Introduction

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The progression of inflammatory reactions depends on the interactions between various chemical mediators and cytokines (Umetsu and DeKruyff, 1997; Litchfield and Lee, 1992). An asthma attack is thought to be a type of airway inflammation. Therefore, the biosynthesis of these lipid mediators is increased in asthmatic subjects (Sladek et al., 1990; Kumlin et al., 1992; Wenzel et al., 1990; Taylor et al., 1991). Many lipid mediator antagonists, such as pranlukast (Nakagawa et al., 1992), zafirlukast (Krell et al., 1990) and seratrodast (Ashida et al., 1989), have been studied in an effort to find a treatment for bronchial asthma (Barnes, 1992).

Both cys-LTs and TXA₂ are well known to be important mediators in allergic responses in the lungs (Henderson, 1991). LTD₄ increases vascular permeability (Peck et al., 1981; Rinkema et al., 1984) and induces airway smooth muscle contraction (Dahlén et al., 1980; Ueno et al., 1982), whereas TXA₂ has potent bronchoconstricting activity (Nagai et al., 1991; Francis et al., 1991) that may contribute to airway hyperreactivity (Jones et al., 1992; Minoguchi et al., 1992; Nagai et al., 1993). Therefore, the roles of LTD₄ and TXA₂ in asthma are thought to be different, which suggests that a multi-pathway inhibitory agent may be a more potent therapeutic agent for bronchial asthma than single-pathway inhibitors (Sakurai et al., 1997).

YM158 (fig. 1) was discovered by focusing on the synthesis of high-affinity, orally effective dual LTD₄- and TXA₂-receptor antagonists. This report, presents the pharmacologic profile of YM158 by means of in vitro functional assays.

	Materials and Methods

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Sources of biological samples. Male Hartley guinea pigs were purchased from Japan S.L.C. (Hamamatsu, Japan) or Charles River Japan (Yokohama, Japan). Human platelets were isolated from blood samples obtained from healthy volunteers.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Chemical structure of YM158.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   LTD4 ()-, U46619 ()- and carbachol ()-induced contraction of isolated guinea pig trachea. Results represent the mean ± S.E.M. of five experiments.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   A) Effect of YM158 on concentration-response curves of LTD4-induced guinea pig tracheae contraction. Results are expressed as a percentage of the response to 3 × 108 M LTD4 and are the mean ± S.E.M. of 7 to 22 experiments. : control (n = 22); : 3 × 109 M YM158 (7); : 1 × 108 M YM158 (8); : 3 × 108 M YM158 (7). B) Schild analysis of YM158 (3 × 109 to 3 × 108 M) antagonism of LTD4-induced guinea pig tracheae contraction.

View larger version (13K): [in this window] [in a new window]   Fig. 4.   A) Effect of YM158 on concentration-response curves of U46619-induced guinea pig tracheae contraction. Results are expressed as a percentage of the response to 3 × 107 M U46619 and are the mean ± S.E.M. of 7 to 21 experiments. : control (n = 21); : 3 × 109 M YM158 (7); : 1 × 108 M YM158 (7); : 3 × 108 M YM158 (7). B) Schild analysis of YM158 (3 × 109 to 3 × 108 M) antagonism of U46619-induced guinea pig tracheae contraction.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effect of YM158 () 1 × 108 (panel A), 3 × 108 (panel B) and 1 × 107 M (panel C) on control () concentration-response curves for contraction to PGD2 in guinea pig tracheae. Results are expressed as a percentage of the corresponding maximum response and are the mean ± S.E.M. of four experiments. The maximum tensions were 2.60 ± 0.08 g at 3 × 105 M PGD2 (n = 12).

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effect of YM158 () 1 × 107 (panel A), 1 × 106 (panel B) and 1 × 105 M (panel C) on control () concentration-response curves for contraction to PGF2 in guinea pig tracheae. Results are expressed as a percentage of the corresponding maximum response and are the mean ± S.E.M. of four experiments. The maximum tensions were 2.11 ± 0.11 g at 3 × 105 M PGF2 (n = 12).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Effect of YM158 on concentration-response curves of guinea pig tracheal relaxation induced by salbutamol. Results are expressed as a percentage of the maximum response to salbutamol used and are the mean ± S.E.M. of four experiments. : control; : 1 × 105 M YM158; : 1 × 106 M YM158.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effect of YM158 on concentration-response curves for guinea pig platelet aggregation induced by U46619. Results are expressed as a percentage of the maximum response to U46619 used and are the mean ± S.E.M. of seven experiments. : control; : 1 × 107 M YM158; : 3 × 107 M YM158; : 1 × 106 M YM158.

