Screening models for evaluation of anti ulcer activity

PHARMACOLOGICAL SCREENING
MODELS USED FOR EVALUATION OF
ANTIULCER AGENTS
SIVASWAROOP YARASI

INTRODUCTION: • A peptic ulcer is a group of disorders which
is responsible for the ulcer formation or
mucosal lesions formation in the
esophageal lining (swallowing pipe),
stomach or duodenum (the first part of the
small intestine).
• The small sores are formed due to the
imbalance between mucosal defensive
factors bicarbonate, mucin, prostaglandin,
nitric oxide, and other peptides and growth
factors) and injurious factors (pepsin,
Helicobacter pylori, NSAIDs gastric acid).

Common types of ulcers are:
• It is an open sore or discontinuation in the lining of mucosa exposed to acid and pepsin
secretion. As it is related to pepsin secretion, hence the name peptic ulcer. The peptic
ulcers are of two types i.e., gastric and duodenal ulcer.
• Types of peptic ulcer based on site:
• Type I: These type of gastric ulcer, have ulcer along the lesser curvature of stomach and
have normal or decreased gastric acid secretion.
• Type II: It includes two types of ulcers, one gastric and one duodenal. This type of ulcer
has normal or increased gastric acid secretion.
• Type III: Type- III ulcers are present on pre-pyloric region of stomach and have normal or
increased gastric acid secretion and are known as Pre-pylori ulcer.
• Type IV: Proximal gastro esophageal ulcer. These types of ulcers are located near the
gastro-esophageal region with gastric acid secretion rate normal or below normal.
• Type V: Anywhere.
Peptic ulcer:

• The esophageal ulcers are the ulcers occurring in the lower end of esophagus due to acid
reflux or GERD.
Esophageal ulcer:
• Bleeding ulcer is untreated peptic ulcer which has not been treated for long time.
Bleeding ulcer:
• The peptic ulcers that haven’t healed after three months of treatment are refractory
ulcers.
Refractory ulcer:
• This type of ulcer consist of group of lesions found in esophagus, stomach, or duodenum.
Stress ulcer:

SIGNS AND
SYMPTOMS OF
PEPTIC ULCER
• Abdominal discomfort and nausea,
• Bloating and abdominal fullness,
• Water brash,
• Hematemesis, Melena,
• Rarely acute peritonitis,
• Dark or black stools,
• Dry tongue,
• Weak or feeble pulse,
• Shortness of breath.

DEFENSIVE FACTORS AGGRESSIVE FACTORS
Adequate blood flow Reactive oxygen species
Secretion of PG, Mucin, Bicarbonate Increased secretion of HCl and Pepsin
Mucus bicarbonate layer Inadequate dietary habits
Cellular regeneration Free oxygen radicals
Mucosal barrier Consumption of NSAID’s,
Surface mucus secretion Alcohol
Secretion of NO Stress, Anxiety
Cytokines, VEGF Infection with H. Pylori, Bile Salts10
Nutritional deficiency, Cortecosteroid use,
Tobacco, Oxidative Stress, Increased lipid
peroxidation, Neutrophil infiltration

SCREENING OF ANTI
ULCER ACTIVITY
IN VIVO & IN VITRO METHODS

Introduction
• All in vivo animal models can be used to investigate the preventive or curative
properties of medicines or drugs depending on the time of induction of the peptic
ulcer.
• For preventive studies, it is advisable to pretreat animals for at least two weeks before
the ulcerogen is administered to induce the peptic ulcer, after which measurement of
the degree of ulcers is taken with the appropriate index to determine the extent of
ulcer prevention achieved.
• In the case of healing or curative models, the ulcers are induced after which the
animals are treated for at least two weeks followed by measurement of the extent of
ulcers with the appropriate index to determine the degree of healing of the ulcers

• In employing these models, it is advisable to decide on an appropriate experimental
design.
• Nyarko et al. recommend that, for in vivo studies, animals should be placed in a
minimum of seven groups of at least six animals in each group.
• Generally, Group 1 is designated as negative control (where animals treated with
ulcerogen are not treated with any reference drug or test material such as plant
extract except for water).
• Group 2 serves as positive controls (animals treated with ulcerogen are given a
reference drug).
• Group- 3 animals are normal controls (the animals are not treated with any
ulcerogen except with the vehicle used to prepare the test agent.
• In this design, animals in Group-4 to 7 treated with the ulcerogen are also treated
with different doses of the extract test material.

• In all experiments, it is important to keep the animals in cages with
raised floors of wire mesh to prevent coprophagy.
• In addition, for preventive models, it is advisable to compare the
potential drug or test material with cytoprotectant reference drugs such
as misoprostol and sucralfate that are known to prevent peptic ulcers.
• In the case of healing, or curative studies, the use of histamine receptor
antagonists such as cimetidine or ranitidine, and proton pump inhibitors
such as omeprazole, is recommended as reference drugs.
• A combination of proton-pump inhibitors with antibiotics such as
clarithromycin could be used when H. pylori is employed as an ulcerogen.

IN VIVO ANIMAL MODELS FOR
SCREENING
Gastric anti-ulcer activity
screening
• Pylorus ligation induced
gastric ulcers in rats (Shay
rat)
• Drug (NSAID ) induced gastric
ulcers in rats
• Ethanol induced gastric
ulcers in rats
• Acetic acid induced gastric
ulcers in rats
• Stress induced ulcers in rats
Duodenal anti-ulcer activity
screening
• Cysteamine induced
duodenal ulcer in rats
• Duclerozine induced
duodenal ulcer in rats
• Indomethacin and histamine
induced duodenal ulcer in
rats
• Histamine induced duodenal
ulcers in guinea pigs
Anti-secretory activity
screening
• Isolated whole stomach
preparation
• GOSH and SHILD perfused rat
stomach preparation

Gastric Anti-Ulcer Activity
Screening

Pylorus Ligation
Induced Ulcer
in Rats (Shay’s
Method)
It is the oldest
method or oldest
animal model for
gastric ulcer which
is developed by
Shay et al. in 1945.
One of the easiest
methods for the
production of
gastric ulceration in
the rat based on
ligature of the
pylorus.
The mucosal
lesions are caused
by the
accumulation of
acidic gastric juice
in the stomach.
These ulcers result
from autodigestion
of the gastric
mucosa leading to
a breakdown of the
gastric-mucosal
barrier.

