Screening models for DM Various screening models

1. INTRODUCTIONTO DIABETES AND ANTI-
DIABETICDRUGSCREENINGMODELS
BYVAISHALICHADDHA
MPHARM1ST YEAR
PHARMACOLGY

2. • Diabetes is a disease that affects your body's
ability to produce or use insulin. Insulin is a
hormone. When your body turns the food you
eat into energy (also called sugar or glucose),
insulin is released to help transport this energy
to the cells.

5. • “Diabetes mellitus can be defined
as a heterogenous group of
chronic disorders of carbohydrate,
lipid and protein metabolism
characterized by high blood
glucose levels due to relative or
absolute deficiency of insulin”

7. • DIABETES INSIPIDUS - a disease in which the
secretion of or response to the pituitary
hormone vasopressin is impaired, resulting in
the production of very large quantities of
dilute urine, often with dehydration and
insatiable thirst.

14. Type 1 diabetes happens when your immune system destroys
cells in your pancreas called beta cells. They’re the ones that
make insulin.

15. Type 2 diabetes is a condition in which cells cannot use blood
sugar (glucose) efficiently for energy. This happens when the
cells become insensitive to insulin and the blood sugar gradually
gets too high.
Type 2 can be caused by:
• Being overweight
• Eating a lot of foods or drinks with sugar and simple
carbohydrates
• Artificial sweeteners (sugar free sodas, sugar free foods)
intake
• Lack of activity (sedentary behavior)
• Lack of exercise
• Stress and stress IQ hormones
• Genetics(MOSTLY)

16. Type 2 diabetes is the most common type of diabetes. It
is a chronic problem in which blood glucose (sugar) can
no longer be regulated. There are two reasons for this.
First, the cells of the body become resistant to insulin
(insulin resistant). Insulin works like a key to let glucose
(blood sugar) move out of the blood and into the cells
where it is used as fuel for energy. When the cells
become insulin resistant, it requires more and more
insulin to move sugar into the cells, and too much sugar
stays in the blood. Over time, if the cells require more
and more insulin, the pancreas can't make enough
insulin to keep up and begins to fail.

18. CLASSIFICATION OF ANTI DIABETIC
DRUGS
ORAL ANTI-DIABETIC DRUGS

19. INSULIN

20. SCREENING MODELS
IN VIVO MODELS-
• For IDDM
1. Chemically induced diabetes
2.Hormone induced diabetes
3.Virus induced diabetes
4.Surgery induced diabetes
5.Insulin antibody induced diabetes
6.Genetic models
• For NIDDM
1.Neonatal STZ induced NIDDM
2.Other chemicals
3.Genetic models

21. Blood glucose lowering effects in rats
Procedure
• Male Wistar rats of 180-240 gm are kept on a
standard diet (Altromin 1324).
• Groups of 4-7 non-fasted animals are treated orally
or IP with various doses of test compounds
suspended in 0.4% starch suspension. One control
group receives vehicle only.
• Blood is withdrawn from the tip of the tail
immediately before and 1,2,3,5 and 24 hours after
administration of test compound.
Evaluation
• Average blood sugar values are plotted versus time
for each dosage.
• Percentage data related to the value before the
experiment are

22. Chemically induced diabetes
Chemicals that induce diabetes can be classified into 3
categories
>>Specifically damage β cell
>>Cause temporary inhibition of insulin production
and/or secretion
>>Diminish metabolic efficacy of insulin target tissues
1. Alloxan induced diabetes
2. Streptozocin induced diabetes

23. ALLOXAN INDUCED DIABETES
>>A cyclic urea analog, the first agent to be
introduced in this category to induce
permanent diabetes in animals
>> Shows a triphasic reponse
>> Species to species variation
ALLOXAN => DIULERIC ACID => ALLOXAN+
FREEACID =>DAMAGES THE DNA OF BETA
CELLS => CELL DEATH

24. • RABBITS:
• weight 2-3.5 kg are taken ear vein – 150 mg/kg
alloxan monohydrate (5g/100 ml, pH 4.5) for 10
minutes resulting in 70% of animals becoming
hyperglycemic and uricosuric.
• The rest either die or are temporarily
hyperglycemic.
• RATS:
• Wistar or Sprague – Dawley strain Weight 150-
200 g
• Inj. Alloxan 100-175 mg/kg SC
• MALE BEAGLE DOGS:
• weight 15-20 kg inj. Alloxan IV 60 mg/kg

25. Drawbacks of alloxan
• High mortality in rats
• Causes ketosis in animals due to free fatty
acid generation
• Diabetes induced is reversible
• Guinea pigs are resistant to alloxan

26. STREPTOZOCIN INDUCED DIABETES
Streptozocin [2-deoxy-2-methyl(3-methyl-3-
nitrosourea)1Dglucopyranose]
is a broad spectrum antibiotic produced from
Streptomyces achromogens.
Mechanism of β cell damage
>>By process of methylation
>>Free radical generation
>>Nitric oxide production

