This document provides an introduction to diabetes and anti-diabetic drug screening methods. It begins by classifying diabetes and defining the main types - type 1, type 2, gestational, and secondary. It then describes the pancreas and its beta cells that produce insulin. Various models for inducing diabetes in animals are discussed for screening anti-diabetic drugs, including chemical agents like alloxan and streptozotocin, viral induction, immune-mediated induction using anti-insulin serum, genetic alteration in mice/rats, pancreatectomy, and hormone-induced using dexamethasone. Common screening methods and their principles, procedures, advantages, and limitations are summarized.

1. INTRODUCTION TO DIABETES & ANTI-
DIABETIC DRUG
SCREENING METHODS
By
ANURAG RAGHUVANSHI
Dept. of pharmacology
(A10654915001)
AMITY UNIVERSITY NOIDA
2015-16
anuragraghuvanshi3@gmail.com

2. CONTENTS
Classification of Diabetes
Definition & types of Diabetes mellitus
Pathological changes & symptoms
Pancreas- types of cell
Screening methods- classification
references

3. CLASSIFICATION OF
DIABETES

4. DIABETES INSIPIDUS
Diabetes insipidus is caused by problems related to the
antidiuretic hormone (ADH) or its receptor and causes frequent
urination.
There are four types of diabetes insipidus;
1) Central diabetes insipidus,
2) Nephrogenic diabetes insipidus,
3) Dipsogenic diabetes insipidus, and
4) Gestational diabetes insipidus.

5. SYMPTOM & DIAGNOSIS
The most common symptom of diabetes insipidus is frequent
urination.
The diagnosis for diabetes insipidus is based on a series of
tests (for example, urinalysis and fluid deprivation test).
The treatment for diabetes insipidus depends on the type of
diabetes insipidus.
Diabetes can lead to chronic kidney disease.
Diabetes is the most common cause of kidney failure in the
US.

6. WHAT IS DIABETES
MELLITUS?
To answer that, you first need to understand the role of insulin
in your body.
When you eat, your body turns food into sugars, or glucose. At
that point, your pancreas is supposed to release insulin.
Insulin serves as a “key” to open your cells, to allow the
glucose to enter -- and allow you to use the glucose for
energy.
But with diabetes, this system does not work.
Several major things can go wrong – causing the onset of
diabetes. Type 1 and type 2 diabetes are the most common
forms of the disease, but there are also other kinds, such as
gestational diabetes, which occurs during pregnancy, as well as
other forms.

7. DIFFERENCE BETWEEN
DIABETES MELLITUS AND
DIABETES INSIPIDUS
Diabetes insipidus should not be confused with diabetes
mellitus (DM), which results from insulin deficiency or
resistance leading to high blood glucose, also called blood
sugar.
Diabetes insipidus and diabetes mellitus are unrelated,
although they can have similar signs and symptoms, like
excessive thirst and excessive urination.
Diabetes mellitus is far more common than diabetes insipidus
and receives more news coverage. Diabetes mellitus has two
main forms, type 1 diabetes and type 2 diabetes. Diabetes
insipidus is a different form of illness altogether.

8. DEFINITION AND TYPES:
Diabetes mellitus is a metabolic disorder characterized by
hyperglycemia, glycosuria, hyperlipidemia, negative nitrogen balance
and sometimes ketonaemia.
Type 1 diabetes mellitus (IDDM):- due to β-cell destruction, usually
leading to absolute insulin deficiency
Type 2 diabetes mellitus (NIDDM) :-due to a progressive insulin
secretory defect on the background of insulin resistance
Gestational diabetes, Type III:- diabetes diagnosed in the second or
third trimester of pregnancy that is not clearly overt diabetes
Maturity-onset diabetes of the young (MODY):- at a young age, and
usually inherited in an autosomal-dominant manner.
Secondary diabetes: accounts for only 1-2% of patients with diabetes
mellitus

