Shriyansh Shrivastav ,M.Pharm (Department of Pharmacology) , Indo Soviet Friendship college of Pharmacy, Moga ,Punjab

1. PRECLINICAL SCREENING OF ANTIULCER
PRESENTED BY
SHRIYANSH SRIVASTAV
M.PHARM 1ST YEAR
DEPARTMENT OF PHARMACOLOGY
SUBMITTED TO
Dr. Shamsher Singh (HOD)
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2. Contents
 INTRODUCTION
 SYMPTOMS OF PEPTIC ULCER
 PATHOPHYSIOLOGY
 CLASSIFICATION
 SCREENING METHODS
 IN VIVO METHODS
 IN VITRO METHODS
 REFERENCE
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3. PEPTIC ULCER DISEASE
 Peptic ulcer disease arises from the imbalance between defensive factor( mucus,bicarbonate and
mucosal blood flow) and aggressive factor ( acid,pepsin,NSAID,Helicobacter pylori)
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4. Contd….
 On average, patients with duodenal ulcers produce more acid than the control subjects
particularly at night (basal secretion)
 Injurious factors (acid and pepsin)
 Although, patients with gastric ulcers have normal or even diminished acid production,
ulcers rarely if ever occur in the complete absence of acid.
 A complex interaction between
H. pylori, and
NSAIDS
is also a cause of peptic ulcer.
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5. Symptoms of peptic ulcer
 Burning abdominal pain
 Nausea
 Vomiting
 Pain in stomach
 Weight loss
 Constipation
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6. Pathophysiology
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7. Classification
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8. Continue…
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9. Screening methods
In vitro method
1) H+/K+ ATPase
inhibition assay
In vivo method
1) Pylorus ligation in rats (Shay rat)
2) Stress ulcer model
3) Histamine induced gastric ulcer
4) Indomethacin induced ulcer in rats
5)Gastric Ischemia Reperfusion injury
in rat
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10. In vitro method
1) H+/K+ATPase inhibition assay
• Proton pump > final step in the synthesis of acid by parietal cell
Procedure -:
Incubation of guinea pig gastric mucosa
buffer ATP and test compound
Add malachite green ( colorimetric agent )
after 30 minutes
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11. Contd….
Add 15% sodium citrate after 10sec.for 45 minutes
Released of orthophosphate from ATP quantified by
colorimeter at 570 nm
Evaluation
• Percentage inhibition of H+/K+ ATPase is calculated.
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12. In vivo method
1) Pylorus ligation in rats ( shay rat)
Purpose:
A simple and reliable method for production of gastric ulceration in the rat based on
ligation of the pylorus.
The ulceration is caused by accumulation of the acidic gastric juice in the stomach.
Procedure
Female Wistar rats weighing 150-170 g are starved for 48 h having access to drinking water ad
libitum.
During this time they are housed single in cages with raised bottoms of wide wire mesh in order to
avoid cannibalism and coprophagy
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13. Contd…
 Ten animals are used per dose and as controls.
 Under ether anesthesia a midline abdominal incision is made.
 The pylorus is ligated, care being exercised that neither damage to the blood
supply nor traction on the pylorus occurs.
 Grasping the stomach with instruments is to be meticulously avoided, else
ulceration will invariably develop at such points.
 The abdominal wall is closed by sutures. The test compounds are given either
orally by gavage or injected subcutaneously
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14. Contd…
 The animals are placed for 19h in plastic cylinders (inner diameter of 45mm) being closed
on both ends by wire mesh.
 Afterwards, the animals are sacrificed in CO2 anesthesia.
 The abdomen is opened and a ligature is placed around the oesophagus close to the
diaphragm.
 The stomach is removed and the contents are drained in a centrifuge tube.
 Along the greater curvature, the stomach is opened and pinned on a cork plate.
 The mucosa is examined with a stereo microscope.
 In the rat, the upper two fifths of the stomach form the rumen with squamous epithelium
and possess little protective mechanism against the corrosive action of gastric juice.
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15. Contd…
 Below a limiting ridge, in the glandular portion of the stomach, the protective mechanisms are
better in the mucosa of the medium two fifths of the stomach than in the lowest part, forming
the antrum.
 Therefore, lesions occur mainly in the rumen and in the antrum.
 The number of ulcers is noted and the severity recorded with the following scores:
0 = no ulcer
1 = superficial ulcers
2 = deep ulcers
3 = perforation
 The volume of the gastric content is measured.
 After centrifugation, acidity is determined by titration with 0.1N NaOH.
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16. Evaluation
 An ulcer index UI is calculated:
UI = UN + US + UP x 10-1
UN = average number of ulcers per animal
US = average of severity score
UP = percentage of animals with ulcers
 Ulcer index and acidity of the gastric content of treated animals are
compared with controls.
 Using various doses, dose response curves can be established for ulcer
formation and gastric acid secretion.
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17.  ID50 values can be calculated by probit analysis, whereby 0% corresponds to no
and 100% to maximal stimulated gastric acid output.
 Special remark
The “Shay-rat” has proven to be a valuable tool to evaluate anti-ulcer drugs with
with various mechanisms of action.
