An orally active inhibitor of leukotriene synthesis accelerates healing in a rat model of colitis.

An orally active inhibitor of leukotriene synthesis accelerates healing in a rat model of colitis JOHN L. WALLACE AND CATHERINE Gastrointestinal Research Group, University

M. KEENAN of Calgary, Calgary, Alberta

WALLACE,JOHN L., AND CATHERINE M. KEENAN. Anordy active inhibitor of leukotriene synthesis accelerates healing in a rat modeZ of colitis. Am. J. Physiol. 258 (Gastrointest. Liver Physiol. 21): G527-G534,1990.-Leukotrienes (LTs) have been implicated as mediators of the inflammation and ulceration associated with ulcerative colitis and Crohn’s disease. In the present study, the effects of a novel, orally active inhibitor of LT synthesis (MK-886) were examined in a rat model of chronic colitis. Colitis was induced by intracolonic administration of trinitrobenzenesulfonic acid. Colonic LTB, synthesis was measured after incubation of tissue samples in vitro and by in vivo equilibrium dialysis. A single dose of MK-886 (10 mg/kg) significantly inhibited colonic LTB, synthesis for ~24 h. Daily treatment with this dose significantly reduced colonic damage, as assessed macroscopically and histologically, when the treatment was performed 2 h before induction of colitis and daily thereafter for 1 wk, but not when treatment was performed during the second week after induction of colitis. A less marked beneficial effect of MK-886 was observed when the pretreatment dose was excluded, suggesting a role for LTs in the early events of the inflammatory process. Inhibition of LT synthesis during the first 24 h after induction of colitis did not alter the extent of infiltration of neutrophils into the colon, as measured by tissue myeloperoxidase activity. Daily treatment with sulfasalazine (100 mg/kg po) either during the first or second week after induction of colitis did not significantly affect the rates of healing. At the dose used, sulfasalazine only produced a transient inhibition of colonic LTB* synthesis. This study therefore demonstrates that a specific, orally active inhibitor of LT synthesis can significantly accelerate healing in this animal model of colitis when the treatment is performed during the early phase of the inflammatory response. leukotrienes; flammation

inflammatory

bowel

disease;

lipoxygenase;

in-

are mediators of inflammation that are derived from membrane phospholipids, primarily through the actions of the enzymes phospholipase A, and 5-lipoxygenase. In addition to their potent chemotactic and chemokinetic properties, several leukotrienes stimulate smooth muscle contraction and can increase vascular permeability (8). In inflammatory bowel disease (IBD), there is now clear evidence supporting a role for leukotrienes in amplifying the disease process once it is initiated by an as yet unidentified event or factor. Synthesis of leukotriene Bq (LTB4) has been shown to be markedly elevated in samples of colon or in rectal dialysates from ulcerative colitis patients (7, 14). Furthermore, several drugs that have been shown to have beneficial actions as treatments for IBD, including corticosteroids, sulfasalazine, and 5aminosalicylic acid, have

LEUKOTRIENES

0193~1857/90

$1.50

Copyright

T2N 4N1, Canada

been shown to reduce the production of leukotrienes by inflamed colon (7, 12). Lobos et al. (9) recently reported that chemotactic activity (for neutrophils) in extracts of inflamed colonic mucosa correlated well with the concentration of LTB, in the extract. They suggested that LTB, may play an important role in the recruitment of neutrophils into the inflamed tissue in IBD. Animal models have also provided evidence that leukotrienes play an important role in the pathogenesis of colitis (1, 15, 19, 20) dne of the problems encountered in trying to further test the hypothesis that leukotrienes are important mediators in IBD has been the absence of specific inhibitors of leukotriene synthesis. Inhibitors that have been tested in the past either have actions on cyclooxygenase as well as &lipoxygenase, or have potent antioxidant activity. This has made the interpretation of data difficult. Oxygen-derived free radicals have been implicated in IBD (5, lo), so it is entirely possible that the antioxidant activity of agents used as 5-lipoxygenase inhibitors may have contributed to beneficial effects observed with those compounds. Furthermore, a general problem with compounds that act on 5-lipoxygenase through redox mechanisms is their insolubility and poor bioavailability (4). We have previously demonstrated that treatment with a 5-lipoxygenase inhibitor resulted in significant acceleration of healing in a rat model of chronic colitis (19). However, the poor bioavailability of that compound after oral administration made it necessary to administer the compound intracolonically. Such an agent would therefore be limited in its clinical usefulness, since it would have to be administered directly at the site of inflammation. In the present study, we have used a rat model of chronic colitis (11) that has histopathological similarity to human Crohn’s disease to assessthe effects of treatment with a specific inhibitor of leukotriene synthesis. This compound, MK-886, does not act via a redox-related inhibition of 5-lipoxygenase and does not inhibit superoxide anion generation by neutrophils (4). Rather, the compound inhibits activation of 5-lipoxygenase through inhibition of enzyme translocation (4). MK-886 is derived from a series of indol-2propanoic acids and is active after oral administration. METHODS

Animals. Male Wistar rats weighing 225-275 g were obtained from Charles River Breeding Farms (Montreal, Quebec) and were housed in standard wire-mesh cages

0 1990 the American

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Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (155.247.166.234) on July 30, 2018. Copyright © 1990 American Physiological Society. All rights reserved.

