Screening of

Peptic Ulcer Drugs

PEPTIC ULCER

Breach in continuity of mucosa of alimentary tract which

extends through the muscularis mucosa into the submucosa or

deeper

18/04/15 2

PROTECTIVE FACTORS

AGGRESSIVE FACTORS

• Acid, pepsin, bile

• Helicobacter pylori

• NSAIDs & other drugs

• Smoking, Alcohol, stress

• Free radicals

• Oily, spicy, irregular dietary habit

• Hereditary factors

Gastric Acid Secretion

Class Drugs H2 receptor antagonist Cimetidine, Famotidine, Ranitidine

Proton pump inhibitors Omeprazole, Pantoprazole, Rabeprazole

Anticholinergics Pirenzepine, Telenzepine

Prostaglandin analogue Misoprostol

Antacids Sodium Bicarbonate, Al/Mg hydroxide

Ulcer protective Sucralfate, Colloidal Bismuth Subcitrate

Anti-H.pylori Amoxicillin, Clarithromycin, Metronidazole

Drugs For Peptic Ulcer

IN VITRO METHODS

• [I125] Gastrin Binding Assay

• Tiotidine Binding Assay

• H+ / K+-ATPase Inhibition Assay

SCREENING METHODS

IN VIVO METHODS

• Pylorus Ligation in Rats

• Stress Ulcer Model

• Histamine-Induced Gastric Ulcer

• Ethanol-Induced Mucosal Damage

• Acetic Acid-Induced Gastric Ulcer

• Reserpine-Induced Chronic Ulcers

• Cysteamine-Induced Duodenal Ulcer

• Demaprit-Induced Duodenal Ulcer

• Mepirizole-Induced Duodenal Ulcers

• Experimental Colitis

In-vitro methods

H+/K+ - ATPASE INHIBITION ASSAY

• 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

microtitre plate for 30 mins at 37°

• 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.

PYLORUS LIGATION IN RATSShay et al. (1945), OLDEST ANIMAL MODEL OF GASTRIC ULCER

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 to avoid coprophagy.

• 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.

• Contents of the stomach are drained into

a graduated centrifuge tube and acidity determined by

titration with 0.1 N NaOH.

• 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.

ULCER CLASSIFICATION: SHAY ET AL (1945)

Grade 4 - Perforation

Grade 3 - Hemorrhagic spots with many ulcers

Grade 2 - Deeper hemorrhagic spots with few ulcers

Grade 1 - Scattered hemorrhagic spots

Grade 0 - Normal gastric mucosa

ULCER INDEX

Method 1:

The ulcer index is calculated as:

Ulcer Index = 10 / X

Where X = Total mucosal area / Total ulcerated area

Method 2:

Ganguly and Bhatnagar extended abovecriteria for inclusion of petechiae.

• Five petechiaes are considered to beequivalent to 1 mm of ulcer area. Ulcerindex calculated as described previously.

Method 3:

An ulcer index U is calculated asU = Un +Us +Up / 10

Where, Un = Average number of ulcers peranimal

Us = Average of severity score(graded from 0 to 3)

Up = Percentage of animals withulcer

ULCER SEVERITY SCORE

0 – no ulcer

1 – superficial erosion

2 – deep ulcer

3 – penetrated or perforated ulcer 18/04/15 21

0 - Normal stomach

0.5 - Red coloration

1 - Spot ulcers

1.5 - Haemorrhagic streaks

2 - Ulcer > 3 mm but < 5 mm

3 - Ulcers > 5 mm

OTHER METHODS ULCER

SEVERITY SCORE

ULCER INCIDENCE & GRADING:WILHELMI AND MENASSE-GDYNIA (1972)

0.5 – minute, sporadic, punctate lesions

1 – several small lesions

2 – one large extensive lesion or multiple moderate sized lesion

3 – several large lesion18/04/15 23

• Quantification of drug induced mucosal damage• Ulcers – necro-haemorrhagic spots > 2mm diameter

• Srivastava et al. (1991)

Shedding of epithelium= 10

Petechial & frank hemorrhages= 20

One or two ulcers= 30

More than two ulcers= 40

Perforated ulcers= 50

Inference

• Ulcer index of test drug compared with control group to detect anti-

ulcer effect of test drug.

