No abstract

1 In the present study we have classi®ed the receptor(s) mediating increases in intracellular calcium concentration ([Ca 2+ ] i ) in human washed platelets and compared the pharmacological pro®le obtained with that observed in Jurkat cells, stably transfected with a bovine P2Y 1 -receptor. 2 The P2Y 1 -receptor antagonist, adenosine-3'-phosphate-5'-phosphate (A3P5P), competitively antagonized agonist responses in both Jurkat cells, and in platelets with similar a nities (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 a nity (pK A ) of 4.9. This value is consistent with its known P2Y 1 -receptor activity. In platelets, AR-C66096 at a concentration (0.1 mM) approximately 100 fold greater than its known P2Y ADP receptor a nity, had no e ect on ADP-induced increases in [Ca 2+ ] i . 4 The ability of adenine nucleotide analogues to elevate [Ca 2+ ] i in the Jurkat cells was also determined. The rank order of agonist potency (p[A] 50 ) was: 2-MeSADP (8.3)42-ClATP (7.8)4ADP (7.5)=2-MeSATP (7.4)4ATPgS (6.5)4ATP (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 ATPgS and ATP were lower in platelets. 6 The operational model of agonism was used to test whether the agonist concentration-e ect pro®les obtained in these two cell types could be explained on the basis of di erences in receptor reserve. The analysis indicated that the data obtained in platelets closely resembled that predicted for a low density or poorly coupled P2Y 1 -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 a nity value for ATP similar to that obtained previously at P2Y 1 -receptors. 8 In summary, the results of this study indicate that human washed platelets contain P2Y 1 -receptors which mediate increases in [Ca 2+ ] i and that this receptor population is pharmacologically distinct from P2Y ADP -receptors.

In normal physiologic responses to injury and infection, inflammatory cells enter tissue and sites of inflammation through a chemotactic process regulated by several families of proteins, including inflammatory chemokines, a family of small inducible cytokines. In neutrophils, chemokines chemokine (CXC motif) ligand 1 (CXCL1) and CXCL8 are potent chemoattractants and activate G protein-coupled receptors CXC chemokine receptor 1 (CXCR1) and CXCR2. Several small-molecule antagonists of CXCR2 have been developed to inhibit the inflammatory responses mediated by this receptor. Here, we present the data describing the pharmacology of 3S)-3,4-dihydroxybutan-2-yloxy)pyrimidin-4-yl)azetidine-1-sulfonamide], a novel antagonist of CXCR2. AZD5069 was shown to inhibit binding of radiolabeled CXCL8 to human CXCR2 with a pIC 50 value of 9.1. Furthermore, AZD5069 inhibited neutrophil chemotaxis, with a pA 2 of approximately 9.6, and adhesion molecule expression, with a pA 2 of 6.9, in response to CXCL1. AZD5069 was a slowly reversible antagonist of CXCR2 with effects of time and temperature evident on the pharmacology and binding kinetics. With short incubation times, AZD5069 appeared to have an antagonist profile with insurmountable antagonism of calcium response curves. This behavior was also observed in vivo in an acute lipopolysaccharide-induced lung inflammation model. Altogether, the data presented here show that AZD5069 represents a novel, potent, and selective CXCR2 antagonist with potential as a therapeutic agent in inflammatory conditions.