9. SECONDARY DIABETES
Pancreatic disease: cystic fibrosis, chronic pancreatitis,
pancreatectomy, carcinoma of the pancreas.
Endocrine: Cushing's syndrome, acromegaly, thyrotoxicosis,
phaeochromocytoma, glucagonoma.
Drug-induced: thiazide diuretics, corticosteroids, atypical
antipsychotics, antiretroviral protease inhibitors.
Congenital lipodystrophy.
Acanthosis nigricans.
Genetic: Wolfram's syndrome (which is also referred to as
DIDMOAD: diabetes insipidus, diabetes mellitus, optic atrophy and
deafness),Friedreich's ataxia, dystrophia myotonica,
haemochromatosis, glycogen storage diseases.

10. PATHOLOGICAL
CHANGES
Increase in vessel wall matrix.
Thickening of capillary basement membrane.
Cellular proliferation resulting in vascular
complication :-
Lumen narrowing
Early atherosclerosis
Sclerosis of glomerular capillaries
Retinopathy ( damage to retina)
Neuropathy (dysfunction of peripheral nerves)
Peripheral vascular insufficiency

11. SYMPTOMS OF
DIABETES

14. NORMAL BLOOD GLUCOS LEVEL – 100mg/dl
You are a diabetic if –
Fasting blood sugar is more than 126 mg/dl Or
2 hours after having food, your blood sugar is more than 200 mg/dl

16. DIABETIC FOOT
Diabetes can lead to foot ulcers and amputation
Your chances of having it are high if you have –
 Diabetes for many years
 Foot injury
 Poor blood sugar control
 Poor blood flow in feet
 Poor sensation in feet

17. Diabetes can affect the eyes in many ways –
 Retinopathy
 Cataract
 Glaucoma
Early intervention reduces risk of blindness

18. PANCREAS
The pancreas is an organ located in
the abdomen. It plays an
essential role in converting the food
we eat into fuel for the body's cells.
The pancreas has two
main functions: an
exocrine function that helps in
digestion and an
endocrine function that regulates
blood sugar.

19. Β CELL The primary function of
a beta cell is to store
and release insulin.
Insulin is a hormone that
brings about effects
which reduce blood
glucose concentration.
Beta cells can respond
quickly to spikes in blood
glucose concentrations
by secreting some of
their stored insulin while
simultaneously
producing more.

20. THE ΒCELLS CONSTITUTE THE
CORE OF THE ISLETS AND ARE THE
MOST ABUNDANT CELL TYPE. THE
A CELLS, COMPRISING 25% OF THE
ISLET CELL MASS, SURROUND THE
CORE AND SECRETE GLUCAGON.
THE D CELLS (5-10%) ELABORATING
SOMATOSTATIN ARE
INTERSPERSED BETWEEN THE A
CELLS. THERE ARE SOME PP (OR F)
CELLS (PANCREATIC POLYPEPTIDE
CONTAINING) ALSO.
• Somatostatin inhibits release of both
insulin and glucagon.
• Glucagon evokes release of insulin as
well as somatostatin.
• Insulin inhibits glucagon secretion.

21. TREATMENT
Conventional Preparations of Insulin
Short Acting :
 Regular Insulin
 Prompt Insulin Zinc suspension (semi lente)
Intermediate Acting
 Insulin zinc suspension (Lente)
 Neutral protamine hagedron or Isophane insulin
Long Acting
 Extended Insulin zinc suspension (Ultra lente)
 Protamine Zinc Insulin
Purified Insulin Preparations
 Single Peak Insulins : Actrapid, Lentard, Actraphane
 Monocomponent Insulins : Monotsard
 Human Insulin : Human Actrapid, Human Monotard

22. ORAL HYPOGLYCEMIC
Sulphonyl Ureas
First Generation Second
Generation
Tolbutamide Gilbenclamide
Chlorpropamide Glipizide
Acetohexamide Gliclazide
Tolazamide
Biguanides
Phenformin
Metformin
Miscellaneous
Acarbose
Guargum