CRITICAL ASSESSMENT OF THE METHOD

• The “Shay-rat” has been proven to be a valuable tool to evaluate anti-ulcer drugs with various mechanisms of action.

STRESS ULCER

• Selye (1936) , for the first time , described the use of restraints

for production of Gastric ulcers

Advantages:

• 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

I. RESTRAINT- INDUCED ULCERS (HANSON AND BRODIE ,1960)

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 are placed in a piece of

galvanized steel window screen of appropriate size.

• Screen is moulded around the animal and

held in place with wire staples.

• To restrain the rats, the limbs are put together in pair

and tightened with adhesive tape.

• Rats are kept under restraint for 24 hours

• Then sacrificed & their stomachs dissected out.

• Ulcer index and ulcer severity are determined.

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

II. Cold water immersion induced ulcer(Takagi et al, 1964)

• 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

• 10 min later, they are sacrificed

• The stomach is removed & ligated at both ends

• Filled with formaline & kept overnight

• On the next day, the stomach is opened along

the greater curvature and examined for ulcerative lesions

III. STRESS & NSAIDS–INDUCED ULCERS

• Procedure :

• Wistar rats; 150-200g

• Fasted for 24-36 hrs

• Test agent (in 1% carboxymethyl cellulose) via gastric intubation

and a NSAID such as aspirin, indomethacin or diclofenac i.p.

• Rats placed in stress cages

• Immersed in water upto level of xiphoid process at 230C for 7 hrs

• Animal sacrificed ; Stomach removed ; Evaluated for Ulcer Index

• Dose of NSAID required to increase gastric erosion by 100%

relative to immobilization is compared with that of NSAID

required to produce 100% increase in gastric erosion under the

protective effect of test drug

IV. SWIMMING STRESS ULCERS

• Procedure :

• Albino rats ; either sex

• Fasted for 24 hrs; free access to water

• Rats forced to swim in deep concrete tube filled with

water at 230C for 5 hrs

• Animals removed

• Sacrificed & stomach removed ; opened along greater

curvature

• Severity grading done ; Ulcer Index calculated

0 - Normal lesion

1 - Lesions with diameter < 1 mm

2 - Lesions with diameter 1 – 2 mm

3 - Lesions with diameter 2 – 4 mm

5 - Lesions with diameter > 4 mm

HISTAMINE-INDUCED GASTRIC

ULCER

(Barrett et al, 1955)

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 sulphate (50 mg base) is administered i.p.

• Promethazine hydrochloride 5 mg is injected i.p.

15 min before and 15 min after histamine to protect

the animals against histamine toxicity.

• The standard/test drugs are administered p.o. or s.c.

45 minutes before histamine injection.

• 4 hours after histamine injection, guinea pigs

are sacrificed and stomach dissected out.

• The gastric contents are subjected to analysis

• Stomach is opened along the greater curvature, ulcers are

identified.

ULCER SCORING : (Barrett et al, 1955)

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

Advantages:

• Produces 100% gastric ulceration

• Increased volume of gastric acid secretion

(Robert et al, 1979)

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.

Ethanol-induced Mucosal damage

• 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

Witt et al. (1985) described a method to quantify the

extent of ethanol-induced gastric lesions.

Using a transmission densitometer to measure the

optical density of the photographic negatives of gastric mucosa.

Damaged areas have lower optical density values.

Advantages :

• Gastric lesions are observed after an hour of

administration of ethanol.

• Reproducible method to produce gastric lesions in

experimental animals.

EVALUATION

• The significance of differences in optical density between control and ethanol-treated tissue is evaluated by nonpaired single-tail Student’s t-test.

