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

ejp ELSEVIER

European Journal of Pharmacology 264 (1994) 317-323

BAY u9773, a novel antagonist of cysteinyl-leukotrienes with activity against two receptor subtypes Stephen R. Tudhope *, Nigel J. Cuthbert, Trevor S. Abram, Michael A. Jennings, Robert J. Maxey, Angela M. Thompson, Peter Norman, Phillip J. Gardiner Pharma Research, Bayer plc, Stoke Court, Stoke Poges SL2 4LY, UK Received 10 March 1994; revised MS received 22 June 1994; accepted 5 August 1994

Abstract

The effects of BAY u9773 (6(R)-(4'-carb•xypheny•thi•)-5(S)-hydr•xy-7(E)•9(E)•••(Z)••4(Z)-eic•satetraen•ic 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 C 4 or leukotriene D 4 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 pA 2 (or pK a) values in the range 6.8-7.4 and also antagonised 'atypical' receptors with pA 2 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 pK i of 7.0 -I- 0.1. In the guinea-pig lung strip, BAY u9773 was found to be inactive at 1 0 - 6 M against leukotriene C 4- 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.

Keywords: BAY u9773; Leukotriene D4; Leukotriene C4; Cysteinyl-leukotriene receptor

1. Introduction

Cysteinyl-leukotrienes are products of the 5-1ipoxygenase pathway of arachidonic acid metabolism (Samuelsson et al., 1979), predominantly produced by human leukocytes associated with the inflammatory response (Lewis and Austen, 1984). They are potent constrictors of smooth muscle, including both central and peripheral human airways (Dahl6n et al., 1980; Hanna et al., 1981) and have been demonstrated to increase vascular permeability (Peck et al., 1981) and stimulate mucus production (Johnson et al., 1983). These leukotrienes have therefore been implicated in a number of pathological inflammatory diseases including asthma, allergic rhinitis, inflammatory bowel disease and psoriasis (Busse and Gaddy, 1991). Inevitably, this has led to an intense search for specific antagonists

* Corresponding author. Tel. 0753 647171, fax 0635 566783. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 4 8 5 - 4

of these agents. However, as different cysteinylleukotriene receptors may be involved in such a variety of pathological conditions, the development of effective therapeutic agents relies on a thorough understanding of these receptors. In the last decade, many potent cysteinyl-leukotriene receptor antagonists have been reported, including SKF 104353 (Hay et al., 1987), ICI 198615 (Snyder et al., 1987) and MK 571 (Jones et al., 1989). These compounds are selective for a subset of cysteinylleukotriene receptors, and can be used to define cysteinyl-leukotriene receptor systems as 'typical' or 'atypical', depending on the activity or inactivity respectively, of these antagonists. In the course of developing a cysteinyl-leukotriene receptor antagonist by systematic modification of leukotriene E 4 (Abram et al., 1993), we identified BAY u9773 (Fig. 1) which was found to antagonise 'typical' and 'atypical' cysteinyl-leukotriene receptors on guinea-pig trachea (Cuthbert et al., 1991b) and 'atypi-

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c

o

2

OH n

Fig. 1. BAYu9773. cal' receptors on human pulmonary vein (Labat et al., 1992). Here we report a more detailed pharmacological profile of BAY u9773 and use it to further characterise the 'atypical' cysteinyl-leukotriene receptor subtype.

2. Materials and methods

were taken from the periphery of the lung and guineapig tracheal spirals were prepared by the method of Constantine (1965). Fresh adult sheep lungs were obtained from a local abattoir and rings of bronchial muscle (3-5 mm diameter) isolated. Adult male ferrets (1.2-1.5 kg) were killed by an overdose of sodium pentobarbitone, the spleen removed and cut into strips (Gardiner et al., 1993). All preparations were suspended in Tyrode's solution containing indomethacin (3 x 10 -6 M), to inhibit cyclooxygenase activity, and L-serine borate (4.5 x 10 -2 M) and L-cysteine (10 -2 M) to inhibit leukotriene metabolism (Krilis et al., 1983). The tissues were set up at a resting tension of 1 g and aerated with 5% CO2 in 0 2 at 37°C. Ferret spleen, sheep bronchus and rat lung tension changes were recorded isometrically, whilst guinea-pig trachea and lung strip contractions were recorded isotonically.