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18. 2. STRESS ULCER via
IMMOBILIZATION STRESS
 PURPOSE AND RATIONALE
 Psychogenic factors, such as stress, play a major role in the pathogenesis of
gastric ulcers in humans.
 The first report of the use of restraint as stress factor came in 1936 from
Selye.
 The precise methodologies to study the effect of anti-ulcers drugs on
immobilization stress in rats were described later by Hanson and Brodie in
1960 and Bonfils et. al in 1966.
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19. Contd…
 PROCEDURE
 Groups of 10 female Wistar rats per dose of test drug and for controls
weighing 150-170 g are used.
 Food and water are withdrawn 24 h before the experiment.
 After oral or subcutaneous administration of the test compound or the
placebo the animals are slightly anesthetized with ether.
 Both lower and upper extremities are fixed together and the animals are
wrapped in wire gaze.
 They are horizontally suspended in the dark at 200C for 24 h and finally
sacrificed in CO2 anesthesia.
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20. Contd…
ADVANTAGES OF THE METHOD
 The experimental model resembles the psychogenic factors in the pathogenesis of
gastric ulcers in patients.
 Therefore, it is not surprising that not only antacids, anticholinergics, H2-antagonists,
proton-pump-inhibitors, but also psychotropic drugs, like neuroleptics have been
found to be effective in this test.
 The test is being used in final drug evaluation only.
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21. 3 HISTAMINE INDUCED GASTRIC ULCER
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22. Continue…
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25. 4. INDOMETHACIN INDUCED ULCERS
IN RATS
 Nonsteroidal anti-inflammatory agents, like indomethacin and acetyl-
salicylic acid induce gastric lesions in humans and in experimental animals by
inhibition of gastric COX – I resulting in less formation of prostacyclin, the
predominant prostanoid produced in the gastric mucosa.
 Groups of 8-10 Wistar rats weighing 150-200g are used.
 The test drugs are administered orally in 0.1% Tween 80 solutions 10 min
prior to oral indomethacin in a dose of 20 mg/kg dissolved in 0.1% Tween 80
solutions.
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26.  Six hours later, the rats are sacrificed in CO2 anesthesia and their stomachs
removed.
 Formol saline (2% v/v) is then injected into the totally ligated stomachs for
storage overnight.
 The next day, the stomachs are opened along the greater curvature, then
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.
 5 (ranThe mean score in control rats is about 2ge 20-28).
 Inhibition of the lesion production is expressed as percentage value.
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27.  Hassan et al. in 1997 described the effect of an endothelin converting enzyme
inhibitor on local gastric ischemia reperfusion injury in rats.
 Endothelin-1 has potent ulcerogenic effects in the stomach.
 Endogenous endothelin-1 has been implicated for ethanol, indomethacin and
hemorrhagic shock induced gastric ischemia reperfusion injuries
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5. GASTRIC ISCHEMIA REPERFUSION
INJURY IN RATS

28.  Male Wistar weighing 200-250 g are fasted for 24 h with free access to water.
 The rats are anesthetized with 1.5 g/kg urethane i.p.
 The stomach is exposed by a medial laparatomy and instilled with 0.15M HCI
(l/100 g) via the forestomach.
 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.
 Pretreatment with test drug or standard is given to groups of 5 rats immediately
before the induction of ischemia.
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29.  At the end of the experiment, the rats are sacrificed by cervical dislocation.
 The stomach is fixed with 10% buffered formalin and photographed for
macroscopic evaluation of injuries.
 For the assessment of microscopic injuries a sample of corpus 0.5 cm below the
limiting ridge containing the entire width of the anterior wall is taken from each
stomach and processed for subsequent histological evaluation.
 A planimeter attached to a computer is used to trace the macroscopic mucosal
injury from color photographs.
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30.  The results are expressed as a percentage of the total glandular
mucosal area.
 Each histological section is stained with hematoxylin-eosin and
examined under light microscope.
 At one cm length of each histological section assessment is made
for;
 epithelial damage (score = 1)
 glandular disruption
 vasocongestion or edema in the upper mucosa (score = 2)
 hemorrhagic damage in the mid to lower mucosa (score = 3), and
 deep necrosis and ulceration (score = 4)
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31. Evaluation
 Each section is evaluated on a cumulative basis to give the histological index, the
maximum score thus being 10.
 Data are expressed as mean ± SME.
 Comparisons between different groups are made by one way analysis of variance
followed by Fisher’s least significant difference test.
 P values of 0.05 are considered as statistically significant
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32. Reference
 Vogels H.Gerhard of Drug Discovery and Evaluation Pharmacological Assay
Second edition Page No. 867-872.
 Isenberg JI, Flemstrom G. Physiologyand pathophysiology of gastro duodenal
bicarbonate secretion.Digestive Diseases. 1991; 23: 27-33.
 Harsh Mohan. Text book of pathology. New Delhi: Jaypee publishers; 2009
 Hojgaard L, Nielsen AM, Rune SJ.Peptic ulcer pathophysiology: Acid bicarbonate,
and mucosal function . Scandinavian Journal
 Slide Share.
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33. THANKS FOR LISTENING
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