
generation
Nitric oxide
production

Procedure
A steady state reached after 10-14 days allowing to use the animal
for pharmacological test
Severity & symptom of DM depend upon STZ dose to be given
Triphasic response: Hyper at 1 hr – Hypo at 6 hr – Hyper at 24-48hr
Inj. STZ [60mg/kg] IV is given
Male wistar rats [15-220g] fed standard diet

STZ induced DM
Advantages over Alloxan
• Greater selectivity towards β-cell
• Lower mortality rate
• Longer-irreversible DM induction
Disadvantages
• GP + Rabbit resistant to
diabetogenic action

Surgically induced animal model

Surgically Induced DM
Partial
pancreatectomy
• > 90% of organ is removed
• Anterior lobe is kept intact
Total
pancreatectomy
All part removed

Surgically induced DM
Disadvantage
• Loss of α/δ cell in addition to β-cell
• Loss of pancreatic enzyme necessary for digestion
• Total resection of pancreas in rat is difficult
• Development & severity of DM is strain specific
Advantage
• Combination with chemical agent produce stable
form of DM in animal
• Combination approach minimize organ damage
due to chemical & reduce the intervention

Chemically Induced DM- Neonatal STZ model of NIDDM
Reversible procedure: Initial hyperglycaemia – normoglycaemia by 3 weeks of age
Increase plasma glucose level
Decrease pancreatic Insulin stores
Severe pancreatic β-cell destruction
Inj. Streptozotocin [80-100mg/kg] IP at birth/within 5days of birth
Neonatal rats of wistar/sprague-dawley strain are used

Other chemically induced animal
model
Rabbit
• Adrenaline
[0.1mg/kg; sc]
• Hyperglycaemia:
Peak-1hr & Last-
4hr
• Oral
hypoglycemic
agent can be
screened
Mice
• Antiserum
produced against
OX-insulin in GP
& Sheep
• Reversible DM
• S/E- mild
pancreatitis in
rats
Rats
(Partial
pancreatectomized
)
• EDTA

Normoglycaemic animal model
Normoglycaemic
animal model
Rabbit model
[animal of
choice]
Rat model Dog model

Rabbit model
Mixed breed rabbit [3-4.5kg] are used
Experimental animal divided into 2 groups of 4-5 each
Insulin like substance
• Food stopped 1 day before experiment
Hypoglycemic agent
• Free access to normal diet till experiment day

Rabbit model
Test Group
Test drug 1ml/kg in
0.4% starch
suspension is given
Control group
Only vehicle is given
Blood sample collection – before and 1,2,3, 4, 5, 24, 48 & 72 hrs
Blood sample taken from Ear vein [10ul]
Blood sugar plotted against time
Blood glucose determination – Hexokinase enzyme method

Rat model
 Male wistar rat [180 – 250 gm]
 Animals are divided into 2 groups of 4-7 each
Test group
Test drug given in 0.4%
starch suspension orally/IP
Control group
Blood sample collection – Before & 1,2,3,5 &24 hr after test drug
Blood sample taken from tip of tail [10ul]
Blood sugar values plotted versus time or each dose

Dog model
 Male beagle dog [15-20kg]
 Animals are divided into 2 groups of 4-7 each
 Food is stopped 18hr prior to test drug administration
Test group
Test drug given in 0.4%
starch suspension orally/IV
Control group
Blood sample collection – Before & upto 48 hrs of test drug administration
Plasma Insulin – Radio-immunological method

Modification of dog model
Blood glucose estimation – before & upto 6hr [1 hourly interval]
32 IU short acting Insulin + Food + Test compound in oral suspension
32IU Insulin (long acting) single dose SC + Vitamin D 1ml every 3
month IM
Given dry feed with 2-3gm pancreatic enzyme
Pancreatectomized dog [upto 2-3years]

Modification of Dog model
Blood glucose estimation – before & upto 6 hr [hourly sampling]
28 IU of short acting Insulin + Food + Test compound in oral suspension
28 IU short acting Insulin single daily dose + Commercial diet
Infusion 1000ml of 5 % glucose + 10 IU Insulin via jugular vein +
Standard diet ad libitum daily for 1 week
Dog is given 60mg/kg Alloxan IV Single dose

In vitro method
 Isolated pancreas of rat
 To study effect of drug on insulin/glucagon/somatostatin secretions without
interference from other organ changes
Isolated pancreas perfused via portal vein cannula at 1.75 ml/min
[Kreb’s ringer bicarbonate buffer with 2% bovine albumin & 5.5 mmol/l
glucose]
Pancreas is removed under pentobarbital [50mg/kg IP] anaesthesia
Animal are fed ad libitum
Male wistar rats [200-250gm]

