This document discusses the preclinical screening methods for antidiabetic drugs, detailing types of diabetes mellitus, the pathophysiology, and various screening methods such as in-vivo and in-vitro models, including chemically and genetically induced diabetes models. It highlights the use of compounds like alloxan and streptozotocin for inducing diabetes in animal models, their mechanisms of action, and the evaluation of drug efficacy through glucose level assessments. Additionally, it mentions spontaneous diabetic rat models that closely resemble human diabetes, emphasizing their relevance for drug testing and understanding diabetes pathology.

1. PRECLINICAL SCREENING
OF ANTIDIABETICS
PRESENTED BY
VINCY DINAKARAN
FIRST YEAR M.PHARM
DPS,CHERUVANDOOR
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2. DIABETES MELLITUS
Diabetes Mellitus is a metabolic disorder characterized by
hyperglycemia, glycosurea, hyperlipidemia, negative nitrogen
balance and sometimes ketonaemia resulting from defects in
insulin secretion, insulin action or both.
TYPES OF DIABETES MELLITUS
 Type 1 : Insulin dependent diabetes mellitus ( IDDM) or
Juvenile onset diabetes mellitus
▪︎ beta cell destruction of pancreatic islets
◇type 1A: Autoimmune antibodies that destruct beta cells are
present in blood
◇type 1B: idiopathic – no antibodies are detected.
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3.  Type 2 :Non insulin dependent diabetes mellitus
(NIDDM)or maturity onset diabetes mellitus
CAUSES
1. Abnormality in gluco-receptor of beta cells
2. Reduced sensitivity of peripheral tissues to insulin
3. Excess hyperglycemic hormones (glucagon) or
obesity
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4. Pathophysiology
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5. 5

6. CLASSIFICATION
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7. SCREENING METHODS
IN-VIVO METHODS
A. MODELS FOR IDDM
1. Chemically induced diabetes
a. Alloxan
b. Streptozotocin
2. Hormone induced diabetes
a. Growth hormone
b. Corticosteroid
3. Virus induced diabetes
4. Genetically diabetic models
a. Spontaneously diabetic rats
●BB rat
● WBN/KOB Rat 7

8. ● Cohen diabetic rat
● Zucker Fatty rat
b. Spontaneously diabetic mouse
● KK mouse
● NOD Mouse
5. Other diabetogenic compounds
a. Dithizone
b. Monosodium glutamate
6. Insulin antibodies
7. Surgically induced diabetes
B MODELS FOR NIDDM ( type 2)
1. Chemically induced diabetes
a. Neonatal STZ Madeleine for NIDDM
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9. 2. Genetic models
a. Monogenic model for NIDDM
♧ Yellow mouse (The Agouty Mouse)
♧ Tubby Mouse
♧ Zucker Diabetic Fatty Rat (ZDF)
b. Polygenic model for NIDDM
♧New Zealand Obese Model (NZO)
♧Japanese KK Mouse
c. Transgenic and knock out models
d. Miscellaneous models
♧Invertebrate animal model
♧ Diet induced metabolic dysregulation 9

10. IN- VITRO
1. Enzyme Inhibition Assay
a. Alpha amylase inhibition assay
b. Alpha glucosidase inhibition assay
2. Glucose uptake assay
a. Glucose uptake assay yeast cell model
b. Glucose uptake assay by adipocytes cell line
3. Isolated islets from pancreas
4. Cultured skeletal muscle cell
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11. In-vivo methods
1. ALLOXAN INDUCED DIABETES
 Alloxan is a cyclic analogue , reported to produce reversible
diabetes in animals.
 Widely used to produce diabetes in rats, mice, rabbits and dogs.
 Selective uptake of the compound due to its structural similarity
to glucose and highly efficient uptake mechanism of pancreatic
beta cells.
PURPOSE AND RATIONALE
》Alloxan have capacity to produce reversible diabetes.
》It is a toxic cyclic urea analogue which destroys beta cells of islets
of Langerhans in pancreas.
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12. 》This compound causes severe necrosis of pancreatic islets.
》Alloxan induces production of H2O2 and some of the free radicals
such as O2 and OH- that first produce damage and later the death of beta
cells.
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Animal used : rabbits
Weight : 2-3 kg
Duration of study: 1 week

