Biochemical and pharmacological characterization of AZD1981, an orally available selective DP2 antagonist in clinical development for asthma.

BACKGROUND AND PURPOSE: The discovery of DP2 as a second receptor for PGD2 has prompted the search for antagonists as potential novel therapies based on the associations between PGD2 and disease. Here we describe the biochemical and pharmacological properties of 4-(acetylamino)-3-[(4-chlorophenyl)thio]-2-methyl-1H-indole-1-acetic acid (AZD1981), a novel DP2 receptor antagonist. EXPERIMENTAL APPROACH: Binding to DP2 , functional receptor pharmacology and selectivity were studied in both human and animal systems. KEY RESULTS: AZD1981 displaced radio-labelled PGD2 from human recombinant DP2 with high potency (pIC50 = 8.4). Binding was reversible, non-competitive and highly selective against a panel of more than 340 other enzymes and receptors, including DP1 (>1000-fold selective). AZD1981 inhibited DP2 -mediated shape change and CD11b up-regulation in human eosinophils, shape change in basophils and chemotaxis of human eosinophils and Th2 cells with similar potency. AZD1981 exhibited good cross-species binding activity against mouse, rat, guinea pig, rabbit and dog DP2 . Evaluation in mouse, rat or rabbit cell systems was not possible as they did not respond to DP2 agonists. Agonist responses were seen in guinea pig and dog, and AZD1981 blocked DP2 -mediated eosinophil shape change. Such responses were more robust in the guinea pig, where AZD1981 also blocked DP2 -dependent eosinophil emigration from bone marrow. CONCLUSIONS AND IMPLICATIONS: AZD1981 is a DP2 antagonist that blocks functional responses in eosinophils, Th2 cells and basophils. It exhibited similar potency irrespective of the cell type, DP2 agonist or species used. This selective orally active agent is currently under clinical evaluation as a potential therapeutic agent in respiratory diseases including asthma.

Formoterol and salmeterol induce a similar degree of ß2-adrenoceptor tolerance in human small airways but via different mechanisms.

BACKGROUND AND PURPOSE: Steroids prevent and reverse salbutamol-induced ß(2)-adrenoceptor tolerance in human small airways. This study examines the effects of the long-acting ß(2) agonists (LABAs) formoterol and salmeterol, and the ability of budesonide to prevent desensitization. EXPERIMENTAL APPROACH: Long-acting ß(2) agonists in the presence and absence of budesonide were incubated with human precision-cut lung slices containing small airways. Tolerance was deduced from measurements of reduced bronchodilator responses to isoprenaline and correlated with ß(2)-adrenoceptor trafficking using a virally transduced, fluorescent-tagged receptor. The ability of the LABAs to protect airways against muscarinic-induced contraction was also assessed. KEY RESULTS: Following a 12 h incubation, both formoterol and salmeterol attenuated isoprenaline-induced bronchodilatation to a similar degree and these effects were not reversible by washing. Pre-incubation with budesonide prevented the desensitization induced by formoterol, but not that induced by salmeterol. Formoterol also protected the airways from carbachol-induced bronchoconstriction to a greater extent than salmeterol. In the epithelial cells of small airways, incubation with formoterol promoted receptor internalization but this did not appear to occur following incubation with salmeterol. Budesonide inhibited the formoterol-induced reduction in plasma membrane ß(2)-adrenoceptor fluorescence. CONCLUSIONS AND IMPLICATIONS: Although both formoterol and salmeterol attenuate isoprenaline-induced bronchodilatation, they appear to induce ß(2)-adrenoceptor tolerance via different mechanisms; formoterol, but not salmeterol, enhances receptor internalization. Budesonide protection against ß(2)-adrenoceptor tolerance was correlated with the retention of receptor fluorescence on the plasma membrane, thereby suggesting a mechanism by which steroids alter ß(2)-adrenoceptor function.

P2Y1-receptors in human platelets which are pharmacologically distinct from P2Y(ADP)-receptors.

