Methods for screening of hypoglycemics

Anti-Diabetic Drug Screening
Models
- Aasifa Shaikh
Sem I, Department Of Pharmacology
SSR COLLEGE OF PHARMACY

•Diabetes is a chronic metabolic disorder characterized
by either the insufficient production or the lack of
response to a key regulatory hormone of the body’s
metabolism, insulin.
•It can be categorized as Type-1 diabetes [insulin
dependent diabetes mellitus (IDDM)] and Type-2
diabetes [non- insulin dependent diabetes mellitus
(NIDDM)].
•The overall prevalence of diabetes is approximately 10%
of the population, of which 90% is Type-2.

•The disease is characterized by hyperglycemia,
hypercholesterolemia, and hypertriglyceridemia,
resulting from defects in insulin secretion or
reduced sensitivity of the tissue to insulin (insulin
resistance) and/or combination of both.
•Characteristically, it is a serious endocrine
syndrome with poor metabolic control and
responsible for increased risk of cardiovascular
diseases including atherosclerosis, renal failure,
blindness or diabetic cataract.

Types of Diabetes
TYPE-1 (IDDM)/Juvenile Onset
Diabetes Mellitus
There is β –Cell destruction in
pancreatic islets.
It is an autoimmune disease.
 It is very rare.
 Risk factors: Age (Children)
Family History
 Incidence is growing Steadily.
Treatment: By Injecting Insulin
into body.
TYPE-2 (NIDDM)/Maturity Onset
Diabetes Mellitus
 There is no loss or moderate
reduction in β-cell mass.
 It usually begins as insulin
resistance.
 It is very common.
 Risk Factors: Obesity, Age above 45
Incidence is rising at an epidemic
rate
Treatment: With diet and exercise,
Monitoring blood sugar level,
Medications.

Current treatment modalities
•Different insulin preparations and different
insulin delivery systems
• Sulfonylureas
• Biguanides
• Meglitinides
• Thiazolidinediones
• α- glucosidase inhibitors

Screening helps us to understand
PATHOGENESIS
SAFETY AND EFFICACY OF
DRUGS
PREVENTION
OF DISEASE COMPLICATIONS
THERAPEUTIC
AGENTS

Methods for screening of Hypoglycemics
1- Methods to Induce Experimental Diabetes
Mellitus.
A. Pancreatectomyin Dogs.
B. Alloxan - Induced Diabetes.
C. Streptozotocin – Induced Diabetes .
D.Growth Hormone-Induced Diabetes.
E. Insulin Deﬁciency Due to Insulin Antibodies.

2- Genetically Diabetic Animals
A. Spontaneously Diabetic Rats.
B. Spontaneously Diabetic Mice.
C. Chinese Hamster.
D.Transgenic Animals and Knockout Mice .
E. Metabolic Systems Biology

3 - Measurement of Blood Glucose-Lowering
and Antidiabetic Activity
A.Blood Glucose-Lowering Effect in Rats.
B. Blood Glucose-Lowering Effect in Mice.
C. Euglycemic Clamp Technique.
D.Effects of Insulin Sensitizer Drugs.
E. Effects of Thiazolidinediones on Peroxisome
Proliferator Activated Receptor-γ.

4-Measurement of Insulin and Other Glucose-
Regulating Peptide Hormones.
A. Radio immunoassays for Insulin, Glucagon and
Somatostatin
B. Amylin
C. Receptor Binding and In Vitro Activity of Glucagon
D. Glucagon-Like Peptide I
E. Insulin-Like Growth Factors.

5-Insulin Target Tissues and Cells
A. Adipose Tissue and Adipocytes.
a. Epididymal Fat Pads of Rats.
b. Primary Rat Adipocytes.
c. Insulin-Resistant Primary Rat Adipocytes.
d. Primary Rat Hepatocytes .

6-Assays for Insulin and Insulin Like Metabolic
activity
a) Assays for Insulin and Insulin-Like Activity Based on Adipocytes
b) Assays for Insulin and Insulin Like Metabolic Activity Based on
Hepatocytes, Myocytes and Diaphragms
c) Assays for Insulin and Insulin-Like Signal Transduction Based on
Adipocytes, Hepatocytes and Myocytes
d) Assays for Insulin and Insulin-Like Regulation of Gene and Protein
Expression
e) Assays for Insulin and Insulin-Like Regulation of Energy Metabolism.