Chemicals. The following drugs and chemicals were used: YM158, zafirlukast and montelukast were synthesized by Yamanouchi Pharmaceutical Co., Ltd. (Tsukuba, Ibaraki, Japan). Pranlukast and seratrodast were purified from the commercially available formulations ONON (Ono Pharmaceutical Co., Osaka, Japan) and BRONICA (Takeda Chemical Industries, Osaka, Japan), respectively. LTD₄, U46619, PGD₂, PGF₂ and indomethacin were purchased from Cayman Chemical Co. (Arbor, MI, USA) or Sigma (St. Louis, MO, USA).

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Obtaining a control cumulative dose-response curve for each agonist (contractile studies on isolated guinea pig tracheae). Guinea pigs weighing 450 to 740 g were sacrificed by exsanguination, and appropriate tissues were immediately removed. Each trachea was cut into segments containing four tracheal cartilage rings, and these segments were cut open on the other side of smooth muscle. Each tracheal strip was suspended in a 10-ml organ bath containing Krebs-Henseleit solution with the following composition: 118.2 mM NaCl, 4.6 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄ · 7H₂O, 25 mM NaHCO₃, 2.5 mM CaCl₂ · 2H₂O and 10.0 mM glucose. For these studies 5 × 10⁶ M indomethacin was added to the Krebs-Henseleit solution to avoid the influence of cyclooxygenase products (Orehek et al., 1973). L-Cysteine (3 × 10³ M) was added to LTD₄-induced contraction experiments to avoid degradation of LTD₄ (Snyder et al., 1984). Serine borate (4.5 × 10² M) was added to LTC₄-induced contraction experiments to avoid degradation of LTC₄ (Charette and Jones, 1987; Snyder et al., 1984). Snyder and co-workers (1984) reported that this L-cysteine and L-serine borate prevented the bioconversion of LTD₄ and LTC₄ to other cys-LTs, respectively, and shifted the LTD₄ and LTC₄ concentration-response curves to the left, respectively, in these assay systems. The organ baths were maintained at 37°C and continuously aerated with 95% O₂-5% CO₂. Tracheal responses were isometrically recorded using a force transducer (TB-611T, Nihon Kohden, Japan) connected to a force amplifier (AP-621G, Nihon Kohden, Japan) and a pen recorder (model 3056, Yokogawa, Japan). Tracheal strips isolated from guinea pigs were placed under tension using a mass of 1 g, and each preparation was equilibrated for 30 min. The preparations were primed twice with 3 × 10⁶ M carbachol, and then a cumulative concentration-response curve for each agonist was obtained by increasing the bath concentration of the agonist approximately 3-fold.

Agonist-induced contraction of trachea. Each tracheal strip was primed by 3 × 10⁶ M carbachol twice within a 60-min interval, and a control cumulative concentration-response curve for LTD₄ or U46619 was obtained. After completion of the first concentration-response curve, each preparation was equilibrated by washing with fresh Krebs-Henseleit solution and allowed to recover to base line. Various concentrations (figs. 3-5) of test compounds were added to the organ bath and incubated for 30 min, after which time a second concentration-response curve for the same agonist was obtained. Experiments using histamine and carbachol generated only one concentration-response curve for each preparation, because similar curves on first and second cumulative additions of these agonists were not obtained.

₂ agonist-induced relaxation of tracheae. Each tracheal strip was primed by carbachol (1 × 10⁶ M) twice within a 60-min interval, and a control cumulative concentration-response curve for salbutamol against contraction induced by 1 × 10⁶ M carbachol was obtained. After completion of the first concentration-response curve, each preparation was equilibrated by washing with fresh Krebs-Henseleit solution and allowed to recover to base line. Various concentrations (fig. 6) of test compounds were added to the organ bath and then incubated for 30 min, after which a second concentration-response curve was obtained in the same way.

