Indian Journal of Ex perimental Biology Vol. 42, July 2004, pp. 667-673

Effect of 5-lipoxygenase inhibition on events associated with inflammatory bowel disease in rats

Vijay Pal Singh, Chandrashekhar S Patil & Shrinivas K Kul karni *

Pharmaco logy Division, Uni versi ty Institute of Pharmaceuti cal Sc iences, Panjab Uni versity, Chand igarh 1600 14

Received 24 October 2003; revised 27 April 2004

Leukot rienes playa part in infl am matory response. The unique ro le of the enzy me 5- lipoxygenase (5-LOX) in the producti on of leukotrienes makes it a li ke ly therapeuti c target fo r inflam matory conditions li ke asthma, rheumatoid arthritis, psori as is, and inflam matory bowel disease (I BD). The aim of the present study was to eva luate the effec t of zil euton, an orally acti ve se lecti ve 5-LOX inh ibitor agai nst the events associated with dex tran sod ium sulphate- induced coliti s in a rat model of IBD. The ani ma ls were admi nistered simultancously zil euton ( I OOmg/kg) or sul phasa lazine ( I OOmg/kg) orall y fo r 7 days. On day eight, rats were sac rificcd, and di stal colon isolated to determine myeloperox ida e ac ti vity, ill piva

superoxide dismutase ac ti vity, prostag landin E2 levels and histologica l examination. Both zileuton and su lphasalazine sign ificantly prevented the developmen t of inflammatory eve nts assoc iated wit h coliti s. The effect of zi leUlon was more pronounced towards reducing myeloperox idase ac ti vity and increas ing PGE2 kvels in distal co lon. The resul ts show that chemotac ti c leukotrienes are responsible for infl am matory surge in da maged colon and, zileuton , significantl y improved hea ling by inh ibit ion of neutrophil rec ru itment and indirectly through increase in prostaglandins at the site of infl ammat ion. It is suggested that inhi bitors of 5-LOX enzy me may have useful therapeutic role in the treatment of chroni c intestinal infl ammati on.

Keywords: Infl ammatory bowel ci isease, 5-Lipoxygenase: 5-LOX , Co liti s, Zileuton

Infl ammatory bowel di sease (lBO) is a general te rm fo r a group of chro ni c infl ammatory d isorders of gastro intestinal tract [Ulcerat ive coliti s (UC) and Crohn 's d isease (CD)] I. The pathological hallmark of IBO is marked by the presence of mucosal ul cerati on, infiltratio n of the mucosa with neut rophil s and abdominal di scomfo rt or pain with a lte red bowel habits (di arrhea, constipation or alte rcating episodes of di arrhea ewd constipationf T hough eti o logic agents as we ll as the chem ical medi ators to thi s multi factoria l disorder are unknown and largely undefined, ro le of numero us inflammato ry medi ators in both acute and chro nic fo rms is recogni zed. Products of arachido ni c ac id (AA) metaboli sm (prostag landins: PGs and leukotri enes: LTs) are medi ators of intestinal inflammato ry response that initi ate and maintain the sequence of patho logical events of lB 0 '.4.

Leukotrienes, the lipoxygenase product of AA, are one of the vita l med iato rs highlighted in infl ammatory cascade a llied to rE05. Leukotrienes (main ly leukotri ene B~: LT B4) have been fou nd in inflamed

*Correspo ncient mil hor TeleFax : +9 1- 172-2779426 E-mai l: skpu@yahoo.com

colon mucosa in co ncentratio n known to induce deleterio us effects (che mo kines is, cell aggregation, increas ing vascular permeability)4 . These concentratio ns are simil ar to those seen in human IB0 6

. Tn addit ion, drugs known to be e ffecti ve in the treatment o f IBO are capable of reducing intestinal leukotriene productio n in both experimental mode ls of co liti s and human IB07-11

. Consequently, the 5-lipoxygenase (LOX) enzy me responsible fo r the synthes is of leukotrienes has become a target fo r the development of new potentia l anti-infl ammatory drug in ulcerative colitis and other infl ammatory conditio ns 12.

Zileuto n, an orally effecti ve 5-LOX inhibitor has been shown to se lecti vel y inh ibit ill vitro synthesis of LTB4 in a va ri ety of intact ce ll and cell free systems l3. In pati ents with active ulcerati ve co liti s, zileuto n 600 mg, qid has shown to reduce the local re lease of leuko trienes into recta l di alysates l4. However, Iipoxygenase inhibitors such as benoxaprofen and MK-59 1 d id not offer meaningful ad vantage in re li ev ing the rED sy mpto ms I5.1(i . Hendel ef 0/. , demo nstrated no signi ficant re latio nship with IBO activity and a lte ration in mRNA for 5-LOX in colonic biopsies l7 . Thi s suggests a further sc rutiny of potential of 5-LOX inhi bito rs in !BO.