23. MODELS FOR ANTI DIABETIC
SCREENING

24. MODELS FOR IDDM
CHEMICAL
INDUCED
HORMONE
INDUCED
VIRAL INDUCES
-ALLOXAN
-STZ-60mg/kg
(STREPTOZOTOCI
N) N-nitro
derivative of
glucosamine)
DEXAMETHASONE ENCEPHALO-
MYLOCARDITIS
SURGICALLY INDUCED GENETIC ALTERATION
Pancreatectomy—
the surgical removal of the entire
pancreas—is similar to type
1 diabetes
-NOD mouse
-BB rat (bio breeding rat)
-LETL rat
-N rat ( nude)
-Sprague-Dawley (SD)

25. CHEMICALS USED
IRREVERSIBLE REVERSIBLE OTHER AGENTS
Alloxan Azide Diphenyl
thiocarbazine
streptozotocin cyproheptadine Oxine-9-
hydroxyquinolone
malonates -Vacor
-somatostatin

26. Models For Insulin Dependent Diabetes Mellitus
[IDDM]
Alloxan induced diabetes
Principle/Mechanism -
Interacts with sulfhydryl groups and inhibit it- Following it’s uptake by the beta
cells, alloxan interacts with sulfhydryl- containing cellular components,
particularly enzymes known to be essential for beta cell function.
Glucokinase , an enzyme which has signal-recognition function in coupling the
glucose concentration to insulin release and it is particularly sensitive to
inhibition by alloxan.
Other proposed mechanism for alloxan cytotoxicity include direct induction of
mitochondrial abnormalities, extreme sensitivity of beta cells to the cytotoxic
effects of free radicals (generated during the reduction/ re-oxidation cycle of
alloxan) & damage to DNA within the beta cell nucleus.

27.  Dose: - In rats Alloxan at dose of 100 mg/kg produces diabetes.
Rabbits- 150mg/kg, beagle dog- 60mg/kg, baboons- 200mg/kg.
Procedure: -
Albino rats of either sex [150-200g] are injected with a single dose of
Alloxan monohydrate [100 mg/kg body weight] dissolved in normal
saline by i.p. route.
Animals showing fasting blood glucose level above 140 mg/dl after 48
hour of Alloxan administration are considered diabetic
For a period of six weeks, drug samples to be screened .
Blood glucose levels show triphasic response with hyperglycemia for 1
hour followed by hypoglycemia that lasts for 6 hours & stable
hyperglycemia after 48 hours
Serum is separated by centrifuge (3000 rpm) at (2-4 °C) for ten
minutes.
The serum glucose level is estimated by glucose oxidase-peroxidase
method [GOD-POD kit] using auto analyser.

28. LIMITATION
 HIGH MORTALITY
KETOSIS
SOME SPECIES ARE RESISTANT TO ALLOXAN e.g.
guinea pig
STREPTOZOTOCIN HAS ALMOST COMPLETELY
REPLACED ALOXAN

29. Streptozotocin induced diabetes
(glucosamine-nitrosourea compound)
Rakieten (1963) reported the diabetogenic activity of the antibiotic
Streptozotocin. The compound turned out to be specifically Cytotoxic to
beta-cells of the pancreas.
Streptozotocin induced diabetes in laboratory animals, mostly in rats,
has become a valuable tool in diabetes research being used by many
investigators.
Principle/ Mechanism
 Streptozotocin: is a broad-spectrum antibiotic, which causes beta
islet cell damage by free radical generation. It induces diabetes in
almost all species of animals excluding rabbits and guinea pigs.
Fragmentation of DNA
Nitric oxide generation
Induces diabetes in all species.

30. o Greater selectivity towards beta
cells
o Low mortality
o Longer and irreversible diabetes
ADVANTAGES

31. Procedure: -
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 diabetic.
After six weeks of treatment blood samples are collected from 6 hr.
fasted animals through caudal vein
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.

