Characterization of three diaminopyrimidines as potent and selective antagonists of P2X3 and P2X2/3 receptors with in vivo efficacy in a pain model.

BACKGROUND AND PURPOSE: P2X3 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 APPROACH: The 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 RESULTS: Compound A potently blocked P2X3 (pIC(50)= 7.39) and P2X2/3 (pIC(50)=6.68) and showed no detectable activity at P2X1, P2X2, P2X4 and P2X7 receptors (pIC(50)< 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 3mg·kg(-1) ). CONCLUSIONS AND IMPLICATIONS: The 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.

Mechanisms of cellular adhesion. IV. Role of serum glycoproteins in fibroblast spreading on glass.

We have investigated the exogenous factors required for the transition from the round shape of suspended fibroblasts to the characteristic spread shape on serum-coated glass. Following the evidence of others that the transition is facilitated by adsorbed component(s) related to CIG/LETS (cold-insoluble globulin/large external transformation-sensitive) proteins, we have isolated 2 such preparations from chick serum. Their influence has been investigated on fibroblast adhesion, spreading and growth and they have been characterized by gel electrophoresis, immunological cross-reactivity, amino acid and carbohydrate residue analysis, sedimentation velocity behaviour and circular dichroism spectroscopy. The preparations have molecular weights of 225/215,000 and 140,000 Daltons respectively and are closely similar in composition and secondary structure. The 225/215 000 Dalton doublet is probably a product of limited proteolysis which almost certainly occurred in the avian circulation. For cells seeded on glass precoated in different ways and in different supplemented media we could detect no change in the extent of attachment but there were profound influences on cell shape following this initial adhesion. We confirm that prior adsorption of either CIG-related preparation to glass does indeed promote fibroblast spreading in the absence of other serum components and that CIG is the sole serum component with this type of activity. We now add 2 important qualifications: (i) the presence of substrate-adsorbed serum CIG is not essential for spreading when other serum components are present in the medium; and (ii) the adhesive organization shown by interference reflexion microscopy is incompletely formed unless the additional serum components are present in the medium. We therefore conclude that 16C fibroblasts have the ability when given the stimulus of soluble serum components other than CIG, but not otherwise, to synthesize all the components necessary for the highly organized contacts with glass, including endogenous CIG/LETS proteins.