• Basically an increase in acid-pepsin accumulation due to pylorus
obstruction may cause subsequent mucosal digestion.
• The model is useful for evaluating the effects of anti-secretory drugs
that reduce secretion of gastric aggressive factors such as acid and
pepsin.
• The model is also useful for assessing the cytoprotective effects of
drugs that increase secretion of mucus.

• Evaluates anti-ulcer drugs with various mechanisms of
action and different doses.
Advantage:
• The ulcers localized in antrum of the stomach
Disadvantage:

NSAIDs
Induced
Gastric
Mucosal
Damage
• Non-steroidal anti-inflammatory drugs (NSAIDs) are used for
the treatment of inflammation but somewhere these NSAIDs
also cause gastrointestinal disease like ulcer formation gastric
acid secretion.
• This phenomenon has been employed in the development of
NSAIDs-induced gastric ulcer models in rats.
• The model is important in investigating the potential
usefulness of anti-secretory and cytoprotective agents since
the underlying pathophysiology involves gastric acid secretion
and mucosal prostaglandin synthesis.
• It is the most commonly used ulcer model in antiulcer studies.
• The frequency of usage could be attributed to the fact that
NSAID induced peptic ulcers are the second most common
etiology of peptic ulcers aside those caused by Helicobacter
pylori.

The pathogenesis of NSAIDs-induced gastric ulceration:
Prostaglandins play a vital protective role via stimulating the secretion of bicarbonate and mucus, maintaining mucosal blood
flow and regulating mucosal cell turnover and repair.
NSAID blocks the activities of the cyclooxygenase enzymes (COX-1 and COX-2) hence leading to inhibition prostaglandin
synthesis.
This leads to reduced mucus and bicarbonate secretion, decreased mucosal blood flow, impaired platelet aggregation,
alteration of microvascular structures leading to epithelia damage, reduced angiogenesis, and increased leukocyte adherence.
Increased production of reactive oxygen species (ROS), increased lipid peroxidation, and neutrophil infiltration also play a role in
oxidative mucosal damage by NSAIDs.

NSAIDs also inhibit gastric peroxidases and may increase mucosal hydrogen peroxide and hydroxyl ion levels that
will cause oxidative mucosal damage.
NSAIDs, particularly those of acidic nature, can directly kill epithelial cells.
• Various mechanisms have been proposed for this cytotoxic action of NSAIDs, including the induction of osmotic lysis subsequent to
trapping of charged NSAIDs with the epithelial cells and death of the epithelial cell subsequent to uncoupling of oxidative phosphorylation.
NSAIDs can also reduce mucus and bicarbonate secretion, thus decreasing the effectiveness of the juxta
mucosal pH gradient in protecting the epithelium.
Furthermore, NSAIDs disrupt the layer of surface-active phospholipids on the mucosal surface, independent of
effects on prostaglandin synthesis. Such an action would render the mucosa less able to resist damage induced by
luminal acid.

• This method gives an effective result in the detection of the effect of the antiulcer drug
on gastric blood flow.
• It is a suitable method for the detection of the effect of COX inhibitors on leukocyte
adherence.
Pros:
• This model is that inhibition of both COX-1 and COX-2 is required for NSAIDS – induced
gastric injury in rats because neither a COX-1 nor a COX-2 inhibitors cause
macroscopically or histologically detectable gastric damage when given individually in
most of the cases.
Cons:

Ethanol-induced Mucosal Damage in Rats
• Ethanol is considered a risk factor for developing gastric ulcers.
• It readily penetrates the gastric mucosa due to its ability to solubilize
the protective mucous and expose the mucosa to the proteolytic and
hydrolytic actions of hydrochloric acid and pepsin, causing damage to
the membrane.
• Moreover, alcohol stimulates acid secretion and reduces blood flow
leading to microvascular injuries, through disruption of the vascular
endothelium and facilitating vascular permeability; it also increases
activity of xanthine oxidase.
• Ethanol also triggers imbalances in cellular antioxidant processes.

Ethanol also produces necrotic lesions in the gastric mucosa of animals by a direct toxic effect
thereby reducing the secretion of bicarbonates and depleting gastric mucus production in animals
Furthermore, ethanol-induced membrane damage is associated with increased permeability of
the plasma membrane by sodium and water.
It also produces massive intracellular accumulation of calcium, which represents a major step in
the pathogenesis of gastric mucosal injury.
This leads to cell death and exfoliation in the surface epithelium.
Also, ethanol-induced ulceration is linked to reduced mucosa microcirculation and to increased
apoptosis.

The model is independent of gastric acid secretion and resembles acute peptic ulcers in
humans
As a model, ethanol-induced ulcer may not be appropriate or useful for the assessment of the
usefulness of antisecretory drugs or testing materials due to the absence of gastric acid
secretion where acid secretion underlies the development of the ulcer.
Instead, the ethanol induced ulcer model is useful for studying the efficacy of potential drugs
or testing agents that have cytoprotective and/or antioxidant activities.
Advantages:
• Gastric lesions are observed within an hour of administration of ethanol.
• Reproducible method to produce gastric lesions in experimental animals.

Evaluation: The significance of difference in optical densities between control and ethanol treated is
evaluated by non-paired single tail test.

Acetic Acid-
induced
Gastric Ulcer
in Rats
• One of the least understood aspects of peptic ulcer is the chronicity
of the disease that is characterized by repeated episodes of healing
and re-exacerbation, a phenomenon which is a challenge to both
patients and physicians.
• Most experimental ulcerative lesions heal quickly in a few days
without scar formation and do not reulcerate spontaneously.
• Takagi et al. developed a model for inducing chronic gastric ulcer in
rats by means of submucosal injection of acetic acid and reported on
the healing process of lesions for extended intervals after the ulcer
preparation.
• The experimental gastric ulcer was considered as chronic due to its
persistence for a long time and resemblance to human chronic ulcer
both grossly and histologically.