13. PROCEDURE
Rabbits of 2 to 3 kg are used
Alloxan infusion (150mg/kg) via ear vein for 10 min.
Animals are given glucose + food ad labitum and regular insulin (28
IU per day single dose) for one week.
70% become hyperglycaemic/ uricosuric and 30% either die or
temporary hyperglycaemic.
Triphasic Response
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14. TRIPHASIC RESPONSE
 Blood glucose level first rise at 2 hour
 Followed by hypoglycemic phase at 8 hour
 Finally and increase at 24 hr probably due to depletion of beta cells of
insulin.
EVALUATION
♤ Glucose level
♤ Insulin level
♤ Hepatic glycogen level
 Compare results of standard with control group animals
DRAWBACKS
□ High motility rate in rats
□ Diabetes induced is revesible
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15. □ Diabetes induced is revesible
□ Some species like Guinea pigs are resistant to its diabetogenic
action.
 Alloxan has been almost completely replaced by streptozotocin
because of its drawbacks.
STREPTOZOTOCIN INDUCED
DIABETES
 It is a broad spectrum antibiotic, which is produced from
Streptomyces achromogens.
 It is used to induce both type 1 diabetes mellitus and type 2
diabetes mellitus at dose of 50mg/kg i.p. for 3 days and 80mg/kg
( i.v., i.p., or s.c.) in neonatal rats for 10 days respectively.
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16. Mechanism
 By process of methylation
 Free radical generation
 Nitric oxide production
PROCEDURE
☆ STZ induces diabetes in almost all species of animals . Diabetic
doses varies with species and the optimal dose required in various
species are
a. Rats 50 to 60 mg/kg (i.p/i. V)
b. Mice 175 to 200 mg/kg (i.p/i.v)
c. Dogs 15 mg/kg for 3 days
☆ It shows the triphasic responses
1. Hyperglycemia at 1 hr 16

17. 2. Hypoglycemia lasting for 6 hrs
3. Stable hyperglycemia by 24 to 48 hr after STZ administration.
ADVANTAGES
 Greater selectivity towards beta cells
 Low mortality rate
 Irreversible diabetes induction
VIRUS INDUCED DIABETES
♧Associated with chronic islet cell inflammation, elevation of islet
cell antibody and prolonged presence of viral RNA in the pancreas.
MECHANISM
 Infecting and destroying beta cells of pancreas
 By eliciting immune auto reactivity to the beta cells 17

18.  Viruses producing systemic effects, not directly affecting beta cells.
☆Various virus used for inducing diabetes
●RNA picornavirus
●Coxsackie – B4
●Encephalomylocarditis (EMC-D and M variants)
●Mengo 27
●Reovirus
●Lymphocytic choriomeningitis (LMCV)
PROCEDURE
6 to 8 weeks old mice are inoculated by 0.1ml of 1:50 dilutions of D
variant Encephalomyocarditis (EMC) through i.p.
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Animal used : mice
Age :6-8 weeks
Duration of study : 6 weeks

