Evaluation of anti ulcer agents_Dr. Mansij Biswas

S

Evaluation of

Anti-ulcer

Agents

Dr. Mansij BiswasSYR, Dept. of Pharmacology & Therapeutics,

Seth G S Medical College & KEM Hospital

18/04/15 1

Definition

“Ulcers are defined as a breach in continuity of mucosa of

alimentary tract which extends through the muscularis mucosa

into the submucosa or deeper”- Robbins

18/04/15 2

3

Physiology of acid secretion

18/04/15

Pathophysiology

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AggressiveS Acid, pepsin, bile

S Helicobacter pylori

S NSAIDs & other drugs

S Smoking, Alcohol, stress

S Free radicals

S Oily, spicy, irregular dietary

habit

S Hereditary factors

Protective S Mucus

S PGE2, PGI2, IL

S Mucosal blood flow

S Bicarbonate

S SDO, catalase, EDRF

History

Development of the first H2 blocker, Cimetidinein 1974 by James W. Black & colleaguesprovided a specific class of anti ulcer followedby PPIs

In latest, Anti-ulcer activity of Cromakalim (BRL34915), Levcromakalim and Nicorandil,potassium-channel openers, againstexperimentally induced gastric and duodenalulcers in rats and guinea-pigs evaluated

18/04/15 5

Classification of available therapy

Class Drugs

H2 receptor antagonist Cimetidine, Famotidine, Ranitidine

Proton pump inhibitors Omeprazole, Pantoprazole, Rabeprazole

Anticholinergics Pirenzepine, Telenzepine

Prostaglandin analogue Misoprostol

Antacids Al/Mg hydroxide, Simethicone

Ulcer protective Sucralfate

Anti-H.pylori Amoxicillin, Clarithromycin, Metronidazole

Bismuth containing compounds

Bismuth subsalicylate (BSS)

18/04/15 6

Need for the newer anti ulcer

drug!!

High morbidity continuous need for newer anti-ulcer drugs

S Cost effectiveness- low cost

S Safety & Efficacy- More efficacious with less side effects

S Heals ulcers and prevent recurrence- useful in suppressing chronic cases as well

S Drug for H. Pylori eradication18/04/15 7

Experimental Evaluation

“The most important undertaking in science

is the establishment of experimental

methods”

- Ivan P Pavlov

18/04/15 8

Criteria for Experimental methods

(Lee & Bianchi)

S Simple, reproducible, easy quantification of

results.

S Make use of a variety of animal species.

S Induce characteristic ulceration in specific

locations (stomach and duodenum).

S Involve different mechanisms.

S Ulcers induced should not spontaneously heal

during the observation period.18/04/15 9

Ideal animal for screening anti ulcer

agents?

S Continuous secretion of acid

S Stomach is analogous to man.

S Being omnivorous resembles man nutritionally

*** Guinea pigs are used when histamine is used to induce ulcers.

RATBecause………

…

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The rat stomach- location &

structure

Approach of Evaluation

S Gastric anti-ulcer activity

S Duodenal anti-ulcer activity

S Anti-secretory activity

S Cyto-protective activity

S Elucidation of mechanism of action

18/04/15 12

How to assess experimental erosions and

ulcers

S Counting the number of hemorrhagic spots

or

S By scoring the number, length and depth of

mucosal lesions

Drawback: alcohol or drug induced confluent

lesions

how to overcome?18/04/15 13

Microprocessor linked planimeter with

a stereomicroscope

18/04/15 14

Ulcer classification: Shay et al

(1945)

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Ulcer scoring: Barret et al (1955)

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Histological grading

Observations

Score

a) Epithelial damage, Glandular disruption

1

b) Vasoconstriction or edema in upper mucosa

2

c) Haemorrhagic damage in middle/lower mucosa

3

d) Deep ulceration and perforation

4

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Ulcer index

Method 1:

The ulcer index is calculated as:

Ulcer Index = 10 / X

Where X = Total mucosal area / Total ulcerated area

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Ulcer index

Method 2:

Ganguly and Bhatnagar extended above criteria for

inclusion of petechiae.

S Five petechiaes are considered to be equivalent to 1

mm of ulcer area. Ulcer index calculated as described

previously.

18/04/15 19

Ulcer index

Method 3:

An ulcer index U is calculated as

U = Un +Us +Up / 10

Where, Un = Average number of ulcers per animal

Us = Average of severity score (graded from 0

to 3)

Up = Percentage of animals with ulcer18/04/15 20

Ulcer severity score

0 – no ulcer

1 – superficial erosion

2 – deep ulcer

3 – penetrated or perforated ulcer

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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

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S 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

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Ulcer incidence & grading:

Wilhelmi and Menasse-Gdynia

(1972)

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• Quantification of drug induced mucosal damage

• Ulcers – necro-haemorrhagic spots > 2mm

diameter

Percentage protection score

C - T% protection = ------------ × 100

C

Where,

C= mean severity of ulcer score in control groupT= mean severity of ulcer score in treatment group

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Other parameters

S Volume of the gastric juice

S Free and total acidity (BAO / MAO levels)

S Occult blood and external appearance of the

stomach

S Mucosal congestion

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Gastric anti-ulcer activity

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Pylorus ligation in rat: Shay et al (1945 )

S Oldest animal model of gastric ulcer

S Wistar rats (150-200 gm), fasted for 48 hours

S Housed in cages with wide mesh wire bottoms to prevent coprophagy.

