Phillip J. Gardiner

BAY u9773, a novel antagonist of cysteinyl-leukotrienes with activity against two receptor subtypes

The effects of BAY u9773 (6(R)-(4-carboxyphenylthio)-5(S)-hydroxy-7(E),9(E), 11(Z),14(Z)-eicosatetraenoic acid), a cysteinyl-leukotriene analogue, were investigated on a variety of smooth muscle preparations in order to determine its profile as a cysteinyl-leukotriene receptor antagonist. The tissues were contracted with leukotriene C4 or leukotriene D4 and their receptor characteristics defined as either typical or atypical according to the activity or inactivity, respectively, of the selective antagonists ICI 198615, MK 571 and SKF 104353. BAY u9773 antagonised typical cysteinyl-leukotriene receptors with pA2 (or pKB) values in the range 6.8-7.4 and also antagonised atypical receptors with pA2 values in the range 6.8-7.7. However, BAY u9773 had no effect at 10(-6) M against a selection of non-leukotriene stimuli in the same preparations. BAY u9773 competitively displaced [3H]leukotriene D4 binding to guinea-pig lung homogenate, with a pKi of 7.0 +/- 0.1. In the guinea-pig lung strip, BAY u9773 was found to be inactive at 10(-6)M against leukotriene C4- and leukotriene D4-induced contractions, which may suggest the existence of a third type of cysteinyl-leukotriene receptor. These data demonstrate that BAY u9773 is a selective cysteinyl-leukotriene receptor antagonist with comparable activity at both typical and atypical receptors and as such represents a valuable tool for the study of cysteinyl-leukotriene receptors.

BAY u3405, a potent and selective thromboxane A2 receptor antagonist on airway smooth muscle in vitro

1 BAY u3405 (3(R)‐[[(4‐fluorophenyl) sulphonyl]amino]‐1,2,3,4‐tetrahydro‐9H‐carbazole‐9‐propanoic acid) has been evaluated on airway smooth muscle, from a number of species including man, for its thromboxane A2 (TXA2) antagonist activity. 2 BAY u3405 was a potent, and competitive, antagonist of the TXA2‐mimetic U46619‐induced contractions of human, guinea‐pig, rat and ferret airway smooth muscle with pA2 values between 8.0 and 8.9 and with no inherent contractile activity (10−9–10−4 m). 3 The TXA2 antagonist activity of BAY u3405 was stereoselective. Its (S)‐enantiomer, BAY u3406, was approximately 50 fold less effective against U46619 on guinea‐pig and human airway smooth muscle. 4 BAY u3405 also competitively antagonized contractions of guinea‐pig airway smooth muscle induced by prostaglandin D2 (PGD2) or its metabolite 9α, 11β‐PGF2. On human and ferret airway smooth muscle it abolished contractions induced by PGD2, PGF2α and 16, 16‐dimethyl‐PGE2. 5 A high concentration (10−6 m) of BAY u3405 had no effect on the contraction, or relaxation, of airway smooth muscle induced by a range of other agonists, nor did BAY u3405 have any effect on other prostanoid receptor types (DP, EP1, EP2, FP or IP). 6 BAY u3405, in contrast to some other TXA2 antagonists, is a potent and selective antagonist on a wide range of airways including human. This high affinity, and the oral activity of the compound described elsewhere, suggest it may be an appropriate tool to investigate the role of prostanoids in airway diseases such as asthma.

Leukotriene receptors on human pulmonary vascular endothelium.

1 Cysteinyl‐leukotrienes cause contractions and/or relaxations of human isolated pulmonary vascular preparations. Although, the localization and nature of the receptors through which these effects are mediated have not been fully characterized, some effects are indirect and not mediated via the well‐ described LT1 receptor. 2 In human pulmonary veins (HPV) with an intact endothelium, leukotriene D4 (LTD4) induced contraction above basal tone. This response was observed at lower concentrations of LTD4 in the presence of nitric oxide synthase inhibitor Nω‐nitro‐L‐arginine (L‐NOARG). Contractions (in the absence and presence of L‐NOARG) were partially blocked by the LT1 antagonists (MK 571 and ICI 198615). 3 LTD4 relaxed HPV previously contracted with noradrenaline. This relaxation was potentiated by LT1 antagonists, but was abolished by removal of the endothelium. LTD4 also relaxed human pulmonary arteries (HPA) precontracted with noradrenaline but this effect was not modified by LT1 antagonists. 4 The results suggest that contraction of endothelium‐intact HPV by LTD4 is partially mediated via LT1 receptors. Further, in endothelium‐intact HPV, this contraction was opposed by a relaxation induced by LTD4, dependent on the release of nitric oxide, which was mediated, at least in part, via a non‐LTi receptor. In addition, LTD4 relaxation on contracted HPA was not mediated by LT1 receptors. 5 The mechanical effects of LTD4 on human pulmonary vasculature are complex and involve both direct and indirect mechanisms mediated via at least two types of cysteinyl‐leukotriene receptors.