Agonist-induced contraction of guinea pig ileum. Guinea pigs weighing 370 to 740 g were sacrificed by exsanguination. The terminal ileum was removed and suspended in Tyrode's solution with the following composition: 136.8 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 1.1 mM MgCl₂, 0.42 mM NaH₂PO₄, 11.9 mM NaHCO₃ and 5.6 mM glucose (pH 7.4). The ileum was divided into segments approximately 40 mm in length and set in a Magnus vessel containing 10 ml of Tyrode's solution aerated with a 95% O₂-5% CO₂ gas mixture. The tissue was placed under tension by a 1-g mass. The force generated by the tissue was isometrically measured. Ileal contractile response against 1 × 10⁹ M LTD₄ was measured first in the absence of the agent and then in the presence of the compound at various concentrations. An IC₅₀ value was calculated with linear regression analysis (maximum-likelihood method) using SAS.

U46619-induced platelet aggregation. Using a syringe containing 1 volume of 3.8% sodium citrate aqueous solution, we collected 9 volumes of blood. Guinea pig and human PRP was obtained by centrifuging the blood 10 min at 270 × g. The remaining blood was further centrifuged at 1100 × g for 10 min to yield PPP. The PRP was diluted with PPP to control the platelet count to 500,000 cells/µl. Platelet aggregation was induced by a stable analog of TXA₂, 1 × 10⁶ M U46619, and was measured using NBS Hema Tracer VI (Nikoh Bio Science, Tokyo, Japan). Various concentrations of the compound were added to the PRP 2 min before the addition of U46619, and an IC₅₀ value (50% inhibition concentration) was calculated from the inhibition ratio on the basis of the maximum light transmittance. All experiments were carried out within 4 h after blood collection to avoid a decrease in platelet sensitivity to U46619.

Statistical analysis. All data are shown as the means ± S.E.M. or the mean with 95% CL. The statistical values described were calculated by linear regression analysis for EC₅₀, IC₅₀ and pK_B values and by Schild plot analysis for pA₂ and slope values using SAS.

	Results

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LTD₄, U46619 and carbachol potently induce contraction of guinea pig tracheae. As shown in table 1, LTD₄ (1 × 10¹⁰ to 1 × 10⁷ M), U46619 (1 × 10⁹ to 3 × 10⁷ M) and carbachol (1 × 10⁸ to 1 × 10⁵ M) concentration-dependently induced contractions of tracheae isolated from guinea pigs (fig. 2). EC₅₀ values of 1.84 ± 0.42 nM for LTD₄, 5.52 ± 1.20 nM for U46619 and 59.60 ± 5.74 nM for carbachol were obtained. The maximum tensions were 2.37 ± 0.15 g at 1 × 10⁷ M LTD₄, 2.30 ± 0.23 g at 3 × 10⁷ M U46619 and 2.67 ± 0.20 g at 1 × 10⁵ M carbachol (table 1).

LTD₄-induced contractions of guinea pig tracheae. YM158 produced concentration-dependent parallel rightward shifts of the control concentration-response curves of LTD₄ in guinea pig tracheae (fig. 3A). As shown in table 2, the pK_B values at 3 × 10⁹, 1 × 10⁸ and 3 × 10⁸ M YM158 were independent of the concentration of YM158. Schild plot analysis (fig. 3B) revealed slopes not significantly different from unity and an average YM158 pA₂ value of 8.87 (8.65-9.24) (table 2). When average pA₂ values were compared, YM158 was shown to be approximately 10-fold less potent than montelukast (pA₂ = 9.68; 8.86-35.04). The pK_B values of pranlukast and zafirlukast at 3 × 10¹⁰ M were 10.19 ± 0.14 and 10.09 ± 0.10 (table 2), respectively.

U46619-, PGD₂- and PGF₂-induced contractions of isolated guinea pig tracheae. YM158 produced concentration-dependent parallel rightward shifts of the control concentration-response curves of U46619 in guinea pig tracheae (fig. 4A). As shown in table 3, the pK_B values at 3 × 10⁹, 1 × 10⁸ and 3 × 10⁸ M YM158 were independent of the concentration of YM158. Schild plot analysis (fig. 4B) revealed slopes not significantly different from unity and an average YM158 pA₂ value of 8.81 (8.63-9.09) (table 3). When average pA₂ values were compared, YM158 was shown to be approximately 2.5-fold more potent than seratrodast (pA₂ = 8.42; 8.11-9.10) (table 3).