S Under light ether anesthesia, Pylorus ligated avoiding damage to its blood supply.

S Sacrificed 19 hours after operation

S Stomach dissected out open along the greater curvature18/04/15 28

S Contents collected & subjected to analysis for volume, pH, free and total acidity, mucin, prostaglandin, total carbohydrate:protein ratio etc

S Inner surface examined for ulceration, and ulcer index is calculated

S Advantage: Evaluates anti-ulcer drugs with various mechanisms of action and different doses.

S Disadvantage: The ulcers localized in the rumen and antrum of the stomach


18/04/15 29

Inference

S Ulcer index of test drug compared with control group to

detect anti-ulcer effect of test drug.

Other parameters help to infer the mechanism of ulcer

protection-

S Decrease in volume, free & total acidity: antisecretory

action

S Rise in pH: acid neutralising action

S Increase in mucin, PGs: cytoprotective effect.18/04/15 30


Stress ulcer

S Experimental counterpart of Curling ulcer in human

S 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 evaluated18/04/15 31

S Albino rats (150-200 gm), Deprived of food for 36

hours.

S Each rat is then placed in a piece of galvanized steel

window screen of appropriate size.

S The limbs are put together in pair and tightened with

adhesive tape so that the animal cannot move.

S Drugs administered 30 min before restraining animal

S At the end of 24 hours the animals are removed from

the screen and sacrificed using overdose of ether.

Restraint Ulcers: (Brodie & Hanson, 1960)

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Restraint Ulcers: (Brodie & Hanson, 1960)

S Used for studying the healing of ulcers

S Lesions do not penetrate the muscularis mucosa

S Technique is species specific.

18/04/15 33

Water immersion induced restraint

ulcer

Principle: It has been shown that in stress, gastric

lesions were significantly enhanced by exposure

to water immersion.

S Male Wistar rats, fasted for 24 hours ,immobilized

in a stress cage and then immersed in a water

bath (23°C) for 16 hours.

S The animals sacrificed by a blow on the head after

injecting i.v. Evan’s blue

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S Stomach removed, filled with 1% formalin.

S The ulcer index is estimated

S Test drugs are administered 30 min prior to stress.

18/04/15 35

Water immersion induced restraint

ulcer

Cold & restraint ulcers: (Vincent et al, 1997 )

Exposure of rats to cold during the restraint period

accelerates the occurrence of gastric ulcers and

shortens time of necessary immobilization

S Wistar rats deprived of food for 12 hours

S Immobilized on a rectangular wooden board by tying

their four limbs to the four corners of the board.

Boards are placed at an angle of 450 in the refrigerator

at 4-60 C with the rats in head low position for 3 hours.

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S Sacrificed by a blow on the head

S Ulcer index calculated as described for restraint

ulcers.

S Test drugs are administered 30 min before

immobilizing the animals for acute and 7 days prior to

stress for chronic models.

18/04/15 38

Advantages:

S Do not require extensive period of starvation

S Do not require that the animal be restrained for

lengthy period of time

S Restricted virtually all the movements of the animals

without respiratory or the circulatory trauma

S Very high and reliable degree of gastric glandular

ulcers can be produced in rodents in a relatively short

period


18/04/15 39

Swimming stress ulcers

Swimming in vertical cylinders filled with water acts as a stress factor for accelerated production of ulcers.

S Rats fasted for 24 -36 hours are forced to swim inside the vertical cylinders (height 30 cm, diameter 15 cm) containing water up to 15 cm height, maintained at 23°C.

S Administer the test drugs 30 min prior to the stress.

S Sacrifice after 3 hours.

S Dissect out the stomach and examine the gastric mucosa for presence of ulceration

18/04/15 40

Activity stress ulcers in rats

S House young adult rats

individually in running

wheel activity cage ,

allowing continuous

access to the wheel and

feed only for one hour

every day.

S Some of these animals will

die within 4-16 days.

Dissect them and examine

the gastric mucosa for

stress ulcers.

18/04/15 41

Activity stress ulcers in rats

Advantage:

S Animals respond to centrally acting agents such as diazepam and imipramine suggesting a role of central neurotransmission in the production of gastric ulcer.

Disadvantage:

S Time consuming and needs continuous supervision of the animals in the activity cages.

