M.V.Ramana et al. / Journal of Pharmacy Research 2012,5(9),4808-4811 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Effect of Sulfasalazine and against dextran induced in female balb/c mice

Dr. M.V.Ramana2, N.S.V.Mukharjee1, Dr. A. Ramesh, M.Manohar, N.Hindu, T.Vanaja, B.Pavani, N.Santhosh, E.Srikanth, M.Venkatesh, K.Phaneendra, *VVSPC Rao.M *Research scholar, BITS PILANI, Hyderabad campus, Hyderabad,India 1. Assistant professor, G.B.N.Institute of Pharmacy, Hyderabad,India 2. Principal, G.B.N.Institute of Pharmacy, Hyderabad,India Received on:12-06-2012; Revised on: 17-07-2012; Accepted on:26-08-2012

ABSTRACT Sulfasalazine is the most widely prescribed drug for ulcerative colitis (1) (5). Following oral administration of sulfasalazine (5) practically unabsorbed reaches the colon where it is split by colonic bacteria to 5-aminosalicylic acid and sulphapyridine. Prednisolone is absorbed into the blood stream so act indiscriminately throughout whole body. Prednisolone is a which is used in ulcerative colitis. Prednisolone works by preventing or reducing inflammation (4). It is used to treat a number of conditions that are characterized by excessive inflammation. Prednisolone suppresses the immune system (6) (4) and so can be used to treat autoimmune diseases (3) (4). If prolong usage of also leads to their immune system can become weak. The objective of this study was to determine effect of sulfasalazine and Prednisolone against dextran sodium sulphate induced colitis in balb/c mice. We observed the parameters like body weight, colon length, colon weight and IL-6 estimation. We observed there is significant reduction in body weight and colon length and increase in colon weight and IL-6 levels with DSS administration. Our findings suggest that sulfasalazine and Prednisolone produces synergistic effect against dextran sodium sulphate induced ulcerative colitis.

Keywords: Dextran sulfate sodium; ulcerative colitis; sulfasalazine; Prednisolone; immune response.