19. 0.1ml of above dilution contain 50 PFU(Plague Forming Units) of
EMC virus. ( Mortality due to this concentration of virus is
approximately 10 to 20%)
A less infecting variant produces a comparable damage by eliciting
autoimmune reactivity to the beta cells.
Infected animals are considered hyperglycemic if their non fasting
levels exceed by 250mg/dl.
Drug samples to be screened are administered orally for a period of
6 weeks.
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20. After 6 weeks of drug treatment , blood glucose estimation is done to
determine the anti-diabetic activity.
GENETICALLY DIABETIC MODELS
SPONTANEOUSLY DIABETIC RATS
 Similar to the human condition, these strains display complex and
heterogeneous characteristics.
 In some of these models, insulin resistance predominate in association
with obesity, dyslipidemia and hypertension, which provides valuable
insights to study some events that are observed in human type 2
Diabetes Mellitus.
 The occurrence of spontaneous diabetes seen in several strains of rat.
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21. BB RAT (Biobreeding Rat)
 The BB rat is model of spontaneous diabetes associated with insulin
deficiency and insulitis due to autoimmune destruction of pancreatic
beta cells.
 Diabetes inherited as an autosomal recessive trait and develops with
equal frequency and severity among males and females.
 The onset of diabetes is sudden and occurs at about 60 to120 days of
age.
 The diabetogenic syndromes of BB rat shares many characteristics with
human IDDM.
PROCEDURE
Rats are bred at the laboratory by sib mating over 20 generations
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22. After 20 generations of sib mating spontaneous development of IDDM
in rats are obtained.
Onset of clinical diabetes is sudden and occurs at 60 to 120 days of age.
Clinical presentation is similar to that of humans with marked
hyperglycemia, glucosurea, and weight loss and decrease plasma insulin
and these results in ketoacidosis if untreated.
Animals are treated with drug samples to be screened for a required
period of time.
WBN/ KOB Rat
 Spontaneous hyperglycemia, glycosuria, and glucose intolerance have
been observed in aged males of an inbred wister strain , named
WBN/KOB Rat. 22

23.  These animals exhibit impaired glucose tolerance and glucosuria at
21 weeks of age.
 Decrease in number and size of islets are found after 12 weeks of age.
 Degeneration of pancreatic tissue are observed in the endocrine part,
mainly around degenerated islets and pancreatic ducts in 16 week old
males.
INVITRO
Alpha amylase Inhibition Assay
 alpha-amylase hydrolyse alpha bonds of large alpha linked
polysaccharides such as starch and glycogen yielding shorter chains
dextrins and maltose.
 It will break large, insoluble starch molecules into soluble starches.
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24. PROCEDURE
Test sample was prepared in dimethyl sulphoxide from 1microg/ml stock
solution and the sample was added to 0.5 mg/ml alpha amylase.
Incubated for 10 min at room temperature
1%starch solution (500micro L) was added and incubated at room
temperature for 10 min.
1ml of Dinitrosalicylic acid was added and heated in a boiling water bath
for 5 min. And cool.
Tested samples were diluted with 10ml of distilled water.
Measure the absorbance at 540nm and percentage of enzyme inhibition
activity of bioactive fractions were calculated . 24

25. EVALUATION
Inhibition activity (%) =(Abs sample- Abs control)×100/Abs sample
Abs = absorbance
All the experiments were carried out in triplicates.
GLUCOSE UPTAKE ASSAY BY ADIPOCYTES
CELL LINE
 Adipose tissue is considered to have a link between obesity and
type 2 diabetes mellitus, elevated intracellular lipid concentrations
and insulin resistance.
 Insulin resistance either at the adipocyte or skeletal muscle levels
contribute to hyperglycemia.
PROCEDURE
☆ Isolated Adipocyte Preparation: Uptake of (3H) glucose into fat
cell and incorporation of this radio labelled glucose in the lipid can
measure insulin like activity.
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26. Male wister rats-Epididymal fat pad are taken.
These pads are cut into pieces and incubated for 20 min. At 370Cwith
1mg/ ml collagenase in KRHB+ 25 mM HEPES/KOH + 0.1 mM
glucose+ 1% w/v BSA.
Cell suspension is filtered through 100mm Nylon screen.
Washed 3 times by flotation with KRHB without glucose and suspended
in same solution.
Suspension is adjusted to a final titre of 4×105 cell/ml.
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27. REFERENCE
 Drug Discovery and Evaluation by H. Gerhard Vogel, Second
Edition. Page number: 317 -328 and 947-1057
 In vivo and In vitro Models for Biological Screening of Anti-
Diabetic Drugs by R. Vijayaraj et.al. International Journal of
Pharmacy and Biological Sciences- Published online on 1 April
2019.
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