S Animals developing activity stress gastric lesions do not respond to histamine H2 blockers.

18/04/15 42

Hemorrhagic shock induced gastric ulcers in

rats

Hemorrhagic shock acts as a stress factor and

is seen to cause gastric erosions in rats.

Endogenous Endothelin-1 has been implicated

in hemorrhagic shock induced gastric

ischemia- reperfusion injury

18/04/15 43

Hemorrhagic shock induced gastric ulcers in

rats

S Following anaesthesia & after stabilization of baseline

measurements, remove 13 ml/kg of blood every l-2

min, from carotid artery cannula, producing

hypotension to a mean arterial pressure of 30-40 mm

Hg.

S Connect a transducer via a three way stopcock to the

same arterial line to monitor the arterial blood

pressure.

S Twenty min after the shock, sacrifice the animals

S Remove the stomach and grade the intensity of the

18/04/15 44

Gastric mucosal damage by NSAIDS in rats

Principle: NSAIDs inhibit cyclooxygenase enzyme in

the gastric mucosa.

S Administer the test compounds 30 min to 1 hour

before the noxious challenge. Sacrifice the animals

after prescribed period (vary with different agents,

usually 4-6 hours). Examine the stomachs for

mucosal lesions.

S The incidence and grading of the severity of lesions

are done according to different methods.18/04/15 45


The following drugs are used as ulcerogens:

Aspirin

administer orally (gavage) in a dose of 500 mg/kg

Produces gastric erosions i.e. superficial mucosal

lesions not penetrating the muscularis mucosa, mainly

in the glandular segment of the stomach in 100% of the

animals.18/04/15 46


Phenylbutazone

S This is given in a similar fashion as aspirin in a dose of

100 mg/kg, per oral (suspension) or intraperitonial

(solution), at an interval of 15 hours.

Indomethacin

S lndomethacin is given in a dose of 20 mg/kg, per oral

while Ibuprofen is given in the doses of 200 mg/kg, per

oral at 15 hours intervals. 18/04/15 47

Gastric erosion following short term stress

& concurrent administration of NSAIDS

Principle:

S Administration of nonsteroidal anti-inflammatory agents

along with exposure to short term stress accelerates

production of gastric lesions in Wistar rats.

Procedure:

S Administer the substances (in 1% CMC) to be investigated

via gastric intubation at the same time as the intraperitoneal

injection of a NSAlD

S Place the rats in a stress cage and immerse to the level of

xiphoid process in a water bath (23°C) for 7 hours, then

sacrifice.18/04/15 48

Gastric erosion following short – term stress

& concurrent administration of Nonsteroidal

anti inflammatory drugs (NSAIDS )

Evaluation:

S The ulcer factor (UF) is the dose of the NSAID, which

increases the ulcer index to 100% against controls

(stress alone).

Advantage:

S Ulcer indices produced are easily reproducible

S Remain constant, suitable for studying the dose

dependent effect of anti-ulcer drugs

18/04/15 49

Histamine induced gastric ulcers in Guinea

pigs

Histamine enhances gastric acid secretion and causes

vasospasm which results into development of gastric

lesions.

S Male guinea pigs weighing 300-400 g.

S Inject 1 ml of histamine acid phosphate (50 mg base)

intraperitoneally.

S Inject Promethazine hydrochloride 5 mg

intraperitoneally 15 min before and 15 min after

histamine to protect the animals against histamine 18/04/15 50

Histamine induced gastric ulcers in Guinea

pigs

S Give the drugs under investigation orally or

subcutaneously 30-45 min before histamine injection

S Sacrifice the animals four hours after histamine

administration and dissect out the stomach

S The gastric contents are subjected to analysis

S Cut open the stomach and assess the ulcers produced

18/04/15 51

Histamine induced gastric ulcers in Guinea pigs

Advantages:

S Produces 100% gastric ulceration

S Increased volume of gastric secretion

S Marked enhancement of free and total acidity.

18/04/15 52

Acetic acid induced kissing gastric ulcer

Yakagi et al, 1969 Okabe et al, 197118/04/15 53

S New method by Tsukimi &

Okabe (1994) give intra-

luminal acetic acid injection

S Acetic acid produces

penetrating ulcers in fundus

area of rat. Ulcerated areas

in anterior and posterior

wall are identical (kissing)18/04/15 54

Acetic acid induced kissing gastric ulcer

Advantages of Acetic acid model

S Simple, readily resulting in ulcers of consistent size

and severity at an incidence of 100%.

S 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.

S The ulcers respond well to various anti-ulcer drugs.

Steroidal and non-steroidal anti-inflammatory drugs

negatively impact healing of the experimental ulcers.18/04/15 55

Reserpine induced solitary chronic gastric

ulcers

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

S Administer reserpine 5 mg/kg/day for 5 days and sacrifice after two weeks

S Solitary chronic gastric ulceration, oval or round situated at the lesser curvature in the pre-antral region.