A new method was developed by Okabe and Pfeiffer, which involves intraluminal application of
acetic acid solution.
The model has become well established and is now used throughout the world by basic and
clinical scientists.
The acetic acid model is suitable for chronic peptic ulcers.
It is used to evaluate the effect of potential drugs or to test materials on the healing process of
chronic peptic ulcers and also to screen for their antisecretory and cytoprotective effects.
The model easily and reliably produces round, deep ulcers in the stomach and duodenum of
mice, rats, Mongolian gerbils, guinea pigs, cats, dogs, miniature pigs, and monkeys.

• The main pros of this model are that it is a simple procedure resulting in ulcers of
consistent size and severity at an incidence of 100%.
• Resembles human ulcers in terms of both pathological features and healing
mechanisms
• Relapse of healed ulcers are frequently observed, just as in peptic ulcer patients.
PROS
• Submucosal injection produced ulcers penetrating entire gastric wall & adherence
of ulcer based on adjacent organs (mainly liver) and it is considered as a pitfall of
this model.
CONS

Stress
Induced
Models:
• Psychogenic factors like stress, play a very important role in the
pathogenesis of gastric ulcers.
• Stress-induced ulcers manifest as single or multiple mucosal defects.
• This type of ulcer is commonly seen in very critically ill patients in the
intensive care unit and sometimes it also causes gastrointestinal
bleeding which is linked with a highly increased morbidity and
mortality despite aggressive acid suppression treatment.
• Stress ulceration is caused due to stress and the conditions of stress
depend upon various factors such as psychological factors, emotional
factors, stressful events like burns, trauma, surgery etc.
• The pathophysiology of stress-induced ulcers is complex.

There are four stress ulcer
methods available for in-
vivo studies

Restrain Induced
Ulcer model
• This method has been successfully used for studying
curling’s ulcers and the healing of ulcers as well.
• In a series of similar investigations Brodie and coworkers
systemically examined the restraint technique and
popularized its use.
• Disadvantages were still present, including the fact that the
lesions did not penetrate the musclaris mucosa and as such
were not ulcers in true sense and the fact that the
technique appeared to be somewhat species specific.
• Hence the restraint method has been modified by various
workers in order to make it less time consuming, less
cumbersome and more reproducible.

Either sex
Divided
into
groups

Coldwater Immersion Induced Ulcer
In this model, gastric ulcers are induced by water-immersion stress or cold restraint stress in
rats or mice.
This model employs the restraint technique developed by Brodie and Hanson coupled with the
cold-water or ordinary-water immersion method by Levine.
Cooling of rats in water during the restraint period accelerates the occurrence of gastric
mucosal lesions formation and shortens the time of necessary immobilization.
Gastric ulcers induced by cold-water-restraint stress (CWRS) or cold-restraint stress (CRS) in rats
or mice are known to resemble human peptic ulcers, both grossly and histopathologically.

The model is widely used and is reported to be useful for assessing or studying the effects
of agents/medicines on the healing of ulcers in rats.
This model doesn’t require an extensive period of starvation prior to use.
Did not require that the animals be restraint for lengthy periods of time, restricted virtually
all the movements of animals without respiratory or circulatory trauma and rapidly
produced very high and very reliable degree of gastric glandular restraint ulcer in rodents.

Stress and NSAIDs Immersion Induced Ulcer
• Stress is responsible for excessive acid secretion while NSAIDs are also responsible for
gastric injury.
• If both are combined together, then their chances of gastric injury may increase also.
• It is assumed that oxygen species production promoted by NSAIDs induces gastric mucosal
apoptosis which is the main reason behind the gastric injury.
• NSAIDs anti-inflammatory drugs cause a gastric ulcer or gastrointestinal injury through both
topical as well as the systemic effect which is caused by inhibition of the cyclooxygenase
enzyme.
• Thus, prostaglandin formation is blocked which is responsible for the gastric injury.
• Non-steroidal antiinflammatory drugs (NSAIDs) such as indomethacin, aspirin and
ibuprofen are known to cause gastric ulcers, especially when abused.

Swimming
Stress Ulcer
As compared to the other stress methods, in swimming stress ulcer method,
more ulcer formation occurs in the animal because it is a more depressed
strain and a highly positive correlation is obtained between depression and
ulcer severity scores.
In this method, animals are forced to swim in deep water.
The individual animal is placed into a cylindrical chamber which is closed on
both sides.
Rats are forced to swim under 30 cm deep water at 23-28°C water so that
feet of the animals cannot touch the surface of the cylinder.
Minimum two swimming sessions were conducted and drug was
administered between these two periods

• During swimming stress, release of histamine causes increase in acid concentration the stomach
and produces an ulcer.
• The gastrointestinal motility increases due to the result of folds in the stomach which is caused
because of stress and when intestine comes in contact with acid, damage chances increases.
• The quality and quantity of mucus also decrease due to stress and cause damage in the mucosal
layer. It also decreases synthesis of the mucosal component.
• This model is useful for screening of antiulcer activity of not only anticholinergic, H2 antagonist
proton pump inhibitor but assessment of psychotropic drugs like neuroleptics (because stress is a
psychogenic factor) can be done by this method.

• Advantages of stress ulcer models:
• Technically simple
• Do not require anesthesia or surgery
• Lesions located in glandular region of stomach
• As psychogenic factors are involved in the pathogenesis of gastric ulcers,
psychotropic drugs could be evaluated

Duodenal anti-ulcer
activity screening

Cysteamine
Induced
Duodenal Ulcer
• A duodenal ulcer in rats induced by cysteamine HCl
was first described by Selye and Szabo in 1973.
• Cysteamine induced duodenal ulcer in rat has been
widely used as a model of peptic ulcer disease.
• This chemically induced ulcer resembles duodenal
ulcer in man with respect to its location,
histopathology and some aspects of pathophysiology.
• Although the mechanism involved in ulcer production
has not been fully elucidated, generally it is reported
that Cysteamine stimulates gastric acid secretion rate
and inhibits the alkaline mucus secretion from
Brunner’s glands in the proximal duodenum resulting
in the formation of duodenal ulcer.
Selye and Szabo (1973)

• Cysteamine also affects processes that increase gastric acid and pepsin
secretion in the gastric mucosa, with a decrease in defensive processes that
lower levels of bicarbonate, mucus, and epidermal growth factors.
• Studies have demonstrated that cysteamine also reduces somatostatin
bioavailability and markedly elevates serum gastrin levels, with an
associated increase in gastric acid secretion, a significant decrease in the
neutralization of acid in the proximal duodenum, decreased dopamine
levels in glandular stomach and duodenum, and inhibition of gastric
emptying and motility.