In vitro method
Hormone/Insulin/Glucagon/Somatostatins are estimated
Radioimmunologically
Sample taken and stored at -20*C
Sample (perfusate) collection – every minute till 30 minute
From 16th till 30th minute glucose is perfused
After 5 min of perfusion test drug is added till 15minute
Temp. 37.50C & Pressure – 100mmHg

In vitro assay
Isolated
adipocyte
preparation
Uptake of (3H)
glucose into fat cell
& incorporation of
this radiolabelled
glucose in the lipid
can measure
Insulin like activity

In vitro assay
Suspension is adjusted to a final titre of 4x105 cell/ml
Suspended in same solution
Washed 3 times by flotation with KRHB without glucose
Cell suspension is filtered through 100mm Nylon screen
These pads are cut into pieces & incubated for 20 min at 37*C with
1mg/ml collagenase in KRHB + 25mM HEPES/KOH + 0.1mM Glucose
+ 1% w/v BSA
Male wistar rats - Epididymal fat pad are taken

Glucose uptake by isolated diaphragm from
mice & rat
To study the effect of Insulin & Insulin
like substance on muscle tissue
To study
glycogen
synthesis
To study glucose
transport
To study
glycogen
synthase activity

To study glycogen synthesis
After 30 min hemidiaphragm are blotted on tissue & frozen
in liquid nitrogen
Incubate in Krebs Hensleit buffer + Carbogen (95:5) +
5mM (U-14C) Glucose + Insulin/test compound
Divided into 2 equal pieces
Diaphragm obtain from Male Sprague dawley rat

To study glycogen synthesis
14C labeled glycogen is determined by liquid
scintillation technique
Glycerin pellets are washed 3 times with 70% ethanol
After freezing at -20*C sample are centrifuged at 2000rpm
for 10min
Powdered tissue is dissolved by heating for 45 min at
100*C in 30% KOH and then 70% ethanol is added

To study glucose transport
Glucose transport is initiated by addition of 50ml of 10mM
(I-3H) deoxyglucose
Incubated for 30 min in 5 ml of above buffer + Test
compound/Insulin
Diaphragm washed 2 times with same buffer without
glucose
Rat diaphragm are incubated for 30 min at 37*C in
HEPES buffered saline + Carbogen (95:5)

To study glucose transport
Glucose transport is calculated as difference b/w diaphragm
associated radioactivity measured in absence/presence of
Cytochalesin B
Sample of supernatant are centrifuged at 1000rpm for 15min
& mixed with scintillation cocktail & counted for radioactivity
Blotted with filter paper & Homogenized
After 15min diaphragm are rinsed 4 times with ice-cold buffer
containing 10mM glucose + 25mM Cytochalesin B

To study glycogen synthase activity
Supernatant is used for glycogen synthase
activity
Homogenate centrifuged at 10000rpm for 20 min
Grounded in porcelain mortar & then homogenized at 0*C in 10 vol of
25mM tris/HCl + 100mM NaF + 5mM EDTA + 0.1mM PMSF
Homogenate prepared by blotting & freezing the diaphragm with liquid
nitrogen
Intact hemidiaphragm from male wistar rat is incubated in DMEM with test
compound/insulin + 5mM glucose + Carbogen (95:5) at 37*C

Insulin Receptor Binding Assay

IRB assay with isolated human adipocyte are performed in 300µl cell
suspension containing about 1x105cell/ml in HEPES buffer
Diameter/volume/surface for every cell is calculated
Adipocyte washed 5 times in HEPES buffer containing 50g/l human
albumin
Adipose tissue finely chopped & incubated for 90min at 37*C in HEPES
buffer
Subcutaneous adipose tissue is obtained from abdomen of patient
undergoing gastroenterological surgery

Non specific binding is measured and subtracted from total binding
Non specific binding measured by incubating tracer in presence of large
excess unlabeled insulin
Reaction is stopped by adding 10ml of chilled 0.154mol/l NaCl and
subsequent centrifugation with silicon oil
Iodine labeled ligand (monoiodinated insulin) is incubated with increasing
conc. of unlabeled human insulin & insulin derivative to be tested
IRB assay with isolated human adipocyte are performed in 300ul cell
suspension containing about 1x105cell/ml in HEPES buffer

Modification of IRB Assay
Receptor
prepared from
Rat liver
Solubilized
purified insulin
receptors from
livers of Zucker
fatty rats &
Sprague-
dawley rats
with dietary
obesity
Receptor
binding &
Tyrosine
kinase
activation by
insulin
analogue in
human
hepatoma
HepG2 cells

References..
 Antidiabetic agents in Gupta SK, Drug Screening methods, 2nd
edition; New Delhi; India.

Antidiabetic screening

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Antidiabetic screening