Effect of Licofelone Against Nsaids-Induced Gastrointestinal Ulceration and Inflammation

Indian Journal of Experimental Biology Vol. 43, March 2005, pp. 247-253

Effect of licofelone against NSAIDs-induced gastrointestinal ulceration and inflammation

Vijay Pal Singh, Chandrashekhar S Patil & Shrinivas K Kulkarni* Pharmacology Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160 014

Received 16 September 2004; revised 16 November 2004

The present study was aimed to evaluate the effect of licofelone, a dual inhibitor of cycloxygenasel/2-5-lipoxygenase against indometh acin-induced gastric damage in rats and mice in order to assess the role of leukotrienes if any, in non­ steroidal anti-inflammatory drugs (NSAIDs)-induced gastrointestinal inflammation. Acute pretreatment with licofelone reversed th e indomethacin-induced gastri c ulceration, neutrophil adhesion in mesentery venules, neutrophil count in blood, lipid peroxides and vascularity in the stomachs of mice and rats. Further, chronic pretreatment of li cofelone also prevented indometh acin-induced gastric morphological changes and cellular infiltration in mesentery venules. Moreover, acute

administration of indomethacin elevated leukotriene B4 levels in gastric mucosa, which was reversed by pretreatment with li cofelone The results suggest th at li cofelone offered gastroprotection against NSAIDs-induced gastropathy through its effect on leukotrienes and by inhibiting extravasation of neutrophils.

Keywords: Gastri c ulceration, NSAIDs, Licofelone, LTB4 . LOX, Neutrophils

Gastrointestinal (Gl) damage induced by nonsteroidal by NSAIDs. The circulating activated neutrophils anti-inflammatory drugs (NSAIDs) is one of the most appeared to be important in the development of frequent associated adverse effects. These changes are gastric erosions, and changes in vascular integrity 9 presumably due to inhibition of cyclooxygenase following NSAIDs treatment . Indeed, depletion of (COX), the enzy me responsible for conversion of blood neutrophils or inhibition of their adherence is 10 arachidonic acid (AA) to prostaglandins (PGs) that reported to prevent NSATD-gastropathy . Also, the are needed to maintain the integrity of gastric role of neutrophil derived oxygen free radicals in mucosa'. However, the postulate that COX inhibition NSAIDs-induced disruption of gastric membrane 11 by NSAIDs diverts AA metabolism to 5-lipoxygenase integrity is documented . (5-LOX) pathway, suggests the possible role of Licofelone (ML3000), a dual inhibitor of COX/5- leukotrienes (LTs) in vascular changes and mucosal LOX, has been reported to possess a potent 2 damage associated with the use of NSAIDs . Thus, antiinflammatory effect and a favorable 12 13 dual inhibitors of COX/LOX pathway, which would gastrotolearbility properties ' • The present study has be gastrofriendly whilst retaining anti-inflammatoy been aimed to evaluate effect of licofelone, dual effect, were developed. However, role of L Ts in COX/LOX inhibitor against the events and on the NSAIDs-induced gastropathy is debatable. Both, effectors associated with NSAIDs-induced gastric supporting and opposing reports on the effect of 5- damage in rats and mice. LOX inhibitor and leukotriene receptor antagonists against NSAIDs-induced gastric damage are Materials and Methods 3 7 available - . LTs cause chemotaxis, degranulation of Animals-Swiss mice (22-25 g), Wistar rats (180- neutrophils, increase vascular permeability and affect 200 g) (Central Animal House, Panacea Biotec Ltd., 8 vascular tone . Neutrophils have been implicated as Lalru) of either sex were used. They were housed in mediators of the epithelial cell and microvascular plastic cages at room temperature (25±0.5°C) and dysfunction associated with several models of 12:12 hr L:D cycle. Animals were given food and gastrointestinal mucosal injury including that induced water ad libitum. Experiments were carried out between 0900 and 1500 hr. The experimental *Correspondent author TeleFax : +91-172-2779426 protocols were approved by the Institutional Animal E-mail:skpu @yahoo. com Ethics Committee. 248 INDIAN J EXP BIOL, MARCH 2005