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Selectivity of YM158. The effects of YM158 on contractile responses of guinea pig tracheae induced by various agonists were examined. As shown in table 4, YM158 at 1 × 10⁶ M did not affect responses to LTC₄, carbachol or histamine. Furthermore, YM158 at 1 × 10⁶ and 1 × 10⁵ M did not affect the relaxation of guinea pig tracheae induced by salbutamol (fig. 7).

LTD₄-induced contraction of guinea pig ilea. YM158 produced a concentration-dependent inhibition of guinea pig ileum contraction induced by 1 × 10⁹ M LTD₄ with an IC₅₀ value of 5.8 × 10¹⁰ M. As shown in table 5, pranlukast, montelukast and zafirlukast also concentration-dependently inhibited LTD₄-induced ileal contraction, with IC₅₀ values of 1.2 × 10¹⁰, 7.9 × 10¹¹ and 7.5 × 10¹¹ M, respectively.

U46619-induced guinea pig platelet aggregation. U46619 induces a concentration-dependent aggregation of guinea pig platelets (data not shown). YM158 shifted these U46619-induced concentration-response curves to the right in a parallel manner without reducing the maximum response (fig. 8). The average pA₂ value for YM158 was 7.08 (6.93-7.31), and the slope of the regression line (1.03, 95% CL: 0.77-1.29) by Schild plot analysis did not significantly differ from unity. The inhibitory effects of seratrodast, daltroban and YM158 were represented as an IC₅₀ value against platelet aggregation induced by a submaximal dose of U46619 (1 × 10⁷ M). As shown in table 6, the IC₅₀ value of YM158 was almost the same as those of seratrodast and daltroban.

U46619-induced human platelet aggregation. As shown in table 6, YM158 and daltroban produced inhibition of human platelet aggregation induced by U46619, and they exhibited these effects in the same concentration ranges as observed in the guinea pig platelet aggregation experiments. In contrast, the anti-aggregating effect of seratrodast in human platelets was about 84-fold less than that in guinea pig platelets.

	Discussion

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The present study has shown YM158 to be a potent and selective LTD₄ and TXA₂ receptor antagonist. The LTD₄ and TXA₂ receptor antagonistic activity of YM158 and other cys-LTs or TXA₂ antagonists, (montelukast, pranlukast, zafirlukast and seratrodast) was examined on LTD₄- or U46619-induced contraction of isolated from guinea pig tracheae. Schild plot analysis of the LTD₄ and TXA₂ antagonism of YM158 revealed slopes not significantly different from unity. Therefore, YM158 is a competitive antagonist for these tracheal LTD₄ and TXA₂ receptors. Furthermore, YM158 is expected to exhibit both LTD₄ (8.87) and TXA₂ (8.81) receptor antagonistic activity in the same dose range in vivo. Comparing pK_B or pA₂ values reveals that the LTD₄ receptor antagonistic activity of YM158 was approximately 20-fold less potent than that of zafirlukast and pranlukast and was 6.5-fold less potent than that of montelukast. However, 1 × 10⁶ M zafirlukast, pranlukast or montelukast did not affect the EC₅₀ value of U46619-induced contraction of isolated guinea pig lungs (data not shown). The TXA₂ receptor antagonistic activity of YM158 was 2.5-fold more potent than that of seratrodast.