• It is also known that certain transcription factors (e.g., early growth
response factor-1, hypoxia-inducible factor-1) and their target genes
play a key role in the pathogenesis of cysteamine-induced duodenal
ulcers.
• There are two types of duodenal ulcers, namely, acute and chronic.
• Acute duodenal ulcers are produced experimentally in rats by a
single administration of cysteamine hydrochloride (400mg/kg
p.o.).
• In the case of chronic duodenal ulcer, cysteamine is administered
twice, first 400mg/kg (p.o.) at an interval of 4 hours and by adding
cysteamine-HCl to drinking water for a period of time.

• Ulcerogenesis is seen with one full dose of cysteamine.
• Easily reproducible
• Suitable method for studying the pathogenesis of duodenal ulcer
• An effective method for anti-ulcerogenic regimes detection.
PROS:
• The ulcers are located on the anterior wall, the walls frequently get perforated resulting in
peritonitis or penetrate into the liver.
• A small ulcer is usually present on the posterior wall of the duodenum which penetrates the
pancreas and results in unfavorable conditions.
CONS:

Dulcerozine
induced
ulcers
• Kurebayashi et al reported that acute perforating
duodenal ulcers can be produced in rats following
single oral administration of dulcerozine, compound
structurally related to non-steroidal anti-
inflammatory drugs such as Phenylbutazone and
others known to cause gastrointestinal damage in
man and animals.
• It has been proposed that prolonged gastric
hypersecretion might be an important factor
contributing to the pathogenesis of duodenal ulcer
in this species.
• The dulcerozine induced duodenal ulcers in rats is a
useful model for studying the pathogenesis of
duodenal ulcer and testing the anti ulcer drugs from
the practical and pathogenic standpoint.

• Because the lesions develop are analogous to the clinical disease with
respect to location.
• The factor producing the pathologic changes is similar in man and
animal used.
• The drug treatment and a surgical operation effective in animals could
be clinically useful.
• It is extremely simple to perform, the massive production is feasible
and the results are obtained within 18 hours.

Histamine-
induced
Gastric Ulcer
in Rats
• Developed by Barrett et al, 1955
• Histamine is one of the major transmitters which is
responsible for gastric acid secretions, thus on this basis,
histamine-induced gastric ulcer model is used for the
determination of the effect of the drug in case of excessive
gastric acid secretion as well as for the study of
antihistaminic drugs.
• Histamine does not only enhance gastric acid secretion,
but it also causes disturbances of the gastric mucosa,
microcirculation, abnormal motility, and reduction in
mucus production.

• The mechanism by which histamine induces gastric ulcers is
through its potent acid stimulating and vasodilating capability,
which leads to increased vascular permeability.
• Histamine released from mast cells and binds with receptors
present on the surface of parietal cells which leads to
activation of adenyl cyclase.
• This adenyl cyclase converts ATP into c-AMP. This conversion
enhances secretion of HCl from parietal cells
• Advantages:
• Produces 100% gastric ulceration
• Increased volume of gastric acid secretion
• Marked enhancement of free and total acidity.

Indomethacin + Histamine-induced Duodenal Ulcer
Described by Takeuchi et al. involves administering indomethacin and histamine to rats.
A single administration of indomethacin and subsequent dosing with histamine consistently
produce lesions at the opposite site of the mesenteric attached in the proximal duodenum of rats.
The development of duodenum lesions by indomethacin + histamine induction in rats is due to
both an increase in gastric secretion and an impairment of acid induced duodenal HCO3 secretion.
This method is one of the useful methods for the determination of antiulcer activity

• It is a simple procedure with a high incidence of ulcer formation and no mortality.
• This model for duodenal ulcers is useful for studying the pathogenesis of duodenal
ulcers and for screening anti-duodenal ulcer drugs or agents
• This model shows that both an increase in gastric acid secretion and an
impairment of HCO3 secretion are responsible for ulcer production.
PROS
• The submucosal injection produced ulcers penetrating entire gastric wall &
adherence of ulcer base to adjacent organs.
CONS

Reserpine-
Induced
Peptic Ulcer
• Reserpine-induced gastric ulceration has been attributed
to the degranulation of gastric mast cells consequent to
liberation of histamine, believed to be mediated by the
cholinergic system.
• Reserpine- induced gastric mucosal lesions were dose
dependent.
• The mucosal lesions which are formed by this method
healed spontaneously within 3 weeks.
• The morphological changes in gastric mucosa are very
similar to the destructive changes found in the mucosa of
human gastric ulcers

Pros:
• This model can be preferentially used for studying the acute as well as
chronic ulcers.
• This method is most suitable for analyzing the ulcer inhibition
mechanism of calcitonin.
Cons:
• This model are that the method is dose-dependent and acid dependent.

Anti-Secretory
Activity Screening

Isolated whole stomach preparation
Immature Wistar rats
weighing around 40 g are
taken.
Anaesthetized with 30
mg/kg pentobarbitone
intra-peritoneally.
The abdomen is opened
and esophagus is ligated
close to the stomach.
An incision is made in the
rumen of the stomach and
the contents are washed
out with warm Krebs-
Henseleit solution.
A second incision is made at
the pyloric sphincter and
polyethylene cannulas are
inserted and tied into the
stomach via these incisions.
The stomach is dissected
out & Place immediately
into 10 ml organ bath
containing Kerbs-Henseleit
solution at 37°C.
The lumen of the stomach
is perfused at a rate of
1ml/min with modified
Kerbs Henseleit solution
(without Na2Co3 and
KH2PO4) at 37°C.
The effluent perfusate from
the stomach is passed over
a micro dual electrode &
change in pH is converted
to a function of H+ ion
activity.
This method is used for evaluation of H2 – receptor antagonists.