1. In the present study we have classified the receptor(s) mediating increases in intracellular calcium concentration ([Ca2+]i) in human washed platelets and compared the pharmacological profile obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y1-receptor. 2. The P2Y1-receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar affinities (pK(B) of 5.8 and 6.0, respectively). 3. The selective P2Y(ADP) antagonist, AR-C66096, exhibited partial agonism in the Jurkat cells with an affinity (pK(A)) of 4.9. This value is consistent with its known P2Y1-receptor activity. In platelets, AR-C66096 at a concentration (0.1 microM) approximately 100 fold greater than its known P2Y(ADP) receptor affinity, had no effect on ADP-induced increases in [Ca2+]i. 4. The ability of adenine nucleotide analogues to elevate [Ca2+]i in the Jurkat cells was also determined. The rank order of agonist potency (p[A]50) was: 2-MeSADP (8.3)>2-ClATP (7.8)>ADP (7.5)=2-MeSATP (7.4)>ATPgammaS (6.5)>ATP (6.2), with ATP appearing to be a partial agonist. 5. The same rank order of potency was observed when similar experiments were performed in platelets. However, the absolute potencies of all the agonists and the intrinsic activities of both ATPgammaS and ATP were lower in platelets. 6. The operational model of agonism was used to test whether the agonist concentration-effect profiles obtained in these two cell types could be explained on the basis of differences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y1-receptor system. 7. The hypothesis that the observed partial agonist behaviour of ATP was the result of receptor activation by contaminating ADP with concomitant receptor blockade by ATP, was tested in the platelet system. This hypothesis was supported by a theoretical analysis, which yielded an affinity value for ATP similar to that obtained previously at P2Y1-receptors. 8. In summary, the results of this study indicate that human washed platelets contain P2Y1-receptors which mediate increases in [Ca2+]i and that this receptor population is pharmacologically distinct from P2Y(ADP)-receptors.

Cloning and characterisation of a bovine P2Y receptor.

Using a chick P2Y1 receptor cDNA probe we have isolated a mammalian P2Y receptor clone from a bovine aortic endothelial cell library. The sequence has a high degree of similarity to the chick P2Y1 clone. When transfected into the Jurkat cell line, the cDNA conferred sensitivity to purinoceptor agonists. Using fura-2 loaded cells the potency order at the receptor was found to be 2-methylthioadenosine 5' triphosphate = adenosine 5' diphosphate > adenosine 5' triphosphate >> alpha,beta-methyleneadenosine 5' triphosphate and uridine 5' triphosphate. This corresponds to the agonist potency order expected for the bovine aortic endothelial cell P2Y receptor.

P2Y purinoceptor and nucleotide receptor-induced relaxation of precontracted bovine aortic collateral artery rings: differential sensitivity to suramin and indomethacin.

We have examined a series of adenine nucleotides and UTP for their ability to cause relaxation of precontracted bovine aortic collateral artery rings. The overall rank order of agonist potency for relaxation was 2-methylthioadenosine 5'-triphosphate (2MeSATP) > adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) > UTP > ADP > ATP. These responses were endothelium-dependent. Interaction studies showed that responses to the selective P2Y purinoceptor agonist 2MeSATP, and to ADP, were mediated by different receptors from those mediating responses to UTP. Suramin, a P2 purinoceptor antagonist that binds to diverse sites for ATP, produced a concentration-dependent shift in the agonist concentration-effect curve to 2MeSATP, with a pKB of 5.45 +/- 0.15 and a slope of 0.94 +/- 0.09. Suramin produced a small, nonsignificant shift in the UTP response curve and had little effect on responses to ATP. Indomethacin (2.8 x 10(-6) M) had no effect on concentration-effect curves to UTP but almost abolished the relaxations produced by 2MeSATP and ADP. The concentration-effect curves to ATP and ATP gamma S showed a significant (P < .05) rightward shift in the presence of indomethacin. These results suggest the presence of separate P2Y purinoceptor and nucleotide receptors mediating endothelium-dependent relaxations of bovine aortic collateral artery smooth muscle. ATP acts at both receptors, whereas ADP acts at only one (P2Y). The effects of indomethacin show that these receptors differentially modulate the release of cyclooxygenase-derived mediators of relaxation.

Further subclassification of ATP receptors based on agonist studies.

Recent studies using agonist analogues of ATP and other nucleotides have generated some surprising observations which may have ramifications for the classification of P2 receptors, particularly for those responses currently attributed to P2Y receptor activation. 2-MethylthioATP (2-MeSATP), the conventional P2Y receptor agonist, does not interact with ATP in the expected fashion in various models of endothelial function, suggesting that it acts by a different mechanism. Furthermore, in certain cell types where responses to ATP are mediated by phospholipase C activation, 2-MeSATP has little or no activity. Interestingly, the pyrimidine uridine triphosphate (UTP) invariably shows similar potency to ATP in systems where 2-MeSATP is inactive. In this article Steve O'Connor and colleagues discuss these data and their significance, and propose that separate receptors may be responsible: one sensitive to 2-MeSATP and the other, a 'nucleotide' receptor, sensitive to UTP.