Drugs and regimen-Licofelone, indomethacin, and were placed on a heated (37°C) platform used to and naproxen (Panacea Biotec Ltd., New Delhi, adapt a microscope for intravital microscopy. A India), were obtained in house. All drugs were midline laparotomy was performed and a segment of suspended in Tween 80 and administered orally in the mid-jejunum was exteriorized and placed over an dose volume of 10 mllkg. Schematic drawings optically clear saline-coated cover slip in such a way representing the drug regimen in early and late events that a clear area of mesentery was within the focal of indomethacin-induced gastroinflammation is given area. The tissue was covered with heated (37°C) in Fig1a and b. The dose selection was based on phosphate buffered saline (PBS) and saline moistened 9 12 earlier studies • . gauze was placed over the exposed intestine to Visual gastric lesions--Gastric score was prevent the ti ssue from dehydrating and to hold the 14 calculated by method of Chan et al • After 4 hr of tissue in the position. Single unbranched post­ dosing, animals were sacrificed and stomach was capillary venules approximately 25-35 J.lm in diameter excised along its greater curvature. After rinsing with were used for these studies, since they are transparent normal saline, the mucosa was examined for the enough to observe blood flow and leukocyte adhering 15 presence of petechiae or frank hemorrhage lesions. to the endothelial wall of the vessel . The preparation Petechi ae were assigned a score of 1, and lesions were was allowed to stabilize for-8-10 min and mesenteric scored according to their length (a score of 5 for circulation was observed using an microscope (Nikon lesions with length between 1 and 3 mm; a score of 10 Eclipse E600). A camera (Nikon DMX 1200F) for lesions greater than 3 mm). The sum of total mounted on the microscope projected the image on scores was used for comparison. All treatment groups the monitor. The area of normal blood flow and that were coded to prevent measurement bi as. with leukocyte adherence in mesentery was traced and Intravital microscopic measurement of neutrophil photographed. adhesion in the rat mesenteric venules-After 60 min Measurement of lipid peroxides and LTB4 in rat of single acute/last chronic dose, the rats were gastric mucosa-Animals were administered vehicle anesthetized with thi opental sodium (25 mg/kg, ip) or test compounds once and killed by cervical dislocation 60 min post-treatment. The gastric mucosa was removed from each stomach and placed in a glass Licofelone (30 mg/kg) tissue homogenezier. Tissue homogenate was t 30 min Tween 80, indomethacin Sacrifice/ Indomethacin (100mg/kg) Anesthetize prepared in a ratio of 1g of wet tissue to 9ml 1.15 % (100mg/kg) KCl for lipid peroxides and in methanol for LTB4

4h estimation. Different group of animals were used for Fasting lipid peroxide and LTB4 measurements. The lipid -4h 60 min peroxides (thiobarbituric acid reactive substances; 16 (TBARS) were measured by method of Wills • For LTB4 measurements the methanol homogenate was Gastric score Visual gastric score, centrifuged (-13°C, 11,000 rpm, 20 min), and L TB.. estimation, and Intravital microscopy for supernatant separated. The LTB4 content was Neutrophil adhesion determined using LCMS/MS (API 3000: PE SCIEX, using turbo ion spray as ion source. Column Anesthetize Tween 80, indomethacin Cl8/5cm/Novopak, mobile phase: 0.001 % (2mg/kg) or Licofelone (10mg/kg) or llcofelone ammonium acetate and acetonitrile in ratio 65:35, (10mg/kg)/indomethacin (2mg/kg) Fasting flow rate 0.2ml/min, injection volume: 50 J.ll). A linearity upto 500 pg concentration was observed. 4days 4h Neutrophil count in blood was carried out at time interval parall el to intravital microscopy. The neutrophil count in blood was determined using auto­ 1. Gastric morphology 2. Intravital microscopy cell-counter (Swelab, Sweden). for Neutrophil adhesion Gastric vascular permeability in mice-The effect Fig. !- Schematic drawin gs representing the experimental design and drug regimen to study (a) early events (b) late events on vascularity was assessed by the formation of associated with indometh acin-induced gastric damage. vascular casts incorporating supra allura red in mouse SINGH et al. : LICOFELONE & NSAIDs-INDUCED ULCERATION AND INFLAMMATION 249

