Prostaglandins 44:261-275, 1992 0N0-4057, A NOVEL, ORALLY ACTIVE LEUKOTRIENE B4 ANTAGONIST: EFFECTS ON LTB4-INDUCED NEUTROPHIL FUNCTIONS K. Kishikawa,

N. Tateishi, T. Maruyama, and T. Miyamoto

R. Seo, M. Toda,

Minase Research Institute, Ono Pharmaceutical Co., Ltd., Sh!m-moto, Mishima, Osaka 618, Japan

ABSTRACT ONO-4057(5-[2-(2-Carboxyethyl)-3-{ 6-( 4-methoxyphenyl )5E-hexenyl } oxyphenoxy ] valeric acid), an orally active leukotriene B4(LTB4) antagonist, displaced the binding of [3HI LTB 4 to the LTB 4 receptor in human neutrophil (Ki = 3.7+0.9 nM). ONO4057 inhibited the LTB4-induced rise in cytosolic free calcium (the concentration causing 50% inhibition (IC50) = 0 . 7 + 0 . 3 ~ M ) a n d inhibited human neutrophil aggregation, chemotaxis or degranulation induced by LTB4 (IC50 = 3.0+0.1, 0.9+0.1 and 1.6_+0.1/.tM) without showing any agonist activity at concentration up to 30~M. ONO-4057 did not inhibit fMLP or C5a-induced neutrophil activation at concentrations up to 30/.tM. In the in vivo study, ONO-4057 given orally, prevented LTB4-induced transient neutropenia or intradermal neutrophil migration in guinea pig (the dose causing 50% e f f i c a c y ( E D 5 0 ) -- 25.6mg/kg or 5.3mg/kg). Furthermore, ONO-4057 given topically, suppressed phorbol-12myristate-13-acetate (PMA)-induced neutrophil infiltration in guinea pig ear (the effective dose = 1 mg/ear). These results indicate that ONO-4057 is a selective and orally active LTB 4 antagonist and may be a potential candidate for the treatment of various inflammatory diseases. INTRODUCTION L T B 4 , one of the arachidonic acid metabolites by the 5lipoxygenase pathway, has been proposed to be an important mediator of inflammation based on the following reports: first, LTB4 is potent stimulant of neutrophil functions including chemotaxis, aggregation, adhesion and degranulation and stimulates neutropenia or neutrophil accumulation in the skin (1, 2, 3), second, elevated tissue levels of LTB4 have been reported in inflammatory diseases such as psoriasis (4), inflammatory bowel

Copyright © 1992 Butterworth-Heinemann

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disease (5), rheumatoid (6), and gout (7). For these reasons, an L T B 4 antagonist might be useful in the treatment of several inflammatory diseases by blocking LTB4 receptor-mediated neutrophil functions. Recently, we have developed an orally active L T B 4 receptor antagonist, ONO-4057(Fig 1), which has high affinity to the LTB4 receptor of human neutrophils. In this report, we studied the effects of ONO-4057 on LTB4-induced neutrophil activation in vitro and in vivo.

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O oMe 5-[2-(2-Carboxyet hyl)-3-{ 6-(4-methoxyphenyl)5E-hexenyl } oxyphenoxy] valeric acid Fig 1 Structure and chemical name of ONO-4057 MATERIALS AND METHODS Materials and Animals L T B 4 was synthesized at ONO Pharmaceutical Co., Osaka, Japan. [3HI LTB 4 (32 Ci/mmole) was purchased from New England Nuclear, Boston, MA, USA. o-Dianisidine, fMLP, human recombinant C5a, cytochalasin B, phorbol-12-myristate-13-acetate(PMA), fura2-AM and horse radish peroxidase were purchased from Sigma, St.Louis, MO USA. Ficoll-paque® was purchased from Pharmacia LKB, Uppsala, Sweden. PVP-coated chemotaxis membrane of 2 gm pore size was obtained from Nucleopore Corp., Pleasaton, CA, USA. Male Hartley guinea pigs, 200-400 g body weight, were purchased from Nippon Rabbit Co. Osaka, Japan