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in a room with carefully controlled ambient temperature (25°C) and photoperiod (14:lO h light-dark cycle). The rats were fed standard laboratory chow and tap water ad libitum. All experimental procedures described in this report were performed in accordance with the guidelines of the Canadian Council on Animal Care. Induction of colitis. Colitis was induced by intracolonic administration of 0.25 ml of 50% ethanol (vol/vol) containing 30 mg of 2,4,6-trinitrobenzenesulfonic acid (TNB), as previously described (11, 19). A rubber cannula with an outside diameter of 2.3 mm was inserted intrarectally into an ether-anesthetized rat so that the tip was 8 cm proximal to the anus. After instilling the TNB-ethanol solution, the cannula was left in place for a few seconds then gently removed. The rats were returned to their cages. In some experiments, a control group was included in which the rats received isotonic saline intracolonically in place of TNB (subsequently referred to as “saline controls”). These animals were included to provide data on eicosanoid production in the absence of colonic damage. Treatment with a leukotriene synthesis inhibitor. Four treatment regimens were used, as illustrated in Fig. 1. In protocol A, rats were treated with MK-886 (3 or 10 mg/ kg po) 2 h before induction of colitis and once daily for Two Week Study Period r

1 2 3 4 5 6 7 8 9 10 11 12 13 14

TNB a

Protocol A:

..B

.

.

.

.

.

l

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the next 7 days. Groups of rats (n > 7) were killed on day 7 and day 14 after induction of colitis for assessment of damage (see below). In protocol B, the treatment regimen was identical to that described above, except that the rats did not receive a dose of MK-886 before induction of colitis. The first dose of MK-886 (10 mg/ kg) or vehicle was given 2 h after administration of TNB and daily thereafter. The rats in these groups were killed 14 days after induction of colitis. In protocol C, the rats were pretreated with MK-886 (10 mg/kg) 2 h before induction of colitis, but no further drug treatment was performed. The rats were killed 14 days after TNB administration for assessment of damage. In protocol D, treatment with MK-886 (3 or 10 mg/kg po) was started on day 7 after induction of colitis and was continued on a once-daily basis until day 14. Administration of MK-886 was always performed between 0800 and 0900 h, whereas assessment of damage was always performed between 1000 and 1100 h. In the TNB-treated and saline control groups, rats received the vehicle for MK-886 (1% carboxymethylcellulose). Assessment of damage and inflammation. All scoring of damage and excision of tissue samples were performed by an observer unaware of the treatment group (J. L. Wallace). The rats in the various treatment groups were randomized before being killed. The rats were weighed and killed by cervical dislocation, and the distal 10 cm of colon was removed. The colon was opened by a longitudinal incision, rinsed with tap water, and pinned out on a wax block. Macroscopically visible damage was scored on a O-10 scale using the scoring system described in Table 1, which takes into consideration the area of involvement and the presence or absence of ulcers. The presence or absence of adhesions between the colon and other organs was also noted. Tissue samples (-100 mg) were excised for subsequent measurement of myeloperoxidase (MPO) activity and LTB, synthesis. Two samples were taken from each colon for each of these parameters. One sample was taken from a region of grossly 1. Criteria and inflammation

TABLE

Protocol

B:

Score 0

Protocol

C:

H 0

Protocol

n BBBBBB

D:

q l

= Damage

Treatment Assessed

for scoring of colonic ulceration Appearance

Macroscopical 0 1 2 3 4 5 6-10

= Drug

1. Schematic diagram illustrating the 4 treatment protocols used in this study. I, days on which the drug MK-886 was administered. l , days when rats were killed for assessment of damage. In all 4 protocols, trinitrobenzenesulfonic acid (TNB) was administered at the beginning of day 1. In protocols A and C, MK-886 was administered 2 h before TNB administration. In studies in which the effects of treatment with sulfasalazine were examined, protocols A and D were used. See METHODS for further details of the times of drug administration and doses used. FIG.

COLITIS

Normal Localized hyperemia, no ulcers Ulceration without hyperemia or bowel wall thickening Ulceration with inflammation at 1 site 2 or more sites of ulceration and inflammation Major sites of damage extending >I cm along length of colon When an area of damage extended >2 cm along length of colon, score was increased by 1 for each additional cm of involvement Microscopical Normal Damage limited to surface epithelium Focal ulceration limited to mucosa Focal, transmural inflammation, and ulceration Extensive transmural ulceration and inflammation bordered by normal mucosa Extensive transmural ulceration and inflammation involving entire section