S This model can be suitably used for studying the acute as well as chronic ulcers.

18/04/15 56

Serotonin induced gastric mucosal lesions

S Serotonin creatinine sulphate dissolved in saline, injected

subcutaneously to wistar rats in the dose of 20 mg/kg.

S In gross observation, gastric lesions are scarcely noticed at

0.5 hour after serotonin injection (UI 1.2), but are obviously

distinguishable at 1 hour (UI 7.5) and reach maximum

intensity at 4 hours (UI 15.2). Decreases to 8.0 at 8 hours

and maintained at this level up to 24 hours after serotonin

injection.

S The lesions are located mainly at the side of the greater

curvature of the corpus.18/04/15 57

Duodenal anti-ulcer activity

Until 1970, rats were considered unusually

resistant to the induction of duodenal

ulcers.18/04/15 58

Cysteamine induced duodenal ulcer in

rat Selye & Szabo (1973)

S Cysteamine inhibits alkaline mucus secretion from

Brunner’s glands (proximal duodenum)

S It stimulates gastric acid secretion

S Delays gastric emptying.

S Also increases serum gastrin concentration.

18/04/15 59

Cysteamine induced acute ulcers

S Cysteamine HCL ( 10 % ) dissolved in normal saline is

given to rats in dose of 28 mg/ 100 g of body weight

orally for 3 times at a interval of 3.5 hours or 40 mg/100

g orally for 2 times at an intervals of 4 hours.

S Rats are sacrificed after 28 hours of 1st dose.

18/04/15 60

Cysteamine induced chronic ulcers

Procedure:

S Use acute ulcerogenic regimen on the first day

S Subsequently, provide access to drinking water

containing 0.2, 0.05, or 0.01% cysteamine HCI.

S Chronic duodenal ulcers develop within 21 to 60 days.

18/04/15 61

Cysteamine induced duodenal ulcer

Advantages:

S Resembles duodenal ulcer in man to its location,

histopathology and some aspects of pathophysiology

S One of the most suitable methods for studying the

pathogenesis

S It is inhibited by the anticholinergic agents, antacids, and

prostaglandin and H2 receptor antagonists.

S This model is the first easily reproducible and economic

model of peptic ulcer in which the ulcerogenic agent can be

maintained for indefinite periods.18/04/15 62

Cysteamine induced duodenal

ulcer

Disadvantage:

S The mechanisms of action are not fully explained.

S It is difficult in a screening programme as multiple

doses are necessary to prevent cysteamine-induced

ulcers.

18/04/15 63

Dulcerozine induced duodenal ulcer in rats:

Kurebayashi et al (1984)

S Dulcerozine, a compound structurally related to NSAID

(phenylbutazone) induces acute perforating ulcers in

rats probably by causing prolonged gastric hyper

secretion

S Single dose of 300 mg/kg suspended in 5% gum acacia

orally

18/04/15 64

Dulcerozine induced duodenal ulcer

Advantages:

S The lesions developed are analogous to the clinical

disease with respect to location and histology

S The factors producing pathologic changes are similar

in man

S It is extremely simple to perform

S The massive production is feasible and results are

obtained within 18 hours18/04/15 65

Dimaprit induced duodenal ulcer:

Del Soldato et al (1985)

Dimaprit is used as specific H2 receptor agonist to induce peptic ulcer. So this model is used to study the anti ulcer activity of some H2 receptor antagonists.

Procedure:

Inject Dimaprit subcutaneously to the 24 hours fasted guinea pigs in the dose of 2 mg/kg every hour (6 times) and sacrifice the animals 1 hour following the last injection. Administer the test drugs 30 min before the first dose of Dimaprit.

18/04/15 66

Dimaprit induced duodenal ulcer

Advantages:

S Induces severe hemorrhagic ulceration in duodenal bulb,

associated with a significant increase in gastric volume &

acid content

S The procedure is extremely simple and rapid.

S It is feasible and specific model for H2 antagonism.

S Pharmacodynamic parameters particularly the duration

of action can be evaluated . 18/04/15 67

Duodenal ulcers following s.c. infusion of

Pentagastrin & Carbachol

S Immobilize rats in individual cages.

S Stimulate acid secretion by a 24 hour subcutaneous

infusion of 1 mg/kg/min pentagastrin plus 0.5

mg/kg/min carbachol in physiological saline (0.01

ml/min).

S Administer the test compounds 5-10 min before the

infusion.

18/04/15 68

S Sacrifice the animals at the end of 24 hours and

assess the intensity of duodenal ulceration by

subjective grading method.

Disadvantage:

S The lesions are distributed over a wide area and are

not restricted to the proximal part of duodenum.