27. • Diabetogenic doses vary with species
• Rats – 50-60 mg/kg IP or IV
• Mice – 175-200 mg/kg IP or IV
• Dogs – 15 mg/kg for 3 days
• Blood glucose levels show triphasic response as seen with
alloxan
• • Hyperglycemia at 1 hour
• • Hypoglycaemia that lasts for 6 hours
• • Stable hyperglycemia by 24-48 hours after STZ administration
• Advantages and disadvantages: :
• • Greater sensitivity towards β cells
• • Lower mortality rates
• • Longer and irreversible diabetes induction
• • But, guinea pigs and rabbits are resistant to its diabetogenic
action

28. GROWTH HORMONE INDUCED
DIABETES
• Growth hormone induced diabetes
• • Cotes and co-workers (1949) described the
diabetogenic action of
• anterior pituitary growth hormone in cats.
• • Repeated administration of GH induces an intensively
diabetic
• condition with all the symptoms of diabetes including
ketonemia and
• ketonuria in intact adult dog.
• • Rats of any age subjected to similar treatment do not
become
• diabetic but grow faster and show hypertrophy of
pancreatic cells

29. GLUCOCORTICOIDS INDUCED
DIABETES
• Ingle (1941) described hyperglycemia and glucosuria in
forced fed rats with
cortisone.
• Hausberger and Ramsay in 1953 showed that
experimental diabetes by
cortisone can be induced in guinea pigs and rabbits
without forced feeding.
(Abelove and Paschkis,1954)
• In the rat, adrenal cortex, stimulated by corticotrophin,
has the capacity to
secrete amounts of steroids which induce diabetes (Ingle
et al 1946)

30. VIRUS INDUCED DIABETES
They may produce DM by:
• Infecting and destroying β cells in pancreas.
• A less infecting or cytologic variant producing a
comparable damage by eliciting immune auto-
reactivity to β cells.
• Viruses that produce systemic effects, not directly
affecting β cells. Viruses used for inducing DM:
• Coxsackie B4
• Encephalomyocarditis (EMC-D and M variants)
• Mengo-2T
• Reoviruses
• Lymphocytic Choriomengitis Virus (LMCV) Armstrong
variant

31. SURGERY INDUCED DIABETES
• • Induction of DM can be achieved through surgical
removal of all or
• part of pancreas.
• • Depending on the amount intact pancreatic cells, DM
may range in
• duration from a few days to several months.
• Disadvantages:
• • Surgical removal of pancreas also causes loss of α and
δ cells in addition to β
• cells.
• • There is also loss of pancreatic enzymes for digestion.
• • The total resection is difficult to achieve and the
severity of DM is strain

32. INSULIN ANTIBODIES-INDUCED
DIABETES

33. GENETICALLY DIABETIC
ANIMALS
• In recent years, various animals have been shown to
exhibit diabetes
• mellitus spontaneously
• • The ob mutation in mice resulted in leptin deficiency
• • The fat mutation in mouse results in biologically
inactive carboxypeptidase E,
• which processes the prohormone conversion of POMC
into MSH-α, which
• activates the hypothalamic MC4 receptor.
• • Agouti yellow mouse exhibit ubiquitous expression of
Agouti protein which
• represents an antagonist of hypothalamic MC4
receptor.

35. TRANSGENIC ANIMALS
• 1. Skeletal muscle and insulin resistance/MIRKO mice
model
• 2. Adipose tissue and insulin resistance/FIRKO mouse
• 3. Mouse with specific disruption of IR gene in β cells which
show a selective loss of insulin
• secretion in response to glucose and progressive
impairment of glucose tolerance
• 4. The liver specific insulin receptor knock-out (LIRKO)
mouse
• 5. CNS specific disruption of IR gene (NIRKO)
• 6. IRS-1 deficient mouse which show genetically
determined insulin resistance
• 7. PPAR γ inactivation

36. STZ INDUCED NIDDM
• • Neonatal rats of Sprague-Dawley strain are taken
• • Treated with STZ 80-100 mg/kg IP at birth or within 5
days of birth
• which leads to severe β cell destruction, deficiency in
pancreatic
• insulin levels and rise in plasma glucose.
• • In contrast to adults, the β cells in neonates partially
regenerate.
• • Following an initial spike in plasma glucose, these
rats become
• normoglycemic by 3 weeks.

