In vivo evaluation techniques for antiulcer

In vivo evaluation techniques for antiulcer

1. In vivo EVALUTION TECHNIC FOR ANTI-ULCER

2. IN VIVO METHODS Pylorus Ligation in Rats Stress Ulcer Model Histamine-Induced Gastric Ulcer Ethanol-Induced M ucosal D amage Acetic Acid-Induced Gastric Ulcer Reserpine-Induced C hronic U lcers Cysteamine -Induced Duodenal Ulcer Demaprit -Induced Duodenal Ulcer Mepirizole -Induced Duodenal Ulcers Experimental Colitis

3. PYLORUS LIGATION IN RATS Shay et al. (1945), OLDEST ANIMAL MODEL OF GASTRIC ULCER Principle: P ylorus 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 .

4. 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.

5. 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 I ndex is calculated and the Ulcer Severity graded.

6. Ulcer classification: Shay et al (1945)

7. Ulcer index Method 1 : The ulcer index is calculated as: Ulcer Index = 10 / X Where X = Total mucosal area / Total ulcerated area

8. Method 2 : Ganguly and Bhatnagar extended above criteria for inclusion of petechiae . Five petechiaes are considered to be equivalent to 1 mm of ulcer area. Ulcer index calculated as described previously.

9. 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 ulcer

10. Ulcer severity score 18/04/15 10

11. Other methods Ulcer Severity Score

12. Ulcer incidence & grading: Wilhelmi and Menasse -Gdynia (1972 ) 18/04/15 12 Quantification of drug induced mucosal damage Ulcers – necro-haemorrhagic spots > 2mm diameter

13. 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

14. Inference Ulcer index of test drug compared with control group to detect anti-ulcer effect of test drug.

15. 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.

16. 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

17. 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

18. 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

19. 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).

20. 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.

21. Demaprit -induced Duodenal Ulcer del Soldato P (1982) Principle: H 2 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.

22. 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.

23. 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

24. Leads to, ulceration in proximal duodenum and erosions in antrum . Anti ulcer therapy started 24 hrs after Mepirizole administration 11 th 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.

25. 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.

26. 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 .

27. 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% NaCl solution, 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.

28. 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

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

30. 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.

31. 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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