Duodenal ulcers following s.c. infusion

of Pentagastrin & Carbachol

18/04/15 69

Indomethacin + Histamine induced

duodenal Ulcers in rats

A single administration of indomethacin and

subsequent dosing with histamine consistently

produces lesions at the opposite side of the mesenteric

attachment in the proximal duodenum in rats.

Procedure:

Indomethacin (5 mg/kg) subcutaneously and

subsequently give histamine dihydrochloride (40 mg/kg)

three times at 2.5 hours intervals, beginning 30 min

after the injection of indomethacin.18/04/15 70

lndomethacin + Histamine induced duodenal

Ulcers in rats

Advantages:

S Simple procedure with high incidence of ulcer formation

and no mortality.

S The physiological factors involved in this model appear

to be relevant to the pathogenesis of human duodenal

ulcer disease and for screening anti-ulcer agent.

S Model shows that both an increase in gastric acid

secretion and an impairment of HCO3 secretion are

responsible for ulcer production. 18/04/15 71

Other duodenal ulcer models

S Few more models have been introduced

for the production of duodenal ulcers in

rats during the last two decades:

a) Mepirizole induced ulcer ( 1982 )

b) MPTP-induced ulcer ( 1985 )

c) Glacial acetic acid induced ulcer ( 1989

)18/04/15 72

Anti-secretory activity

18/04/15 73

Pavlov’s and Heidenhain pouch

(1878)

S The Pavlov’s gastric pouch with intact vagal and

sympathetic nerve supply whereas Heidenhain pouch

is denervated.

S Prepared from the dog (Beagle or Mongrel, 20 kg)

stomach.

S Stimulate gastric secretion by injecting the fasting

animal with 100 mg/kg/h of 2-desoxy-d-glucose (2-DDG),

8 mg/kg/h of pentagastrin or by 80 mg/kg/h of histamine

dihydrochloride, intravenously for 3.5 hour. 18/04/15 74

Pavlov’s and Heidenhain pouch (1878)

S Gastric secretion is also induced in dogs with

Heidenhain pouch using a mixture of pentagastrin (8

mg/kg/h) & carbachol (1 mg/kg/h) given

intravenously.

S Give the substances to be investigated intravenously

as a single bolus injection 60 min after the infusion

of 2-DDG or 90 min after beginning the infusion of

other secretogogues and measure gastric secretion

a further 180 or 120 min, respectively.

18/04/15 75

Continent gastric fistula in dogs:

Foschi et al (1984)

Surgically created anti-reflux flap of the gastric wall

characterizes this method.

Advantages:

S It does not require a large laparatomy incision or gastric

sutures

S Simple and takes only 30 minutes

S Stable reproducible secretion18/04/15 76

Ghosh and Schild perfused rat stomach

preparation (1958)

This method is introduced for the continuous recording

of gastric acid secretion.

S The acid secretion can be stimulated by histamine,

acetyl choline and gastrin. In this method several doses

of drugs can be evaluated in the same animal by a

quantitative assay.

S Pyloric end of stomach is cannulated and polythene tube

is passed down the esophagus and stomach is washed

out thoroughly by passing distilled water18/04/15 77

Ghosh and Schild perfused rat stomach

preparation (1958)

S Perfuse continuously at 1 ml/min with N/4000 sodium

hydroxide

S The perfusate bathes a microflow glass electrode

connected to a direct reading pH meter

S The drug under investigation is injected prior to each

dose of a secretogogue

S Any inhibition or potentiation of the effect is recorded.

18/04/15 78

Chronic gastric fistula in rat:

Komarow (1963)

S A Pavlov’s pouch type of cannula is used in this method

.

S In male Wistar rats (280-300g) under anesthesia insert a

beveled stainless steel cannula into ruminal part of

stomach close to the greater curvature and away from

the secretory portion.

S The animals are restrained in a cage for 2-3 weeks and

are fasted 18 hours before the experiment.

S Hourly collections of the gastric juice are done. 18/04/15 79

Chronic denervated gastric pouch:

Alfin and Lin (1959)

S A denervated pouch is created in male Wistar or Sprague

Dawley rats weighing 250-350g and connected to the

exterior by a steel cannula.

S The animals receive procaine penicillin 150 MU and

pasted diet for some days and are strictly in air-

conditioned rooms.

18/04/15 80

Chronic denervated gastric pouch:

Alfin and Lin (1959)

S The animals are deprived of food but not water

before the experiment and are kept in restraining

cages.

S The gastric juice is collected in graduated

centrifuged tubes and the amount of secretions are

measured after administration of secretogogues

alone or with antisecretory agents after 2 hours.

18/04/15 81

82

Isolated whole stomach preparation of

rat

(Bunce & Parsons, 1976)

S Modification of Ghost and Schild method.

S Dissected stomach placed in 10 ml organ

bath containing Krebs-Henseleit solution

with carbogen gas (95% O2 + 5% CO2) at 370

C.