The test compounds
or secretogogues are
added in a volume of
0.5 ml to the Kerb’s
Henseliet solution
bathing the serosal
surface of the
stomach.
After setting up the
stomach preparation,
the basal H+ output is
allowed to stabilize,
both under control
and treated.
Secretogogue
response is measured
by measuring the
amount of acid
secreted at peat
response above the
predicting basal level.
The rate of acid
secretion is measured
and expressed as (H+)
moles * 108 per
minute Both solutions
are gassed with
carbogen (95% O2 and
5% CO2)
Compare the test and
standard with that of
the control to access
the anti-ulcer activity
of the drug.

GOSH AND SHILD PERFUSED RAT
STOMACH PREPARATION
• PRINCIPLE:
• The anti-ulcer agent screening is done by continuous reading of acid
secretion in the rat, which is stimulated by histamine, acetylcholine and
gastrine and that acid secretions can be blocked by anti-ulcer drugs.
• PROCEDURE:
Male Sprague Dawley
or Wistar rats of
weighing 180 g are
taken and starved
overnight
Rats are randomly
grouped as four rats in
a group.
The animals are
anesthetized with 25%
urethane solution (0.6
ml/100 g, i.m.).
The body temp is artificially
stabilized by means of a heating
coil using a rectal thermometer
and also trachea is exposed and
cannulated artificial respiration.

The external jugular
vein is exposed and
cannulated with
polythene tubes
leveled at the tip.
The abdomen is opened
through midline
incision, and pyloric end
of the stomach is
cannulated.
A polythene tube is
passed down the
oesphagus and tied in
the cervical region.
The stomach is
washed out
thoroughly by passing
distilled water to the
tube.
Perfuse the stomach
with N/4000 sodium
hydroxide at a uniform
rate.
The concentration of
sodium hydroxide is
adjusted so that the
perfusate under basal
conditions has a pH.
The perfusate emerges
out bathes the micro
electrodes which are
directly connected to
the pH meter.
Changes in acid
secretion are accessed
by pH changes.
• The gastric secretion is stimulated by continuous i.v. infusion of 100
microgram/kg/hr of pentagastrin or 3 mg/kg/h of histamine hydrochloride or 30
microgram/kg/h of carbachol.
• The test is injected either prior or after the infusion and measure the inhibition of
acid secretion and compare the results with that of standard.

Measurement of Gastric Lesions

• Measurement of gastric ulcerations following their induction is done by first
dissecting the stomach along its greater curvature and fixing on a board or
transparent glass.
• Examination can be carried out macroscopically with a hand lens and by
tracing on a transparent paper after which the transparent paper is placed
onto a graph sheet and sizes of ulcers are measured.
• Examination can also be carried out microscopically using a light or scanning
microscope.
• The stomachs can also be scanned using a camera, and later the presence of
ulcers are quantified using computer-assisted image analysis programmes
such as Scion, Image J, EARP Image analysis software, or by any other
appropriate software.

• Several methods have been designed to assess the extent of ulcerations and
subsequently the calculation of an ulcer index as well as the protective
and/or curative ratios for the ulcers.
• In all of these ways of determining ulcer indices, attempts are made to find
a solution to the problem of incomplete quantification of gastric and
duodenal ulcers.

Method described by
Takagi and Okabe
• The following scores/ratings used to
evaluate the ulcer index as well as the
severity of gastric lesions:
Ulcer
index
Lesion property
0 No lesion
1 Mucosal oedema and petechiae
2 One to five small lesions (1-2mm),
3 More than five small lesions or one
intermediate lesion (3-4 mm),
4 Two to more intermediate lesions or one
gross lesion (>4 mm),
5 Perforated ulcers

The ulcer index, the
percentage protective ratio,
and the percentage curative
ratio are, respectively, given
by the following equation:

• The number of ulcers rather than the size of ulcers is given importance
for assessing ulcer severity.
• The approach may yield results that may be statistically correct in terms
of the number of ulcers but will be biologically irrelevant.
• Several researchers, through their studies, have tried to reduce the level
of biological incorrectness in ulcer quantification.

Method described by Ganguly:
• Total ulcerative area in relation to the total
area of each stomach is used in determining
the ulcer index.
• The relative area is used to assign the ulcer
index according to the scale shown
Relative area/mm2 Ulcer index
No ulcer 0
91-100 0.1
81-90 0.2
71-80 0.3
61-70 0.4
51-60 0.5
41-50 0.6
31-40 0.7
21-30 0.8
11-20 0.9
1-10 1.0
Perforation 1.0

This method although quiet old, would be better than others as it takes into
consideration the total area of the stomach in relation to the total area of the
ulceration in determining the ulcer index.
Although this procedure also overlooks the level of erosion histopathologically
in the quantification of ulcer.
Some studies report that the method described by Ganguly produces ulcer
indices that are close to being both biologically and statistically relevant.

Method described by
Dekanski et al.
The severity of the mucosal lesions can
also be assessed and the ulcer index is
scored as follows:
Ulcer index Lesion
0 No damage
1 Blood at the lumen
2 Pinpoint erosions
3 One to five small erosions <2 mm
4 More than five small erosions <2 mm
5 One to three large erosions >2 mm
6 More than three large erosions >2 mm

percentage protective
ratio, and the percentage
curative ratio are,
respectively, given by the
following equation:

Method used
is described
by Desai et al.
• In this method which is based on the intensity of lesions, ulcers are given
scores as follows:
Ulcer index Lesion
0 No ulcer
1 Superficial mucosal erosion
2 Deep ulcer or transmural necrosis
3 Perforated or penetrated ulcer

percentage protective
ratio, and the percentage
curative ratio are,
following
equations:

According to
method by
Nwafor et al.
• The scores are multiplied by
2 when the width of the
erosion is larger than 1 mm.
• Measures only the length of
the erosive ulcers without
considering the total area of
ulcerations in relation to the
total mucosal area.
Scores Observation of erosions
1 Small round hemorrhagic
erosion
2 Hemorrhagic erosion <1
mm
3 Hemorrhagic erosion of 2-
3mm
5 Hemorrhagic erosion >4mm
The observation of erosions and scores made as 1–5 as
follows:

The ulcer index, the percentage
protective ratio, and the
percentage curative ratio are,
following equation:

According to the
method by Kulkarni
• The ulcer index can be measured or registered using the following scores
involving the number and severity of ulcers:
Ulcer index Lesions
0.0 Normal colored stomach
0.5 Red coloration
1.0 Spot ulcers
1.5 Hemorrhagic streaks
2.0 Ulcers with area >3 but ≤5mm2
3.0 uUcers > 5mm2
where UI = ulcer index, UN = average number of ulcers per animal, US = average of severity score, and UP = percentage of
animals with ulcer.

