EVALUATION OF EFFICACY FOR THE TEST FORMULATIONS IN RAT MODEL OF GASTROESOPHAGEAL REFLUX DISEASE

1.0  TEST SYSTEM DETAILS:

Species              : Rattus norvegicus (Rats)

Strain               : Albino Wistar

Age                  : 8-12 weeks

Body Wight      : 250-300 g

Sex                   : Male

No. of animals  : 8 Animals in each group

Total animals    : 56

Study Duration:  28 Days

2.0   ALLOCATION OF GROUPS:

Group No.	Group Description	Disease Induction	Treatment administered	Dose Volume and Route
G1	Sham Control	(Surgery + No Ligation of pylorus and corpus)	0.5% MC	10 ml/kg, p.o.
G2	Disease Control	(Surgery + Ligation) of pylorus and corpus)	0.5% MC
G3	Reference Control		Lansoprazole-1 mg/kg, q.d. in 0.5% MC
G4	Treated with low dose of   Test-X1		Test-X1 mg/kg, b.i.d.  in 0.5% MC
G5	Treated with intermediate dose 1 of  Test-X2		Test -X2 mg/kg,    b.i.d. in 0.5% MC
G6	Treated with intermediate dose 2 of  Test-X3		Test -X3 mg/kg,   b.i.d. in 0.5% MC
G7	Treated with high dose of  Test-X4		Test-X4 mg/kg,  b.i.d. in 0.5% MC

    X1, X2, X3, and X4 are defined as the incremental doses of the Test formulations. MC-Methyl

Cellulose, p.o.-per os. The dose range will be from 10 mg/kg to 1000 mg/kg, q.d: quaque die, bid: bis in die 

3.0  METHOD:

- Animals will be procured, randomized based on body weight and allocated into 7 different groups consisting of 8 animals each.

- Group of animals G1 will serve as sham control group and will be treated with only 0.5% MC, p.o..

- Group of animals G2 will serve as a disease control group and will be treated with only 0.5% MC, p.o..

- Group of animals G3 will serve as reference control group and will be treated with Lansoprazole-1mg/kg, p.o.

- Animals of groups G4 to G7 will be administered Test, orally at different dose levels (X1, X2, X3, and X4) ranging from 10 to 1000 mg/kg, b.i.d.

 -Treatment of vehicle or standard drug or test compounds will be given prophylactically for two weeks.

 -At the day of experiment, animals will be fasted for 24 h prior to surgery, but animal will be provided free access to water.

- After 1h of drugs treatment on the final day, the animal will be anaesthetized with ketamine and xylazine and the stomach will be exposed after a 1 cm incision will be made at the left upper quadrant of the abdomen.

- The duodenal pyloric ring and the border between the forestomach and glandular area of the exposed stomach will be ligated by surgical suture.

- The stomach will be placed to its original position, and the abdomen will be closed using autoclips. After surgery, rats will be not allowed to eat or drink for 4 h.

- After this, rats will be sacrificed by CO2 asphyxiation and their abdomens will be incised. The esophagus will be exposed up to the neck region, and the portions of esophagus near the neck and just below the ligated pylorus near the duodenum will cut to excise both the esophagus and stomach.

 -The gastric contents will be collected and centrifuged at 3,000 rpm for 5 min. Using the supernatant, the amount and pH of the gastric juice will be measured, free and total acid output will be calculated.

 -The stomach will be incised along the greater curvature extending to the esophagus, and a photo will be acquired with a digital camera.

 -The esophagus epithelial tissues will be scraped using a slide glass on ice. After adding lysis buffer (200 mM NaCl, 10 mM Tris, 5 EDTA, 1 mM PMSF, and 10% glycerin, 1% protease inhibitor cocktail), the tissue will be lysed for 1 h on ice.

 -The lysate will be centrifuged for 1,500 ´g, 4°C for 15 min, and the supernatant will be collected for the protein assay.

4.0  END POINT PARAMETER(S):

·        pH of gastric contents

·       Total Acidity

·       Free acidity

·       Determination of Gastrin, Motilin, Vasoactive peptide level in serum.

·       Cytokines in esophageal epithelium (IL1β, IL6 and IL8).

·       Myeloperoxidase Level in Esophagus.

·       Histopathology of Esophagus & Stomach.

 

5.0  REFERENCE(S):

5.1  Mahattanadul S, Radenahmad N, Phadoongsombut N, Chuchom T, Panichayupakaranant P, Yano S, Reanmongkol W. Effects of curcumin on reflux esophagitis in rats. Journal of natural medicines. 2006 Jul;60(3):198-205.

5.2  Kim DK, Lee KH, Kim SJ, Kim SJ, Lee SJ, Park CH, Kim BT, Song GS, Moon BS, Ryu SY. Effects of Tegoprazan, a Novel Potassium-Competitive Acid Blocker, on Rat Models of Gastric Acid–Related Disease. Journal of Pharmacology and Experimental Therapeutics. 2019 Jun 1;369(3):318-27.

5.3  Qiu Y, Hu JL, Zhao CC, Zhang JQ, Wu F, Ma BL, Feng Y, Ruan KF. Zhujie Hewei granules ameliorated reflux esophagitis in rats. Evidence-Based Complementary and Alternative Medicine. 2019 Dec 26;2019.

5.4  M. Salehi, H. Karegar-Borzi, M. Karimi, and R. Rahimi, “Medicinal plants for management of gastroesophageal reflux disease: a review of animal and human studies,” The Journal of Alternative and Complementary Medicine, vol. 23, no. 2, pp. 82–95, 2017.

5.5  D. Asaoka, A. Nagahara, Y. Izumi et al., “Effect of lansoprazole on extraesophageal manifestations of GERD in a rat chronic acid reflux esophagitis model,” Gastroenterology, vol. 134, no. 4, pp. A170–A171, 2008.

5.6  S. Terashima, Y. Matsusaka, H. Nishio, S. Kato, and K. Takeuchi, “Orally administered glycine is highly effective against acid reflux esophagitis in rats,” Gastroenterology, vol. 132, no. 4, p. A490, 2007.

                                                       END OF THE DOCUMENT

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