18/04/15

83

Isolated whole stomach preparation of

rat

(Bunce & Parsons, 1976)

S The test compounds or secretogogues

added in a volume of 0.5 ml to the organ bath

bathing the serosal surface of stomach.

S Rate of acid secretion expressed as (H+)

moles × 10-8 per min

18/04/15

Cyto-protection activity

Robert et al (1979) introduced a concept thatprior administration of PGs protect the ratstomach against various noxious agents.

18/04/15 84

Necrotizing agents used

S 30 mg of aspirin suspended in 0.15 M HCI

S Absolute ethanol

S 0.6 M HCI, 0.2 M NaOH

S 25% NaCI

S 80 mM of sodium taurocholate

S Boiling water (thermal injury)

S Chili extract & Capsaicin18/04/15 85

Gastric Cytoprotection

S 0.6 M HCI and 0.2 M NaOH - Elongated black and red

patches

S 25% NaCl - Red dots grouped in transverse bands

S Boiling water - Red elongated bands usually parallel

to the long axis of the stomach.

18/04/15 86

Gastric Cytoprotection

S Lesions are located mostly in the corpus (the

portion of the stomach secreting acid and pepsin),

the antrum is less affected.

S The damage induced by burning or by hypertonic

solutions is characterized by severe congestion of

the entire mucosal surface and it is distinctly

different from that induced by other agents.

18/04/15 87

Ethanol induced gastric

lesions

The lesions are preceded by early vascular damage and increased

vascular permeability, the probable mechanisms for which are

decreased level of glutathione (GSH) and generation of free radicals.

Procedure:

S After 24 h of food deprivation, give 100 gm per 1ml of ethanol orally

to Wistar rats and 15 min later, kill the animals by cervical

dislocation.

S Absolute ethanol lesions appear as blackish lesions grouped in

patches of varying size, usually to the major axis of the stomach.

S Pretreatment with ulcer protective agents have been shown to

prevent damage caused by ethanol.18/04/15 88

Chili induced gastric damage

Crude chili and capsaicin, a constituent of chilies have been

shown to produce dose dependent early vascular damage.

Procedure:

S Wistar rats either sex (150 - 180g)

S Anaesthetize them with Pentobarbitone sodium (15 mg/kg).

Inject Evan’s Blue dye (1 mg/kg) into the exposed femoral

vein

S 5 min later give chili extract orally to all groups at a dose 8

mg/kg18/04/15 89


S Sacrifice the animals 10 min later.

S Remove the stomachs and keep the gastric

contents and glandular portion of stomach in

concentrated HCI after noting the weight.

S After allowing 18 h. digestion of tissues in the acid,

extract Evan’s Blue in chloroform and estimate

spectrophotometrically at 620 nm.

18/04/15 90


Evaluation:

S Compare the results of spectrophotometry in the test

and control groups and plot the dose response curve.

Advantages:

S Misoprostol, free radical scavengers (dimethyl

sulphoxide and superoxide dismutase) and allopurinol

protect against chilly induced vascular damage.

S Sucralfate, Cimetidine and ranitidine can be investigated

for their protective effects.18/04/15 91

Quantitive assessment of mucus content

S Wistar rats of either sex and between the weight of 150

– 200 grams.

S 4 hours after administration of either distilled water or

NSAIDs, sacrifice the animals and dissect out the

stomachs.

18/04/15 92

S For mucus barrier estimation remove the glandular part of stomach

, weigh and immediately transfer to 10 ml of 0.1 % w/v buffered

Alcian Blue solution.

S The gastric glandular tissue is stained for 2 hrs at room

temperature. Excess uncomplexed dye is removed by 2 successive

rinses in 10 ml of 0.25 M sucrose solution.

S Extract the dye complexed with gastric wall mucus with 10 ml of 0.5

M magnesium chloride solution which is intermittently shaken for 1

min at 30 min intervals for 2 hrs.


18/04/15 93

S 4 ml of Alcian Blue extract is then vigorously shaken

with an equal volume of diethyl ether.

S The amount of Alcian Blue in the aqueous layer is

measured spectrophotometrically at 540 nm.


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S The quantity of Alcian blue extracted per gram weight

glandular tissue is calculated from a standard curve

obtained by using various dilutions of 0.1 % Alcian

Blue.

Optical density X std Alcian blue (µg)

Mucus content

=________________________________________

(µg/ gm) Wt of glandular part (gm)


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Other models

S Isolated gastric mucosal preparation (Main and Pearce-

1978)

S Primary culture of rat gastric epithelial cells (in vitro

model– Zheng et al, 1994.

S Determination of the prostacyclin levels of the gastric

mucosa in rats.

S Measurement of gastric mucosal blood flow

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Elucidation of mechanism of

action

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H2 blocker evaluation- in vitro

H2 antagonism in isolated guinea pig atria (Reinhardt,

1974)

S Compounds that inhibit the positive chronotropic effect

mediated by histamine H2 receptors in isolated guinea pig

atria can be classified as specific H2 histamine antagonists.