7-Measurement of Glucose Absorption
A.Inhibition of Polysaccharide Degrading
Enzymes
eg - Assay for α-Amylase
B. Assays for GLUT2 Transport Activity
eg - Perfusion of Jejunal Loops
C. Evaluation of Glucose Absorption In Vivo

8- Monitoring of Diabetic Late
Complications
A. Aldose Reductase Activity.
B. Nerve Conduction Velocity.
C. Nerve Blood Flow (Doppler Flux)
D. Electroretinogram.
E. Streptozotocin-Induced Cataract.

9- Insulin Analogs: Assessment of
Insulin Mitogenicity and IGF-I Activity.
A. Introduction and Application to Insulin
Analogs
B. Signaling Via Insulin Receptor
C. IGF-I Receptor Afﬁnity.
D. Insulin Receptor Afﬁnity
E. Assessment of Hypoglycemic Activity In Vivo.

1. Methods to Induce Experimental
Diabetes Mellitus
A - Alloxan induced Diabetes
•Alloxan is a cyclic urea (tetra- oxo – hexa hydro
pyrimidine)
Purpose : Alloxan has direct cytotoxic action on beta
cells of pancreas. It selectively destroy beta cells and
produce insulin deficiency and hyperglycemia.

Mechanism of action
-Free radical damage beta cell DNA
- Reacts with protein SH group causing cell necrosis
Procedure
• Rats - Sprague Dawley (SD) strain weighing 150-200
gms
Alloxan standard solution is prepared at a strength
of 5gm/100ml kept at pH 4.5

Alloxan is injected by :
-Intravenous - 65mg/kg
- Subcutaneous 100–175 mg/ kg
- Intraperitoneal 100–175 mg/ kg
•Triphasic response:
- at 2hr, hyperglycemia
- at 8hr, hypoglycemia
- at 24 hr, hyperglycemia

Drawbacks
•Risk of high mortality (up to 50%)
• Unstable at physiological pH, so care is taken to see
that it is preserved at a pH of 4.5.
•Dosage variation with age & species.
•This process has been almost completely replaced by
streptozotocin for inducing diabetes because of
these drawbacks.

B -Streptozotocin induced diabetes
PURPOSE
AND
RATIONALE
Rakieten and coworkers (1963) reported
the diabetogenic activity of the antibiotic
streptozotocin. The compound turned out
to be speciﬁcally cytotoxic to beta-cells of
the pancreas.

•2-deoxy-2-[3-methyl-3-nitrosourea] 1-D
glucopyranose
•Initially used for antibacterial & anti-tumor
activity
•From fungus Streptomyces achromogenes
Induces permanent DM :
By methylation
Free radical generation
Nitric oxide production

Three phases of changed blood glucose level are observed.
Initially after 3hrs glucose level increased upto 150- 200mg/dl.
After 8hrs, serum insulin values are increased upto 4 times
Male Wistar rats weighing 150–220g fed
with a standard diet, are injected with
60mg/kg streptozotocin prepared in
citrated buffer.(4.5pH)
Procedure

After 24- 28 hrs hyperglycemia already occur reaching values
800mg/dl with glycosuria & ketonemia. Histologically beta
cells are degranulated.
After 14 days animal is used for pharmacological effect
Hypoglycemic phase followed by hyperglycemia

Triphasic response
• 1 hr : Hyperglycemia
• 6 hr : Hypoglycemia
• 24-48 hr : Hyperglycemia

Advantages
Almost replaced Alloxan because of
greater selectivity towards β cells
Lower mortality rate
Longer duration diabetes induction

Disadvantages
 Highly unstable at room temperature
(preserved at 20 0C)
 Single dose may not give results
therefore streptozotocin might also be
given 2 divided doses 4 hrs apart
 Necessary to maintain cold
temperature

Some modifications with STZ
• Multiple low doses of STZ induce immune
mediated insulitis mimicking type 1 DM.
• Enhancement of streptozotocin induced
diabetes in CD-1 mice by cyclosporin A.
• Streptozotocin combined with complete
Freund’s adjuvant, incomplete freund’s adjuvant,
Mycobacterium butyricum,Listeria
monocytogenes, or endotoxin all produce
hyperglycemia.