37. • Other chemical methods
• • For NIDDM in rabbits - adrenaline (0.1
mg/kg SC).
• • The hyperglycemia is seen at 1 hour and
lasts for 4 hours. Oral
• hypoglycemic agents can be screened by this
method.
• • Other chemicals are 8-hydroxy quinoline,
biphenyl thio carbazine,
• EDTA (partially depancreatized rats), thiazides,
chlorthiazide,
• hydrochlorthiazide, diazoxide and furosemide

38. GENETIC MODELS FOR NIDDM
• Monogenic models of obesity and NIDDM
• 1. Yellow mouse (the Agouti mouse)
• 2. Obese and diabetic mouse
• 3. Tubby mouse
• 4. Fat mouse
• 5. Zucker diabetic fatty rat
• 6. Koletsky and JCR: LA-Corpulent rats
• Polygenic models of obesity and NIDDM
• 1. New Zealand obese mouse
• 2. Japanese KK mouse
• 3. Nagoya – Shibata – Yasuda mouse
• 4. PBB/Ld mouse
• 5. Otsuka-Long-Evans-Tokushima Fatty rat
• 6. Goto-Kakisaki rat
• 7. Chinese Hamster
• 8. Djungarian (Siberian) hamster
• 9. South African Hamster

41. Blood glucose lowering effects in rats
• Procedure
• • Male Wistar rats of 180-240 gm are kept on a standard
diet (Altromin 1324).
• • Groups of 4-7 non-fasted animals are treated orally or IP
with various doses of test compounds suspended in 0.4%
starch suspension. One control group receives vehicle only.
• • Blood is withdrawn from the tip of the tail immediately
before and 1,2,3,5 and 24 hours after administration of test
compound.
• Evaluation
• • Average blood sugar values are plotted versus time for
each dosage.
• • Percentage data related to the value before the
experiment are calculated.

42. • Modifications:
• • Studies in glucose loaded rats – in this method,
glucose 1 gm/kg body
• weight is given following the test compound
either orally or SC
• • Studies in streptozocin diabetic rats – in this
method, diabetes is
• induced with streptozocin which leads to fall in
plasma insulin levels.
• Compounds which release insulin from islets as
the sole hypoglycemic
• mechanism of action are not effective in these
animals

43. • Hypoglycemic seizures in mice
• The biological assay of insulin using hypoglycemic seizures in mice has been
suggested in 1923 by Fraser. In most Pharmacopoeias, the biological assays
have been replaced by chemical methods now.
• Animals – 96 mice of either sex weighing 20 ± 5 gm randomly distributed into 4
groups
• Procedure:
• The mice are deprived of food 2-20 hours immediately preceding the test.
• Insulin solutions standard and test are prepared by diluting 30 and 60
• mIU/ml in NS with pH 2.5 and 0.5 ml/20 g injected SC.
• The mice are kept at a uniform temperature, between 29-35⁰C in transparent
• containers within an air incubator with a transparent front.
• The mice are observed for 1.5 hours and no of mice that are dead, convulse
• or lie still for more than 2-3s when placed on backs are noted.
• Evaluation: The percentage of mice of each group showing the above
mentioned symptoms is calculated and relative potency of test solution is
calculated using a 2+2 point assay.
• Modifications: Suggested by Young and Lewis in 1947, also done by Vogel in
1964 The rotating drum method

44. ISOLATED RAT PANCREAS
Purpose Used for studying the effect of drug on Insulin, glucagon,
somatostatin secretion.
Procedure
• Adult Wistar rat (150-200 gm) are fed ad libitum
. • Pancreas are removed under Pentobarbital (50 mg/kg IP)
• Through a portal vein canula Krebs-ringer bicarbonate buffer with
2% bovine albumin & 5.5 mmol/l glucose is perfuse at rate of 1.75
ml/min. at pressure 100mmHg.
• Perfusate is collected every min. for 30 min. after first 5 min. test
drug added till 15 min. next 16-30 min glucose is perfused.
Evaluation
Insulin, Glycogen, Somatostatin are estimated using
Radioimmunoassay. The effect of test drug on hormone secretion
of pancreas in response to elevated glucose level is compared with
the control.

45. ISOLATED RAT DIAPHRAGM
• Purpose:
• Determination of Insulin based on the stimulation of glucose
uptake by the isolated diaphragm from rat .
• Procedure:
• Animal - Male Sprague Dawley (70-100 gm) Animal sacrificed
during anesthesia and diaphragm are carefully removed, spread
out and divided into two equal pieces Hemi diaphragm are
incubated in Krebs buffer solution with carbogen with 5µM glucose,
insulin or compound to be tested.
• After 30 min hemidiaphragm are blotted on tissue, grounded on
porcelin mortar pestle chilled with liquid nitrogen
• After 4 hour at -200C Sample centrifuged for 10 min.
• Evaluation
• The concentration dependent of glucose uptake and conversion into
glycogen and concentration of insulin or insulin mimetic compound
are determined.

46. CONCLUSION
• • For an animal model to have relevance to study of type 2
DM in
• humans, either the characteristics of the animal models
should
• mimick the pathophysiology and natural history of diabetes
or it
• should develop complications of diabetes similar to that in
humans.
• • No single animal model encompasses all these
characteristics and
• there are various models in use that mimick various
conditions as
• seen in humans.

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