INTRODUCTION: Ulcerative colitis (UC) is a typical inflammatory intestinal disease (8) be- creased in the inflamed mucosa of patients with UC. Nuclear factor (NF)-kb longing to the class of inflammatory bowel diseases (IBD) (7). The pathogen- (13) performs a crucial function in the expression of many genes involved in esis of UC is believed to involve the interaction of genetic, immune, and immune and inflammatory responses. Active NF-kb-ikb complexes (14) are environmental factors. UC is associated with intestinal inflammation and sequestered in the cytoplasm. On induction by a variety of stimuli, NF-kB often results in weight loss, diarrhea accompanied by blood and mucus, fever, translocates into the nucleus where it can bind to specific DNA sequences gastric dysmotility and shortening of the colon. Pathogenic hallmarks of UC located in the promoter regions of target genes and activate gene transcrip- include ulceration of the mucosa, blunting and loss of crypts, infiltration of tion, thus, by controlling the transcription of inflammatory cytokine genes, it inflammatory cells. The pathologic changes frequently cause epithelial dys- plays a pivotal role in the regulation of immune and inflammatory cytokine plasia and DNA damage with microsatellite instability. Additionally, pro- genes, it plays a pivotal role in the regulation of immune and inflammatory longed and chronic UC (10) may progress to a colorectal cancer .Thus, to responses (15). Increased DNA-binding capacity of NF-kB associated with develop measures to prevent cancer development in UC patients, it is neces- the secretion of high levels of IL-1 and IL-6 has been reported in macroph- sary to understand the pathogenesis of UC at the molecular and cellular ages of UC patients. NF-kB has been recognized as an ideal target for mo- levels. Current studies on UC have reported that proinflammatory cytokines lecular therapies (16) employed to treat inflammatory diseases. Generally, (11) are involved in the initiation of the inflammatory response in colitis (12). UC is characterized clinically by acute exacerbation and corticosteroids may It was reported that mucosa from patients with UC shows increased expres- not be effective even when administered in high doses. In addition, long term sion of interleukins (IL-1 and IL-6), which are believed to play an integral role use of corticosteroids is often associated with serious adverse effects such as in the pathogenesis of UC. Hence, there is currently a strong interest in hormonal disturbances, peptic ulcers, liver dysfunction and psychological agents that block the generation or activities of inflammatory cytokines (18). problems. The appearance of these adverse effects may necessitate discon- Cyclooxygenase (COXs) have been implicated in a number of physiological tinuation of corticosteroid treatment, there by resulting in acute exacerbation events, including the progression of inflammation, immunomodulation (14) (21). Therefore, an alternative treatment for active UC is necessary to elimi- (9) and transmission of pain. Two COX enzymes have been identified. COX- nate the clinical problems associated with corticosteroid therapy (19). 1 is a constitutive enzyme that is required for the formation of (PGs) protecting the stomach and kidney from damage. COX-2, which is MATERIALS & METHOD: normally expressed at very low levels, can be rapidly induced by a variety of stimuli such as cytokines, growth factors, hormones, and carcinogens and is Animal care and use believed to be responsible for the production of prostaglandins associated Female Balb/c mice of (18-22gm) were purchased from national centre for with mediation of inflammation. COX-2 expression is reported to be in- laboratory sciences, National institute of nutrition, Tarnaka, Hyderabad, In- dia. Animals were housed conventionally in an animal room under controlled *Corresponding author. conditions (18-220c, 55±10 humidity, 12 h day/night cycle) and fed with VVSPC Rao standard diet (NCLAS, Hyderabad). BITS, Pilani Hyderabad Campus, Jawahar Nagar, Acute Model: Shameerpet Mandal, Animals were acclimatized for 4 to 5 days prior to the start of the experi- Hyderabad, 500078, ment. Animals were randomly distributed to various groups based on their Andhra Pradesh.India body weight. They were divided into five experimental groups with five Journal of Pharmacy Research Vol.5 Issue 9.September 2012 4808-4811 M.V.Ramana et al. / Journal of Pharmacy Research 2012,5(9),4808-4811 animals in each group, housed in clean filter top cages under COLON LENGTH standard conditions in a 12 hr dark/12 hr light cycle and fed with standard mouse chow. 4 3.42 3.28 2.9 2.86 Group-1 animals were received normal drinking water,

E 3 Group-2 animals were received DSS-5%; 2.14 R

Group-3 animals were treated with sulfasalazine (50 mg/kg) along with DSS- T 5%. E M 2 I

Group-4 animals were treated with predinisolone (0.25 mg/kg) along with T DSS-5%. N E

Group-5 animals were treated with sulfasalazine (25mg/kg) & predinisolone C 1 (0.125 mg/kg) along with DSS-5%

Induction of colitis: 0 i Animals were housed in a clean filter-top cage in a specific pathogen-free L S e e n A S in n d M D z lo e environment, and fed with standard chow and water. Animals were acclima- la o r R a s P O i + tized for about 4 to 6 days before starting the experiment. Animals were N as n a l f d lf u r e u randomly distributed into various groups based on their body weights. S P S + + + s s s s s s 1. Animals were divided into 5 groups, of each group containing 5 D D D animals. Group 2. Acute colitis in mice was induced by providing drinking water ad Fig no.2:Changes in colon length of different groups of dextran sulphate libtium containing 5% DSS for 7 days. sodium induced colitis in mice model.Results were as mean+/-SEM. 3. Mice were checked daily for the loss of body weight & Symp- COLON WEIGHT toms. 0.524 4. Sulfasalazine and Predinisolone diluted with water were orally 0.6 administered once a day from day 0 of DSS treatment. 5. On the 7th day, animals were euthanized and the colon was re- 0.382

moved for the measurement of colon length, colon weight and IL- ) 0.352 m 0.4 0.29

6 levels. g ( t

h 0.204 RESULTS AND DISCUSSION: g i e 0.2

Table no:1 Different groups of animals and changes in different W parameters.