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visible damage (2-3 cm proximal to the anus), whereas the other was taken from the region 8-10 cm proximal to the anus, which was invariably devoid of damage. Additional tissue samples (5 mm x 1 cm) were excised from these two regions for histological evaluation. The samples were immersed in neutral, buffered Formalin and were then processed by routine techniques before embedding in paraffin. Thick sections (4-8 pm) were mounted on glass slides and stained with hematoxylin and eosin. Histological scoring of ulceration and inflammation was performed separately and blindly by both authors using the criteria outlined in Table 1. The scores for each colon were then averaged. There was a highly significant linear correlation between the scores assigned by the two observers (r = 0.94; n = 70; P < 0.001). Leukotriene synthesis and myeloperoxidase activity. The tissue samples taken for measurement of MPO activity were weighed then frozen on dry ice. The samples were stored at -20°C for no more than 10 days. We have previously found that MPO activity decreases by -15% per week during storage under these conditions (unpublished data). In the present study, samples from control and test groups were always stored for the same period of time and were assayed for MPO activity in the same assay. MPO activity was measured using the technique of Bradley et al. (2). MPO is an enzyme found primarily in the azurophilic granules of neutrophils, and it has been used extensively as a biochemical marker of neutrophi1 infiltration into intestinal tissue (1, 5, 6, 16, 17, 19). The tissue samples for measurement of LTB, synthesis were weighed and then incubated in vitro as described in detail previously (18). The amount of LTB, released by the tissue during a ZO-min incubation period was determined using a specific radioimmunoassay (18). The antiserum used cross-reacts at ~0.05% with the other major leukotrienes. Onset and duration of inhibition of leukotriene synthesis by MK-886 and sulfasalaxine. To determine if once daily dosing with MK-886 or sulfasalazine was sufficient to give 24 h inhibition of leukotriene synthesis, rats were given MK-886 (3 or 10 mg/kg po) or sulfasalazine (100 mg/kg po) 2 h before intracolonic administration of TNB. In vivo dialysis, as originally described by Edmonds (3) and modified by Rampton et al. (13) and Zipser et al. (ZO), was performed on these rats 4 or 24 h after TNB administration. Immediately after dialysis was performed, the rats were killed and tissue samples were excised for measurement of MPO activity, as stated above. Dialysis was performed at the same times in a group treated with the vehicle in place of MK-886 (TNB controls) and with a group receiving saline in place of TNB (saline controls). In addition to measuring LTB, in the dialysate, the cyclooxygenase product, thromboxane B, (TxB& was measured by radioimmunoassay. The measurement of this eicosanoid, which is a stable metabolite of TxA2, would provide data indicating whether MK-886 had any inhibitory action on cyclooxygenase in this model. After performing the second dialysis, the rat was killed and two samples (-100 mg each) of grossly inflamed colonic tissue were excised. One sample was frozen at -20°C for subsequent determination of MPO activity, whereas the second sample was incubated in

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vitro for subsequent measurement of LTB, and TxB2 synthesis. In saline control rats, the samples were taken from the region of the distal colon where damage was normally observed in TNB-treated rats (2-3 cm proximal to the anus). A separate experiment was performed in which groups (n = 4) of rats were pretreated orally with either MK886 at doses of 3 or 10 mg/kg or with the vehicle 2 h before induction of colitis by administration of TNB. One hour after TNB administration, the rats were killed and tissue samples were excised, as described above, for measurement of LTB, synthesis. This experiment was performed to demonstrate that inhibition of LTB, synthesis by MK-886 was achieved within the first hour after TNB administration. Gillard et al. (4) have previously demonstrated that MK-886 inhibits leukotriene synthesis in the rat within 2 h of oral administration at doses of 3 or 10 mg/kg. In vivo dialysis. The dialysis tube was constructed by placing a sheath of dialysis tubing (7.6 cm diam when filled; mol wt cut off of 1,000; Spectrum Medical Industries, Los Angeles, CA) over a rubber cannula similar to that used to administer TNB. The dialysis tubing was tied around the cannula at both ends and a drop of silicon rubber sealant was placed on the end of the tube to be inserted into the colon to reduce the abrasiveness of the tube. The length of the portion of the tube that would be filled with dialysate was 6 cm. The rats were anesthetized with pentobarbital sodium (60 mg/kg ip), and the dialysis tube was inserted. One milliliter of dialysate (120 mM NaCl, 30 mM KHCOS, and 0.3% bovine serum albumin, adjusted to pH 7.9 with NaOH) was then injected into the tube by a syringe connected to the rubber cannula. The syringe was then taped to the rat’s tail to secure the dialysis tube in place. One hour later the dialysate was withdrawn by syringe and frozen for subsequent measurement of LTB, and TxB2 by radioimmunoassay (18). The dialysis tube was removed, and the rat was returned to its cage. Materials. Trinitrobenzenesulfonic acid was obtained from Sigma Chemical (St. Louis, MO). MK-886 {3-[l(4-chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-Z-y1)2,2-dimethylpropanoic acid) was kindly supplied by Dr. John Gillard of Merck-Frosst Canada (Pointe Claire, Quebec, Canada) and was prepared freshly each day as a suspension in 1% carboxymethylcellulose. Sulfasalazine (Azulfidine Oral Suspension; 50 mg/ml) was kindly supplied by Pharmacia Laboratories (Piscataway, NJ). The reagents for the radioimmunoassays were obtained from Amersham Canada (Oakville, Ontario, Canada). The reagents used in the MPO assay were obtained from Sigma. All other chemicals were obtained from Fisher Scientific (Don Mills, Ontario, Canada). Statistical analysis. All data are expressed as means t SE. Comparisons between groups of nonparametric data, such as the damage scores, were made with the Wilcoxon rank-sum test. Differences in the incidence of adhesions between groups were compared using a median test and x2. Comparisons between groups of parametric data were made with Student’s t test for unpaired data. With all statistical analyses, an associated probability (P value) of ~5% was considered significant.