Modification by Hattory (1990)

S Rabbit papillary muscles

S H2 antagonists inhibit the histamine induced decrease in

action potential duration18/04/15 98


H2 antagonism in isolated rat uterus

S Histamine inhibits spontaneous and electrically stimulated contractions of rat uterus horns, this effect is antagonized by H2 antagonists but not by H1 antagonists.

S Virgin Wistar rats (180 – 200 gms), Sacrificed and uterine horn are taken and kept in de Jalon solution

S Contractions of preparation are recorded, log response curve plotted

S H2 antagonists cause reduction in Histamine induced inhibition. 18/04/15 99


Histamine stimulated adenylate cyclase from gastric

mucosa

S This is inhibited by H2 antagonists.

Histamine H2 receptor binding

S Can be determined by 3H-tiotidine as ligand binder

S Using polymerase chain reaction

S Cloning for gene coding for H2 receptor 18/04/15 100

H2 blocker evaluation- in vivo

Activity at Histamine H1 and H2 receptors

(Owen and Pipkin, 1985)

S Simultaneous and quantitative assay of the action of

antagonists and agonists at H1 and H2 receptors in

anesthetized guinea pigs

S H1 causes bronchoconstriction and H2 causes

tachycardia.

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Proton Pump Inhibitors evaluation

Gastric vesicles prepared from porcine fundic mucosa

(Hongo et al)

S Increase permeability of vesicles to K+ by storing at -

80oC

S Vesicles pre-treated with compound and 2mM ATP

added

S H+ ion transport is monitored from the distribution of

fluorescent probe acridine orange18/04/15 102


H+/K+-ATPase inhibition in membrane vesicles of

stomach mucosa of pigs (Lee and Forte, 1978)

S H+/K+-ATPase inhibitor (Sulfhydril group - sulphenic

acid + Sulphenamide )

S The decrease in fluorescence is taken as measure

for the intravesicular proton uptake.

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Effect of H+/K+ ATPase inhibitiors on serum gastrin levels

S Proton pump inhibitors block total acid output- causes increase in gastrin levels, which is measured by RIA.

Aminopyrine uptake and oxygen consumption in isolated rabbit gastric glands

S The ability of the gastric glands to form acid is measured based on aminopyrine accumulation using liquid scintillation counter.

S Oxygen consumption of the gastric glands measured by Warburg respirometer18/04/15 104

H. pylori models

S High levels of colonization (106-107 cfu/g tissue) were

achieved consistently in C57BL/6 mice, varied with

BALB/c, DBA/2, and C3H/He mice by Sydney strain of

H. pylori (strain SS1, cagA and vacA positive),

Marchetti et al. (1995) & Konturek et al. (1999)

S Experiments developed using Mongolian gerbils have

demonstrated that H pylori infection is clearly

responsible for gastric carcinogenesis.

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Clinical Evaluation

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Phases of Clinical Trial

Phase 0: Human microdosing studies

S Single subtherapeutic dose of the study drug

administered to a small number of subjects (10 to

15)

S Gives no data on safety or efficacy

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Phase I

S Assessment of safety, tolerability, PK, PD

S Small no. of healthy volunteers (20- 40), Patients in remission from their disease may be used

S Volunteers with high acid output and familiar with procedure (to avoid anxiety)

S Dose escalation study to determine dose for therapeutic use.

S No blinding, no comparator


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Observations

S PK parameters: Cmax, Tmax, AUC (0- 24 hrs), t1/2,

elimination rate constant, clearance, distribution

- Effect of food on pharmacokinetics

S Tests for safety & tolerability of the IND:

-B/P, pulse rate/ respiratory rate, body

temperature, body weight, ECG, laboratory tests

(hematology/ blood biochemistry/ urine-analysis)

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Phase II (Therapeutic exploration):

Assessment of safety & efficacy.

- Phase IIA: assess dosing requirements for subsequent

studies.

- Phase IIB: assess efficacy.

Provides basis for confirmatory study design, endpoints &

methodology

Phase IIA: Small no. of pts.- 20 – 200, Phase IIB: Larger no. of

pts.- 50 – 300.


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Phase III (Therapeutic conformation):

S Randomized controlled multicentric trials.

S Confirm efficacy & compare with the gold std. drug.

S Safety assessment

S Large no. of pts. 250- 1000 or more

S Parallel group / Cross over


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Phase IV: Post marketing Surveillance Trial

S Detect rare adverse effects, drug interactions.

S Discover previously unanticipated benefits or uses.

S Quality of life

S Large patient population taking the drug.

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Key Inclusion Criteria

S Age 18-65 years

S Those with duodenal ulcer (DU) or chronic gastritis (CG) confirmed by UGI endoscopy

S Positive for H. pylori by rapid urease test (RUT), serology, Giemsa staining and histological examination.