Groups Change in body Colon length Colon 0.0 weight(gms) (cms) weight(gms) i L S e e n A S in n d M D z lo e Normal (negative control) 1.84 3.42 0.204 l a o r R a s P O i + Dss-5% (positive control) 0.86 2.14 0.524 N a s n a lf d lf Dss + Sulfasalazine (50mg/kg) 1.2 2.9 0.332 u re u S P S Dss + Prednisolone (0.25mg/kg) 1.32 2.86 0.352 + + + s s s s s s Dss+Sulfa+Predni(25mg/kg;0.125mg/kg) 1.58 3.28 0.29 D D D Change in body weight GROUP Fig no.3:Changes in colon weight of different groups of dextran sulphate 2.0 sodium induced colitis in mice model.Results were as mean+/-SEM.

250 IL-6 1.5 200

1.0 150 *** *** 100 *** 0.5 50

0.0 0 Chan ge in body weight (gms) Conc entration (Pg/ mg t iss ue)

DSS DSS Normal Normal

Sulfasalzine Sulfasalzine Prednisolone Combination Prednisolone Combination Group Group Fig no.1:Changes in body weight of different groups of dextran sulphate Fig 4:Effect of sulfasalazine and prednisolone on IL-4 in colon.Results were sodium induced colitis in mice model.Results were as mean+/-SEM. expressed as mean=/-SEM.P<0.05 significantly different fron the control group by dunnet’s multiple comparison test. Journal of Pharmacy Research Vol.5 Issue 9.September 2012 4808-4811 M.V.Ramana et al. / Journal of Pharmacy Research 2012,5(9),4808-4811 DISCUSSION: x. PMID 15233681. UC is a chronic inflammatory (2) (4) condition of the colonic mucosa of 5. Orholm M, Binder V, Sørensen TI, Rasmussen LP, Kyvik KO unknown etiology and pathogenesis. Oral administration of DSS (22) (23) (2000). “Concordance of inflammatory bowel disease among Dan- induces colitis resembling UC in humans. In the present study, acute colitis ish twins. Results of a nationwide study”. Scand. J. was induced in balb/c mice with 5% DSS. UC is an idiopathic disease charac- Gastroenterol. 35 (10): 1075–81.Doi: 10.1080/003655200 terized by intestinal inflammation. UC is commonly treated with glucocorti- 451207. PMID 11099061. coids, sulfasalazine and so on, however these drugs cause serious adverse 6. Tysk C, Lindberg E, Jarnerot G, Floderus-Myrhed B effects such as hormonal disturbances, formation of peptic ulcer, liver dys- (1988).”Ulcerative colitis and Crohn’s disease in an unselected function and psychological problems. UC is a chronic intestinal inflamma- population of monozygotic and dizygotic twins. A study of heritabil- tory condition (25) manifested by symptoms including weight loss and ity and the influence of smoking”. Gut 29 (7):990–996.Doi: 10.1136/ bloody diarrhoea. gut.29.7.990. PMC 1433769. PMID 3396969. 7. Baumgart DC, Carding SR (2007). “Inflammatory bowel dis- At the site of inflammation, the recruited cells are activated to release a host ease: cause and immunobiology”. The Lancet 369 (9573): 1627– of inflammatory mediators, including tumor necrosis factor (TNF-alpha), 1640. Doi: 10.1016/S0140-6736(07)60750-8.PMID 17499605. IL-6, and COX-2. These mediators contribute the initiation and progression 8. Baumgart DC, Sandborn WJ (2007). “Inflammatory bowel dis- of the inflammatory process (24) (26). The levels of inflammatory media- ease: clinical aspects and established and evolving therapies”. tors, including COX-2 and IL-6, have been reported to be elevated in colitis The Lancet 369 (9573): 1641–57. Doi: 10.1016/S0140- group animals. 6736(07)60751-X. PMID 17499606. 9. Fauci et al. Harrison’s Internal Medicine, 17th Ed. New York: During DSS treatment, inflammation of colon is enhanced, includes rises in McGraw-Hill Medical, 2008. ISBN 978-0-07-159991-7. the extent of diarrhea and rectal bleeding. Colonic inflammation (27) (20) (25) 10. Roediger WE, Moore J, Babidge W (1997). ”Colonic sulfide in is also characterized by severe lesions throughout the mucosa (3) (21), alter- pathogenesis and treatment of ulcerative colitis”. Dig. Dis. ation of epithelial structure, high-level neutrophil and lymphocyte infiltra- Sci.42 (8): 1571–9. Doi:10.1023/A:1018851723920. PMID tion (28) into the mucosal and sub mucosal areas, and loss of crypts. We 9286219. explored how DSS, a negatively charged polymer of glucose with engrafted 11. S. Kane (2006). ”Asacol - A Review Focusing on Ulcerative Coli- sulfate groups, induces colitis in mice. tis”. 