2.1. Materials Organ bath preparations were bathed in Tyrode's solution of the following composition (M): NaC1, 1.4 x 10-1; KCI, 2.7 X 10-3; CaCI2, 1.8 x 10-3; MgCI 2, 10-3; NaH2PO4, 4 x 10-4; NaHCO3, 1.2 x 10-2; glucose, 5.6 × 10 -3. Leukotriene C 4 and leukotriene D 4 were synthesised in our laboratories and determined to be 90-97% pure by analytical reverse-phase high performance liquid chromatography. BAY u9773 (6(R)-(4'carboxyphenylthio )-5( S )-hydroxy- 7( E ),9( E ),l l(Z),14 (Z)-eicosatetraenoic acid) was synthesised as previously described (Abram et al., 1993) and stored in dimethylsulphoxide (10 -2 M) under argon at -70°C. SKF 104353 (2-(S)-hydroxy-3-(R)-((2-carboxyethyl) thio)-3-(2-(8-phenyloctyl)phenyl) propanoic acid) was synthesised by Dr. H. Kluender (Bayer AG, Wuppertal, Germany), MK 571 ((+)-(E)-5-(3-(2-(7-chloroquinolin-2-yl)ethenyl-phenyl)-4,6-dithianonane dicarboxylic acid N,N-dimethylamide), by Dr. J. Butler (Bayer AG, Wuppertal, Germany) and ICI 198615 ((1((2-methoxy-4(((phenylsulfonyl)amino)carbonyl)phenyl) methyl)-lH-indazol-6-yl) carbonic acid cyclopentyl ester), was generously supplied by Dr. R. Krell (Zeneca Pharmaceuticals, Delaware, USA). Indomethacin, L-cysteine, L-serine, Tris hydrochloride, Polypep and noradrenaline were purchased from Sigma. Boric acid and histamine acid phosphate were purchased from BDH and prostaglandin F2~ and U46619 from Cascade Biochem. [3H]leukotriene D4 was supplied by Dr. M. Bye (Amersham International). All salts used were of AnalaR grade.

2.2. Organ bath studies 2.2.1. Male Dunkin Hartley guinea-pigs (350-500 g) and male Wistar rats (300-400 g) were killed by cervical dislocation followed by exsanguination. Lung strips

2. 2. 2. Dosing procedure The preparations were left to equilibrate for at least 1 h with regular washing at 15 min intervals. Tissue viability was determined by addition of KC1 (10-1 M) in all preparations except ferret spleen where noradrenaline (10 -5 M) was used. After washing and reequilibration, the tissues were incubated with antagonist or vehicle for 30 min before constructing cumulative concentration-responses to the cysteinyl-leukotriene. Control and test responses were determined on paired preparations from the same animal. At the end of the cumulative dosing procedure, maximal contractile responses were determined as before. One concentration-response curve was obtained from each preparation and 4-15 preparations were used to obtain each curve. 2.23. Data analysis The contractile responses of paired tissues were normalised to the final maximal responses obtained with KCI or noradrenaline and expressed as a percentage of the maximum agonist-induced contraction of the control tissue. ECs0 values (the concentration producing 50% of the maximal response) were determined by least squares non-linear regression to a sigmoidal curve and converted to pD 2 values by taking the negative logarithm. The pD 2 values of control and antagonisttreated tissues were compared for significance using a one-tailed, paired Student's t-test. Where significance was reached (P < 0.05), the dose ratio (DR) of the antagonist treated tissues, compared to their controls, was used to calculate the pK B values from the following equation: pK B = log ((DR - 1)/[Antagonist](M)) For 'n' paired tissues, the mean + standard error (S.E.M.) was determined, pA 2 values were calculated by the method of Arunlakshana and Schild (1959),

S.R. Tudhope et al. / European Journal of Pharmacology 264 (1994) 317-323

constraining the slopes to unity after least squares linear regression analysis had shown the slopes not to be significantly different from unity.