Pharmacological estimation of agonist affinity: detection of errors that may be caused by the operation of receptor isomerisation or ternary complex mechanisms.

1. Recent theoretical studies have questioned the pharmacological estimation of agonist affinity. They showed that when receptor isomerisation or ternary complex mechanisms operate, the receptor inactivation method can substantially overestimate affinity, whereas methods for partial agonist analysis are more accurate. We previously suggested that the operation of such mechanisms and therefore the presence of errors could be detected by analysing the same partial agonist by the receptor inactivation and comparative methods. This paper describes the practical application of this test. 2. The ternary complex mechanism was simulated for a partial agonist under various conditions relating receptor (R) and transducer (T) concentrations, one of which also corresponds to the receptor isomerisation mechanism. The theoretical data so generated were then analysed by the inactivation and comparative methods to quantify the magnitude of error of affinity estimation that could occur. 3. This analysis showed that for a partial agonist with approximately 85% of the activity of a full agonist, the inactivation method could produce an affinity (pKA) estimate up to 0.7 log10 units higher than that produced by the comparative method. This difference would occur when the total receptor concentration ([R0]) is less than or equal to the total transducer concentration ([T0]). It also showed that the overestimation of affinity by the inactivation method was accompanied by drastic overestimation of Em, the maximal effect parameter. 4. The test was then exemplified using the muscarinic receptor system in the guinea-pig isolated left atrial preparation, where there is evidence that a ternary complex mechanism operates. The test agonist was pilocarpine, which produced on average 83% of the activity of the full agonist, carbachol. Pilocarpine was analysed in comparison with carbachol and by receptor inactivation in the same tissue resulting in small and statistically insignificant differences in Em (96.7% and 97.3% respectively) and pKA (5.03 and 4.95 respectively). 5. In conclusion, in this experimental system, there was no evidence for the errors in agonist affinity estimation predicted by theory. Although this conclusion only applies to this system and application of the test to others is necessary to establish the generality of the present results, further examination of the theoretical basis for the predicted errors is required.

The influence of the initial stretch and the agonist-induced tone on the effect of basal and stimulated release of EDRF.

1. The effects of initial stretch and degree of agonist-induced tone on acetylcholine-induced relaxations were examined in rings of rat isolated aorta. The relaxation to acetylcholine was antagonized by atropine and almost completely abolished by haemoglobin. Relaxation to sodium nitroprusside was similar in rings with an intact or disrupted endothelium but that to isoprenaline was greater in intact preparations. 2. In preparations with either an intact or disrupted endothelium there was a similar length-dependent increase in the resting tension of the aortic rings. The size of the contractile response to phenylephrine (1 microM) was dependent on the initial length (and hence degree of stretch) of the preparation in both rubbed and unrubbed tissues. The absolute difference in contractile response between rubbed and unrubbed was greatest at 1.8 mm and less at the other lengths tested, including the optimum degree of stretch for contraction i.e. 2.4 mm. 3. The absolute acetylcholine-induced relaxation (only seen in rings with an intact endothelium) was dependent on the initial length (and hence degree of stretch) of the preparation and was maximum at 2.4 mm. The proportionate relaxation (i.e. expressed as a percentage of induced tone) was also length-dependent being optimal at 1.5 mm. 4. The sensitivity of the vessels to acetylcholine varied depending on the level of agonist-induced tone. When tone was low, acetylcholine sensitivity was high (at [NA] 0.03 microM: pIC50 = 7.36 +/- 0.07), when the concentration of noradrenaline was increased the tone increased and the acetylcholine sensitivity was low (at [NA] 0.3 microM: pIC50 = 6.57 +/- 0.07). 5. The absolute sensitivities and maximum relaxations induced by acetylcholine are discussed in relation to the initial degree of stretch (and hence length of the preparation) or the degree of agonist-induced tone.

Interactions of palmitoyl carnitine with the endothelium in rat aorta.