2-Genetically Diabetic Animals
A - Transgenic Animals and Knockout Mice
Purpose and
rationale :
The genes controlling various aspects of metabolism and insulin
secretion are manipulated to produce animal models of diabetes
mellitus.
Transgenic animals are animals (most commonly mice) that have had a
foreign gene deliberately inserted into their genome.

There is an another method which is commonly used
to manipulate a single gene, in most cases and this
involves removing or 'knocking out' a target gene..
The end result is what is known as a ‘knockout’
animal.
This results in the genetically modified offspring
The progeny are then bred with other transgenic
offspring to establish a transgenic line.

The genes are manipulated :
To cause insulin resistance
insulin receptor
insulin receptor substrate 1 & 2
Glucose transporters
Hexokinase ІІ
Tumor necrosis factor- α
 Fatty acid-binding protein
RAS associated with diabetes

To cause defective insulin secretion
GLUT-2 (trans membrane carrier protein that
enables protein facilitated glucose movement
across the cell membrane)
Glucokinase
Hepatic nuclear factors
Islet amyloid polypeptide
Or genes that increase body fat
Knock out of uncoupling proteins
Knock out of β3-adrenergic factors

3 - Measurement of Blood Glucose-Lowering and
Antidiabetic Activity
A – Blood Glucose-Lowering Effect in Rats
PURPOSE
AND
RATIONALE
Rats are used for screening as well as for
quantitative evaluation of blood glucose
lowering agents.

Male Wistar rats weighing 180 – 240g are kept on standard diet
Procedure
Groups of 4–7 non-fasted animals are treated orally or intraperitoneally
with various doses of the test compounds suspended in 0.4% starch
suspension
One control group receives the vehicle only

Blood is withdrawn from the tip of the tail immediately before, and 1, 2, 3,
5, and 24h after administration of the test compound.
Blood glucose is determined in 10µl blood samples with the hexokinase
enzyme method (Gluco quant test kit).

EVALUATION
•Average blood sugar values are plotted versus time
for each dosage.
•Besides the original values, percentage data related
to the value before the experiment are calculated.
•Mean effects over a time period are calculated
using the trapezoidal rule.
•Statistical evaluation is performed as:

•The values of the experimental group are compared
statistically with the t-test for each time interval with
those of the control group.
• Differences between several treated groups and the
control group are tested using a simultaneous
comparison.

B - In vivo-Euglycemic clamp technique
• Useful method of quantifying in vivo insulin
sensitivity in male wistar rats
•In this technique, a variable glucose infusion is
delivered to maintain euglycemia during insulin
infusion
•Whole-body tissue sensitivity to insulin, as
determined by net glucose uptake, can be
quantitated under conditions of near steady state
glucose & insulin levels

Male Wistar rats weighing 150–200g are fasted overnight and
anesthetized with pentobarbital (40mg/ kg, i.p.).
Catheters are inserted into a jugular vein and a femoral vein for blood
collections and insulin and glucose infusion, respectively.
To evaluate the insulin action under physiological hyperinsulinemia (steady
state plasma insulin concentration during the clamp test around
100µU/dl),and maximal hyperinsulinemia (under which maximal insulin
action may appear) two insulin infusion rates, 6 and 30mU/kg/min, are used.
The blood glucose concentrations are determined from
samples collected at 5-min intervals during the 90-min clamp
test.

The glucose infusion rate is adjusted so as to maintain the blood
glucose at its basal level during the clamp test.
The ﬁnal glucose infusion rate is calculated from the amount of
glucose infused for the last 30min (from 60 to 90min after start of
the clamp) in which the blood glucose levels are in a steady state.
The glucose metabolic clearance rate is obtained by dividing the
glucose infusion rate by the steady state blood glucose
concentration.
The steady state plasma insulin concentration is calculated from the
insulin concentrations at 60and 90min after the start of the clamp.
At the start and end of the euglycemic clamp test, free fatty acid
concentration is also determined and the free fatty acid suppression
rate is calculated.