2.3. [3H]Leukotriene D 4 binding assay Guinea-pig lung membranes were prepared as previously described (Norman et al., 1990). Binding assays were performed at 20°C in 250 /zl buffer containing Tris HCI (5 x 10 -2 M) pH 7.4, L-cysteine (10 -2 M) and Polypep (1%). Assays were initiated by addition of 50/zl (100-500/xg protein) lung membranes and terminated after 15 min by addition of 3.5 ml ice-cold buffer (5 x 10 -2 M Tris HC1 pH 7.4) and rapid filtration through Whatman G F / B filters, which were washed twice with 3.5 ml ice-cold buffer. Inhibition experiments were performed by incubating [3H]leukotriene D 4 ( ~ 7 × 10-to M) with increasing concentrations of BAY u9773 between 10 -9 M and 10 -5 M. Non-specific binding was defined as binding in the presence of 2 x 10 -6 M leukotriene D 4 and was typically 15% of total binding. After subtraction of nonspecific binding, ICs0 values were obtained from a Hill plot by linear regression and transformed into K i values as described by Cheng and Prusoff (1973).

3. Results

3.1. Organ bath studies 3.1.1. Reference cysteinyl-leukotriene receptor antagonists The reference antagonists ICI 198615, MK 571 and SKF 104353 were potent inhibitors of leukotriene D ainduced contractions of guinea-pig trachea and both leukotriene D a and leukotriene Ca-induced contractions of rat lung (Table 1). They were much less potent,

319

or inactive, on all other test systems examined (Table 1). With the exception of guinea-pig trachea, the antagonist potencies on the other tissues were not significantly different whether leukotriene D a or leukotriene C a was used to contract the tissues (t-test, P > 0.05).

3.1.2. B A Y u9773 BAY u9773 had no intrinsic activity on any of the tissue preparations in the presence of indomethacin, which was routinely added to remove any effects of stimulated or endogenously released cyclooxygenase products (Mong et al., 1987). However, in the absence of indomethacin, BAY u9773 (10 -6 M) produced a significant contactile response on guinea-pig lung (40 + 2% of maximum tissue contraction induced by leukotriene Da, n = 4 observations). BAY u9773 antagonised leukotriene D a- and leukotriene Ca-induced contractions of all test tissues with the exception of guinea-pig lung (Table 2). Illustrative dose-response curves are shown in Fig. 2. In all preparations, BAY u9773 produced parallel shifts in the dose-response curves with no significant depression of the maxima, and the Schild plot gradients were not significantly different from unity. Furthermore, in each preparation, the results were not significantly different whether leukotriene D 4 or leukotriene C a was used to contract the tissues (t-test, P > 0.05). However, one-way analysis of variance (ANOVA) calculations indicated that there was a marginally significant difference (P < 0.05) between the effects of BAY u9773 on the guineapig trachea and ferret spleen preparations, and a highly significant difference (P < 0.001) between the ferret spleen and sheep bronchus pA 2 values. BAY u9773 had no significant effect on a variety of non-leukotriene-induced contractions of the test tissues (Table 3).

Table 1 The effect of reference antagonists on cysteinyl-leukotriene-induced contractions of test tissues Tissue

Agonist

p K B of antagonists (concentration (M)) ICI 198615

MK 571

SKF 104353

Guinea-pig trachea Guinea-pig trachea Rat lung Rat lung Guinea-pig lung Guinea-pig lung Ferret spleen Ferret spleen Sheep bronchus Sheep bronchus

LTD 4 LTC 4 LTD 4 LTC 4 LTD 4 LTC 4 LTD 4 LTC 4 LTD 4 LTC 4

8.7 + 0.4 (10 -7) 5.5 + 0.2 (10 -5) 7.5 _+ 0.1 (10 -7) 7.6 + 0.1 (10 -7) 5.7 + 0.2 (10 -5) 5.9 + 0.3 (10 -5) I (10 -5) 5.6 + 0.1 (10 -5) I (10 -6) I (10 -6)