17 stomach • Briefly, mice were fasted for 4 hr and marked cellular infiltration with in the mesenteric administered indomethacin (10 mg/kg, po) or venules (Fig 2). licofelone (10 mg/kg, po)/indomethacin (10 mg/kg, Intravital microscopic evaluation of effects of COX, po). Indomethacin was administered 30 min after or dual COX/5-LOX inhibitor on the neutrophil licofelone. After 30 min of indomethacin, mice were adhesion in the rat mesenteric venules-Single oral anesthetized with thiopental sodium (25 mg/kg, i.p) administration of indomethacin (1 OOmg/kg), produced and peripheral vasodilation was achieved by placing significant number of gastric lesions at 4hr after in an heated jacket at 40°C for 10 min. The induction dosing. In comparison, 30 min prior administration of of peripheral vasodilation in the mice is important in licofelone (30 mg/kg, po) was able to significantly overcoming alterations in peripheral vasomotor tone reduce the indomethacin-induced gastric lesion score and tissue perfusion related to anesthesia and ambient (Fig 3). 18 temperature that may invalidate the results • The cast In another group, I hr after oral administration of was formed by iv injection of 1 ml of 5% supra allura indomethacin (1 00 mg/kg, po) neutrophil adherence red in 10% gelatin at 40°C into the warmed mice. The to mesentery venules was microscopically recorded. carcasses were chilled and the stomach isolated. The Indomethacin induced a significant adherence of tissue was oven dried at 56°C for 48 hr and weighed. neutrophils when compared to the control group (Fig To overcome dye bleaching by the digestion of tissue 4a and b). To study the effect of dual COX and LOX in sodium hydroxide, the dried tissue was instead inhibition, on indomethacin-induced neutrophil papain digested for 24 hr at 56°C in 0.9ml of digestive adhesion, rats were pretreated with licofelone buffer (dithiothreitol 2 mM, disodium hydrogen orthophosphate 20 mM, EDTA 1mM, papain 12 19 U/ml) according to method of Farndale et al • The dye was then dissolved by the addition of 0.1ml of 5M sodium hydroxide, and the digest were centrifuged at 2000 g for 10 min and filtered through a 0.45 p.,m nitrocellulose filter. The dye content of the filtered samples was spectrophotometrically determined at 504 nm. The result was expressed as either mg of dye content per sample or vascularity Fig. 2-Intravital microscope photog;·aphy of rat mesenteric (V.I) as p.,g dye/mg dry weight of tissue. venules (40x) taken after 4 days treatment with indomethacin Statistical analysis-Data presented as mean ± SE (2 mg/kg). [+ cellular infiltration]. was analyzed using one-way ANOV A followed by 35 p < 0 001 post hoc Dunnett's test. P<0.05 was accepted as the p < 0.001 * - 30 IT I level of significant difference compared to time ::;; matched controls. w "'1!.. 25 .§."' 20 Results E 0 Effect of chronic administration of indomethacin, ~ 15 8 and its modification by licofelone of gross :; morphology of stomach and neutrophil adhesion in -~ 10 ii! the rat mesenteric venules---Chronic administration of .. (!) 5 a indomethacin (2 mg/kg, po) for 4 consecutive days was associated with mortality ( 40%) and intestinal 0 n Vehicle lndorrethacin (100) Licofelone (30) Licofelone (30) + perforations (100%) in surviving rats. Licofelone (10 lndorrethacin (100) mg/kg, po) administered daily 30 min prior to Treatment {mg/kg) indomethacin prevented any death and completely inhibited the intestinal perforation. In earlier studies, Fig. 3--Mean gastric lesion scores in rats. following acute administration of indomethacin, licofelone or pretreatment with licofelone (10mg/kg, po) per se is reported to be free licofelone. 1 as compared to vehicle control; "as 20 P