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Onset and duration of action of MK-886. Administration of TNB resulted in marked changes in colonic eicosanoid synthesis and MPO activity within 4 h. As shown in Fig. 2, colonic synthesis of LTB, and TxBZ, as measured by in vivo equilibrium dialysis, was markedly elevated in TNB-treated rats when compared with saline controls. At 4 h after TNB administration, MPO activity was increased by approximately four times over that of saline controls (4.8 t 1.8 vs. 1.1 t 0.1 U/mg; P < 0.05). Figure 3 illustrates the marked increase in MPO activity 24 h after TNB administration. Eicosanoid synthesis and MPO activity in grossly normal tissue samples from the TNB-treated rats did not differ significantly from those of saline controls at any time studied. The two doses of MK-886 tested (3 and 10 mg/kg) produced comparable (-50%; P c 0.05) inhibition of LTB, synthesis 1 h after TNB administration, as measured after in vitro incubation of tissue samples. When LTB, synthesis was measured 24 h after induction of colitis (26 h after dosing with MK-886), there was a marked reduction with both doses, but the inhibition was only statistically significant with the higher dose (>90% reduction compared with the TNB controls; P < 0.05). Similar results were obtained when LTB* synthesis was measured by in vivo equilibrium dialysis (Fig. 2). Despite this inhibition of LTB, synthesis for 26 h, infiltration of neutrophils was not significantly affected (Fig. 3). MK886 did not significantly affect colonic synthesis of TxBZ (Fig. 2). Pretreatment with sulfasalazine (100 mg/kg) 7. A

I Saline Control m TNB Control cca TNB + MK-886

4 24 HOURS AFTER TNB Bq (A) and thromboxane

FIG. 2. Leukotriene B, (B) content in 1 ml of dialysate after in vivo intracolonic dialysis. Rats were given saline (control) or TNB intracolonically and dialysis was performed 4 and 24 h later. In one group, rats were pretreated with MK-886 (10 mg/kg po) 2 h before administration of TNB. Each bar represents the mean t SE of at least 4 experiments. * Significant differences (P < 0.05) from saline-treated control group.

T

15 B

T

a a

T

Saline Control

TNB Control

MK-886 + TNB

FIG. 3. Leukotriene B, synthesis (A) and myeloperoxidase activity (B) in samples of distal colon excised 24 h after intracolonic administration of saline or TNB. In a 3rd group, rats were pretreated with MK-886 (10 mg/kg po) 2 h before TNB administration. LTB, was measured after in vitro incubation of the tissue, as described in METHODS. Myeloperoxidase activity was measured as a biochemical marker of neutrophil content of the tissue sample. Each bar represents the mean t SE of at least 4 experiments. Letters over bars denote significant differences (P < 0.05) from asaline control group or bTNB control group.

produced a significant reduction of LTB4 in dialysates collected at 4 h after TNB administration (mean inhibition of 69%; P c 0.05), but there was no significant effect at 24 h after TNB administration. When LTB, synthesis was measured from tissue samples incubated in vitro 24 h after TNB administration, there was also no significant effect of sulfasalazine pretreatment. LTB, synthesis in the TNB control group was 85 t 16 rig/g while that in the group pretreated with sulfasalazine was 82 -+ 18 rig/g. Pretreatment with sulfasalazine did not significantly affect the infiltration of neutrophils into the colon, as measured by MPO activity, during the first 24 h after TNB administration. The use of in vivo dialysis to measure colonic LTB, synthesis confirms that the colon did produce elevated levels of this eicosanoid in the absence of any mechanical or chemical stimuli. Measurements of release of LTB4 from tissue samples are, in fact, a measure of the capacity of the tissue to generate this substance in response to mechanical stimulation. There was a significant linear correlation (P c 0.05) between the levels of colonic LTB, synthesis measured by the two techniques in this study.


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The observation that MK-886 at a dose of 10 mg/kg could significantly inhibit colonic LTB, synthesis but did not modify the extent of neutrophil infiltration was somewhat surprising. To determine if this compound might inhibit neutrophil infiltration in a more “mild” form of colitis, experiments were performed in which rats (n = 4 per group) were pretreated with either MK-886 (10 mg/kg) or the vehicle 2 h before intracolonic administration of 50% ethanol alone. The rats were killed 24 h later for assessment of colonic LTB, synthesis (in vitro) and MPO activity. As in the case of TNB/ethanol-induced colitis, the colitis induced by the ethanol vehicle alone was accompanied by a marked increase in tissue myeloperoxidase activity (approximately sevenfold) and LTB, synthesis (approximately fivefold). Pretreatment with MK-886 (10 mg/kg) resulted in significant (P < 0.05) inhibition of n m Em

5- A T

TNB Control MK-886 (3 mg/kg) MK-886 (10 mg/kg)