The percentage protective ratio, and the percentage curative
ratio are, respectively, given by the following equation:

According to
the method by
Andrade et al.
• Made an attempt to improve
on the biological correctness
of ulcer indices factored into
their assessment the areas of
ulceration and categorized
them into levels I, II and III
based on size of the ulcers.
• Ulcers are classified as:
Ulcer Area
Level I ulcer area < 1 mm2
Level II ulcer area = 1–3mm2
Level III ulcer area > 3mm2
• The following parameters are determined:
• Ulcerative lesion index (ULI)
• Percentage protective ratio
• Percentage curative ratio

In vitro models
• H+/K+-ATPase Inhibition Assay
• Gastrin binding Assay
• Tiotidine Binding Assay
• Neutralization Effects of Prepared
Preparation on Artificial Gastric Acid
• Determination of the Duration of
Neutralization Capacity of Prepared
Preparation on Artificial Gastric Acid
• Using a Titration Method of Fordtran's
Model for the Determination of Neutralizing
Capacity In-vitro
• Acid Neutralizing Capacity

• These models are easy to use and less time consuming.
Pros:
• These models are that we can't totally depend upon the result which is obtained
from these models because there are so many laboratory conditions which can
affect the result of these models like if proper procedure is not followed during the
preparation of artificial gastric juice then it leads major errors in results.
Cons:

H+/K+-ATPase
Inhibition
Assay
• H+/K+-ATPase also called a proton pump is
responsible for acid secretion.
• In canaliculi of parietal cell, it exchanges
intracellular H+ with extracellular K+ in response
to the stimulation by histamine acetylcholine
and gastrin.
• H+/K+ - ATPase or proton pump is final step in
the synthesis of acid by parietal cells.

Lesser the orthophosphate released, more is the inhibition of H+/K+ - ATPase by test
compound.
Several H+/K+-ATPase inhibitors contain a sulfhydryl group.
At lower pH-values, the compounds are protonated and rearrange to a sulphenic
acid and a sulphenamide that reacts with sulfhydryl groups in the enzyme.
Therefore, the In-vitro assays are performed both at neutral and at acidic pH levels.

[125I] Gastrin
binding assay
• Gastrin is a major physiologic regulator of gastric acid
secretion and growth of the oxyntic mucosa.
• Biologically active radio labelled hormones may be used
to characterize and localize receptors for peptide
hormones.
• Gastrin-
• Bind to Cholecystokinin B receptor (CCK2) receptors
on parietal cells resulting in release of HCl
• Bind to CCK2 receptors on Enterochromaffin-like cells
(ECL) releases Histamine that act on H2 receptors of
parietal cells resulting in release of HCl.
• Compounds with gastrin receptor antagonistic activity
can be potential antiulcer agents

Procedure:
Fundic gland suspension
(Guinea pig stomach)
Incubated with 50μl [125I]
Gastrin-
•In buffer alone (for total binding)
•In presence of unlabeled gastrin
(for non-specific binding)
•In presence of test compound (for
competition assay)
Incubated 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.

Tiotidine Binding Assay
• Histamine H2-receptor binding can be determined using homogenates from
guinea pig cerebral cortex and 3H-tiotidine as labelled ligand.
• Procedure:
Cerebral cortex
homogenate is incubated
with Tiotidine for 90 min at
40C in the presence of :
•Na2HPO4/ KH2PO4 buffer alone - to
determine total binding
•Unlabeled Ranitidine and buffer - to
determine non-specific binding
•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

Neutralization
Effects of
Prepared
Preparation on
Artificial
Gastric Acid
• Acidity is one of the major gastrointestinal problems
which cause functional disorder like peptic ulcer and
ultimately effect mucosal layer of stomach.
• To the neutralization of this type of acidity, we use
various types of antacids either allopathic antacids
or herbal antacids and these antacids are
neutralizing gastric acids by reducing the gastric pH.
• For the assessment of such antiulcer drugs in the
laboratory, following In vitro methods have been
developed which are helpful to determine the
capacity and effect of the drug in peptic ulcer or
gastric ulcer or other diseases.

• In this method, acid is prepared in laboratory and pH is adjusted like pH - 1.2 - 2.5.
• For the preparation of artificial gastric juice, take NaCl (2 gram) and pepsin (3.2 mg) and
dissolve them in distilled water (500 ml).
• Then after that, hydrochloric acid (7.0 ml) is added and the sufficient amount of water is
added to increase the volume.
• Highly acidic condition damage mucosal layer and if this pH is not controlled in time then it is
responsible for the cause of the gastric ulcer.
• The test drug is added to this artificial acid to estimate its neutralization potential.
The plant extract or prepared preparation (drug) and
reference drug is added separately in artificial gastric juice at
pH 1.2 - 2.5 and then this mixture examined the neutralizing
effect on artificial gastric juice by titration method.
Excess acid is neutralized in titration time and we determined
that the exact amount of prepared preparation neutralizes
the artificial gastric juice.

Determination of
the Duration of
Neutralization
Capacity of
Prepared
Preparation on
Artificial Gastric
Acid
• The determination of the duration of neutralization
of gastric acid is very well explained in the vatier's
artificial stomach apparatus model.
• The apparatus consists of three parts, an artificial
stomach, a peristaltic pump for creating the
environment same as the human body and a pH
recording system for the determination of duration
neutralization of acid.
• In apparatus, the artificial stomach is made up of:
• Secretary flux,
• Reservoir, and
• Gastric emptying flux.

In the reservoir of
an artificial
stomach, a pH
meter is also
attached which is
helpful to
determine the
changes of pH.
Artificial gastric
juice at 3ml/min is
pumped out and 3
ml/min is again
added in a
reservoir at the
same time.
Continuous stirring
at 30 rpm is done
by the help of
magnetic
apparatus.
In the reservoir
portion of the
stomach 90 ml of
prepared test or
reference drug is
added in 100 ml of
gastric juice whose
pH is 1.2 at 37°C.