1. Palmitoyl carnitine (10-1000 microM) resembled Bay K 8644 (10-1000 nM) in that it directly contracted rat aortic rings which were partially depolarized with K+ (12 mM). However, the effects of Bay K 8644 were reduced in the presence of endothelium whereas the presence of the endothelium hardly affected the palmitoyl carnitine-induced contractions, which occurred at high concentrations (greater than 10 microM). 2. Lower concentrations of palmitoyl carnitine (0.3-30 microM; EC50 1.1 microM), but not Bay K 8644, carnitine or palmitic acid, antagonized the relaxant effects of acetylcholine in rat aorta. The antagonism was specific for endothelium-dependent relaxations, in that the relaxations to ATP and the calcium ionophore A23187 were also non-competitively antagonized, albeit at slightly higher concentrations, whereas the direct relaxant effects of sodium nitroprusside were unaffected. Palmitoyl carnitine therefore antagonizes the effects or the release of endothelial-derived relaxant factor (EDRF). The inhibitory effects were reversed on prolonged washout, indicating that the effects were not due to destruction of the endothelial cells. 3. In superfusion experiments, palmitoyl carnitine inhibited the release of EDRF from rat aorta but did not affect the responsiveness to exogenous EDRF, indicating a site of action at the endothelial cell. In superfusion experiments, palmitoyl carnitine, and lysophosphatidyl choline, caused direct relaxations of the aorta, indicating EDRF release, prior to inhibition of release evoked by receptor stimulation. These substances may modulate vascular responsiveness under certain conditions.

Errors in agonist affinity estimation: do they and should they occur in isolated tissue experiments?

Two plausible theories of agonist action - the isomerization and ternary complex mechanisms - predict that agonist affinity measured using the inactivation method may be subject to overestimation. Moreover, the greater the intrinsic efficacy, the greater the predicted error. But are these predictions correct, and are they borne out by experimental data? Paul Leff and colleagues argue that accurate agonist affinity constants can be measured despite these predictions, but that effort must be made to detect any potential errors that might be anticipated from theory.

The interaction of methoctramine and himbacine at atrial, smooth muscle and endothelial muscarinic receptors in vitro.

1. The action of methoctramine and himbacine at muscarinic receptors has been studied using guinea-pig isolated trachea, oesophageal muscularis mucosae, paced left atria, and rat aortic preparations. 2. Methoctramine (1 x 10(-6)-3.2 x 10(-4) M), but not himbacine, elicited positive inotropic responses. These responses were enhanced by pretreating the animals with reserpine. The responses in reserpine-treated animals were not antagonized by phentolamine (1 x 10(-6) M) but were antagonized by propranolol (1 x 10(-6) M). 3. Methoctramine, but not himbacine, exhibited allosteric inhibitory effects at cardiac muscarinic receptors, resulting in a curvilinear Schild plot. Deviations from competitive antagonism were also observed in combination dose-ratio experiments using atropine and methoctramine. At 1 x 10(-6) M, the pKB value for methoctramine was 7.88 +/- 0.15 (mean +/- s.e.mean, n = 5). The pA2 value for himbacine at cardiac muscarinic receptors was 8.52 +/- 0.06 (n = 3). 4. At tracheal and oesophageal muscularis mucosal smooth muscle receptors, the Schild plots for both antagonists were linear. The pA2 values for methoctramine at receptors in these two preparations were similar (6.08 +/- 0.05 and 6.03 +/- 0.09 respectively, n = 4) and were approximately 60 fold less than those values observed at atrial receptors. Himbacine, also exhibited similar values at muscarinic receptors in the trachea and oesophageal muscularis mucosae (7.61 +/- 0.05 and 7.57 +/- 0.04 respectively, n = 4). 5. Muscarinic receptors mediating relaxation of the rat aortic endothelium exhibited pA2 values for methoctramine (5.87 +/- 0.12, n = 6) which were similar to those observed in the smooth muscle, but not the atria. The pA2 values for himbacine at endothelial muscarinic receptors were approximately 0.5 pA2 units lower than those observed at muscarinic receptors in smooth muscle (6.92 + 0.80, n = 6). In addition, the Schild slopes for methoctramine and himbacine at these receptors were significantly (P < 0.05) less than unity. 6. Methoctramine, and to a lesser extent himbacine, are potent and selective antagonists for cardiac muscarinic receptors. However, caution should be used in interpretation of the data with methoctramine in view of the inhibitory allosteric properties and direct inotropic actions of this compound.