12. Calkins BM (1989). “A meta-analysis of the role of smoking in Dextran sodium sulphate induced colitis in Balb/c mice is a good animal inflammatory bowel disease”. Dig. Dis. Sci. 34 (12): 1841–54.Doi: model to study drug action on experimental ulcerative colitis (29). Since 10.1007/BF01536701. PMID 2598752. impaired colonic regeneration can lead to chronic intestinal inflammation. 13. Lakatos PL, Szamosi T, Lakatos L (2007). “Smoking in inflam- The DSS model in Balb/c mice can be useful to study colonic regeneration and matory bowel diseases: good, bad or ugly?” World J thus contribute to unravelling the pathogenesis of human IBD (17). Gastroenterol. 13 (46):6134–9. Doi:10.3748/wjg.13.6134.PMID 18069751. In the present study a significant reduction in body weight, colon length and 14. Guslandi M (October 1999). ”Nicotine treatment for ulcerative increase in colon weight and IL-6 levels due to inflammation. The change in colitis”. Br J Clin Pharmacol 48 (4):481–4. Doi:10.1046/j.1365- body weight of Prednisolone , sulfasalazine, combination, normal, positive 2125.1999.00039.x. PMC 2014383. PMID 10583016. control groups was found to be 1.32, 1.2, 1.58, 1.84, 0.86 in grams respec- 15. Sandborn WJ, Tremaine WJ, Offord KP et al (March 1997). tively, change in colon length of Prednisolone, sulfasalazine, combination, “Transdermal nicotine for mildly to moderately active ulcerative normal, positive control groups was found to be 2.86, 2.9, 3.28, 3.42, 2.14 in colitis. A randomized, double-blind, placebo-controlled trial”. Ann. cms respectively, change in colon weight of Prednisolone, sulfasalazine, com- Intern. Med. 126 (5): 364–71. PMID 9054280. bination, normal, positive control groups was found to be 0.352, 0.332, 0.29, 16. Bibiloni R, Fedorak RN, Tannock GW et al (2005). “VSL#3 0.204, 0.524 in grams respectively) and IL-6 levels of Prednisolone, probiotic-mixture induces remission in patients with active ulcer- sulfasalazine, combination, normal, positive control groups was found to be ative colitis”. Am. J. Gastroenterol. 100 (7): 1539–46.Doi: 112, 135, 84, 51, 203 pg/mg tissue respectively. 10.1111/j.1572 0241.2005.41794.x. PMID 15984978. 17. Borody TJ, Warren EF, Leis SM, Surace R, Ashman O, Siarakas CONCLUSION: S (2004). ”Bacteriotherapy using fecal flora: toying with human The current results are supportive of the clinical evidence of the combined motions”. J. Clin. Gastroenterol. 38 (6): 475–83.Doi: 10.1097/ treatment of ulcerative colitis with sulfasalazine and Prednisolone had greater 01.mcg.0000128988.13808.dc. PMID 15220681. benefit than sole treatment with these agents alone. 18. Borody TJ, Warren EF, Leis S, Surace R, Ashman O (2003).”Treatment of ulcerative colitis using fecal REFERENCES: bacteriotherapy”. J. Clin. Gastroenterol. 37 (1): 42–7. Doi: 1. Ulcerative colitis at E medicine. 10.1097/00004836-200307000-00012. PMID 12811208. 2. Robert Löfberg. Retrieved Oct 2010 Inflammatory tarmsjukdom, 19. Bensoussan M, Jovenin N, Garcia B et al (January kronisk, IBD 2006).”Complementary and alternative medicine use by patients 3. Hanauer SB; Hanauer, Stephen B. (1996). “Inflammatory bowel with inflammatory bowel disease: results from a postal disease”. N. Engl. J. Med. 334 (13): 841–8.Doi: 10.1056/ survey”.Gastroentérologie Clinique et Biologique 30 (1): 14– NEJM199603283341307. PMID 8596552. 23.doi:10.1016/S0399-8320(06)73072-X. PMID 16514377. 4. Kornbluth A, Sachar DB (2004). “Ulcerative colitis practice 20. Terry PD, Villinger F, Bubenik GA, Sitaraman SV (January guidselines in adults (update): American College of Gastroenterol- 2009). “Melatonin and ulcerative colitis: evidence, biological mecha- ogy, Practice Parameters Committee”. Am. J. Gastroenterol- nisms, and future research”. Inflamm. Bowel Dis. 15 (1): 134– ogy. 99 (7): 1371–85. Doi: 10.1111/j.1572 0241.2004. 40036. 40.Doi: 10.1002/ibd.20527. PMID 18626968.

