Screening of anti ulcer agents

SCREENING OF ANTI-
ULCER DRUGS
SUBMITTED TO-
MR. JASMIN KUBAVAT
ASSISTANT PROFESSOR
IRD, GFSU
SUBMITTED BY-
DEEPMALYA GHOSH
000RDMPFP1920007
DRUG DISCOVERY
2ND SEM, 1ST YEAR

DEFINITION OF ULCER
 Peptic ulcer is one of the most prevalent chronic gastrointestinal disorder.
 “A disruption of the mucosal integrity of the stomach and/or duodenum leading to a
local defect or excavation due to active inflammation.”

PATHOPHYSIOLOGY-
 Faulty dietary
habits
 Smoking
 Alcohol
 Drugs
 Hereditary factors
 H.Pylori
Cause of Ulcer

TREATMENT-
1. PHARMACOLOGICAL TREATMENT-
I. H2 receptor antagonist
II. Proton pump inhibitor
III. Mucosa Protective agents
IV. Anti H.Pylori drugs
2. NON-PHARMACOLOGICAL TREATMENT-
I. Avoid spicy food
II. Avoid xanthin containing beverage
III. Avoid alcohol
IV. Avoid smoking
V. Avoid heavy metals
VI. Avoid NSAIDs, corticosteroid, parasympathomimetics
VII. Encourage small frequently low caloric metals.

SCREENING OF ANTI ULCER
AGENTS

 IN VIVO METHODS-
1. Pylorus Ligation in Rats.
2. Stress ulcer Model
I. Through immobilization stress.
II. Cold water immersion induced
ulcer.
3. Histamine-induced Gastric Ulcer.
4. Ethanol-induced mucosal damage.
5. Indomethacin(NSAIDs)-induced
gastric lesions.
6. Subacute gastric ulcer in rat.
7. Gastric ischemia-reperfusion injury
in rats.
8. Acetic acid induced gastric ulcer.
 IN VITRO METHODS-
1. [I125] Gastrin Binding Assay.
2. Tiotidine BindingAssay.
3. H+ / K+-ATPase InhibitionAssay

PYLORUS LIGATION IN RATS
PRINCIPLE:
 Pylorus is ligated over a certain period of time.
 accumulation of acidic gastric juice in the stomach,cause ulcer.
PROCEDURE:
150–200 g Wistar rats are fasted for 48 h, only with water, libitum
They are housed singly in cage with raised bottoms of wide wire mesh to
avoid coprophagy
Under anaesthesia, a 1 inch midline abdominal incision is given below
xiphoid process

Pylorus is ligated without damaging its bloodsupply. Stomach is replaced and
abdominal wall closed with sutures.
Test compounds are given either orally or injected S.C.
The animals are placed in plastic cylinders with an inner diameter of 45 mm
being closed on both ends by wire mesh, after 17-19 h the animals are
sacrificed by CO2 anesthesia.
The stomach is removed, Contents of the stomach are drained into
a graduated centrifuge tube and subjected to analysis for , pH, free and total
acidity, mucin, prostaglandin, total carbohydrate: protein ratio etc.

Stomach is opened along the greater curvature, pinned on a cork plate, inner
surface is examined for ulceration with a stereomicroscope.
The ulcer index is calculated and the Ulcer severity graded.
EVALUATION:
1.Ulcer severity- 0 = No ulcer
1 = Superficial ulcer
2 = Deep ulcer
3 = Perforation
2.Ulcer index (UI)-
UN = Average number of ulcers per animal, US = Average of severity scores,
UP = Percentage of animals with ulcers
UI = (UN + US + UP )× 10-1

 ADVANTAGE:
1.Evaluates anti-ulcerdrugs with various mechanisms of action and differentdoses.
 DISADVANTAGE:
1.The ulcers localized in antrum of thestomach.
 INFERENCE:
1. Ulcer index of test drug compared with control group to detect anti-ulcer effect of
test drug.
2.Other parameters help to infer the mechanism of ulcer protection-
1. Decrease in volume, free & total acidity: ant secretory action
2. Rise in pH: acid neutralizing action
3. Increase in mucin, PGs: cytoprotective effect.