8.2 + 0.2 (10 -8) 5.6 __.0.1 (10 -6) 7.5 + 0.1 (10 -6) 7.8 + 0.4 (10 -7) 5.1 + 0.2 (10 -5) ND 4.7 + 0.1 (10 -5) I (10 -5) I (10 -6) I (10 -6)

7.8 + 0.3 6.3 + 0.1 8.0 + 0.3 8.2 -I- 0.3 ND ND I (10 -5) I (10 -5) I (10 -6) I (10 -6)

(10 -5) (10 -5) (3 × 10 -8) (3 x 10 -7)

p K B values are + S.E.M. for n = 4-15 observations. ND - not determined. I - inactive, control and antagonist-treated tissues not significantly different.

S.R. Tudhope et al. / European Journal of Pharmacology 264 (1994) 317-323

320

3.2. BAY u9773 displacement of [3H]leukotriene D 4 binding A typical displacement curve of [3H]leukotriene D 4 binding to guinea-pig lung membranes by BAY u9773 is shown in Fig. 3. In four similar experiments the Hill slope of the displacement curve was not significantly different from unity, which is consistent with the Law of Mass Action for interaction at a single site. The K i

A. Guinea-pig

values were calculated as described earlier and gave a m e a n p K i of 7.0 ___0.1.

4. Discussion The heterogeneity of cysteinyl-leukotriene receptors is now well documented. In a mixed receptor population, such as guinea-pig trachea (Snyder and Krell,

B. Guinea-pig

trachea

trachea

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C. Rat lung

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Log [leukotriene D4] (M)

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I

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i -9

i -8

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i -8

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Log [leukotriene D4] (M)

Fig. 2. (continued on next page). Effects of BAY u9773 on leukotriene C 4- or leukotriene D4-induced contractions of guinea-pig trachea (A and B), rat lung (C), guinea-pig lung (D), ferret spleen (E) and sheep bronchus (F). Open symbols represent control curves; o = leukotriene D4; [] = leukotriene C a. Closed symbols represent concentration-response curves in the presence of BAY u9773 at 10 -7 M (a), 10 -6 M ( , t ) 3 × 10 -6 M ( l l ) and 10 -5 M (*). Each point is the mean of 4-13 observations. Vertical lines are the standards errors.

S.R. Tudhope et aL / European Journal of Pharmacology 264 (1994) 317-323 E. Ferret spleen

F. Sheep bronchus

f~

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321

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Log [leuko~iene C4] (M) Fig. 2 (continued)

1984) and guinea-pig ileum (Gardiner et al., 1990), leukotriene D 4 selectively activates 'typical' cysteinylleukotriene receptors and leukotriene C a selectively activates 'atypical' receptors. However, in homogeneous cysteinyl-leukotriene receptor populations, leukotriene D 4 and leukotriene C 4 are both full agonists whether the receptors are 'typical' (Buckner et al., 1986; Falcone and Krell, 1992) or 'atypical' (Snyder and Krell, 1986; Gardiner et al., 1993; Cuthbert et al., 1991a). Leukotriene E 4 is a partial agonist in many preparations (Gardiner et al., 1990; Labat et al, 1992; Gardiner et al., 1993), only behaving as a full agonist in tissues with a high receptor reserve. As the naturally occurring full agonists are not generally specific for the receptor subtypes, it would be misleading to refer to -

'leukotriene D 4 receptors' or 'leukotriene C 4 receptors'. Gardiner et al. (1993) has therefore proposed that the 'typical' cysteinyl-leukotriene receptor be termed PL1 (PL denoting peptido-leukotriene, i.e. the full agonists) and that the 'atypical' cysteinylleukotriene receptor be termed PL2. This receptor nomenclature is currently being reviewed by the I U P H A R committee for receptor nomenclature (P.J. Gardiner, Bayer plc, private communication), and in the absence of a universally accepted nomenclature, this terminology will be used henceforth in this discussion.