LTD₄ is thought to be a strong mediator of ileal smooth-muscle contraction. YM158 also inhibited contractile response to LTD₄. The potency of YM158, zafirlukast, pranlukast and montelukast correlated well between the two tissues, trachea and ileum. Although the receptors for cys-LTs, (except for LTB₄; Yokomizo et al., 1997) have not been cloned, these results suggest that the LTD₄ receptors in trachea and ileum are the same type. YM158 also exhibited antagonistic activity on PGD₂- and PGF₂-induced contraction of guinea pig tracheae. Because it has been reported that the bronchoconstrictor activity of PGD₂ and FGF₂ is linked to TP receptor activation (Mais et al., 1985; McKenniff et al., 1988; Gardiner, 1990), this inhibitory effect of YM158 is thought to work through its TXA₂ receptor blocking activity. The rightward shift of PGD₂-evoked responses occurred in a parallel fashion, whereas the concentration-response curves for PGF₂ were shifted to the right in a nonparallel manner. This nonparallel shift of PGF₂-evoked response may be explained by the PGF₂-induced stimulation via EP₂-relaxant receptor (Gardiner, 1986; Honda et al., 1993). PGF₂ is a full agonist at FP-contractile receptor (Giles et al., 1991; Sugimoto et al., 1994) and also has an agonistic activity to EP₂-relaxant and TP-contractile receptors (Coleman et al., 1994), which indicates that PGF₂-induced tracheal contraction is the total response of its effects via TP- and FP-contractile receptors and EP₂-relaxant receptors. Therefore, it is possible that YM158 only antagonizes the TP receptor-mediated contraction among PGF₂-mediated contractile responses and that it shows a noncompetitive inhibition. On the other hand, PGD₂ is a full agonist at DP-contractile receptors (Giles et al., 1991) and also has an agonistic activity to EP₂-relaxant and TP- and FP-contractile receptors (Coleman et al., 1994). YM158, which is a competitive inhibitor to TP receptor (table 3, fig. 4), exhibited a competitive inhibition on PGD₂-induced tracheal contraction, which indicates that TP receptor-mediated contraction was mainly involved in the PGD₂-evoked tracheal contractile response. Coleman and co-workers (1994) reported that these receptors' distribution in the lungs were suggested, and the expression of cloned cDNAs of EP₂ and FP receptors in the lungs were also reported (Honda et al., 1993; Sugimoto et al., 1994). Therefore, various types of receptors were related to these responses, and it was difficult to determine the inhibitory pattern from these functional studies on PGD₂- and PGF₂-induced tracheal contractions.

YM158 showed almost the same potency of antagonistic activity as seratrodast and daltroban in inhibiting the aggregation of guinea pig platelets induced by U46619. Schild plot analysis of YM158 inhibiting U46619-induced guinea pig platelet aggregation revealed that YM158 is a competitive antagonist for the TXA₂ receptor in platelets. Furthermore, YM158 and daltroban showed the same potent antagonistic activity on human platelet aggregation as in guinea pig platelet aggregation. In contrast, the antagonism of platelet aggregation by seratrodast against human platelets was approximately 80-fold lower than that against guinea pig platelets. One of the reasons for the decrease in the antagonistic activity of seratrodast is that the protein-binding rate in human plasma is higher than that in guinea pig plasma. It has been reported that the amount of seratrodast unbound in plasma is 3.6% in guinea pigs and 0.1% in humans (Miwa et al., 1993).

YM158 did not antagonize LTC₄-, histamine- and carbachol-induced contraction of guinea pig tracheae, which indicates that the action of YM158 is highly selective. YM158 also showed low efficacy in inhibiting ₂ stimulant-induced relaxation of guinea pig tracheal smooth muscle. Taken together, these data suggest that YM158 is a novel, selective and dual antagonist for LTD₄ and TXA₂ receptors.

Both LTD₄ and TXA₂ play important roles in the pathogenesis of asthma. LTD₄ induces potent bronchoconstriction and enhances the release of mediators from inflammatory cells, whereas TXA₂ induces bronchial hyperreactivity and bronchoconstriction. LTD₄ receptor antagonists (Taniguchi et al., 1993; Dahlén et al., 1994; Spector et al., 1994) and synthetase inhibitors (Busse, 1996), as well as TXA₂ receptor antagonists (Fujimura et al., 1991) and synthetase inhibitors (Kurashima et al., 1992), have proved to be effective treatments in patients with bronchial asthma. As described previously, the mechanisms by which LTD₄ and TXA₂ contribute to the pathogenesis of bronchial asthma are different. Therefore, a dual antagonist for both LTD₄ and TXA₂ receptors is expected to show strong anti-asthmatic effects in most patients. Thus YM158 is thought to be a leading potential candidate as a novel therapeutic agent for the treatment of bronchial asthma.