Screening Methods for Antiulcer Agents

SCREENING METHODS FOR ANTIULCER AGENTS

An ulcer is a local defect, or excavation of the surface of an organ, or tissue, which is produced by the sloughing of inflammatory necrotic tissue. The term ‘peptic ulcer’ refers to a group of ulcerative disorders of the upper GIT, which appears to have in common, the participation of acid pepsin in their pathogenesis. A peptic ulcer probably results due to an imbalance between aggressive (acid, pepsin, and H. pylori) and defensive (gastric mucous, bicarbonate secre-tion, prostaglandins, innate resistance of the mucosal ceils) factors. In gastric ulcers, acid secretion is normal or low.

Pylorus Ligation in Rats

This principle is based on ulceration induced by accu-mulation of acidic gastric juice in the stomach (Shay et al. 1945).

The requirements include: stereo microscope, adult albino rats (150–170 g), anaesthetic ether, plastic cylinder, 0.1 N sodium hydroxide.

Adult albino rats weighing 150–170 g are starved for 48 h although they have access to drinking water. Normally ten animals are used per dose and as control. After they are ether anaesthetized, a midline abdominal incision is made and the pylorus ligated. The abdominal wall is closed by sutures and test compounds are given either orally by gavage or injected subcutaneously. The animals are placed for 19 h in plastic cylinders with an inner diameter of 45 mm being closed on both ends by a wire mesh. The animals are then sacrificed using carbon dioxide anaesthesia. The abdomen is opened, and a ligature is placed around the aesophagus close to the diaphragm. The stomach is removed and the contents drained into a centrifuge rube. Along the greater curvature, the stomach is opened and pinned onto a cork plate. The mucosa is then examined with a stereo microscope.

The evaluation is done by counting the numbers of ulcers and the severity graded according to the following scores:

0 = no ulcers; 1 = superficial ulcers; 2 = deep ulcers; 3 = perforations

The volume of gastric content is measured after cen-trifugation. Acidity is determined by titration with 0.1 N NaOH. Ulcer index U is calculated using the following formula:

U₁ = U_n + U_s + U_p × 10^–1

where U_n = the average number of ulcers per animal, U_s = average of severity score, and

U_p = percentage of animals with ulcers

Ulcers Through Immobilization Stress

The principle behind this method involves psychogenic factors, such as stress, which play a major role in the pathogenesis of gastric ulcers in man.

The requirements include: adult albino rats, anaesthetic ether, CO₂ anaesthesia, and a stereo microscope.

A group of 10 adult albino rats (150–170 g) per dose of the test drug and for controls are used. Food and water are withdrawn 24 h before the experiment. After oral and sub-cutaneous administration of the test compound or a placebo solution, the animals are slightly anaesthetized with ether. Both the upper and lower extremities are fixed together and the animals wrapped in wire gauge. They are horizontally suspended in the dark at 20°C for 24 h and finally sacrificed using carbon dioxide anaesthesia. The stomach is removed, fixed on a cork plate, and the number and severity of the ulcers registered with a stereo microscope.

Stress Ulcer by Cold Water Immersions

The principle behind this assay is that cooling the rats in water when they are restrained according to the previous model accelerates the occurrence of gastric ulcers and shortens the time of necessary immobilization. In this model, the gastric ulcer formation is mainly due to gastric hypermotility, which could lead to mucosal over-friction.

The requirements include: wistar rats, cages, Evans blue dye, 2% formol saline, CO₂ anaesthesia, and a magnifier.

Groups of 8–10 wistar rats weighing 150–200 g are used. After oral administration of the test compounds, the rats are placed vertically in individual restraint cages in water at 22°C for 1 h. They are then removed, dried, and injected intravenously through a tail vein with 30 mg/ kg Evans blue. After 10 min, they are sacrificed using CO₂ anaesthesia and their stomachs removed. Formol saline (2% v/v) is then injected into the totally ligated stomachs for storage overnight. The next day, the stomachs are opened along the greatest curvature, washed in warm water, and examined under a threefold magnifier.

The evaluation is done by measuring the lengths of the longest diameter of the lesions. This is summated to give a total lesion score (in mm) for each animal; the mean count in control rats should be about 25 (range 20–28). Inhibition of the lesion production is expressed as a per-centage value.

Indomethacin-induced Ulcers in Rats

This assay is based on the fact that use of nonsteroidal anti-inflammatory agents like indomethacin and acetyl salicylic acid induces gastric lesions in man and in experimental animals by inhibition of gastric cyclooxygenase.

The requirements include: wistar rats, 0.1% tween 80, 2% formol saline, and CO₂ anaesthesia.

Groups of 8–10 wistar rats weighing 150–200 g are used. The test drugs are administered orally in 0.1% tween 80 solutions 10 min before an oral administration of indo-methacin (20 mg/kg). After 6 h, the rats are sacrificed in CO₂ anaesthesia and their stomachs removed. Formol saline (2% v/v) is then injected into the totally ligated stomachs for storage overnight. The next day, the stomachs are opened along the greatest curvature, washed in warm water, and examined.

The mean score is calculated in control rats. It should be about 25 (range 20–28). Inhibition of the lesion production is expressed as a percentage value.