4 -3 -2 -1 --

0

6 --

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colonic LTB, synthesis but did not significantly affect colonic MPO activity (7.8 t 1.6 U/mg in the ethanol control group vs. 8.0 t 0.9 U/mg in the group pretreated with MK-886). Effects of treatment with MK-886 during week 1 (protocols A and B). With the higher dose of MK-886 (10 mg/kg), treatment beginning 2 h before TNB administration and continuing daily for 1 wk (protocol A) resulted in a significant reduction of colonic damage score, assessedboth macroscopically (Fig. 4A) and microscopically (Table 2). In rats killed on day 7, 2 h after the last dose of MK-886, there was a significant reduction of LTB, synthesis (in vitro measurement) by samples of inflamed colon with both doses of MK-886 (Fig. 4C). However, LTB, synthesis from samples taken from rats killed 14 days after TNB administration (7 days after the last dose of MK-886) was not significantly affected by treatment with MK-886 during the first week. These data are somewhat skewed, however. In the group treated with the higher dose of MK-886, five of the seven rats had LTB, synthesis in the range of saline-treated control rats (Cl5 rig/g) and had colonic damage scores of 0 or 1. The remaining two rats had damage scores of 4 and 5 and colonic LTB, synthesis of X00 rig/g. Thus the rats in which there was a marked reduction of colonic damage were also the rats in which significant reductions of colonic LTB, synthesis were observed. With both doses of MK-886, colonic MPO activity was significantly reduced in the rats killed on day 14 after TNB administration (Fig. 4B). However, the lower dose (3 mg/kg) did not significantly reduce colonic damage, as assessedmacroscopically (Fig. 4) or microscopically (Table 2). Adhesions between the affected portion of the colon and other organs, usually the small intestine, were frequently observed in TNB-treated rats. There was a strong association between the presence of adhesions and the presence of severe ulceration. Adhesions were observed in 61 rats killed 7 or 14 days after TNB administration. Of these, 59 (97%) had colonic damage scores of 3 or more. Adhesions were invariably located very close to a site of ulceration. Treatment with MK-886 before TNB administration and during the first week after TNB (protocol A ) resulted in a significant reduction in the incidence of adhesions between the colon and 2. Histologically assesseddamage and incidence of adhesions induced by TNB: effects of treatment with MK-886 TABLE

Week of Treatment

Treatment

7

DAYS

14

AFTER

TNB

FIG. 4. Effects of MK-886 or vehicle (TNB control) on colonic damage score (A), colonic myeloperoxidase (MPO) activity (B), and colonic leukotriene B, synthesis (C) 7 and 14 days after induction of colitis by intracolonic administration of TNB. Drug treatment was started 2 h before TNB administration and continued daily for 7 days (see protocol A on Fig. 1). Colonic damage was scored by an observer unaware of the treatment using the criteria outlined in Table 1. Each bar represents the mean t SE of at least 7 experiments. Significant differences from the TNB control group: * P < 0.05; ** P < 0.01.

Vehicle MK-886 MK-886 Vehicle MK-886 MK-886 Vehicle MK-886 MK-886

(3 mg/kg) (10 mg/kg) (3 mg/kg) (10 mg/kg) (3 mg/kg) (10 mg/kg)

Values are groups in which 886 was given protocol A in vehicle-treated

Day Killed After TNB 7 7 7

Histological Damage Score

1 1 1 1 1

14 14

1 2

14 14

3.9t0.5 4.6t0.2 2.8t0.8 3.8t0.3 2.7~10.7 1.3rkO.6" 3.8t0.4

2 2

14 14

3.1t0.6 2.9t0.5

Incidence of Adhesions 10/14 (71%) 417 (57%) 217 (28%)* 8/14(57%) 217 (28%) l/7 (14%)" 10/19 (53%)

7/13

(54%)

s/20

(45%)

means t SE. TNB, trinitrobenzenesulfonic acid. In rats were treated during week 1, the first dose of MK2 h before intracolonic administration of TNB (see Fig. 1). * P < 0.05 compared with the corresponding control group.


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other organs (Table 2). In rats killed on day 7, 2 h after the last dose of MK-886 or vehicle, the incidence of adhesions in the TNB control group was 71%, whereas that in the group treated with MK-886 (10 mg/kg) was only 28% (P < 0.05). Treatment with MK-886 before and during the first week after TNB administration also had significant effects on the changes in body weight that accompanied the development of colitis. During the 3 days after administration of TNB, the rats lost an average of 28 t 5 g (Fig. 5). Thereafter, body weight increased at a mean rate of -6 g/day. Treatment with MK-886 did not significantly affect the rate of body weight gain during the period 3-14 days after TNB administration. However, treatment with MK-886 dose dependently attenuated the decrease in body weight during the first 3 days after induction of colitis. In the group treated with MK-886 at 10 mg/kg, body weights were significantly greater than those in the TNB control group at 3,7, and 14 days after TNB administration. In the group in which MK-886 treatment was started 2 h after induction of colitis and was continued for 1 wk (protocol B), there were significantly lower colonic damage scores in the group treated with MK-886 (10 mg/kg), but the differences were not as great as when a pretreatment dose was also performed (at day 14: damage scores of 4.3 t 0.2 in the TNB control group vs. 3.2 t 0.7 in the MK-886 group; P < 0.05). The incidence of severe ulceration (damage scores of ~3) was 83% in the TNB control group compared with 43% in the group treated with MK-886 (P < 0.05). With the other parameters, there was a consistent trend of a reduction in the group treated with MK-886, but the differences were not statistically significant. For example, the data for the TNB group vs. the group treated with MK-886 for MPO were 9.4 -+ 1.2 vs. 5.8 -+ 1.7 U/mg, for LTB, synthesis (in vitro measurement) were 85 t 16 vs. 59 t 18 rig/g, and for the incidence of adhesions were 57% vs. 43%. Effects of pretreatment with MK-886 (protocol C). Pretreatment with MK-886 without subsequent drug adminOl -