Using a Titration Method of Fordtran's Model for
the Determination of Neutralizing Capacity In-vitro:
• 250 ml beaker is taken and 90 ml of prepared preparation and reference drug in different beakers are
placed at 37°C.
• Continuous stirring is done with the help of magnetic stirrer at 30 rpm for creating stomach
movements or stomach environment.
• Then these (prepared test drug and standard reference) are titrated separately with artificial gastric
juice to the endpoint of pH 3.
• The consumed volume of artificial gastric juice for test is measured and compared with that of a
reference drug.
• Sodium bicarbonate or the combination of magnesium hydroxide and aluminium hydroxide are used
as reference.

• Acid Neutralizing Capacity
• The Acid Neutralizing Capacity (ANC) of a drug is the amount of acid that it can
neutralize by the drug.
• In laboratory acid, neutralizing capacity can be measured by a process known as
back titration.
• In this process, prepared preparation dissolves in the excess of artificial gastric
acid and then this acidic solution titrated against a known amount of base until
the endpoint is reached.
• The number of moles of acid neutralized is equalized to the difference between
the moles of base required and the moles of acid added for the back titration.

Moles of acid neutralized = moles of HCl added – moles of
NaOH required
= (Volume of HCl X Molarity of HCl) - (Volume of NaOH X
Molarity of NaOH)
Acid neutralizing capacity per gram of antacid = moles of HCl
neutralized grams of antacid

Other models which are also
employed in different studies:

Serotonin-
induced
gastric
ulcer:
• Serotonin ulcer, one of the chemically induced experimental gastric
ulcers, has been described by Wilhelmi and Hedinger and Veraguth.
• Since then it has been widely used for the investigation of the etiology of
peptic ulcer disease and as a tool in the search for new antiulcer drugs.
• Male rats of wister strain are used for the experiments.
• The animals are fasted for 24 h prior to the experiments, water being
provided.
• Serotonin creatinine sulphate is found to induce a moderate but evident
gastric lesion.
• In gross observation, gastric lesions are scarcely noticed at 0.5 h after
serotonin injection (ulcer index 1.2), but are obviously distinguishable at 1
hr (ulcer index 7.7) and reach maximum intensity at 4 h (ulcer index 15.2).
• The lesions are located mainly at the side of the greater curvature of the
corpus.
• The ulcer index decreases to 8.0 at 8 h and is maintained at this level upto
24 h after serotonin injection

Haemorrhagic
shock induced
gastric ulcers
• Bleeding acute gastric erosions have now become the second
most common cause of upper gastrointestinal haemorrhage.
• The increased use of alcohol, NSAIDs, as well as the increased
incidence of major trauma, burns and surgery, has resulted in
a great increase in the frequency of gastric erosions.
• The understanding of the pathogenesis of these erosions has
been hampered by the lack of a suitable experimental model.
• In order to investigate the relationship between haemorrhagic
shock and acute gastric erosions rats are anaesthetized with
i.p. urethane (125 mg/ 100g).
• After 20-30 min of stabilization and baseline measurements,
13 ml/kg of blood is removed every 1-2 min, from a cannula
inserted into the carotid artery, producing hypotension to a
mean arterial line to monitor the arterial blood pressure.
• Twenty minutes after the shock, the animals are killed, the
stomachs are removed and the intensity of the macroscopic
lesions is graded by a suitable method.

Dimaprit
induced
gastric ulcers
(del SoldatoP
1982)
• On the basis of universally recognized hypothesis about
the involvement of H2 receptors with dimaprit as specific
H2 receptor agonist.
• Dimaprit was administered 24 h fasted rats and the
animals were sacrificed 4 hours after injection.
• The drugs for studying their gastroprotective effects
were given 30 min before dimaprit.
• The procedure is extremely simple and rapid.
• Its feasibly and specificity are added advantages.
• It is very useful for evaluating not only the absolute
potency of a drug given by any route but also of other
pharmacodynamic parameters particularly the length of
action which seems to be an important criterion in
selecting new potentially H2 antogonistic drugs.

Endotoxin (Lipopolysaccharide) induced gastric
mucosal damage
• Gastric mucosal ischemia is recognized as one of the major factors in the pathogenesis of acute gastric
ulceration.
• Consequent to the impaired gastric blood flow during shock, the clearance of back diffusing H is reduced.
• The intramural pH declines and ulceration occurs.
• These observations based on the circulation have led to developed of another gastric ulcer model using
endotoxin shock.
• Administration of endotoxin (20 mg/kg, i.p.) produced a moderate degree of gastric mucosal damage in rats.
• The lesions remained confined to the glandular mucosa and consisted of small punctiform lesions, erosions
was a typical submucosal ecchymosed in the glandular stomach observed in about 30% of the animals.
• Pretreatment with ranitidine, pirenzepine, proglumide, sucralfate and naloxone provided significant
protection.

Dimaprite
induced
duodenal
ulcers
• Recently Del Soldato et al, have used the model of
dimaprit induced duodenal ulcers in the guinea pigs
to study activity of some H2 receptor antagonists.
• Dimaprit is injected sub cutaneous to the 24 hours
fasted guinea pigs in the dose of 2 mg/kg every hour
and the animals are sacrificed 1 hour following the
last injection.
• It variably induces severe hemorrhagic ulceration
primarily in the duodenal bulb which is associated
with a significant increase in gastric volume and acid
content.
• The test drugs are administered 30 min before the
first dose of Dimaprit.

Duodenal ulcers
following s.c.
infusion of
pentagastrin
and carbachol
• Female rats are immobilized in individual Bollman cages.
• Acid Secretion is stimulated by a 24 h subcutaneous infusion of 1
l/kg/min pentagastrin plus 0.5 mg/kg/min carbachol in physiological
saline (0.01 ml/min).
• The test compounds are administered 5-10 min before beginning
the subcutaneous infusion.
• The animals are sacrificed at the end of 24 h and the intensity of
duodenal ulceration by a suitable grading method followed by
Robert et al, 1970.
• The main drawback of this method is that the lesions are distributed
over widespread areas of the duodenum and are not restricted to
the proximal part of duodenum.