CRITICAL ASSESSMENT OF THE METHOD

• Several prostaglandins provide cytoprotection, particularly in rats, in a dose-range which has no antisecretory activity.

• However, clinical experience with prostaglandins showed that ulcer healing is only achieved at anti-secretory doses (Lindberg et al. 1990).

• 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 antisecretory potential (Herling and Weidmann 1994).

ACETIC ACID-INDUCED GASTRIC

ULCER

Takagi et al. (1969)

A model for inducing chronic gastric ulcer in rats

by means of submucosal injection of acetic acid.

(Okabe et al, 1972)

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.

Procedure (Takagi et al, 1969)

• Albino rats used

• 0.05 ml of Acetic acid (1-30%) injected in the

submucosal layer of the stomach

• Penetrating peptic ulcers : adhered to Liver

• Chronic ulcers with repeated healing and re-aggravation

• Effect of test drug given twice daily for 10-15 days is noted.

Procedure: (Okabe and Pfeiffer, 1972)

• 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) is instilled into the tube

and allowed to remain for 1 minute on the gastric wall .

• After removal of Acetic acid solution, the abdomen is 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.

Advantages:

• Simple procedure: resulting in ulcers of consistent size

and severity at an incidence of 100%.

• Resemble human ulcers in terms of both

pathological features and healing mechanisms.

• Relapse of healed ulcers is frequently observed,

just as in peptic ulcer patients.

Disadvantages:

• Submucosal injection produced ulcers penetrating entire

gastric wall & adherence of ulcer base to adjacent organs

(mainly Liver).

RESERPINE – INDUCED CHRONIC ULCERS

• The mechanism of ulcer formation has been attributed to cholinergic mediated degranulation of gastric mast cells and liberation of histamine.

• Procedure :

• Female Sprague – Dawley rats; 130-180g

• Fasted for 48 hrs, Free access to 0.8% sucrose in 0.2% NaCl w/v

• Liquid diet withdrawn 1hr before starting

• Animals injected with Test drug i.p.

• ½ hr later, Reserpine(5mg/kg) or Vehicle injected i.p.

• 4 hrs later, Animal sacrificed, Stomach removed, Examined for mucosal lesions

CYSTEAMINE-INDUCED DUODENAL ULCER

Selye and Szabo (1973)

Cysteamine HCl (β-mercaptoethylamine HCl)

Principle:

Pathogenesis :

Inhibition of alkaline mucus production

Increased gastric acid secretion

Increased serum gastrin levels

Delayed gastric emptying

Procedure:

• Female Sprague-Dawley rats weighing 150-200

grams are taken

• Test drug and the standard drug are

administered 45 min prior to Cysteamine

administration.

MODEL ANIMAL ROUTE DOSE DURATION

Cysteamine

induced

duodenal

ulcers

(Cysteamine)

10% in N.S.

Sprague-Dawleyrats 150-200

grams

Orally

S.C.

28mg/100g

20mg/100g

3 times at intervals of

3.5 hours --> animals

sacrificed after 28

hours of 1st dose

2 times at 4 hours

interval--> animals

sacrificed after 40

hours of 1st dose

• Perforating duodenal ulcers are produced ;

• Located 2-4 mm from the pylorus, mainly on

the anterior wall of duodenum

• Necrotic material and acute inflammatory

response is present at ulcer crater

EVALUATION

• The intensity of the duodenal ulcer is evaluated using scores from 0 to 3.

• 0 = no ulcer

• 1 = superficial mucosal erosion

• 2 = deep ulcer usually with transmural necrosis

• 3 = perforated or penetrated ulcer

CRITICAL ASSESSMENT OF THE METHOD

• In view of the development of modern gastric K+/H+-ATPase inhibitors the predictive value of methods using experimental ulcers in the rat for clinical healing rates in man has been challenged (Herling and Weidmann 1994).

Advantages:

Ulcerogenesis is seen with one full dose of cysteamine.