Journal of Pharmacy Research Vol.5 Issue 9.September 2012 4808-4811 M.V.Ramana et al. / Journal of Pharmacy Research 2012,5(9),4808-4811 21. Akhtar MS, Munir M (November 1989). “Evaluation of the endoscopy in the diagnosis and treatment of inflammatory bowel gastric anti-ulcerogenic effects of Solanum nigrum, Brassica disease”. Gastrointest. Endosc. 63 (4): 558–65.Doi: 10.1016/ oleracea and Ocimum basilicum in rats”. Journal of j.gie.2006.02.005. PMID 16564852. Ethnopharmacology 27 (1–2): 163–76. Doi: 10.1016/0378- 26. R. Koch (2003). Page 481 in: “Colonic diseases”. By 8741(89)90088-3.PMID 2515396. “Brassica oleracea (leaf) pow- Timothy.ISBN 978-0-89603-961-2. der did not affect the ulcer index significantly but its aqueous 27. Reddy D, Siegel CA, Sands BE, Kane S (2006). “Possible associa- extract lowered the index and increased hexosamine levels, suggest- tion between isotretinoin and inflammatory bowel disease”. Am. J. ing gastric mucosal protection.” Gastroenterol. 101 (7): 1569 22. “Fish oil”. MedlinePlus. 28. Corrao G, Tragnone A, Caprilli R et al (1998). ”Risk of inflam- 23. Brzezinski A, Rankin G, Seidner D, Lashner B (1995). “Use matory bowel disease attributable to smoking, oral contraception of old and new oral 5-aminosalicylic acid formulations in inflam- and breastfeeding in Italy: a nationwide case-control study. Coop- matory bowel disease”. Cleve Clin J Med 62 (5): 317– erative Investigators of the Italian Group for the Study of the 23.PMID 7586488. Colon and the Rectum (GISC)”. Int J Epidemiol 27 (3): 397– 24. Gupta, I.; Parihar, A.; Malhotra, P.; Singh, G.; Lüdtke, R.; 404. Doi: 10.1093/ije/27.3.397.PMID 9698126. Safayhi, H.; Ammon, H. (1997). “Effects of Boswellia serrata 29. Dieleman LA, Ridwan BU, Tennyson GS, et al. Dextran sulfate gum resin in patients with ulcerative colitis”. European journal of sodium (DSS)-induced colitis occurs in severe combined immuno- medical research 2 (1): 37–43. PMID 9049593. deficient (SCID) mice. Gastroenterology. 1994;107:1643– 25. Leighton JA, Shen B, Baron TH et al (2006). “ASGE guideline: 52.[PubMed]

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.5 Issue 9.September 2012 4808-4811