MPTP induced
ulcers
• Parkinsonism inducing agent l-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine (MPTP) given in multiple daily doses, either p.o.
or s.c. induces solitary or double duodenal ulcers in the rat in a dose
dependent manner.
• MPTP decreases the gastric secretion of acid and pepsin as well as
pancreatic bicarbonate, trypsin and amylase.
• Thus it produces duodenal ulcers that are possibly associated with
impaired defence in the duodenal bulb (e.g. decreased availability
of duodenal and pancreatic bicarbonate)
• However, there is very limited experience with model as yet in
order to establish its suitability for routine use in the evaluation of
antiulcer drugs.
• Only some domapine agonists like bromocriptine and lergotrile and
monoamine oxidase inhibitors like pargyline and deprenyl have
been shown to prevent MPTP induced duodenal ulcers in rats

Diethyldithioc
arbamate-
Induced
Gastric Ulcer
• The diethyldithiocarbamate model is used to assess the antioxidant activities
of drugs in the prevention of gastric damage.
• This model is also used to assess the cytoprotective actions of potential
drugs.
• Diethyldithiocarbamate has been reported to induce antral lesions through
the mobilization of superoxide and hydroxyl radicals.
• Superoxide radical and hydroxyl radicals play a pathogenic role in the
induction of this ulcer.
• Acute glandular lesions are induced by subcutaneous administration of 1mL
of diethyldithiocarbamate in saline (800mg/kg body weight) followed by 1mL
oral dose of 0.1N HCl.
• In this model, food is also withdrawn 24 hours and water 2 hours just before
the commencement of the experiment.

Methylene
Blue-Induced
Ulcer.
• Methylene blue (MB), a synthetic drug is known to uncouple ATPases
and generate superoxide radical ions. MB has been used as an
ulcerogenic agent to study antiulcer agents and their mechanisms of
action.
• It can be used as a pharmacological tool to screen various antiulcer
agents, which modulate the H+/K+ ATPase system.
• When administered to animals, MB induces gastric and duodenal
lesions.
• The compound also inhibits nitric oxide synthase activity and hence
reduces the bioavailability of nitric oxide.
• Additionally, MB has affinity for acetylcholine or muscarinic receptors
and has been reported to inhibit cholinesterase activity.
• This implies that the model could be used for assessing antiulcer
agents with anticholinergic effects and proton-pump inhibitory
activity.
• Methylene blue decreases blood supply to gastric mucosa, which
causes oxidative stress and subsequently produces erosion and
ulceration of gastric mucosa.
• To induce ulcers with MB, animals are fasted 24 hours before MB
administration at a dosage of 125mg/kg body weight p.o. followed by
the administration of the drug(s) or substances under investigation.
• Animals are sacrificed after 4 hours of MB administration, and ulcer
index determined.

Ferrous Iron-Plus
Ascorbic Acid-
Induced Gastric
Ulcer
• This type of gastric ulcer model is induced by
the local injection of ferrous iron with ascorbic
acid (Fe/ASA) solution into the gastric wall. The
ulcers produced resemble human gastric ulcers
that penetrate the muscularis mucosa.
• Lipid peroxidation mediated by oxygen radicals
plays a crucial role in the pathogenesis of the
gastric ulceration induced by the Fe/ASA
solution.

Acetic Acid-
Plus H. pylori-
Induced Ulcer
Model.
• Rats can also be ulcerated with acetic acid according to the method
described by Takagi et al. Under anesthesia, laparotomy is
performed in rats through a midline epigastric incision, the
stomach is exposed, and 20% of acetic acid (0.03 mL) is injected
into the sub serosal layer of the glandular portion, using a micro
syringe (0.05 mL).
• After closing the abdominal incision, the animals are maintained in
individual cages, with daily access to commercial food restricted to
the time periods of 9-10 a.m. and 5-6 p.m.
• This allows for adequate fasting for administration of Helicobacter
pylori drugs or agents under investigation as well as standard drugs
(amoxicillin (AMX) 50mg/kg + clarithromycin (CLR) 25mg/kg with a
proton pump inhibitor such as omeprazole 20mg/kg).

• According to Konturek et al., 24 hours after ulcer induction by acetic acid, animals are
inoculated intra gastrically with 1mLof confirmed pathogenic strain of Helicobacter pylori
such as ATCC 43504 (9 × 108) suspended in Mueller- Hinton broth or Brain-Heart Infusion
Broth by using a cannula appropriate for orogastric gavage.
• For the animals in the control, and Acetic Acid-induced ulcer groups without Helicobacter
pylori infection, only Mueller-Hinton or Brain Heart Infusion Broth is administered orally.
• The orogastric inoculation with Helicobacter pylori is done twice a day for 7 days, whereas
the test drugs/agents, control and standard drugs, are administered twice a day, for 14
consecutive days, starting from the third day after ulcer induction by acetic acid.
• After treatment, the animals are sacrificed by cervical dislocation, blood is collected from
the inferior vena cava, and the stomachs are removed for evaluation of gastric lesions.

REFERENCES:
• Adinortey MB, Ansah C, Galyuon I, Nyarko A. In vivo models used for evaluation of potential antigastroduodenal ulcer
agents. Ulcers. 2013;2013.
• Vogel HG, Vogel WH, editors. Drug discovery and evaluation: pharmacological assays. Springer Science & Business Media;
2013 Apr 17.
• Meena DK, Jayanthi M. In-Vivo Models Used for Pre-Clinical Evaluation of Anti-Ulcer Activity. Austin Pharmacol Pharm.
2018;3(2):1017.
• Mishra AP, Bajpai A, Chandra S. A Comprehensive Review on the Screening Models for the Pharmacological Assessment of
Antiulcer Drugs. Current Clinical Pharmacology. 2019 Dec 1;14(3):175-96.
• Patra JK, Das SK, Das G, Thatoi H. Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals. InA Practical Guide to
Pharmacological Biotechnology 2019 (pp. 29-53). Springer, Singapore.
• Umre R, Ganeshpurkar A, Ganeshpurkar A, Pandey S, Pandey V, Shrivastava A, Dubey N. In vitro, in vivo and in silico
antiulcer activity of ferulic acid. Future Journal of Pharmaceutical Sciences. 2018 Dec 1;4(2):248-53.

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