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istration had no significant effect on any of the measured parameters when the rats were examined at day 14 after TNB administration. Damage scores, MPO activity, LTB, synthesis, and the incidence of adhesions were comparable to those seen in the control groups in the other studies. Effects of treatment with MK-886 during week 2 (protocol D). Daily oral treatment with MK-886 (3 or 10 mg/ kg) during the second week after induction of colitis (protocol D) was without significant effect on colonic damage score, colonic MPO activity, or the incidence of adhesions (Fig. 6, A and B; Table 2). However, colonic LTB, synthesis (in vitro measurement) was significantly reduced in the rats treated with either dose of MK-886 (Fig. 6C). Treatment with MK-886 during the second week did not significantly affect the body weight of the rats. Effects of treatment with sulfasalazine. Oral treatment with sulfasalazine, whether started before induction of 5T A

T

‘OTBI

1

0 TNB Control MK-886 (3 mg/kg) B---H MK-886 (10 mg/kg) l

325

2751 250 225

i

DAYS AFTER TNB ADMINISTRATION 5. Changes in body weight after induction of colitis by intracolonic administration of TNB, and effects of treatment with MK-886. Oral drug treatment was started 2 h before TNB administration and was continued daily for 7 days (see protocol A on Fig. 1). Control rats were treated daily with the vehicle. Each point represents the mean t SE of at least 7 rats. * Significant differences from control group (P < 0.05). FIG.

FIG. 6. Effects of daily oral treatment with MK-886 or sulfasalazine (SULF; 100 mg/kg) on the 7th through 14th days after TNB administration (protocol D on Fig. 1) on colonic damage score (A), colonic myeloperoxidase (MPO) activity (B), and colonic leukotriene Bq synthesis (C). Each bar represents the mean t SE of 7-20 experiments. * Significant differences (P < 0.01) from TNB control group, which was treated with vehicle.


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colitis and continued for 1 wk (protocol A) or performed during rueeh 2 after induction of colitis (protocol D), was without significant effects on colonic damage score, MPO activity, incidence of adhesions, or colonic LTB, synthesis (in vitro measurement). Figure 6 shows the data for treatment with sulfasalazine during coeeh 2 after induction of colitis. Treatment with sulfasalazine did not significantly affect the body weight of the rats. DISCUSSION

Substantial evidence has accumulated in support of a role of lipoxygenase products of arachidonic acid in the pathogenesis of IBD (6, 7, 12, 14, 15, 19), although the specific contribution of these mediators to the disease process remains unclear. The present study adds further support to this hypothesis and demonstrates that treatment with an orally active inhibitor of leukotriene synthesis can accelerate healing in an animal model of chronic colitis. A single administration of MK-886 resulted in significant inhibition of colonic LTB, synthesis for ~24 h while not significantly affecting synthesis of the cyclooxygenase metabolite of arachidonic acid, thromboxane B,. MK-886 has a mechanism of action distinct from previously reported inhibitors of %lipoxygenase. Although most inhibitors work through a redox mechanism of inactivating &lipoxygenase, MK-886 appears to inhibit the translocation of the enzyme from the cytosol to the plasma membrane. Hence, this compound is an inhibitor of the activation of 5-lipoxygenase (4). The period of time in which the treatment with MK886 was performed greatly influenced its effectiveness in accelerating healing. Pretreatment with MK-886 2 h before induction of colitis did not, by itself, accelerate healing. However, pretreatment did greatly increase the effectiveness of treatment with the drug during the first week after induction of colitis. These observations suggest an important role for leukotrienes in the earliest phase of the inflammatory response. Both LTB, and TxB2 synthesis were shown to be significantly elevated as early as 4 h after administration of TNB, corresponding to the period in which neutrophil infiltration was first evident. Thus, without the pretreatment dose of MK-886, the inflammatory response would already have been initiated before any effects of the first dose of the drug (2 h after TNB) on leukotriene synthesis would have been apparent. The present data also suggest that a 24-h suppression of leukotriene synthesis was essential for beneficial effects of this compound to be apparent. Although leukotrienes may be involved in the acute response to TNB, the present data suggest that they do not play a very important role in the chronic phase of the inflammatory response in this model. Treatment with MK-886 during week 2 after induction of colitis was completely ineffective, despite significantly reducing coionic LTB, synthesis. It is noteworthy that inhibition of LTB, synthesis for the first 24 h after administration of TNB did not significantly alter the extent of neutrophil infiltration into the colon, as determined by tissue MPO activity. This observation suggests that LTB, is not an important chemotactic factor for neutrophils in this model during the acute phase. When colitis was induced by intraco-