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preparation

Human Venous blood was drawn by venipuncture from healthy volunteers using heparin as anticoagulant and neutrophils were purified by dextran sedimentation, centrifugation on Ficoll-paque®, followed by hypotonic erythrocyte lysis (8). The neutrophils enriched to 95% purity were resuspended in ice-cold Hank's balanced salt solution (HBSS) pH 7.4 (for binding assay) or in icecold HBSS pH 7.4 containing bovine serum albumin (BSA)(for other in vitro assays). Guinea pig Male Hartley guinea pigs (300-500g) were intraperitoneally administered 1% casein in saline (100ml/kg). After 18 hr, animals were sacrified and peritoneal fluid was collected by washing with 30 ml of saline containing 10 lU/ml of heparin. The neutrophils enriched to 95% purity were centrifuged at 300 x g for 5 min and the resultant pellet was resuspended in ice-cold HBSS pH 7.4. Binding assay for Neutrophil LTB 4 receptor Neutrophils obtained from humans or guinea pigs(107 cells) were incubated with 1 nM 13H] LTB 4 in the presence or absence of ONO-4057 at 0 °C for 20 min in 1 ml HBSS pH 7.4. The reaction was terminated by the addition of ice-cold HBSS (2.5 ml), followed by vacuum filtration through glass fiber filters (Whatman CF/C). Specific binding was calculated as the difference between total binding and binding not displaced by 3 ~tM unlabeled LTB 4 ( n o n specific binding). Calcium mobilization Human neutrophils(2 x 107cells/ml containing HBSS-0.1% BSA pH 7.4) were incubated at 37°C with 2~M fura-2-AM for 15 min. After washing, the cell pellets were resuspended at 5 x 106 cells/ml of HBSS-0.1% BSA. The fluorescence of fura-2-1oaded neutrophils was measured using the Ca ++ analyzer(model CAM-220, JASCO, Tokyo, Japan.) which generated dual wavelength excitation beams (340 nm and 380 rim) using a pair of interference filters. Fura-2-1oaded neutrophils were preincubated with ONO-4057 for 2 min at 37°C under constant stirring (600 rpm), followed by

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addition of LTB4 to the cells through a rubber septum. The ratios of emitted fluorescence at 500 nm over the 340 and 380 nm excitation wavelengths were continuously monitored and recorded (9). Cytosolic free calcium concentrations were calculated as described by Grynkiewicz et al (10). A~re~ation Human neutrophils(107 cells/ml) in HBSS containing 0.5% BSA pH 7.4 were preincubated in the presence or absence of ONO-4057 at 37 °C for 1 min prior to the addition of 10 nM LTB4. Aggregation was monitored as the change in light transmission using the multichannel aggregometer (HEMA TRACER 1, Nikou Bio Science, Tokyo, Japan.). Chemotaxis The chemotaxis assay was performed in Boyden chambers (11). The upper chamber contained human neutrophils(106 cells/0.8 ml in HBSS containing 0.5% BSA, pH7.4) and the lower chamber(0.2 ml), separated by a PVP-coated chemotaxis membrane (2 I.tm), contained HBSS alone or LTB 4 in the presence or absence of ONO-4057. After the incubation at 37 °C for 60 min in 5% CO 2, neutrophils, which adhered on chemotaxis membranes, were stained with Giemsa's solution and counted under a microscope (x 400). Degranulation assays Human neutrophils( 2.7 x 107 cells in HBSS containing 0.05% BSA pH 7.4) were preincubated with cytochalasin B (5 I.tg/ml) at 37°C for 6 min in the presence or absence of ONO-4057, followed by incubation with 100 nM LTB 4, 1 ~M fMLP or 100 nM C5a for 10 min at 37°C. Myeloperoxidase (MPO) activity(12) in the cell pellet and supernatant was assayed spectrophotometrically. After subtracting the basal MPO activity in the supernatant, the degree of degranulation was expressed as a percentage of total MPO activity released into the supernatant. The percentage of the total release of MPO activity induced by 100nM LTB4, 100 nM fMLP and 10 nM C5a were 20-30, 40-70 and 40-60%, respectively.

LTB.4-induced transient neutropenia in the guinea pig

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Guinea pigs(male Hartley 200-400g) were anesthetized with pentabarbital(30 mg/kg i.p.) 10 min before LTB 4 injection. L T B 4 ( 1 0 0 ng/kg) in 0.9 % NaCI was injected intravenously through the cannulae into a jugular vein. Thirty sec later, blood was taken from a cannulae inserted into a carotid artery. The number of neutrophils were measured using a multi-blood cell counter (SYSMEX E-2500. Sysmex, Tokyo, Japan). ONO-4057 as a sodium salt in 0.9% NaCI was injected intravenously 1 min before LTB 4 injection or it was orally administered as a suspension of 0.05% aqueous Tween 80 solution 30 min before LTB 4 injection. LTB4_.-induced neutrophil accumulation in the guinea pig After shaving the backs of guinea pigs(male Hartley 200400g), L T B 4 ( 1 0 0 n g / s i t e ) was injected intradermally into the shaved area. After 2 hr, the animals were sacrified by decapitation, their skins removed and frozen on dry ice. The skin punches(10 mm diameter) were processed to determine MPO(neutrophil marker enzyme) levels. The punches were minced with scissors and homogenized in 0.5 % hexadecyltrimethylammonium bromide and sonicated. After centrifugation (10000 x G for 10 min), extracted MPO was m easured spect rophotometrically. Data were expressed as MPO activity(U/g wet weight, one unit of MPO activity was defined as 0.001 unit of horse radish peroxidases). ONO-4057 was administered orally as a suspension of 0.05% aqueous Tween 80 solution 30 min before L T B 4 injection. A solution of LTB 4 (1000 ng/ml) was made up in 0.9% NaCI with less than 0.5 % ethanol which did not induce neutrophil infiltration. PMA-induced skin inflammation in the guinea pig ear PMA(10 nmol/site in 0.1 ml acetone) was applied topically to the ears of guinea pigs (male Hartley 200-400g). After 18 hr, the animals were sacrified by decapitation and ears were removed and frozen on dry ice. Ears were punched (10 mm diameter) and processed to determine MPO levels as the index of skin inflammation. Data were expressed as MPO activity(U/g wet weight). ONO-4057 was dissolved in 0.1 ml acetone and applied topically to the ears I hr before PMA application.