(30 mg/kg, po). Licofelone pretreated animals showed indomethacin (100 mg/kg, po) administration, but not response similar to that of control group (Fig 4c). in licofelone (30 mg/kg, po) or licofelone pretreated Licofelone per se did not induce any neutrophil group (Table 1). adhesion administered upto 100 mg/kg, po. The level of thiobarbituric acid reactive substances Pretreatment with another NSAID, naproxen (100 (TBARS, index of lipid peroxidation) of gastric mg./kg, po) also did not produce any significant mucosa was 0.529 ± 0.05 nmol/mg protein in increase in adhesion when compared with indomethacin group vs. 0.303±0.03 nmollmg protein indomethacin treatment alone (data not shown). in control group. Licofelone per se did not alter the Pretreatment with licofelone (30 mg/kg, po) also TBARS level (0.295 ± 0.04 nmollmg protein). significantly inhibited the indomethacin-induced Pretreatment with licofelone significantly reduced the increase in blood neutrophil count (1.45 ± 0.72 x generation of TBARS (0.319 ± 0.02 nmol/mg protein 103/Jll vs. 4.72 ± 0.98 x 103/Jll in indomethacin group) vs. 0.529 ± 0.05 nmol/mg protein in indomethacin Effect of orally administered indomethacin, or group) (Figs 5,6). licofelone on LTB4 concentration and lipid peroxide Effect of licofelone pretreatment against in gastric mucosa-LTB 4 levels were el evated after ind01iwthacin-induced vascular perrneabilitr--The vascularity (VI) of stomach derived from supra allura red administered with 10% gelatin (iv), was assessed after acute administration of indomethacin (10 mg/kg, po), and pretreatment with licofelone (10 mg/kg, po). Indomethacin resulted in significant (P<0.05) increase in VI when treated to control animals which was significantly reversed by pretreatment with licofelone. Licofelone per se did not affect the VI of mice stomach (Table 2).

Discussion In the present study pretreatment with licofelone reversed the indomethacin-induced morphological changes, gastric ulceration, neutrophil adhesion in mesentery venules. Prior administration of licofelone also caused a significant reduction of blood neutrophil count, and lipid peroxides in rats. In addition, a reduction in mice stomach vascularity was observed. Further, indomethacin treatment caused an

elevation in LTB4 levels in gastric mucosa, which was prevented with licofelone pretreatment. It is suggested that inhibition of COX enzyme by NSAIDs leads to increased flux of AA through lipoxygenase pathway

Table !-Effect of indometh ac in , licofe lone and

licofelone/indomethacin on gastric LTB4 levels [Values are mean± SE]

Treatment (mg/kg) LTB ~ ng/g ti ssue

Vehicle 25.9 ± 3.0 Indomethacin (I 00) 40.2±2.1 * Licofelone (30) 23.2 ± 4.1 Fi g. 4---Intravital microscope photography of rat mesenteric venules (40x) taken after 60 min treatment with a) vehicle, b) Licofelone (30)/ Indomethacin (100) 28 .0±4.21" indomethacin (100 mg/kg), and c) licofelone(30)/indomethacin (100). [Straight arrow:~ clear blood flow through mesenteric P valu es: <0.05''' as compared to vehi cle control; "as compared venule;+ accumulated neutrophils seen as white debris]. to indomethacin SINGH eta/.: LICOFELONE & NSA!Ds-INDUCED ULCERATION AND INFLAMMATION 251

21 with a concomitant increase in LTs (LTB4, and thereby promoting the rolling of leukocytes . This 1 peptidoleukotrienes: LTC4) production . LTB4, a very indicated that licofelone conferred gastroprotection by potent chemotaxin can promote leukocyte recruitment inhibiting the indomethacin-induced increase in 5- by upregulating expression of ~2 integrins on these LOX effectors. Since neutrophils are the major source 21 21 cells . of LTB4 and extravasated neutrophils are implicated Licofelone, a dual inhibitor is reported to suppress in the pathogenesis of gastric damage-induced by 9 in vitro both 5-LOX (IC50 = O.l8fLM) and COX NSAIDs , effect of licofelone on neutrophils was also 2 (IC50 = 0.21 fLM) activit/ , reduce LTB4 1evels in rat studied. paw22 and did not increase the gastric mucosal damage 23 . 1800 in aspirin treated rats In the present study licofelone 10.15 also attenuated indomethacin-induced increased 1600 gastric vascularity in mice. Licofelone is reported to inhibit LTC4 formation in polymorphonuclear cells 1400 24 (IC50 = 3.8 fLM) . Peptido-leukotrienes are known to increase the permeability of the vascular endothelium 1200 and increase P-selectin expression on these cells 1000 0.7 p < 0.001 p = 0.005 0.6 800 c I I * l :s 0.5 - 600 e Q. .§' 04 a 0 400 T T ..L ~03

