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BACKGROUND AND PURPOSEP2X3 and P2X2/3 receptors are highly localized on the peripheral and central pathways of nociceptive signal transmission. The discovery of A-317491 allowed their validation as chronic inflammatory and neuropathic pain targets, but this molecule has a very limited oral bioavailability and CNS penetration. Recently, potent P2X3 and P2X2/3 blockers with a diaminopyrimidine core group and better bioavailability were synthesized and represent a new opportunity for the validation of P2X3-containing receptors as targets for pain. Here we present a characterization of three representative diaminopyrimidines. EXPERIMENTAL APPROACHThe activity of compounds was evaluated in intracellular calcium flux and electrophysiological recordings from P2X receptors expressed in mammalian cells and in a in vivo model of inflammatory pain (complete Freund's adjuvant (CFA) in rat paws). KEY RESULTSCompound A potently blocked P2X3 (pIC50 = 7.39) and P2X2/3 (pIC50 = 6.68) and showed no detectable activity at P2X1, P2X2, P2X4 and P2X7 receptors (pIC50 < 4.7). Whole-cell voltage clamp electrophysiology confirmed these results.Compounds showed good selectivities when tested against a panel of different classes of target. In the CFA model, compound B showed significant anti-nociceptive effects (57% reversal at 3 mg·kg -1 ). CONCLUSIONS AND IMPLICATIONSThe diaminopyrimidines were potent and selective P2X3 and P2X2/3 receptor antagonists, showing efficacy in vivo and represent useful tools to validate these receptors as targets for inflammatory and neuropathic pain and provide promising progress in the identification of therapeutic tools for the treatment of pain-related disorders.

The phosphorylation and desensitization of metabotropic glutamate receptor type 1c in response to agonist and phorbol esters has been studied. Specific immunoprecipitation of mGluR1c from cells treated with agonist or PMA showed a timedependent increase in the phosphorylation of a membrane protein with the same molecular weight as the dimeric form of the receptor. Measurements of inositol phosphate production showed a rapid functional desensitization of about 90% after agonist treatment, whereas treatment with PMA caused only a 30% loss in the same time. The extent of receptor phosphorylation following the different treatments paralleled the desensitization of the receptor. These results strongly suggest that phosphorylation of the dimeric form of mGluR1c, as a functionally active form, may play a role in its rapid desensitization.z 1999 Federation of European Biochemical Societies.

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