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ionic administration of the ethanol vehicle alone, pretreatment with MK-886 was again found to suppress colonic LTB, synthesis without markedly altering neutrophil infiltration. The identity of the factor responsible for the massive recruitment of neutrophils into the TNBtreated colon are as yet unidentified. Since LTB, is a potent chemotactic factor for neutrophils in humans (8), it remains possible that LTB4 plays an important role in the recruitment of neutrophils in human IBD. While not influencing the initial neutrophil infiltration, treatment with MK-886 before and during week 1 after TNB administration resulted in a significant reduction of the MPO activity when measured 1 wk after completion of the treatment (day 14 after induction of colitis). It is likely that this reduction of neutrophil infiltration was a consequence of the accelerated healing of colonic ulcers. The use of the in vivo dialysis method to measure colonic LTB, and TxB2 synthesis confirms that the inflamed colon does in fact ‘produce elevated levels of these eicosanoids. A valid criticism of the measurement of eicosanoid synthesis from tissue samples incubated in vitro is that this is actually a measure of the capacity of the tissue to produce eicosanoids in response to mechanical stimulation. The failure of sulfasalazine to accelerate healing in the present study may be in part attributed to the relative weakness of this compound as an inhibitor of leukotriene synthesis. Although sulfasalazine produced a significant reduction of colonic LTB4 synthesis when measured 6 h after administration, the inhibition was no longer apparent 18 h later. Therefore, unlike the 10 mg/kg dose of MK-886, sulfasalazine did not significantly inhibit coionic LTB, synthesis for the 24-h period between doses. Interestingly, a lower dose of MK-886 (3 mg/kg), which did not produce 24-h suppression of LTB4 synthesis, also did not significantly accelerate healing. An alternative explanation for the failure of sulfasalazine to affect healing is that there may have been insufficient conversion of this compound to the putative active moiety 5-aminosalicylic acid. We have previously demonstrated significant beneficial effects of intracolonic treatment with 5-aminosalicylic acid in the TNB model of colitis (19). The observation of significant inhibition of LTB, synthesis with sulfasalazine suggests that there was some conversion to 5-aminosalicylic acid during the passage through the gut. Although apparently not involved in the initial recruitment of neutrophils into the colon after TNB administration, leukotrienes do appear to play an important role in the inflammatory process and in adhesion formation in this model. The dose of MK-886 (10 mg/kg) that suppressed leukotriene synthesis for 24 h significantly accelerated the healing of ulcers, reduced colonic MPO activity, and reduced the incidence of adhesions. Leukotrienes have many other actions in addition to their chemotactic actions that could conceivably contribute to the production of ulcers or inflammation in this model. For instance, leukotrienes can promote the activation of neutrophils to release free radicals and proteases that have the potential to cause tissue necrosis. Leukotrienes can also affect vascular permeability and tone as well as smooth muscle contractility (see Ref. 8 for review). In


G534

LEUKOTRIENE

INHIBITION

the TNB model of colitis, MK-886 was superior as an anti-inflammatory agent to any other compound we have previously tested, including 5-aminosalicylic acid (19), sulfasalazine, and prednisolone (unpublished data). From a clinical standpoint, it would clearly be advantageous for a drug to improve healing when given in the chronic phase of the inflammatory process. However, if the hypothesis that drugs that are effective in the treatment of human IBD, such as 5-aminosalicylic acid, sulfasalazine, and glucocorticoids, work through a mechanism involving reduction of leukotriene synthesis, then it follows that more specific and potent inhibitors of leukotriene synthesis should have clinical value. Orally active compounds such as MK-886 offer several advantages over poorly bioavailable 5-lipoxygenase inhibitors, the most important of which is the ability to inhibit leukotriene synthesis in regions of the gastrointestinal tract that are not easily accessible. For example, a compound that must rely on topical activity for effectiveness as an inhibitor of leukotriene synthesis would likely be of little value in the treatment of inflammation in the jejunum or ileum. Obviously, appropriate clinical studies on compounds such as MK-886 are necessary to test the hypothesis that such agents will have beneficial effects in the treatment of IBD. The authors are grateful to W. MacNaughton and W. McKnight for their assistance in performing these studies, and to Drs. A. FordHutchinson and J. Gillard of Merck-Frosst Research Laboratories for their helpful comments and support of this project. This work was supported by grants from the Medical Research Council (MRC) of Canada, the Canadian Foundation for Ileitis and Colitis, and Merck-Frosst Canada. J. L. Wallace is the recipient of scholarships from the MRC of Canada and the Alberta Heritage Foundation for Medical Research. Address for reprint requests: J. L. Wallace, Dept. of Medical Physiology, Univ. of Calgary, Calgary, Alberta TZN 4N1, Canada. Received

16 August

1989; accepted

in final

form

7 November

1989.