4-Measurement of Insulin and Other Glucose-
Regulating Peptide Hormones
A - Amylin
•Amylin, also named islet amyloid polypeptide, is a
pancreatic islet peptide consisting of 37 amino acids
with a role in the maintenance of glucose
homeostasis.

•The peptide is predominantly present in the β-cells of
the pancreas and to a lesser extent in the
gastrointestinal tract and in the nervous system, where
amylin mRNA is also present along with speciﬁc binding
sites.
PURPOSE
AND
RATIONALE
Binding sites with high afﬁnity for amylin are present in
several brain regions, with the nucleus accumbens and
surrounding tissue containing more than twice as many
banding sites as any other regions.

Procedure
Membranes are prepared from male Sprague-Dawley rats (150–200g).
Following decapitation ,the basal forebrain regions (nucleus accumbens) are
removed to PBS (pH 7.4) at 4°C
The tissues are weighted, then placed in 10ml/g tissue of ice-cold 20mM
HEPES/KOH (pH 7.4) and homogenized with a Polytron

An additional 30ml of cold HEPES/KOH is added, and the homogenate
centrifuged (48,000×g, 15min)
After discarding the supernatant ﬂuid, membrane pellets are resuspended
by homogenization in 40ml of fresh HEPES/KOH and centrifuged as before.
Membranes are washed again by homogenization in buffer and
centrifugation.

The ﬁnal membrane pellet is resuspended in a volume of 20mM
HEPES/KOH containing 0.2mM PMSF added immediately before use from
a stock 0.2M solution in ethanol.
A volume of buffer is used to yield a concentration of about 80mg original
tissue/ml.
Membranes are kept frozen at –80°C until use.

Binding assay
Membranes from 4mg of original wet weight of tissue are incubated with
125I-BH-amylin in 20mM HEPES/KOH (pH 7.4), containing:
0.5mg/ml bacitracin,
0.5mg/ml BSA and
0.2mM phenyl methyl sulfonyl ﬂuoride (PMSF),
for 60min at 23°C.
Incubations are carried out in duplicate tubes and are started by addition
of membranes

Incubations are terminated by filtration through glass fiber filters that have
been presoaked in 0.3% polyethylene-imine, followed by washing with
15ml of cold PBS.
Competition curves are generated by measuring binding of
13pM 125I-BH-amylin in the presence of 10−11 to 10−6
unlabeled peptide. Data are fitted to a four parameter logistic
equation to derive half-maximal inhibitory concentrations(IC50
values)and slope factor.
EVALUATION

A - Insulin-Resistant Primary Rat Adipocytes
PURPOSE
AND
RATIONALE
• Primary rat adipocytes can be maintained
in culture for long-term treatment with
compounds/drug candidates to study their
insulin-like activity upon chronic challenge
or for the induction of insulin resistance.
5 - Insulin Target Tissues and Cells

Procedure
For induction of insulin resistance, primary rat adipocytes
are incubated in buffer S
25mM glucose, 0.5mM sodium pyruvate and 10nM insulin for 4 to 20h
at 37°C under slow shaking

When assayed for insulin-stimulated glucose transport following a
15- to 30-min incubation period in buffer S but lacking insulin
(which enables down regulation of the glucose transport system
from the chronically insulin stimulated to basal levels).

Evaluation
The adipocytes made insulin-resistant in vitro show a right-ward
shift of the concentration-response curve (decreased insulin
sensitivity) and a reduced maximal glucose transport velocity at
constant or only slightly elevated basal glucose transport
(decreased insulin responsiveness)
During prolonged incubation in suspension culture, the adipocytes
tend to increase their basal glucose uptake which might reﬂect
elevated energydemands due to stress conditions or loss of plasma
membrane integrity

It is therefore most critical to choose culture conditions which
preserve the energy status of the rat adipocytes and are
compatible with 5- to 6-fold stimulations of glucose transport, at
least, at low glucose concentrations corresponding to the normal
insulin sensitive state.

B – Primary rat hepatocytes
Sprague-Dawley rats (100–200g) are fed prior
to the hepatocyte isolation.
Hepatocytes are isolated from rat livers
thoroughly perfused with buffer containing
collagenase, hyaluronidase and trypsin
inhibitor.
The rat livers are perfused for 4min at
25ml/min ﬂow rate with perfusion buffer

And then with collagenase buffer
until the digestion is complete
(∼6min).
At the end of the perfusion,
connective tissue and large blood
vessels are removed.
The hepatocytes are then passed
through a 100-µm nylon mesh
sieve.

