Introduction to Screening models for Antiulcers Screening methods, Invivo methods, Invitro methods Presented by N. Ramya Department of Pharmacology

1. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 1
A Seminar as a part of curricular requirement
for I year M. Pharm I semester
Presented by
N. Ramya
(Reg. No. 20L81S0101)
Department of Pharmacology
Under the guidance/Mentorship of
Dr. K. SOMASEKHAR REDDY M.Pharm, Ph.D,
Associate Professor And
Head Dept. of Pharmacology
Screening models for antiulcers

2. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 2
Contents
• Screening methods
• Invivo methods
• Invitro methods
• References

3. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 3
SCREENING METHODS
IN VIVO METHODS IN VITRO METHODS
• Pylorus Ligation in Rats
• Stress ulcer Model
oRestraint- induced ulcers
oCold water immersion induced ulcer
• Histamine-induced Gastric Ulcer
• Ethanol-induced mucosal damage
• NSAIDs-induced gastric lesions
• Acetic Acid-induced gastric Ulcer
• Dimaprit-induced Duodenal Ulcer
• [I125] Gastrin Binding Assay
• Tiotidine Binding Assay
• H+ / K+-ATPase Inhibition
Assay

4. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 4
PYLORUS LIGATION IN RATS
Principle:
 Pylorus is ligated over a certain period of time
 Accumulation of gastric acid causes ulceration
Procedure:
• Wistar rats weighing 150-200 grams
• Fasting : 48 hours ; water ad libitum.
• Housed singly in cages with raised bottoms of wide wire mesh.
• Under anaesthesia, a one-inch midline abdominal incision is given below the
xiphoid process.
• Pylorus is ligated without damaging its blood supply.
• Stomach is replaced and abdominal wall closed with sutures.
• Test compounds are given either orally or injected s.c.
• About 17-19 hours after pyloric ligation, rats are sacrificed and stomachs are
dissected out.

5. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 5
• About 17-19 hours after pyloric ligation, rats are sacrificed and
stomachs are dissected out.
• Contents of the stomach are drained into a graduated centrifuge tube and
subjected to analysis for volume, pH, free and total acidity, mucin,
prostaglandin, total carbohydrate:protein ratio etc.
• Stomach is opened along the greater curvature, pinned on a cork plate.
• Its inner surface is examined for ulceration with a binocular microscope.
• The ulcer index is calculated and the Ulcer severity graded.
EVALUATION OF THE TEST
• Ulcer severity
 0 = No ulcer
 1 = Superficial ulcer
 2 = Deep ulcer
 3 = Perforation

6. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 6
Stress Ulcer Model
1)Restraint- induced ulcers
Principle
Stress plays a significant role in the pathogenesis of gastric ulcers.
Procedure
• Albino rats weighing 150-200 grams are taken
• Fasted for 36 hours before experiment
• Drug is administered orally or subcutaneously
• 30 min later animals are subjected to restraint
• For restraint, the rats were placed in a piece of galvanized steel window screen
of appropriate size.
• Screen was moulded around the animal and held in place with wire staples.
• To restrain the rats, the limbs were put together in pair and tightened with
adhesive tape.
• Rats were kept under restraint for 24 hours
• Rats were then sacrificed & their stomachs dissected out.
• Ulcer index and ulcer severity were determined.

7. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 7
Cold water immersion induced ulcer
Principle
• Exposure of cold conditions to restrained animals accelerates the occurrence
of gastric ulcers.
• Shortens the immobilization time.
Procedure
• Wistar rats weighing 150-200 grams are used.
• After fasting the animals for 16 hours, the test sample is administered orally.
• Rats are then placed individually in restraint cages vertically, and then
immersed in water upto the xiphoid process, at 22°C for 1 hour.
• Then rats are removed from the cages, dried and Evan’s blue injected i.v. via
the tail vein, 10min later, they are sacrificed
• The stomach is removed & ligated at both ends
• It is filled with Formol saline & kept overnight
• On the next day, the stomach is opened along the greater curvature and
examined for ulcerative lesions

8. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 8
Histamine-induced Gastric Ulcer
Principle
• Gastric acid secretion is increased when histamine is administered
intraperitoneally.
Procedure
• Guinea pig weighing 300-400 grams are taken
• Fasted for 36 hours before experiment; water ad libitum
• 1 ml of histamine acid phosphate (50 mg base) was administered i.p.
• Promethazine hydrochloride 5 mg was injected i.p. 15 min before and 15
min after histamine to protect the animals against histamine toxicity.
• The standard/test drugs were administered p.o. or s.c. 45 minutes before
histamine injection.
• 4 hours after histamine injection, guinea pigs were sacrificed and stomach
dissected out.
• The gastric contents were subjected to analysis
• Stomach was opened along the greater curvature, ulcers were identified.

9. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 9
ULCER SCORING
Type 0 : No visible ulcers
Type 1 : 10 or less small ulcers, 1-3 mm in diameter
Type 2 : 11 or more ulcers, 1-3 mm in diameter
Type 3 : 1 or more ulcers, 4-6 mm in diameter
Type 4 : 1 or more ulcers, 7 mm or more in diameter
Type 5 : Perforation of the gastric wall

10. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 10
Ethanol-induced Mucosal damage
Principle
• Ethanol, being a necrotizing agent, damages the superficial epithelial layers &
inhibits the release of mucosal prostaglandins.
Procedure
• Wistar rats weighing 150-200 grams are taken
• Fasted for 18 hours before experiment; water ad libitum.
• Rats are given test drugs or standard drug orally.
• 30 mins later 1 ml/200gm of 99.80% alcohol is administered orally.
• After 1 hour, Rats are sacrificed and stomachs dissected out.
• Severity score and ulcer index are calculated

11. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 11
Indomethacin-induced gastric lesions
Principle
• NSAID induced gastric damage ; by blocking COX enzyme, endogenous
prostaglandin production inhibited.
Procedure
• Rats fasted for 36 hours before Indomethacin administration (20 mg/kg,
orally)
• 30 min prior to the administration of the Indomethacin, standard/test drug is
administered.
• 1 hour after Indomethacin administration, Rats are sacrificed, their stomach
dissected out and examined for the number of lesions under the microscope.
Ulcer index and ulcer severity are determined.

12. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 12
Acetic Acid-induced gastric Ulcer
• A model for inducing chronic gastric ulcer in rats by means of submucosal
injection of acetic acid.
• New method which involves temporary instillation of acetic acid solution.
Principle
• Acetic acid enhances the ulceration in stomach by increasing the acidity of
stomach contents.

13. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 13
Procedure
• Wistar rats weighing 150-200 grams are taken
• Fasted for 24 hours before experiment.
• Pentobarbital anaesthesia
• A cylindrical glass tube of 6 mm diameter : tightly placed upon the
anterior serosal surface of stomach 1 cm away from the pyloric end.
• 50% Acetic acid (0.06 ml per animal) was instilled into the tube and
allowed to remain for 1 minute on the gastric wall .
• After removal of Acetic acid solution, the abdomen was closed.
• Animals were caged and fed normally.
• Test drugs were given orally on Day 1 twice daily, 4 hours after
application of acetic acid and continued upto 10 days after induction of
ulcer
• Animals were sacrificed after 18 hours of the last dose to assess ulcer size
and healing.
• Ulcer index and Severity score calculated.

14. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 14
Dimaprit-induced Duodenal Ulcer
Principle:
• H2 receptor agonist
• Induced gastric erosion in rats after single i.v. dose.
• Duodenal ulcer in guinea pigs after repeated s.c. dose.
Procedure:
• Wistar rats weighing 150-200 g or guinea pigs 250-300 g are taken.
• Fasted for 24 hours before experiment; free access to water
• Test drug or standard drug is given orally 60 min before injecting Dimaprit in
rats and 30 min before injecting it in guinea pig.
• Dimaprit is given in a dose 100 mg/kg i.v. in rats and 2 mg/kg s.c. every hour
for 6 hours in guinea pig.
• After 1 hour of Dimaprit injection, Animal is sacrificed and stomach dissected
out.
• Stomach is opened along the greater curvature and examined for ulceration.

15. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 15
In-vitro methods
[125I]Gastrin binding assay
Principle:
• Gastrin ( G cells of gastric antrum)
 Bind to CCK2 receptors on parietal cells release HCl
 Bind to CCK2 receptors on ECL cells
 Histamine act on H2 receptors of parietal cells release HCl
• Compounds with gastrin receptor antagonistic activity --> can be potential
antiulcer agents

16. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 16
Procedure:
• Fundic gland suspension (Guinea pig stomach)
• Incubated with 50μl [125I] Gastrin
1)In buffer alone (for total binding)
2) In presence of unlabeled gastrin (for non-specific binding)
3)In presence of test compound (for competition assay)
• For 90 minutes at 37°C
• Ice cold buffer, in Microcentrifuge tubes, is layered with incubated mixture
• Centrifuged for 5 minutes at 10,000 g
• Radioactivity is quantified in pellet after discarding the supernatant.
Evaluation:
• Total binding, non-specific binding and specific binding are determined.
• Percentage of specifically bound [125I] Gastrin displaced by a given
concentration of the test compound calculated.
• The higher the displaced [125 I] Gastrin , more is the gastrin antagonistic
activity of test compound.

17. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 17
Tiotidine Binding Assay
Assay is done using cerebral cortex homogenate obtained from
guinea pigs.
Procedure:
• Cerebral cortex homogenate is incubated with Tiotidine for 90 min at 40C in
the presence of Na2HPO4/ KH2PO4 buffer alone and Unlabeled Ranitidine
and buffer.
• Test compound in buffer (for competition assay)
• 5 ml of ice cold phosphate buffer is added to terminate the incubation.
• Subsequently reaction mixture is filtered under vacuum through glass fiber
filters that are presoaked with buffer
• Filters are then washed with 5ml of ice cold buffer twice.
• Radioactivity measured by liquid scintillation counting.
Evaluation:
 Specific binding is measured.
 Specific binding = Total binding – Non specific binding

18. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 18
H+/K+ - ATPase inhibition assay
H+/K+ - ATPase or proton pump are final step in the synthesis of
acid by parietal cells
Procedure:
• Homogenate of 80 ng Microsomal gastric H+/K+ - ATPase (pig gastric
mucosa) incubated with 100µl buffer, 1mM ATP and Test compound in
microtitre plate for 30 mins at 37°C
• Reaction is stopped by adding Malachite green (colorimetric agent)
• After 10 seconds, 15% sodium citrate is added for 45 minutes
• Release of orthophosphate from ATP quantified by colorimeter at 570 nm.
Evaluation
• Percentage inhibition of H+/K+ - ATPase is calculated.
• Lesser the orthophosphate released, more is the inhibition of H+/K+ - ATPase
by test compound.

19. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 19
References
Maity S, Chaudhuri T, Vedasiromani JR, Ganguly DK: Cytoprotection mediated
antiulcer effect of tea root extract. Indian J pharmacol 2003;35: 213-19.
Singh S: Evaluation of gastric antiulcer activity of fixed oil of ocimum
basilicum Linn. and its possible mechanism of action. Indian J Expl Biol 1999;
253-257.
Prabha T, Dora BM, Priyambada S, Debnath PK, Goel RK: Evaluation
of Pongamia pinata root extract on gastric ulcers and mucosal offensive and
defensive factors in rats. Indian J Expel Biol 2003; 304-10.

20. RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721 20