Easily reproducible

Disadvantage:

• Ulcers, located on the anterior wall, frequently perforate,

resulting in peritonitis, or penetrate into the liver.

• A small ulcer is usually present on the posterior wall

(“kissing ulcer”) of the duodenum , penetrates the pancreas.

DEMAPRIT-INDUCED DUODENAL ULCER

del Soldato P (1982)

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 Demaprit in

rats and 30 min before injecting it in guinea pig.

• Demaprit 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 Demaprit injection, Animal is

sacrificed and stomach dissected out.

• Stomach is opened along the greater curvature

and examined for ulceration.

MEPIRIZOLE–INDUCED DUODENAL ULCERS

Okabe et al(1982)

• Model useful for :- - Screening of Anti ulcer drugs

- Studying pathogenesis of Duodenal Ulcers

Procedure :

• Male Sprague – Dawley rats (200-220g)

• Mepirizole(200mg/kg) in 1% carboxymethyl cellulose solution

via gastric intubation is administered

• Subsequently, rats kept in cages with raised mesh bottom and

deprived of food and water for 24 hrs

• Leads to, ulceration in proximal duodenum and

erosions in antrum.

• Anti ulcer therapy started 24 hrs after

Mepirizole administration

• 11th day, Animal sacrificed

• Duodenum and Stomach evaluated for ulcer

area under microscope

• Ulcer or erosion indices are calculated from the

sum of area of ulcers & erosions respectively.

EXPERIMENTAL COLITIS

•

PURPOSE AND RATIONALE

Inflammatory bowel diseases, ulcerative colitis and Crohn’s disease, represent chronic alteration of the gastrointestinal tract of unknown etiology perhaps involving immunological events.

The immunological parameters have been described as secondary but may possibly be attributed to the chronicity of the disease.

PROCEDURE

• A three-step concept is realized to mimic the human disease, using 2,4,6-trinitrobenzene sulfonic acid (TNBS) as a defined hapten:

• 1. specific hypersensitivity by active immunization,

• 2. local inflammation by local challenge,

• 3. chronicity by chronic application of the immunogen.

Female Sprague Dawley rats weighing 150–200 g are sensitized by intradermal injection of 0.8% TNBS solution into a shaved area on the back once daily for three consecutive days.

After 18 days, the animals receive a further intradermal booster injection.

Intradermal challenge of 0.08% TNBS in 0.05 ml 0.9% NaClsolution, is given 14 days later in order to determine the type and specificity of the immunological reaction.

Ten days after the intradermal challenge, a flexible polyethylene tube of 0.5 mm diameter is implanted under ketamine (100 mg/kg i.p.) anesthesia 15 cm proximal to the cecum and emerging at the neck for TNBS or drug administration.

After a 10-day recovery phase, the animals are treated daily for 3 weeks with 0.08% TNBS in saline (0.2 mg/rat) given through the catheter.

Control groups receive only saline.

Drugs are applied either by gavage twice a day. suspended in carboxymethyl cellulose, or intraluminally once a day, suspended in saline.

The animals are sacrificed by CO2 inhalation 24 h after the last intraluminal application of TNBS.

The distal 10 cm of small intestine anterior to the ileo-caeco-colic junction (5 cm distance to the open end of the catheter) including Peyer’s patches are dissected, cut open longitudinally and rinsed with saline

Immediately after dissection, the distal small intestine is visually assessed for inflammation according to the following scores:

EVALUATION

• Results are expressed as means ± SEM of (n) experiments.

• Differences between control and inflamed tissue, and influence of drug treatment are compared.

• Statistical significance is calculated by Wilcoxon-MannWhitney U-test for unpaired data.

• The level of significance is taken as p < 0.05.

CRITICAL ASSESSMENT OF THE METHODS

• The relevance of animal models for the pathogenesis and treatment of human inflammatory bowel disease was reviewed by Dieleman et al. (1997) and by Sartor (1997).

• A critical review of in vitro models in inflammatory bowel disease was given by McKay et al. (1997).

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