32. Virus induced diabetes
Principle: -
Various human viruses used for inducing diabetes include
RNA picorno virus
encephalomyocarditis [EMC-D]
coxsackie B4 [CB-4].
Renovirus
Mengo-2t

33. PROCEDURE
6-8 week old mice are inoculated by 0.1 ml of 1:50 dilutions of D-
variant encephalomyocarditis [EMC] through i.p.
0.1ml of above dilution contains 50 PFU [ plaque forming units] of
EMC virus. (mortality due to this concentration of virus is
approximately 10-20%)
A less infecting variant produces a comparable damage by eliciting
autoimmune reactivity to the beta cells.
Infected animals are considered hyperglycaemic if there non fasting
levels exceed by 250mg/dl
Drug samples to be screened are 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.

36. Principle: - A transient diabetic syndrome can be induced by injecting
guinea pigs with anti insulin serum. Diabetes persists as long as
antibodies are capable of reacting with insulin remaining in the circulation.
Preparation of antibody :-
Bovine insulin, dissolved in acidified water [ph 3.0] at a dose of 1mg is
injected to guinea pigs weighing 300-4oo gm. Anti insulin sera is collected
after two weeks of antigenic challenge.
Procedure: -
Adult albino rats are injected with 0.25-1.0 ml of guinea pig anti- insulin
serum.
Insulin antibodies induce a dose dependent increase of blood glucose
level up to 300 mg/ dl.
The drug sample to be screened is administered by a suitable root and
blood glucose level is analysed to determine the activity.
Insulin antibodies induced
diabetes

37. LIMITATIONS
Effect persist as long as antibodies remain in the circulation
Large doses and prolonged administration- ketonaemia, ketonuria,
glycosuria and acidosis are fatal to animals

38. SURGICALLY INDUCED
METHODS
PRINCIPLE: Surgical removal of pancreas result in insulin
dependant form of DM state

39. SURGICALLY INDUCED
ANIMALS
Partial pancreatectomized animals
e.g. dog, primate, pig & rats
Advantages Disadvantages
Avoids cytotoxic effects of chemical
diabetogens on other body organs
cumbersome technical and
post 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

40. HORMONE INDUCED DM
PRINCIPLE/MECHANISM
DEXAMETHASONE is a long acting glucocorticoid possessing
immunosuppressant action in the islets and produces type 1 diabetes.
PROCEDURE
•Adult rats 150-200gm dexamethasone (2-5mg/kg i.p.)
•Repeated injection of same dose level is carried out for a period of 0-
30 days resulting in IDDM.
•The sample to be screened is administered through a suitable route,
blood glucose is measured.
LIMITATION
Long standing procedure.

41. Genetic models
Non obese diabetic mouse [NOD MOUSE]
NOD mouse is model for IDDM.
Hypoinsulinemia is developed which is
caused by autoimmune destruction of
pancreatic beta cells in association with
autoantibody production.
Procedure: -
Mice are breed at laboratory by sib mating
over 20 generations.
After 20 generations of sib mating,
spontaneous development of IDDM in mice
is obtained. Diabetes develops abruptly
between 100-200 days of age.
[Characterized by weight loss, poly urea,
severe glycosuria]
Animals are treated with the drug sample to
be screened.
N
BB RAT
NOD MUSE

42. The non-obese diabetic(NOD) mouse
is an animal model for human insulin-
dependent diabetes mellitus(IDDM)
that spontaneously develops a T-cell
mediated autoimmune disease
characterized by massive infiltration
of pancreatic Islets and several other
organs(namely the submandibular
salivary & thyroid glands) by CD4 and
CD8 T cells.
LIMITATION
Ketosis
Glycosuria
Weight loss
High mortality

43. COHEN DIABETIC RAT
Diabetes in Cohen rats is characterized by hyperglycemia, glucosuria, and
hyperinsulinemia, with late development of hypoinsulinemia, insulin
resistance, and decrease in the number and sensitivity of insulin receptors.
The rats develop overt diabetes and diabetes related complications when fed a
diet rich in sucrose or other refined sugars and poor in copper content.