(/) ~ 0.2 200 al (/) f-- c. u 1.81 2.02 <.06 6.65 8.70 0.1 Do ·u;~ 2 4 6 8 14 16 18 0 c Ql 1750 9.36 Vehicle hdorrelhacin (100) Licofelone (30) Licofelone (30) + .E hdorrethacin (100) G) 1600 Treatment (mg/kg)

Fig. 5--Effect of different treatments on thiobarbituric acid 140Q reacti ve substances (TBARS) in rat gastric mucosa. ? <0.001* as compared to vehicle control; ?=0.005 "as compared to 1200 indomethacin per se treatment. Vertical lines represent mean ± SE (n=5-6). 1000 Table 2- The effect of indomethacin, and pretreatment with licofelone on the tissue vascularity derived from supra allura 800 red dye in mice gastric mucosa [Values are mean± SE] 600 Treatment (mg/kg) Concentration Vascularity index of dye (Jlg) (V. I) Amount of dye 400 (Jlglmg tissue)

200 Vehicle 0.975 ± 0.08 0.0285 ± 0.003 Indometh ac in (10) 4.56 ± 0.23* 0.071 ± 0.003* 0 Licofelone ( I 0) 1.20 ± 0.05 0.030 ± 0.005 2 4 6 8 10 12 14 16 Licofelone (I 0)/ 2.8 ± 0.49" 0.037 ± 0.004" Time, min Indomethacin (10)

Fig. 6--LCMS/MS chromatogram of (a) leukotri ene B4 (RT I 0.15 P va lu es: <0.05* as compared"to vehicle control; "as compared min, Mass selecti on 337.5 to 249.5) (b), leukotri ene internal to indomethacin standard (RT 9.36 min, Mass selecti on 411.1 to 380. 1). 252 INDIAN J EXP BTOL, MARCJ-12005

The effect on neutrophils was observed parallel to diapedesis of inflammatory cells during NSAID the effect on L TB4 levels. Indomethacin caused therapy. However, as observed here and earlier by significant neutrophil accumulation in postcapillary Wallace et al. 13 that licofelone per se did not change mesenteric venules, and increased neutrophil count in gastric leukotriene contents raises the possibilities that blood, which was completely inhibited following 5-LO>;.. could have other likely implication viz. (i) pretreatment with licofelone. Neutrophils by several formation of oxygenation products, and/or (ii) direct possible mechanism contribute to NSAID-induced oxygenation of membrane phospholipids without gastric ulceration. Neutrophils generated free radicals participation of phospholipase during gastric injury. significantly contribute by a (i) direct tissue necrotic Recently, Smolka et al. 32 reported that licofelone 5 26 effece · (ii) and indirectly via oxygen free radicals. inhibits gastric H, K ATPase enzyme and interleukin- These free radicals influence the inactivation of 8 secretion. This could also be an important endothelium-derived relaxing factor and alters the mechanism in licofelone-persuaded amelioration of 27 resistance of gastric mucosa to damage . Also, NSAIDs-induced gastropathy. neutrophils can release protease and lipid mediators that contribute to gastrointestinal ulceration, changes Acknowledgement in mucosal permeability and affect the vascular tone 28 Grant support provided by Panacea Biotec Ltd., and permeability, respec-tively . In the present study, New Delhi, INDIA to one of the au thors (VPS) as licofelone while inhibiting the indomethacin-induced Research Associate (RA) and for LCMS/MS facility neutrophil adhesion also signi !'icantly reduced the is appreciated. corTesponding increase in TIP, RS levels. LTB4 can stimulate the release of reactive oxygen metabolites from neutrophils. Lipid peroxidation (TBARS) References degrades polyunsaturated fatty acids of the cellular 1 Whittle B J R, Arachidonic acid metabolites and the gastrointestinal toxiCity of anti-inflammatory agents, membranes with subsequent disruption of membrane Prostaglandins, 21 (Suppl.) (1981) 113. integrity and its role in pathogenesis of indomethacin­ 2 Rainsford K D, The effects of 5-lipoxygeanase inhibitors and 29 30 induced gastric ulceration is reported · . 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