668 INDIAN J EXP SIOL, JULY 2004

The present study has been aimed to evaluate the effect of zileuton in compari son to sulphasalazine in an experimental model of dex tran sod ium sulfate (DSS)-induced colitis in rats.

Materials and Methods Anill1als- Wi star rats weighing 150-200 g body

weight (Central Animal House, Panacea Biotec Ltd. , Lalru) of either sex were used. They were housed in plastic cages in a room at 25°±0.5 °C and 12: 12 hr L:D cycle. Animals were given food and water ad IibitulII . Experimen ts were carried out between 0900 and 1500 hI's. The ex perimental protoco ls were approved by the Instituti onal Animal Eth ics Committee.

Drugs and regilllen-Sulphasalazine (Panacea Biotec Ltd ., New Delhi , Indi a), zil euton (A rchechem, Mumbai India), dextran sodium sulphate (DSS, mol wt 5000) and nitro blue tetrazolium ( BT, Himedia Laboratories Ltd, India) were used. Sulphasalaz ine (100 mg/kg) and zi leu ton ( 100 mg/kg) was suspended in tween 80 and admin istered orally once dai ly in the dose volume of 10 mllkg, 24 hr before the induction of col iti s until the day of sacrifice. DSS (5%w/v) was administered in drinking water.

Induction of inflallllllatory bOlVel disease/experilllental colitis in rats and drug regilllen--For the induction of experimental coliti s, rats received standard meal with normal drinking water replaced with water containing 5% w/v DSS for 7 days . Sulphasalazine and zileuton treated group received standard meal, DSS containing water, and respective drugs orally, once a day. Control rats were given same food, plain water without DSS , and 0.5% CMC orally .

Histopathological evaluations-The dista l colon segments from 0.5 % CMC, DSS, sulphasalazine, and zileuton treated animals were iso lated and immersed in 10% neutral buffered formalin till further process ing. Sections were stained with hemotoxyl in­eosin and in vest igated by li ght microscopy for the presence of inflammatory changes. At least three specimens per treatment were viewed to study the hi stopathological changes.

Myeloperoxidase (M PO) activity--Myeloperoxi­dase activity was determined fo llowing technique reported earlier by Singh et al.18

. The isolated segments from different treatment groups were individually homogenized in 5 ml of phosphate buffer (0.0 I M) . Homogeni zed tissue was centrifuged at

10,000 rpm. Supernatant was mixed with 0-

phenylenediamine (600 ~g/ml in phosphate buffer) and 300 mM of H20 2 was added to initiate the reaction. Absorbance was observed at 492 nm at an interval of 30 sec for 5 min. Change in the optical density per minute was calcu lated and the results were expressed as percentage increase of myeloperox idase activity over the control.

III situ detection of superoxide (levels of oxidative stress)-Tissue superox ide es timation was perfo rmed as per Hagen et al. 19 using itro Blue Tetrazoliu m (NBT) perfusion. In the presence of superox ide, BT forms an insoluble blue formazan precipitate withi n the tissues. The method was adapted as follows. The animals from different treatment groups were anes thetized (thi opental sod ium 25 mg/kg ip) , a mi dline laprotomy was performed and a solution of 5ml 1% NBT in 0.9% sod ium chloride (NaCI) at 3r C containing 0.2 mg/ml heparin was slowly (w ithin 2 mi n) perfused in the dorsa l aorta. This was fo ll owed by perfusion of 5 ml 0.9% NaCI at 37°C. The animals were killed immediately, the distal colon regions isolated and placed on a non-absorbent surface. They were then flu shed with 0.9% NaCI (4°C) and fixed in neutral buffered forma lin till fu rther processi ng. Sections were stained with hemotoxy lin-eosin for hi stological examination. The number of formazan prec ipitates in a total of -270 mm2 colonic mucosa was counted and expressed as number of cell s containing formazan prec ipitates/lOO mm" of the mucosa.

PGE2 levels ill distal colon- The PGE} were determined by modification in the method reported earl ier by Tries et al.2o for determination of eicosanoids in the infl amed paw. Briefl y, seven days after DSS or drug treatment the distal colon was iso lated, and homogeni zed in ice cold buffer containing 33% (v/v) acetonitrile and 67% (v/v) of di sodium hydrogen orthophosphate (Na2HPO.j). The fin al solution was brought to pH 3 with 100 mM/o­phosphoric ac id. After centri fugat ion for 30 mi n (4000 g, 4°C), the supernatants were collected and kept at -I3°C until extraction of eicosanoids. Extraction of eicosanoids was performed using C 18 columns (Orochem Technologies, USA). The columns were initially saturated wi th the mob ile phase, sample applied and then washed with water, ethanol 10% and hexane successively twice and finally eicosanoids were eluted with methyl formate. The solvent was evaporated with N2 under pressure

SINGH ef af.: 5-L\POXYGENASE INHIBITION AND INFLAMMATORY BOWEL DISEASE 669

and the residual thus obtained was resolved using reverse phase HPLC (Agilent Technologies, USA) and PGE2 levels were quantified. The results are expressed as ng/gm of colon weight.