Inhibitory effects of BAY u3405 on prostanoid-induced contractions in human isolated bronchial and pulmonary arterial muscle preparations

1 The thromboxane‐mimetic, U46619, was a more potent contractile agonist than prostaglandin D2 (PGD2), PGF2α or histamine in human isolated bronchial and pulmonary arterial muscle preparations. 2 Human isolated proximal bronchial muscles were less sensitive to contractile agents than distal bronchial preparations. However, the former tissues developed a greater contractile force (Emax) when compared with results obtained in the latter tissues. 3 BAY u3405 attenuated the contractions induced by U46619, PGF2α and PGD2 in both human isolated bronchial and pulmonary arterial muscle preparations. 4 BAY u3405 did not alter histamine concentration‐effect curves nor the relaxation induced by Butaprost (TR4979) in human isolated bronchial muscle preparations. In addition BAY u3405 did not modify the relaxation induced by PGI2 in isolated pulmonary arterial muscle preparations. 5 The contractions induced by different prostaglandins were blocked by BAY u3405, suggesting a common functional site for these agents found both on human bronchial and pulmonary arterial muscles.

BAY u3405 an antagonist of thromboxane A2‐ and prostaglandin D2‐induced bronchoconstriction in the guinea‐pig

1 The novel thromboxane (TX) antagonist, BAY u3405, has been evaluated against bronchoconstriction induced by the TXA2 mimetic U‐46619, prostaglandin D2 (PGD2), 5‐hydroxytryptamine (5‐HT), leukotriene D4 (LTD4) and histamine in the guinea‐pig in vivo by use of a modification of the model described by Konzett & Rössler. 2 When given intravenously (i.v.) at 30 or 100 μg kg−1, U‐46619 caused 80% maximal bronchoconstriction in most animals. In contrast, PGD2 caused a smaller 40%‐50% maximal bronchoconstriction at the highest dose tested (300 μg kg−1, i.v.). 3 BAY u3405, given intravenously, orally (p.o.) or by aerosol antagonized U‐46619‐induced bronchoconstriction in a dose‐related manner. The approximate ID50 values were 600 μg kg−1, i.v., 1.7 mg kg−1 p.o. and 0.1% w/v 20 breaths by aerosol. 4 BAY u3405 had similar inhibitory activities against U‐46619‐induced bronchoconstriction and hypertension suggesting that it had no preferential activity on the airways. 5 When given intravenously BAY u3405 antagonized the bronchoconstrictor effect of intravenous PGD2 with ID50 values between 30–100 μg kg−1. 6 The action of BAY u3405 (10 mg kg−1, p.o.) was long lasting, causing significant inhibition of U‐46619‐induced bronchoconstriction 7 h after dosing. 7 At 1 mg kg−1, i.v., a dose that abolished the response to U‐46619 and PGD2, BAY u3405 had no effect on histamine‐, 5‐HT‐ or LTD4‐induced bronchoconstriction. 8 BAY u3405 potently and selectively antagonized U‐46619‐ or PGD2‐induced bronchoconstriction in the Konzett‐Rössler model of guinea‐pig lung function. It should therefore prove to be a useful tool for defining the role of TXA2 and PGD2 in airway diseases such as asthma.

Evidence for two leukotriene receptor types in the guinea-pig isolated ileum

Leukotriene (LT) receptors in the guinea-pig ileum were characterized using LTB4, LTC4, LTD4 and LTE4 and the LT antagonists FPL 55712, ICI 198615 and (+/-)SKF 104353. LTB4 was inactive but the other LTs induced concentration-related contractions. LTC4 responses differed to those induced by LTD4 or LTE4. Inhibitors of LT metabolism had no significant effects on any LT responses. LTD4 contractions were inhibited by all three antagonists but a resistant response was apparent at concentrations of ICI 198615 greater than 10(-8) M. All three antagonists were weak/inactive against LTC4. LTE4 was a partial agonist which antagonized LTD4 responses but had little or no activity against LTC4 or histamine. These results suggest that two distinct LT receptor types exist on guinea-pig ileum. One type is predominantly activated by LTD4 and is antagonized by three structurally distinct LT antagonists and the partial agonist LTE4. The second type is predominantly activated by LTC4 and is insensitive to the LT antagonists.