The cells are washed twice with 125ml of wash buffer
and collected by centrifugation (50×g, 2min).
The hepatocytes are suspended in plating medium
(DMEM, 10% FBS, 100nM insulin, 25nM
dexamethasone, 6.3µg/ml transferrin, 22µg/l
gentamycin) and passed through a 100-µm nylon mesh
sieve.
Cells (1.6×105) are plated in 48-well cell culture
plates for the various metabolic assays.

6 - Assays for Insulin and Insulin Like Metabolic
activity
A - Method Based on the Incorporation of
Radiolabeled Glucose
PURPOSE AND
RATIONALE
This assay measures the complete pathway of lipid
synthesis (lipogenesis) encompassing the transport of
the radiolabelelled glucose across the plasma membrane
of the adipocytes, its conversion into glycerol and/or
fatty acids and their subsequent esteriﬁcation into
predominantly neutral TAG and phospholipids --

and ﬁnally the deposition of TAG in LD in the
cytoplasm of the adipocytes.
Since in adipocytes each of these steps is
stimulated by insulin, albeit to varying
degrees…….
the lipogenesis assay is perfectly suited for the
analysis of effects of compounds/drugs on the
complex insulin signaling cascade regulating
lipogenesis.

The cells are lysed and the total lipids separated from water-
soluble products and the incubation medium including the
unincorporated[3H]glucose by addition of toluene-based
scintillation cocktail.
For measurement of lipogenesis monitoring effects on both
glucose transport and esteriﬁcation, isolated rat adipocytes are
incubated with D-[3H]glucose (0.55mM ﬁnal concentration,0.1–
1µCi).
Procedure

The reaction is started by the transfer of 0.2ml of adipocyte suspension (3.5×105
cells/ml) in KRHB to scintillation vials containing 0.1ml of [3-3H] glucose
(2µCi/ml,4.4mM),0.4 ml of 2-fold KRHB and 0.3ml of insulin or compound/drug with
insulin-mimetic activity dissolved in vehicle (e.g. DMSO) and diluted with KRHB to the
appropriate concentration of compound and vehicle (e.g. 3% DMSO).
After phase separation, radioactivity incorporated in to total lipids/phospholipids is
determined by liquid scintillation counting directly without removal of the lipid
phase based on determination of the radiolabel of the lipidic products partitioned
into the toluene phase containing the scintillator rather than of the [3H]glucose
left in the aqueous phase lacking scintillator.

The 3H-radioactivity is determined with a liquid scintillation counter
The vials are mixed rigorously using a vortexer and subsequently left standing
for 2–4h to allow phase separation.
After incubation for 90min, the reaction is terminated by the addition of 10ml
of toluene-based scintillation cocktail.
The scintillation vials are placed under a stream of carbogen for 10s, then
closed and placed in a very slowly shaking water bath (37°C).

Evaluation
The blank values lacking adipocytes (usually500–600dpm) are subtracted from
the values measured for the corresponding set of test mixtures containing
adipocytes to correct for 3H-radiation originating from the aqueous phase (i.e.
[3H]glucose left in the incubation medium).
Fold stimulations reﬂecting the responsiveness of the glucose transport
and/or esteriﬁcation systems of the adipocytes toward
insulin/compound/drug are calculated as ratio between the corrected test
values (presence of insulin/compound/drug)and basal values (absence of
insulin/compound/drug).
Typically, insulin induces 15to 20-fold, 8- to 12-fold and 2.5- to 4-fold
stimulations in lipogenesis at 50µM, 0.55mM and 2mM glucose, respectively.

Reference
•Appropriate insulin concentrations are
0.01,0.02,0.04,0.06,0.08,0.1,0,12,0.15,
0.2,0.5,1,5nM (ﬁnal concentration in the assay).
•Typically, the EC50 for human insulin is 0.06–
0.10nM.
•The insulin-like activity of compounds/drugs can be
expressed as % of the maximal insulin stimulation

7-Measurement of Glucose Absorption
Evaluation of Glucose Absorption In Vivo
PURPOSE AND
RATIONALE
The inhibition of glucose absorption can be
determined by measuring blood glucose
after administration of starch or
disaccharides with and without the inhibitor.
In addition, non-absorbed starch or
disaccharides can be determined in the
intestine.