STRESS ULCER THROUGH IMMOBILIZATION
STRESSPRICIPLE:
 Stress plays a significant role in the pathogenesis of gastric ulcers.
 This methods to study the effect of anti-ulcer drugs on immobilization stress in rats.
PROCEDURE:
Ten 150–170 g Wistar rats are used, before the experiment they are fasted for
24 h
The animals are anesthetized with ether, after oral or S.C. administration of
the test compound or the placebo solution
The animals are wrapped in wire gaze & both lower and upper extremities are
fixed together. They are suspended in the dark at 20 °C for 24 h and finally
sacrificed in CO2 anesthesia

Stomach is removed, pinned on a cork plate, ulceration examined with help of
stereomicroscope.
Calculated the ulcer index & the Ulcer severity graded.
EVALUATION:
2 = Deep ulcer
3 = Perforation
2.Ulcer index (UI)-
UI = (UN + US + UP )× 10-1

STRESS ULCER THROUGH 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 compound 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 & Evan’s blue(30mg/kg) injected
i.v. via the tail vein
After 10 min later, they are sacrificed in CO2 anesthesia
The stomach is removed & ligated at both ends, It’s injected with Formol
saline(2% v/v) & kept overnight
On the next day, the stomach is opened along the greater curvature and washed in
warm water, and examined under a 3-fold magnifier
The lengths of the longest diameters of the lesions are measured and summated
to give a total lesion score (in mm) for each animal & the mean count for each
group being calculated.

EVALUATION:
The mean score in control rats is about 25 (range 20–28). Inhibition of the
lesion production is expressed as percentage value.
CRITICAL ASSESSMENT OF THE METHOD:
Like other stress models, the test resembling the psychogenic factor for ulcer
disease in human beings, is used for final drug evaluation only.

HISTAMINE-INDUCED GASTRIC ULCER
PRINCIPLE:
 Gastric acid secretion is increased when histamine is administered
intraperitoneal.
PROCEDURE:
300-400 grams guinea pig are taken, fasted for 36 hours before experiment;
only give water & libitum
1 ml of histamine acid phosphate (50 mg base) was administered by i.p.
Promethazine hydrochloride 5 mg was injected i.p. 15 min before and after
15 min histamine to protect the animals against histamine toxicity.

45 minutes before histamine injection the standard/test drugs were administered
p.o. or s.c.
After 4 hours 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.

EVALUATION:
1.Ulcer scoring-
• Type 0 : No visible ulcers
• Type 1 : 10 or less small ulcers, 1-3 mm indiameter
• Type 2 : 11 or more ulcers, 1-3 mm in diameter
• Type 3 : 1 or more ulcers, 4-6 mm indiameter
• Type 4 : 1 or more ulcers, 7 mm or more in diameter
• Type 5 : Perforation of the gastricwall
ADVANTAGES:
1. Produces 100% gastric ulceration.
2. Increased volume of gastric acid secretion.
3. Marked enhancement of free and total acidity.

ETHANOL-INDUCED MUCOSAL DAMAGE
PRINCIPLE:
• Ethanol, being a necrotizing agent, damages the superficial epithelial layers &
inhibits the release of mucosal prostaglandins.
PROCEDURE:
250-300 grams Wister rats are taken & fasted for 18 hours before
experiment; only give water ad libitum
The drug is administered intragastrally 30 min prior to administration of 1
ml/200gm of 99.80% alcohol(Ethanol) and untreated animals are included as
controls
After 1 h administration of ethanol, the rats are scarified by CO2 anesthesia

Stomachs are removed, cut along the greater curvature, & rinsed under tap
water
Stomach are stretched on a piece of foam core mat, a circular full thickness area,
about 13 mm in diameter is cut
4 rows with six holes 13 mm in diameter is placed on a sheet of clear glass,
with smooth side up, and bound the glass with photographic tape, previously
cut portion is placed on hole
The light from the source is illuminated on the holes & the negatives from
photographic plate are taken by the camera

A light transmission densitometer is used to evaluate the negatives and optical
density is evaluated
Hemorrhagic or damaged areas appear bright on the negative, whereas
undamaged tissue appears dark
Damages are indicate at lower optical density values, while higher optical
densities are indicate the little or, damage less
EVALUATION:
1. The significance of differences in optical density between control and
ethanol-treated tissue is evaluated by non paired single-tail Student’s t-test.
2. The subjective scores of the treated tissues are recorded; the graded response
is reflecting the least to most damage.