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Key Exclusion Criteria

S Barrett esophagus

S Esophageal stricture requiring dilation

S Scleroderma

S History of gastrointestinal bleeding or gastric, duodenal, or

esophageal surgery

S Clinically significant diseases involving major organs

S Clinically significant abnormal laboratory values

S Use of a PPI or H2 blocker within 30 days prior to initiating

study treatment

S Pregnant and lactating women18/04/15 114

Trial design

S Single dose for H2 blockers, multiple doses for PPIs

S Crossover design is preferred

S Possible relation to food (a stimulant of acid

secretion)

S Methods to assess anti secretory effects:

- Aspiration of gastric secretion

- Continuous intragastric pH monitoring

- Assessment of the endoscopic appearance of mucosa.

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Trial design

S Aspiration of gastric secretion

S Nasogastric tube positioned in to antrum of the

stomach under fluoroscopic control

S Aspiration performed manually with syringe or by

continuous or intermittent aspiration with low

pressure suction pump.

S The basal gastric acid secretion rate is variable.

S Circadian rhythm variable in healthy subject

S Maintain the I.V rehydration and urinary flow18/04/15 116

Trial design

S Patient aspiration technique is difficult especially

after antisecretory drug (stimulus used: Histamine or

Pentagastrin)

S Aspiration secretion is normally collected in to 15

minutes collection period and pH and volume are

noted

S Useful for measuring pepsin and IF also.

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Trial design

S Continuous intragastric pH monitoring

S Nasogastric electrode tip is positioned in the body of

stomach under fluoroscopic control

S Intragastric content not interfered with aspiration

S Subject can be ambulatory and food / drink may be

taken.

S Inability to measure minute change in the volume of

secretion

S Marked intersubject and intrasubject variability-

larger number of the subject required18/04/15 118

Trial design

S Assessment of the endoscopic appearance of the

mucosa:

S Examination performed at the beginning and end of

treatment period

S Documented by photography

S Even healthy asymptomatic volunteer may show

gastric lesion (15 – 30 % )

S No universal accepted scale for quantification of

mucosal damage

S Study can not be easily compared- subjective18/04/15 119

Trial design for Evaluation of

Cytoprotective agent

S The protective action is independent of any inhibition of acid secretion

S Result of therapeutic studies generally disappointing

S Methods used:

1. Inhibition of aspirin induced gastric bleeding.

2. Endoscopic appearance of mucosa.

3. Inhibition of changes in mucosal potential difference

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Trial design

S At the entry, clinical symptoms, demographic data and

medical history were recorded, and gastroscopy was

performed to establish the endoscopic diagnosis and

status of H pylori infection.

S During the gastroscopy examination, four biopsy

specimens were taken from stomach: one for a rapid

urease test (RUT), one for silver or modified Giemsa

staining, and two for histological examination. Serum

anti-H pylori IgG antibodies were also detected

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S Patients were followed up on the 7th day to check

clinical symptoms, side effects and compliance.

S Study assessments were made at baseline, after

completion of the 7-day study treatment, and at 28

days after treatment completion

** Drug-drug interactions while studying efficacy of

H. pylori combination treatment

Trial design

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Trial design

S At endoscopy (baseline and end of treatment),

two antral biopsies were taken at 5cm from the

pylorus on the greater curve and placed

together on a CLOtest® slide

S All patients underwent 13C-UBT, which was

performed at least 2 hours after endoscopy at

baseline and 4-8 weeks after completion of the

therapy.

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Outcome measures

S Healing of ulcer confirmed by GI

endoscopy.

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Safety Assessment

S Complete physical examination

S Adverse events monitoring

S Routine laboratory evaluations (including

hematologic testing, serum biochemistry

testing, urinalysis, pregnancy testing in

women etc)

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Efficacy assessment

S Daily occurrence of heartburn

S Complete resolution of heartburn

S Average daily antacid use

S Rate of recurrence- for long term

assessment

S Quality of life analysis- LPR-HRQL18/04/15 126

Fallacies of Clinical Methods

S Ulcer patients secrete more acid than control

population.

S studies are usually completed quicker than in

patients

S No universal accepted scale

S Study can not be easily compared- subjective

criterias, anxiety of participants due to

procedures18/04/15 127

Conclusion

S Animal models have provided an invaluable insight into the patho-physiology of the disease and contributed to the discovery of number of therapies.

S We must remain vigilant for any off-label use or any misuse/overuse potential

S The future is likely to see safer and more effective therapeutic options to treat subjects with PUD

S The field is ripe for more research and the market is open for further drug developments

Thank You!!

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Evaluation of anti ulcer agents_Dr. Mansij Biswas

Health & Medicine

Transcript of Evaluation of anti ulcer agents_Dr. Mansij Biswas