Statistical allalysis--Data presented as mean ± S.E. were analyzed using one-way ANOYA followed by post hoc Dunnett's test. P<O.OS was accepted as the level of significant difference compared to time matched controls.

Results Morphological chal/ges · and histopathological

examination-All the rats treated with DSS developed noticeable inflammation in the distal colon;

the middle and the proximal colon had fewer lesions . DSS-evoked colitis was characterized by thickening of muscles, shorten'i'ng of crypts, edema, a massive immune cells infiltration, ulceration and significant areas of complete epithelial rupture (Fig. 1 a,b).

Following sulphasalazine or zileuton treatment for 7 days, the hyperemic and ulcerative changes were almost normal. The crypts length was normali zed with absence of inflammatory changes (Fig, 1 c,d).

MPO actlvlty and oxidative stress-Myeloperoxidase activity in animals with DSS was found to be 2.5 times higher than the control value. The increase 1Il the myeloperoxidase activity was significantly (P < 0.05) reduced 1Il zileuton

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;, '::-~ rJ~N""U- ~ ..... ~:-. ~----________ ~ ________ . ___ ,J __ ~ __

Fi g. I- Mi cropholographs of di slal colonic segments from (a) cont rol rats, (b) rats treated with DSS, (c) DSS and sulphasal az ine (100 mg/kg), and (d) DSS and zileuton (100 mg/kg), [m: mu sc le, small arrow heads denote edema, large arrow heads indicate inflammatory infiltrate. x 125J,

670 INDI AN J EXP BIOL, JULY 2004

(100 mg/kg) or sulphasalazi ne (100 mg/kg) treatments. However, zileuton was more effective in reducing MPO actlVlty when compared to sulphasalazine (Fig. 2).

NBT especially forms insoluble form azan precipitates in presence of superoxide l 9

. NBT perfusion led to the formati on of formazan precipitates in the distal colon segment of OSS-treated rats. These precipitates were strictly localized inside the goblet cells of the colonic mucosal (Fig. 3). Formazan precipitates were increased twice the control value with OSS. Co-administration of sulphasalazine significantly prevented the increased formation of formazan precipitates by NBT, whereas zileuton had no significant effect as compared to OSS-treated group (Table 1).

Eicosanoid lellels-PGE2 levels were estimated in the distal colon segments isolated after 7 days . Co­admin istration of zileuton significantly increased PGE2 levels to 132.25±8.80 ng/g tissue (liS. OSS treated rats; 99.22 ± 3.32 ng/g ti ssue). The change in the PGE2 levels with co-administration of sulphasalazine was not significant (106.28±6.03 ng/g tissue liS. OSS-treated rats 99.22 ± 3.32 ng/g tissue) (Fig. 4) .

Discussion The etiology of inflammatory bowel disease

remains undefined, but it is recognized that during IBO, inflammatory mediators (prostaglandins,

350 ...

~ 300 e..-1/1 Qj 250 > a ~ 0 200 a.. a ~

150 .£ Q) 1/1 100 ro r=-Q)

n E 50

0 Control OSS 5% Ziieulon (100) Sulphasalaz ine

(100) Treatment (mg/kg,po)

Fig. 2-Effect of DSS and co-admin istration of sulphasalazine and zileuton on myeloperoxidase activity (MPO) in d istal colon segment. P*<O.05 as compared to plain water (contro l) group, a < 0.05 as compared 10 DSS-treated group. The numbers in the paren thesis are dose in mgfkg body weight (Basal MPO levels considered 100%) .

th romboxanes and leukotrienes) are generated within the colonic mucosa2.3. Recent experimental studies have demonstrated the role of 5-LOX enzyme metabolites in IBO because of their potent stimulatory activity on inflammatory cells. Indeed, elevated concentration is detected il~ dialysates and biopsies of patients with active ulcerative co liti s2o.21. In particular, LTB4 has been reported to account for majority of chemotactic activities detected in rectal dialysis4

•

Therefore, in the present study zileuton, a spec!fic 5-LOX inhibitor was evaluated in well-characterized model of DSS-induced .colitis in rats. This model shares many of the histopathological features of human ulcerati ve colitis and Crohn's disease. Oral administration of OSS to rodents results in overt inflammation in the mid di stal colon that is reminiscent of human IB022.

Fig. 3-Formazan blue precipitates after 7 days in distal colonic mucosa of rats treated with DSS. [The anesthe ti zed an imals were perfused with a solution contain 1% NBT before being ki lled. x250].