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Statistical analysis A comparison of the difference between the mean values of the various groups of experiments was made by a one-way analysis of variance or the Student's t-test. A difference with a P value of less than 0.05 was considered to be statistically significant. IC50 and ED50 values were determined from linear regression analysis. Results were expressed as the mean ±s.e.m. or the mean(95 % confidence limits). RESULTS Binding assay for neutrophils of humans or guinea pigs Binding assays were performed using intact neutrophils from humans and guinea pigs. On intact neutrophils from humans or guinea pigs, 1 nM [3H| LTB 4 bound exclusively to high affinity sites (Bmax; 11438+1305 or 2541+450 sites/cell, respectively(n=5)) with a dissociation constant of 0.62+0.12 or 4.3+I,0 riM, respectively(n=5). ONO-4057 inhibited 1 nM [3HI LTB 4 binding and the Ki value for the neutrophil LTB 4 receptor from humans and guinea pigs were 3.7+_0.9 and 8.8+_0.8 nM, respectively(n=5). Calcium mobilization L T B 4 induced an increase in fura-2 fluorescence intensity, which expressed intracellular calcium concentration, within 15 sec, which was followed by a slow decay. The maximal effect of intracellular calcium rise was observed at 1 nM LTB 4. ONO-4057 inhibited the LTB4(1 nM)-induced intracellular calcium rise with an IC50 of 0.7+0.3 p.M (n=3) (Fig 2). ONO-4057 alone did not cause any intracellular calcium rise at concentrations up to 3 ~tM (Fig 2). Human neutrophil aggregation and chemotaxis LTB4 induced human neutrophil aggregation with the maximal effect observed at 10 nM. ONO-4057 inhibited LTB4(10 nM)-induced aggregation with an IC50 value of 3.0+0.1 ~M, n=4 (Fig 3), however, it showed no antagonist activity against fMLP(100 riM) at concentrations up to 10 ~M (11.3% inhibition at 10 ~tM, n=2). Less than 5 % of LTB4-induced maximal aggregation was observed

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by ONO-4057(30 ~tM). Furthermore, LTB 4 stimulated human neutrophil chemotaxis in modified Boyden chambers with the maximal effect observed at 10 nM. ONO-4057 inhibited LTB4(10 nM)-induced maximal chemotaxis with an IC50 of 0.9_+0.1 /aM, n=3 (Fig 3).

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Fig 3 Inhibitory effect of ONO-4057 on L T B 4 - i n d u c e d human n e u t r o p h i l a g g r e g a t i o n ( l e f t panel) and c h e m o t a x i s ( r i g h t panel). Aggregation: neutrophils ( 1 0 7 c e l l s / m l ) were preincubated in the presence or absence of ONO-4057 at 37 °C for 1 rain prior to the addition of 10 nM LTB 4. Aggregation was monitored as a change in light transmission. Chemotaxis: neutrophils(106 cells/0.8 ml) in the upper chamber were incubated with L T B 4 in the presence or absence of ONO-4057 in the lower chamber at 37°C for 60 min in 5% CO 2. Neutrophils, which adhered on the chemotaxis membrane, were stained and counted under a m i c r o s c o p e . Data were e x p r e s s e d as a mean+_s.e.m, of % inhibition of the m a x i m a l aggregation or chemotaxis induced by 10 nM LTB 4 alone (n=3-4). Human

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L T B 4 induced human n e u t r o p h i l degranulation in a concentration dependent manner with the maximal effect of LTB 4 occurring at 100 nM. ONO-4057 inhibited L T B 4 ( 1 0 0 nM)-induced degranulation with an IC50 of 1.6+_0.1 ~tM (n=6). Furthermore,

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fMLP and C5a also stimulated human neutrophil degranulation with maximal effects at 100nM and 10nM, respectively. However, ONO4057 had little effect on fMLP or C5a-induced degranulation up to 30 I.tM (Fig 4).

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after administration and neutrophil counts returned to control level within 2 rain. Significant changes in peripheral platelet counts were not observed. ONO-4057 suppressed neutropenia when given intravenously 1 rain, before LTB 4 injection or when given orally 30 min before LTB 4 injection(Fig 5) with ED50 values of 0.7 (0.5-1.1, n=4-6), or 25.6 (19.6-33.4, n=4-6) mg/kg, respectively.

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assessed by MPO activity (data not shown). ONO-4057 suppressed neutrophil accumulation in the skin with an ED50 value of 5.3 (3.28.8, n=10-20) mg/kg when given orally 30 min before LTB4 injection (Fig 6) and its suppression (30 mg/kg) lasted for more than 6 hours (data not shown).

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ONO-4057, a novel, orally active leukotriene B4 antagonist: effects on LTB4-induced neutrophil functions.

ONO-4057(5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E- hexenyl]oxyphenoxy]valeric acid), an orally active leukotriene B4(LTB4) antagonist, displace...
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