REFERENCES 1. BOUGHTON-SMITH, N. K., J. L. WALLACE, G. P. MORRIS, AND B. J. R. WHITTLE. The effect of anti-inflammatory drugs on eicosanoid function in a chronic model of inflammatory bowel disease. Br. J. Pharmacol. 94: 65-72, 1988. 2. BRADLEY, P. P., D. A. PRIEBAT, R. D. CHRISTENSEN, AND G. ROTHSTEIN. Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J. Intest. Dermatol. 78: 206-209, 1982. 3. EDMONDS, C. J. Absorption of sodium and water by human rectum measured by a dialysis method. Gut 12: 356-362, 1971. 4. GILLARD, J., A. W. FORD-HUTCHINSON, C. CHAN, S. CHARLESON, D. DENIS, A. FOSTER, R. FORTIN, S. LEGER, C. S. MCFARLANE, H. MORTON, H. PIECHUTA, D. RIENDEAU, C. A. ROUZER, J. ROKACH, R. YOUNG, D. E. MACINTYRE, L. PETERSON, T. BACH, G. EIERMANN, S. HOPPLE, J. HUMES, L. HUPE, S. LUELL, J. METZGER, R. MEURER, D. K. MILLER, E. OPAS, AND S. PACHOLOK. L-663,536 (MK-886) (3-[I-(4-chlorobenzyl)-3-t-butyl-thio-5

IN

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isopropylindol-%yl] -2,2-dimethylpropanoic acid), a novel, orally active leukotriene biosynthesis inhibitor. Can. J. Physiol. Pharmacol. 67: 456-464, 1989. GRANGER, D. N., M. B. GRISHAM, B. J. ZIMMERMAN, AND C. VON RITTER. Reactive oxygen metabolites as mediators of cell injury in the intestine. In: Inflammatory Bowel Disease: Current Status and Future Approach, edited by R. P. MacDermott. New York: Elsevier, 1988, p. 255-260. KRAWICZ, J. E., P. SHARON, AND W. F. STENSON. Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models. GastroenteroZogy 87: 1344-1350, 1984. LAURITSEN, K., L. S. LAURSEN, K. BUKHAVE, AND J. RASKMASDEN. Effects of topical 5-aminosalicylic acid and prednisolone on prostaglandin E2 and leukotriene Bq levels determined by equilibrium in vivo dialysis of rectum in relapsing ulcerative colitis. Gastroenterology 91: 837-844, 1986. LEWIS, R. A., AND K. F. AUSTEN. The biologically active leukotrienes. Biosynthesis, functions, and pharmacology. J. Clin. Inuest. 73: 889-897, 1984. LOBOS, E. A., P. SHARON, AND W. F. STENSON. Chemotactic activity in inflammatory bowel disease. Role of leukotriene Bq. Dig. Dis. Sci. 32: 1380-1388, 1987. MILLER, M. J. S., AND D. A. CLARK. Participation of free radicals and eicosanoids in experimental necrotizing enterocolitis, a neonatal inflammatory bowel disease. In: Inflammatory Bowel Disease: Current Status and Future Approach, edited by R. P. MacDermott. New York: Elsevier, 1988, p. 267-271. MORRIS, G. P., P. L. BECK, M. S. HERRIDGE, W. DEPEW, M. R. SZEWCZUK, AND J. L. WALLACE. Hapten-induced model of chronic inflammation and ulceration in the rat colon. Gastroenterology 96: 795-803,1989. PESKAR, B. M., K. W. DREYLING, B. A. PESKAR, B. MAY, AND H. GOEBELL. Enhanced formation of sulfidopeptide-leukotrienes in ulcerative colitis and Crohn’s disease: inhibition by sulfasalazine and 5-aminosalicylic acid. Agents Actions 18: 381-383, 1986. RAMPTON, D. S., G. E. SLADEN, AND L. J. F. YOULTEN. Rectal mucosal prostaglandin E2 release and its relation to disease activity, electrical potential difference, and treatment in ulcerative colitis. Gut 21: 591-596,198O. SHARON, P., AND W. F. STENSON. Enhanced synthesis of leukotriene Bq by colonic mucosa in inflammatory bowel disease. Gastroenterology 86: 453-460, 1984. SHARON, P., AND W. F. STENSON. Metabolism of arachidonic acid in acetic acid colitis in rat: similarity to human inflammatory bowel disease. Gastroenterology 88: 55-63, 1985. SMITH, J. W., AND G. A. CASTRO. Relation of peroxidase activity in gut mucosa to inflammation. Am. J. Physiol. 234 (Regulatory Integrative Comp. Physiol. 3): R72-R79, 1978. WALLACE, J. L. Release of platelet-activating factor (PAF) and accelerated healing induced by a PAF antagonist in an animal model of chronic colitis. Can. J. Physiol. Pharmacol. 66: 422-425, 1988. WALLACE, J. L., P. L. BECK, AND G. P. MORRIS. Is there a role for leukotrienes as mediators of ethanol-induced gastric mucosal damage? Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17): G117Gl23,1988. WALLACE, J. L., W. K. MACNAUGHTON, G. P. MORRIS, AND P. L. BECK. Inhibition of leukotriene synthesis markedly accelerates healing in a rat model of inflammatory bowel disease. Gastroenterology 96: 29-36, 1989. ZIPSER, R. D., C. C. NAST, M. LEE, H. W. KAO, AND R. DUKE. In vivo production of leukotriene B, and leukotriene Cq in rabbit colitis. Relationship to inflammation. Gastroenterology 92: 33-39, 1987.


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