Significant progress in understanding the pharmacological characteristics and physiological importance of homomeric and heteromeric P2X channels has been achieved in recent years. P2X channels, gated by ATP and most likely trimerically assembled from seven known P2X subunits, are present in a broad distribution of tissues and are thought to play an important role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. The known homomeric and heteromeric P2X channels can be distinguished from each other on the basis of pharmacological differences when expressed recombinantly in cell lines, but whether this pharmacological classification holds true in native cells and in vivo is less well-established. Nevertheless, several potent and selective P2X antagonists have been discovered in recent years and shown to be efficacious in various animal models including those for visceral organ function, chronic inflammatory and neuropathic pain, and inflammation. The recent advancement of drug candidates targeting P2X channels into human trials, confirms the medicinal exploitability of this novel target family and provides hope that safe and effective medicines for the treatment of disorders involving P2X channels may be identified in the near future.

1 Three fully-de®ned a 1 -adrenoceptors (a 1A , a 1B and a 1D ) have been established in pharmacological and molecular studies. A fourth a 1 -adrenoceptor, the putative a 1L -adrenoceptor, has been de®ned in functional but not molecular studies, and has been proposed to mediate contraction of human lower urinary tract tissues; its relationship to the three fully characterized a 1 -adrenoceptors is not known. 2 In the present study, binding a nities were estimated by displacement of [ 3 H]-prazosin in membrane homogenates of Chinese hamster ovary (CHO-K1) cells stably expressing the human a 1A -, a 1B -and a 1D -adrenoceptors and were compared with a nity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline (NA)-stimulated accumulation of [ 3 H]-inositol phosphates. 3 For the a 1A -adrenoceptor, binding studies revealed a pharmacological pro®le typical for the classically de®ned a 1A -adrenoceptor, such that prazosin, RS-17053, WB 4101, 5-methylurapidil, Rec 15/ 2739 and S-niguldipine all displayed subnanomolar a nity. A di erent pro®le of a nity estimates was obtained in inositol phosphates accumulation studies: prazosin, WB 4101, 5-methylurapidil, RS-17053 and S-niguldipine showed 10 to 40 fold lower a nity than in membrane binding. However, a nity estimates were not`frameshifted', as tamsulosin, indoramin and Rec 15/2739 yielded similar, high a nity estimates in binding and functional assays. 4 In contrast, results from human a 1B -and a 1D -adrenoceptors expressed in CHO-K1 cells gave antagonist a nity pro®les in binding and functional assays that were essentially identical. 5 A concordance of a nity estimates from the functional (inositol phosphates accumulation) studies of the a 1A -adrenoceptor in CHO-K1 cells was found with estimates published recently from contractile studies in human lower urinary tract tissues (putative a 1L -adrenoceptor). These data show that upon functional pharmacological analysis, the cloned a 1A -adrenoceptor displays pharmacological recognition properties consistent with those of the putative a 1L -adrenoceptor. Why this pro®le di ers from that obtained in membrane binding, and whether it explains the a 1L -adrenoceptor pharmacology observed in many native tissues, requires further investigation.

Background and purpose: Purinoceptors containing the P2X3 subunit (P2X3 homotrimeric and P2X2/3 heterotrimeric) are members of the P2X family of ion channels gated by ATP and may participate in primary afferent sensitization in a variety of pain-related diseases. The current work describes the in vitro pharmacological characteristics of AF-353, a novel, orally bioavailable, highly potent and selective P2X3/P2X2/3 receptor antagonist. Experimental approach: The antagonistic potencies (pIC50) of AF-353 for rat and human P2X3 and human P2X2/3 receptors were determined using methods of radioligand binding, intracellular calcium flux and whole cell voltage-clamp electrophysiology. Key results: The pIC50 estimates for these receptors ranged from 7.3 to 8.5, while concentrations 300-fold higher had little or no effect on other P2X channels or on an assortment of receptors, enzymes and transporter proteins. In contrast to A-317491 and TNP-ATP, competition binding and intracellular calcium flux experiments suggested that AF-353 inhibits activation by ATP in a non-competitive fashion. Favourable pharmacokinetic parameters were observed in rat, with good oral bioavailability (%F = 32.9), reasonable half-life (t1/2 = 1.63 h) and plasma-free fraction (98.2% protein bound). Conclusions and implications:The combination of a favourable pharmacokinetic profile with the antagonist potency and selectivity for P2X3 and P2X2/3 receptors suggests that AF-353 is an excellent in vivo tool compound for study of these channels in animal models and demonstrates the feasibility of identifying and optimizing molecules into potential clinical candidates, and, ultimately, into a novel class of therapeutics for the treatment of pain-related disorders.