1. Several prostaglandins provide cytoprotection, particularly in rats, in a dose-
range which has no anti-secretory activity.
2. However, clinical experience with prostaglandins showed that ulcer healing is
only achieved at anti-secretory doses.
3. Therefore, it seems very likely that the cytoprotective property of a compound
in rats has very limited relevance to prediction of its ulcer healing potential in
humans if cytoprotection is really separated from its anti-secretory potential.
ADVANTAGES :
1. Gastric lesions are observed after an hour of administration of ethanol.
2. Reproducible method to produce gastric lesions in experimental animals.

INDOMETHACIN(NSAIDS)-INDUCED GASTRIC
LESIONS
PRINCIPLE:
 NSAIDS like, indomethacin and acetyl-salicylic acid, induce gastric damage ; by
blocking cyclo-oxygenase(COX).
 This is due to formation of Prostacyclin, the predominant prostaglandin produced in
the gastric mucosa(loss of gastric mucosal defense).
PROCEDURE:
(Groups of 8–10) 150–200 g Wistar rats weighing are used & fasted for 36 hours
before Indomethacin administration
The test drugs are administered orally in 0.1% Tween 80 solution, 10 min before to
indomethacin (orally 20 mg/kg)
After 6 h, they are sacrificed in CO2 anesthesia

Stomachs removed, Formol-saline (2% v/v) is then injected into the totally
ligated stomachs for storage overnight
On the next day, the stomach is opened along the greater curvature and washed
in warm water, and examined for the number of lesions under a 3-fold
magnifier microscope
Ulcer index and ulcer severity are determined
EVALUATION:
1. The mean score in control rats is about 25 (range 20–28). Inhibition of the
lesion production is expressed as percentage value.
1. Like other stress models, the test resembling the psychogenic factor for ulcer
disease in human beings, is used for final drug evaluation only.

SUBACUTE GASTRIC ULCER IN RAT
PRINCIPLE:
 A method for producing standard subacute gastric ulcers in rats and for the
quantitative evaluation of the healing process.
PROCEDURE:
(Group of 8 – 15) 120–150 g Wistar rats are taken & fasted for 24 h only give
them water
The rats are anesthetized and orally inserted a polyethylene catheter
including a fine steel wire with a needle tip (1.2 mm diameter) at the lower
end into the stomach and the gastric wall
30 min or 24 h after puncture, test drug are administered orally, & free access
to food and water is provided from 2 h up to the end of the experiment

The rats are sacrificed by overdose of ether and stomach is dissected
Stomach opened along the lesser curvature than rinsed with water and fixed to the
end of a polyethylene pipette of 10 mm diameter in a position with the
punched ulcer in the center
The end of the pipette with the gastric wall is suspended in a beaker with
physiological saline
the pressure in the tube is gradually increased with a valve rubber ball
The tensile strength is measured using a tonometer where the bubble appears at
the gastric ulcerous wall, which is expressed as mm Hg.

EVALUATION :
1. The extent of the healing of gastric ulcers can be characterized by the healing
rate (HR) according the following equation:
Where,
A = tensile strength (mm Hg) at C time-point after puncture
B = tensile strength 30 min after puncture (the average value is 143 mm Hg)
C = time course (h) of the experiment
HR = (A – B) / C (mmHg/h))

GASTRIC ISCHEMIA-REPERFUSION INJURY IN
RATSPRINCIPLE:
 Endothelin-1 has potent ulcer genic effects in the stomach.
 Endogenous endothelin-1 has been implicated for ethanol-, indomethacin- &
hemorrhagic shock induced gastric ischemia-reperfusion injuries.
PROCEDURE :
(Group of 5) 200–250 g Wistar rats are fasted for 24 h, give water than rats are
anesthetized with urethane i.p. (1.5 g/kg)
The test/standard is given to all rats, then stomach is dissected and instilled with
0.15 M HCl
The left gastric artery is clamped by a small vascular clamp for 5 min to induce
ischemia and 30 min of reperfusion is done by releasing the clamp