Inhibition of antigen-induced contraction of guinea-pig airways by a leukotriene synthesis inhibitor, BAY x1005

BAY x1005 ((R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid), an inhibitor of leukotriene synthesis, was evaluated, both in vitro and in vivo, for inhibition of antigen-induced airway contraction in the sensitised guinea-pig. Antigen (ovalbumin 0.001-10 micrograms/ml) challenge of tracheae in the presence of pyrilamine and indomethacin induced contractile responses which were inhibited by BAY x1005 with an IC50 value of 0.36 (0.2-0.8) microM. Using the same test system BAY x1005 (1 microM), ICI D2138 (0.3 microM) or AA 861 (1 microM) had similar inhibitory activities, whereas MK 886, MK 591, and Zileuton (A64077) all tested at 1 microM and REV 5901 (10 microM) had no significant effect. Using tracheae from non-sensitised (control) guinea-pigs the calcium ionophore A23187 (1 microM) induced a maximal contraction which was significantly inhibited by BAY x1005 at 1 microM, whereas MK 886 was only active at 3 microM. BAY x1005 tested at 10 microM and 3 microM had no effect against leukotriene D4- or KCl-induced contractions of guinea-pig tracheae respectively. In the anaesthetised ovalbumin sensitised guinea-pig BAY x1005 caused a dose-related inhibition of ovalbumin-induced bronchoconstriction, with approximate ID50 values of 0.85 mg/kg i.v. and 6.3 mg/kg p.o. In the same model MK 886, MK 591, AA 861 and ICI D2138 each given at 10 mg/kg p.o. had no significant inhibitory activity against antigen challenge. Six hours after administration BAY x1005 (10 mg/kg p.o.) was still effective against the antigen-induced response.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibition of [3H]leukotriene D4 binding to guinea-pig lung membranes. The correlation of binding affinity with activity on the guinea-pig ileum

Guinea-pig lung membranes contain high affinity (KD = 0.8 nM) binding sites for [3H]leukotriene D4 [( 3H]LTD4). The binding is inhibited by leukotriene antagonists, such as ICI 198615 and SK&F 104353, in a manner consistent with the Law of Mass Action at a single site. It is also inhibited by a range of leukotriene analogues in a dose-related manner. Inhibition by some of these e.g. LTC4 suggests that the [3H]LTD4 binding sites are heterogeneous. The binding affinity of the leukotriene analogues is significantly correlated (P less than 0.001) to their spasmogenic activity on guinea-pig ileum but not on guinea-pig lung strip. The binding affinity of the leukotriene antagonists is also correlated to their antagonist activity on guinea-pig ileum (P less than 0.05) but not on guinea-pig lung. These results indicate that the [3H]LTD4 binding site in guinea-pig lung is similar to the leukotriene receptor activated by LTD4 and LTE4 on guinea-pig ileum. The contractile response of guinea-pig lung strips to leukotrienes, in the presence of indomethacin, is mediated by a distinct type of leukotriene receptor.

Characterisation of the peptido-leukotriene receptor PL2 on the ferret spleen strip.

The peptido-leukotriene receptor(s) (PL) on the ferret isolated spleen strip have been characterised by functional studies using the naturally occurring leukotrienes (LTs), a range of structurally distinct PL antagonists, and by ligand binding studies. LTB4 (0.01-10 microM) was inactive on ferret spleen whereas LTC4, LTD4 and LTE4 produced concentration-related contractions with maximal responses, relative to noradrenaline, of 57% (EC50 0.28 microM), 60% (EC50 0.5 microM) and 7% respectively. The leukotriene responses were unaltered by L-serine borate, L-cysteine, indomethacin, phentolamine, propranolol, mepyramine, methysergide or atropine, suggesting that the peptido-leukotrienes were acting through distinct PL receptors. The PL1 antagonists, FPL 55712 (0.01-10 microM), ICI 198615 (10 microM), SK&F 104353 (10 microM) and MK541 (10 microM) were all inactive against LTC4- or LTD4-induced contractile responses. LTE4 was a partial agonist with respect to LTC4 and LTD4 with pKB values of 5.8 and 5.5 respectively. Nifedipine (0.1 microM) produced a rightward shift of the concentration-response curves to both LTC4 and LTD4 and depressed their maximal responses. An unacceptably high level of non-specific binding of [3H]LTD4 to membrane preparations of ferret spleen prevented characterisation of this receptor by ligand binding. These results suggest that the ferret spleen has a homogeneous population of a PL receptor type which is insensitive to existing PL1 receptor antagonists. The functional characteristics of this PL receptor type are similar to those of the PL2 receptor on other tissues. The absence of PL1 receptors on this tissue makes it particularly useful in identifying new and selective drug tools for the PL2 receptor.

The thromboxane receptors of rat and guinea-pig lung.

The thromboxane (TXA2) receptors on rat and guinea-pig lung strips were compared using TXA2 agonists and TXA2 receptor antagonists. On rat lung strip several TXA2 mimetics were full agonists whilst the primary prostanoids lacked agonist activity. On guinea-pig lung strip the same agonists displayed markedly different efficacies. Both preparations contained homogeneous populations of TXA2 receptors as evidenced by BAY u3405 giving comparable pA2 values against four TXA2 mimetics. However, the observed pA2s of nine different TXA2 receptor antagonists, determined against U46619, did not correlate between the two preparations. These results point to the existence of TXA2 receptor subtypes.