Procedure
Male Wistar rats are kept on a standard diet with
free access to tap water at constant
temperature(24±1°C).
Sixteen hours prior to the experiment food but
not water is with held.
Groups of rats receive by stomach tube 2.5g/kg
raw starch in a water suspension without or
with various doses of the α-amylase inhibitor.

After 10, 20, 30, 60, 120and 240min, blood
is withdrawn for determination of blood
glucose and non-esterified fatty acids.
The animals are sacrificed after these
intervals and the residual starch in the
stomach and the intestine determined.
Definitely more starch is found in the
intestine after simultaneous application of
the α amylase inhibitor.

Evaluation
•The values of starch content in stomach and
intestine, as well as the blood glucose-, serum
insulin and NEFA-values are compared between
control and treated animals.

8- Monitoring of Diabetic Late
Complications
Electro-retinogram
PURPOSE AND
RATIONALE
Diabetic neuropathy is one of the important
symptoms of long-lasting diabetes. Several animal
studies with aldose reductase inhibitors have been
performed. Segawa and coworkers measured the
development of electro-retinogram abnormalities
and the possible role of polyol pathway activity in
diabetic hyperglycemia and galactosemia in the
rat.

Procedure
Using a contact lens-type electrode, electro-retinograms (ERG) evoked by strong flushes are
amplified by a preamplifier, displayed on an oscilloscope and summed using a signal averager
which also provides a copy of the averaged ERG.
Photic stimulation is delivered with an intensity of one J in 20s interstimulus intervals.
Electroretinography is performed monocularly with the pupil maximally dilated.
Male Sprague-Dawley rats weighing 310–400g are dark-adapted for 20min and anesthetized with
i.p injections of ketamine, 50–80mg/kg and atropine sulfate, 2mg/kg.

For inducing galactosemia, male Sprague-Dawley rats weighing 140–185g at the beginning
of the study, receive a diet of 30% galactose. Test compounds are given as 0.1% to the diet.
The sum of these amplitudes is expressed as the wavelet index.
The oscillatory potentials (designated O1,O2, and O3 in order of appearance on the
ascending limb of the b-wave) are added together.
The peak latencies are measured as the intervals between the stimulus onset and the peak
of the corresponding a- and b-waves and oscillatory potentials.
Theamplitudesofthe oscillatory potentials are measured.
The amplitudes of the a- and b-waves are measured from the base line to the trough of the
a-wave and from the trough of the a-wave to the crest of the b-wave, respectively.

Evaluation
•Data are calculated as mean ± SEM and signiﬁcance
levels are estimated using the Wilcoxon rank sum
test for unpaired data (two-sided).
•Linear regression is calculated by the least square
method. A p-value of <0.05 is regarded as
statistically signiﬁcant.

9- Insulin Analogs: Assessment of
Insulin Mitogenicity and IGF-I Activity.
A- Introduction and Application to Insulin
Analogs
PURPOSE AND
RATIONALE
The structure and approach to the evaluation of new
insulins is directed towards efﬁcacy in terms of
glucose lowering, in the predictive manner to
differentiate between monomeric insulins with fast
absorption kinetics and long acting insulins with
delayed absorption from the subcutaneous injection
site.

PROCEDURE
The initial steps are performed in vitro, focused on receptor interaction.
Subsequent steps in-vitro to explore the receptor-mediated
signaling, which is however at the present time difﬁcult to attribute
precisely to biological and clinical effects.
The main aim of the in vitro evaluation is to establish the relation of
metabolic activity to mitogenic activity, as previously done for structure
activity studies on insulin analogs, and subsequently directed to predicting
clinical relevance of these observations, when comparing new compounds
with the established biochemical and toxicological proﬁle of [B10-Asp]
insulin, and the clinically used fast acting and long acting insulin analogs.

Methods for screening of hypoglycemics

Methods for screening of hypoglycemics

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Methods for screening of hypoglycemics