The rats are sacrificed by cervical dislocation & stomach is fixed with 10%
buffered formalin and photographed for histological evaluation of injuries
with Planimeter
Each histological section is stained with hematoxylin/eosin and examined under
light microscope
EVALUATION:
1. An one cm length of each histological section is assessed by following scores
:
1 = Epithelial damage,
2 = Glandular disruption, Vasocongestion or edema in the upper mucosa
3 = Hemorrhagic damage in the mid to lower mucosa
4 = Deep necrosis and ulceration.
2. Data are expressed as mean ±SEM.

ACETIC ACID INDUCED GASTRIC ULCER
PRINCIPLE:
 Acetic acid enhances the ulceration in stomach by increasing the acidity of
stomach contents.
PROCEDURE:
150-200 grams Wistar rats are fasted for 24 hours before experiment
Rats are anesthesia with Pentobarbital
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 & allowed to
remain for 1 minute on the gastric wall

After removal of Acetic acid solution, the abdomen was closed
Test drugs were given orally on Day 1 twice daily, 4 hours after application
of acetic acid & continued up to 10 days after induction of ulcer
After 18 hours of the last dose rats were sacrificed to assess
ulcer size and healing.
Ulcer index and Severity score calculated

EVALUATION:
2 = Deep ulcer
3 = Perforation
2.Ulcer index (UI)-

ADVANTAGES:
1. Simple procedure: resulting in ulcers of consistent size and severity at an
incidence of 100%.
2. Resemble human ulcers in terms of both pathological features and healing
mechanisms.
3. Relapse of healed ulcers is frequently observed, just as in peptic ulcer
patients.
DISADVANTAGES:
1. Submucosal injection produced ulcers penetrating entire gastric wall &
adherence of ulcer base to adjacent organs (mainly Liver).

[I125] GASTRIN BINDING ASSAY
PRINCIPLE:
Gastrin ( G cells of gastric antrum) MOA-
GASTRIN
BIND TO CCK2 RECEPTOR ON
PARENTAL CELLS
RELEASE
HCl
GASTRIN
BIND TO CCK2 RECEPTOR ON
ECL CELLS
HISTAMIN
RELEASE
ACT ON H2 RECEPTOR OF
PARENTAL CELL
RELEAS
E HCl

PROCEDURE:
Fundic gland suspension (Guinea pig stomach)
For 90 minutes at 37°C incubated with 50μl [125I] Gastrin with-
a. In buffer alone (for total binding)
b. In presence of unlabeled gastrin (for non-specific binding)
c. In presence of test compound (for competition assay)
In Micro centrifuge tubes, ice cold buffer is layered with incubated mixture
Mixture centrifuged for 5 minutes at 10,000 g
Radioactivity is quantified in pellet after discarding the supernatant

EVALUATION:
1. Total binding, non-specific binding and specific binding are determined.
2. Percentage of specifically bound [125I] Gastrin displaced by a given
concentration of the test compound calculated.
3. The higher the displaced [125I] Gastrin, more is the gastrin antagonistic
activity of test compound.

TIOTIDINE BINDING ASSAY
PRINCIPLE:
 H2 receptor blocker.
 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:
a.Na2HPO4/ KH2PO4 buffer alone (to determine total binding)
b. Unlabelled Ranitidine and buffer (to determine non-specific binding)
c. Test compound in buffer (for competition assay)

5 ml of ice cold phosphate buffer is added to mixture 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:
1. Specific binding is measured.
2. Specific binding = Total binding – Non specific binding

H+/K+ - ATPASE INHIBITION ASSAY
PRINCIPLE:
 H+/K+ - ATPase or proton pump, 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 microlitre
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:
1. Percentage inhibition of H+/K+ - ATPase is calculated.
2. Lesser the orthophosphate released, more is the inhibition of H+/K+ -
ATPase by test compound.

Screening of anti ulcer agents

Screening of anti ulcer agents

Screening of anti ulcer agents

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