120

Table 2 The effect of BAY u9773 on cysteinyl-leukotriene-induced contractions of test tissues Tissue

Agonist

pA 2 (Schild gradient)

Guinea-pig trachea Guinea-pig trachea Rat lung Rat lung Guinea-pig lung Guinea-pig lung Ferret spleen Ferret spleen Sheep bronchus Sheep bronchus

LTD 4 LTC 4 LTD 4 LTC 4 LTD 4 LTC 4 LTD 4 LTC 4 LTD 4 LTC 4

7.4+0.1 7.4 5:0.1 6.8 + 0.3 7.2 + 0.4 I I 6.8 _+0.1 6.9 -I-0.2 7.7 _+0.1 7.6_+0.1

'

(1.0+0.2) (1.1 _+0.1) a a

(1.0 _+0.2) (0.9 5: 0.2) (1.0 5: 0.2) (0.8___0.2)

p A 2 values are 5:S.E.M. for n = 4-12 observations at three or four

different concentrations of BAY u9773. I - inactive, control and BAY u9773 (10 -6 M)-treated tisues not significantly different. a p K a 5: S.E.M. at 10 -6 M BAY u9773 for n = 4 observations.

100

4o 2O

Log [BAY u9773] (M)

Fig. 3. Inhibition of 7 × 1 0 -1° M [3H]leukotriene D 4 binding to guinea-pig lung membranes by BAY u9773. Each point was determined in triplicate and represents the m e a n + S . E . M . A simple mass action curve was fitted to the data using an iterative curve fitting routine. The data shown are from a single experiment representative of four similar experiments•

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Table 3 The effect of BAY u9773 on non-leukotriene stimuli in test tissues Tissue

Guinea-pig trachea Guinea-pig trachea Rat lung Guinea-pig lung Ferret spleen Ferret spleen Sheep bronchus Sheep bronchus

Agonist

Histamine Prostaglandin F2,~ U46619 Histamine Noradrenaline Prostaglandin F2~ Carbachol 5-Hydroxytryptamine

pD 2 values Control

BAY u9773 (concentration (M))

5.2 + 6.4 + 5.0 + 4.9 + 5.7 + 5.0 + 6.4 + 5.4 +

5.0 + 0.2 (10 -5) 6.3 + 0.1 (10 -5) 5.2 -t- 0.1 (10 -6) 5.1 + 0.3 (10 -6) 5.6 + 0.1 (10 -6) 4.8 + 0.2 (10 -6) 6.5 + 0.2 (10 -5) 5.1 + 0.3 (10 -5)

0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1

pD 2 values are + S.E.M. for n = 2-6 observations.

The reference PL1 receptor antagonists, ICI 198615, MK 571 and SKF 104353 were shown here to be very potent (pK B > 7.5) on the guinea-pig trachea contracted by leukotriene D4and the rat lung stimulated by either leukotriene D 4 or leukotriene C 4. This confirms that leukotriene D 4 activates PL1 receptors on guinea-pig trachea, and that the rat lung contains a homogenous population of PL1 receptors. The much weaker activity of the PL1 receptor antagonists in the other tissue systems confirm that leukotriene C 4 activates PL2 receptors in guinea-pig trachea, and that guinea-pig lung, ferret spleen and sheep bronchus display a homogeneous PL2 receptor profile, under the conditions described. The only anomaly was SKF 104353, which was significantly more potent than ICI 198615 and MK 571 on the guinea-pig trachea PL2 receptor system ( P < 0.002). This may relate to the fact that SKF 104353, like BAY u9773, shares many features of the natural cysteinyl-leukotrienes. However, SKF 104353 was inactive on the homogeneous PL2 receptor systems of ferret spleen and sheep bronchus at the concentrations studied. It was also noted that ICI 198615 was significantly ( P = 0.014) more potent on the leukotriene Da-induced contractions of guineapig trachea (pK B = 8.7 + 0.4) than on rat lung (pK B = 7.5 + 0.1). These discrepancies may indicate the existence of subtypes of the PL1 and PL2 receptors or may just reflect small structural differences of the receptors between species. In contrast to the reference antagonists, BAY u9773 inhibited both PL1 and PL2 receptors in a concentration-dependant manner with pA 2 (or p K B) values in the range 6.8-7.7. It was, however, inactive against leukotriene D 4- and leukotriene C4-induced contractions of the guinea-pig lung preparation. BAY u9773 antagonised the PL1 and PL2 receptors of guinea-pig trachea equally (pA 2 = 7.4 + 0.1). Indeed, in all preparations, there was no significant difference ( P > 0.3) between the antagonist potency against leukotriene D 4 and leukotriene C 4. There was, however, a significant difference ( P < 0.001) between the pA 2 values obtained on the homogeneous PL2 receptor populations of ferret spleen (6.8-6.9) and the sheep bronchus (7.6-