Table I- Number of formazan precipitates per 100mm2 of colonic mucosa [ values are mea ± S.E.J

Treatment

Contro l DSS Zileuton (100 mgfkg. po) Sulphasalazine ( IOOmgfkg, po)

No. formazan prec ipitates per

I 00mm2 of colonic mucosa

438.2± 67 .33 840.6 ± 82.44* 6 16.0 ± 55.89

457.0 ± 49.87 "

*P: <0.05 as compared to control, "<0.05 as compared to DSS treated rats

SINGH el al.: 5-L1POXYGENASE INHIBITION AND INFLAMMATORY BOWEL DISEASE 671

160 ~

E 140 Cl

'Qi 120 ~

c: 0

100 '0

'" '0 80 ~ .s 60 III

j 40 ~

:g 20 Il.

0 Control DSS 5% Zil-3uton (100) Sulphasalazine

(100) Treatment (mglkg, po)

Fig. 4--Effect of sulphasalazine, and zileuton on PGE2 levels (n = 5-6). [Vertical lines show mean ± S.E. p ' < 0.05 as compared to DSS-treated group. The numbers in the parenthesis indicate dose in mg/kg body weight.]

In the present study, chronic administration of zileuton normalized the colonic tissue architecture and significantly reduced MPO without significantly affecting SOD activity. The assessment of MPO actIvIty as an index to quantify intestinal inflammation and leukocyte accumulation has been proposed23

. MPO is an enzyme found in the neutrophils and in smaller quantities in the monocytes and macrophages . It has been reported that MPO activity is directly proportional to the number of neutrophils seen in histological sections24 . The reduction in the MPO activity by zileuton suggests a reduction in neutrophil infiltration in the colon. In fact, neutrophils appear to be affected by increased concentration of the powerful chemotactic LTs (most notably LTB4)24. Therefore, it appears that zileuton ameliorates inflammation in colitic rats through down-regulating the recruitment of neutrophils to colon via inhibiting production of 5-LOX pathway metabolites.

However, chronic administration of zileuton also caused an increase in colon PGE2 concentration. It therefore appears that zileuton treatment leads to a shift in arachidonic metabolism towards production of prostaglandins (PGs), which in addition to reduction in MPO activity, may have played a role in the accelerated mucosal healing seen with thi s agent. A role of prostaglandins (namely PGE2) has been indicated in OSS-induced colitis25 . The PGs playa key role in the maintenance of the mucosal integrity.

Administration of minute quantities of prostaglandins has been shown to provide protection against several experimental colitis models, which is suggestive of protective role of PGs in mucosal defense26.27 . This shift of AA metabolism with 5-LOX inhibitor has been identified in some of the studies28.29 but not significantly in others3o,31.This discrepancy in reports may relate to the time period at which eicosanoid levels were measured. Furthermore, whether this represents a true shift in AA metabolism or stimulation of other enzymes by zileuton further along AA biosynthesis cascade remains to be determined.

To further confirm the hypothesis that a relation exists between leukotrienes and leukocyte recruitment in lBO, rats were subjected to OSS colitis and sulphasalazine, which is well established in the management of human IBO. It acts as an inhibitor of prostaglandin synthase, 5-LOX and as a free radical scavenger32,33. In the present study sulphasalazine significantly reduced MPO and SOD activity and increased PGE2 contents. However, the effect of sulphasalazine was less marked for MPO activity and PGE2 contents than that of zileuton. These findings further support the notion that the main ' eff~ct of zileuton is on the synthesis of LTs and indirectly on the neutrophil accumulation.

Randomized, double blind clinical trials with zileuton (600mg qid and 800 mg bid) have shown that the drug is well tolerated and is associated with a trend toward clinical benefit34 . Zingarelli et al. showed zileuton to be effective in attenuating the coli tic lesions in trinitrobenzene sulphonic ac id (TNBS)-induced IBO in rats35. Other studies with zileuton have shown only marginal benefits, which could be due to low doses that were used36. Preliminary study with other 5-LOX inhibitor, BWA4C has shown in vitro reduction in LTB4 in colorectal biopsy specimens37.

In conclusion, the results of the present study clearly show that chemotactic leukotrienes are responsible for inflammatory cascade in damaged colon and, zileuton a 5-LOX inhibitor, significantly improved healing in OSS-induced colitis in rats by inhibition of neutrophil recruitment and indirectly through increase in prostaglandins (which , may be protective) at the site of inflammation.

Therefore, inhibitors of 5-LOX enzyme may be useful therapeutic tools in the treatment of chronic intestinal inflammation .

672 INDIAN J EX P BlOL, JUL Y 2004

Acknowledgement Grant support provided by Panacea Biotec Ltd. ,

New Delhi, India to the author (YPS) as Research Associate (RA) is appreciated .

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