44. Models For NIDDM
Streptozotocin induced neonatal model for NIDDM
Streptozotocin causes severe pancreatic beta cells destruction,
accompanied by decrease in pancreatic insulin stores and rise in
plasma insulin levels.
Procedure: -
Neonatal rats are treated with streptozotocin [90 mg per kg body
weight] (for NR & SD rats150mg/kg STZ was injected intraperitoneally)
prepared in citrate buffer [pH 4.5] by i.p at birth or within the first five
days following birth.
After six weeks rats develops symptoms similar to NIDDM.
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 analysed to determine the activity.

45. ADRENALINE INDUCED ACUTE
HYPERGLYCAEMIA
Adrenaline is a counter regulatory hormone to insulin. It increases
the rate of glycogenolysis and the glucose levels in blood causing
hyperglycaemia.
Procedure:-
Adult albino rats are injected at a dose level of 0.1 mg / kg through
s.c. route
The dose produces peak hyperglycaemic effect after one hour and
lasts up to four hours.
The drug sample to be analysed is administered through a suitable
route.
Blood glucose is determined.(The oral hypoglycaemic agents can
be screened by this method)

46. Assay for insulin & insulin like activity
This assay involves comparing two standard solutions of insulin with
the test drug for it’s insulin like activity
Procedure:-
Four groups of six rabbits weighing at least 1.8 kg are used
Two standard solutions of insulin containing one unit and two units
respectively and two dilutions of sample whose potency is to be
examine are prepared.
Each of the prepared solution (0.5 ml) is injected subcutaneously.
After one hour and 2.5 hr. of each injection, a suitable blood sample is
taken from the ear vein of each rabbit and blood sugar determined
preferably by glucose oxidase method.

47. Blood sugardeterminations in mice:-
Purpose and rationale:-
Eneroth and Ahlund (1968, 1970a,b) recommended a twin crossover
method for bio-assay of insulin using blood glucose levels in mice instead
of hypoglycemic seizures giving more precise results.
Procedure:-
Non-fasting mice of the same strain and sex are used having body
masses such that the difference between the heaviest and lightest mouse
is not more than 2 g. The mice are assigned at random to four equal
groups of not less than 10 animals. Two dilutions of a solution of the
substance or of the preparation to be examined and 2 dilutions of the
reference solution are prepared using as diluent 0.9% NaCl solution
adjusted to pH 2.5 with 0.1 N hydrochloric acid and containing a suitable
protein carrier. In a preliminary experiment, concentrations of 0.02 IU and
0.10 IU are tested.

48. Each of the prepared solutions (0.1 ml/10 g body weight) is injected
subcutaneously to one group of mice according to a randomized block
design. Not less than 2.5 h later, each solution is administered to a second
group of mice following a twin crossover design. Exactly 30 min after each
injection, a sample of 50 μl of blood is taken from the orbital venous sinus
of each mouse. Blood glucose concentration is determined by a suitable
method, such as described by Hoffman (1937).
Evaluation:-
The potency is calculated by the usual statistical methods for the twin-
cross-over assay.

49. REFERENCES:-
Dr Hayley Willacy- Diabetes Mellitus, Document ID:
2048 (v30)http://patient.info/doctor/diabetes-mellitus
http://www.diabetesresearch.org/what-is-diabetes
Drug screening methods :- By S.K Gupta
http://www.ijrpbsonline.com/files/59-3290.pdf
http://www.pharmaceutical-tech.com/articles/id/antidiabetes
http://www.medicinenet.com/diabetes_insipidus/article.htm

50. THANK YOU
By
ANURAG RAGHUVANSHI
Dept. of pharmacology
(A10654915001)
AMITY UNIVERSITY NOIDA
2015-16
anuragraghuvanshi3@gmail.c
om