7.7). Again, this may reflect subtypes of the P L 2 receptor; however, as BAY u9773 did not distinguish between the PL1 and PL2 receptor subtypes of guinea-pig trachea, we consider that it is unlikely to distinguish between subtypes of the PL2 receptor. It is therefore probable that the differing pA 2 values reflect species differences between the sheep and ferret. The guinea-pig lung preparation was earlier classified as PL2, due to the lack of cysteinyl-leukotriene inhibition by the reference PL1 receptor antagonists. However, BAY u9773 was also inactive in this preparation, whereas it effectively blocked both PL1 and PL2 receptor subtypes in the trachea of the same species. We conclude that this receptor is neither PL1 nor PL2, and assuming that the direct effects of cysteinyl-leukotrienes on guinea-pig lung (in the presence of indomethacin) are mediated by a cysteinyl-leukotriene receptor, then this receptor may be termed PL3. For the same reasons as given earlier, we consider it unlikely that this PL3 receptor is a subtype of PL1 or PL2. However, confirmation of a PL3 receptor must await the identification either of a PL3 receptor antagonist, a selective agonist, or protein sequence determination by molecular biology. BAY u9773 was demonstrated to be a specific antagonist of cysteinyl-leukotrienes, having no effect on a range of non-leukotriene stimuli at concentrations that had a marked effect on cysteinyl-leukotriene-induced responses. BAY u9773 also displaced [3H]leukotriene D 4 binding to guinea-pig lung membranes (pK~ = 7.0 + 0.1) in a manner consistent with the Law of Mass Action for interaction at a single site. The Hill slope of the displacemement curve was not significantly different from unity, which is consistent with an antagonist interaction (Norman et al., 1990). In the presence of indomethacin, BAY u9773 had no intrinsic spasmogenic effect on any of the examined tissues, and thus is probably not inhibiting by virtue of partial agonism. We therefore conclude that BAY u9773, under the described conditions, is a competitive antagonist of both PL1 and PL2 cysteinyl-leukotriene receptors. It was noted, however, that in the absence of indomethacin, BAY u9773 was a partial agonist on the

S.R. Tudhope et al. / European Journal of Pharmacology 264 (1994) 317-323

guinea-pig lung preparation. This is consistent with the findings of Labat et al. (1992), where, in the absence of indomethacin, BAY u9773 was shown to be a partial agonist on human bronchus and pulmonary vein. This 'residual' agonism of BAY u9773 has also been observed in vivo, causing a dose-related bronchoconstriction when administered intravenously to anaesthetized guinea-pigs (H.P. Francis, Bayer plc; unpublished observation). Furthermore, it was discovered that BAY u9773 induced human neutrophil adhesion in vitro (R.G. Sturton, Bayer plc; unpublished observation). This agonist activity of BAY u9773 is difficult to explain, as it was not observed in the presence of indomethacin in the examined tissues. We speculate that BAY u9773 is a partial agonist of a subtype of PL1 receptors that is coupled to the release of cyclooxygenase products (Mong et al., 1987), and not responsible for the direct contractile activity of cysteinyl-leukotrienes. If this is the binding site detected on guinea-pig lung, then the binding affinity of BAY u9773 reported herein may reflect an agonist (or partial agonist) interaction, rather than an antagonist interaction. Although the 'residual' agonism of BAY u9773 limits its usefulness, it is nevertheless a valuable tool for the study of cysteinyl-leukotriene receptors. In this paper we have demonstrated the presence of PL2 receptors in three species and postulated the existence of a third type of leukotriene receptor.

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