A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat. Proc Natl Acad Sci U S A

University of Iowa, Iowa City, Iowa, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2002; 99(26):17179-84. DOI: 10.1073/pnas.252537299
Source: PubMed


P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22-92 nM) and was highly selective (IC50 >10 microM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 micromolkg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10-15 micromolkg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 micromolkg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.

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Available from: James S Polakowski, Sep 15, 2014
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    • "P2X 3 knockout mice lose their voiding reflex and have a distended bladder (Cockayne et al. 2000). P2X 3 and P2X 2/3 antagonists attenuate thermal hyperalgesia and mechanical allodynia following chronic nerve constriction injuries (Jarvis et al. 2002). Microglia are activated following peripheral nerve injuries and upregulate the expression of P2X 4 . "
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    ABSTRACT: P2X receptors are cation-permeable ligand-gated ion channels that open in response to the binding of ATP. These receptors are present in many excitable cells, including neurons, striated muscle cells, epithelial cells, and leukocytes. They mediate fast excitatory neurotransmission in the central and peripheral nervous systems and are thought to be involved in neuropathic pain, inflammation, and cell damage following ischemia-reperfusion injuries. P2X receptors are thus a target for the development of new therapeutics to treat chronic pain and inflammation. In this study, we characterized the inhibition caused by pyridoxal-5'-phosphate, a natural metabolite of vitamin B6 (MC-1), of P2X2, P2X4, P2X7, and P2X2/3 receptors stably expressed in HEK293 cells using the patch-clamp technique in the whole-cell configuration. We also tested a new approach using VC6.1, a modified cameleon calcium-sensitive fluorescent protein, to characterize the inhibition of P2X2 and P2X2/3. MC-1 blocked these two P2X receptors, with an IC50 of 7 and 13 μmol/L, respectively. P2X2 exhibited the highest affinity for VC6.1, and the chimeric receptor P2X2/3, the lowest. The patch-clamp and imaging approaches gave similar results and indicated that VC6.1 may be useful for high throughput drug screening. Pyridoxal-5'-phosphate is an efficient P2X blocker and can be classified as a P2X antagonist.
    Full-text · Article · Mar 2014 · Canadian Journal of Physiology and Pharmacology
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    • "Calcium imaging has been successfully used for concentrationinhibition determinations on the wt P2X3 receptor (Jarvis et al., 2002), but the fast-desensitizing properties hamper the evaluation of antagonist competitiveness (Jarvis et al., 2002; Burgard et al., 2000) and the signals can be low (He et al., 2003). We demonstrate in this study that the S 15 V-rP2X3 receptor and the wt- rP2X3 receptor share a virtually identical pharmacological profile. "
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    ABSTRACT: The homotrimeric P2X3 subtype, one of the seven members of the ATP-gated P2X receptor family, plays a role in sensory neurotransmission, including nociception. To overcome the bias resulting from fast desensitization of the P2X3 receptor in dose-response analyses, a non-desensitizing P2X2-X3 receptor chimera has been repeatedly used as a surrogate for the P2X3 receptor for functional analysis. Here, we show that only three of the P2X2-specific amino acid residues of the P2X2-X3 receptor chimera, (19)P(21)V(22)I, are needed to confer a slowly desensitizing phenotype to the P2X3 receptor. The strongest delay in desensitization of the P2X3 receptor by a single residue was observed when (15)Ser was replaced by Val or another hydrophobic residue. Pharmacologically, the S(15)V-rP2X3 mutant behaved similarly to the wt-P2X3 receptor. Analysis of the S(15)V-rP2X3 receptor in 1321N1 astrocytoma cells by a common calcium-imaging-based assay showed 10-fold higher calcium transients relative to those of the wt-rP2X3 receptor. The S(15)V-rP2X3 cell line enabled reliable analysis of antagonistic potencies and correctly reported the mechanism of action of the P2X3 receptor antagonists A-317491 and TNP-ATP by a calcium-imaging assay. Together, these data suggest that the S(15)V-rP2X3 mutant may be suitable not only for automated fluorescence-based screening of molecule libraries for identification of lead compounds but also for facilitated pharmacological characterization of specific P2X3 receptor ligands. We suggest that the mechanism of desensitization of the P2X3 receptor may involve the movement of an N-terminal inactivation particle, in analogy to the "hinged-lid" or "ball and chain" mechanisms of voltage-gated Na and K channels, respectively.
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    • "These effects of ATP on somatosensory systems form part of a significant body of evidence that P2X3 receptors contribute to increased nocifensive behaviors in many models of musculoskeletal and neuropathic pain, as extensively reviewed (see Khakh and North, 2006; Burnstock, 2013). The impact of selective antagonists to inhibit behaviors in these models is impressive allowing justified speculation about the potential for benefit in human musculoskeletal and neuropathic pain conditions (Jarvis et al., 2002; Ford, 2012; North and Jarvis, 2013). However, although somatic pain conditions seem to capture more attention, it is in the viscera, where sensory symptoms are so poorly addressed, that a greater breadth of evidence has evolved from a wide range of investigations that place ATP and the P2X3 receptor mechanism at the heart of pathological sensitization and where therapeutic potential may be most appealing. "
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    ABSTRACT: A sensory role for ATP was proposed long before general acceptance of its extracellular role. ATP activates and sensitizes signal transmission at multiple sites along the sensory axis, across multiple synapses. P2X and P2Y receptors mediate ATP modulation of sensory pathways and participate in dysregulation, where ATP action directly on primary afferent neurons (PANs), linking receptive field to CNS, has received much attention. Many PANs, especially C-fibers, are activated by ATP, via P2X3-containing trimers. P2X3 knock-out mice and knock-down in rats led to reduced nocifensive activity and visceral reflexes, suggesting that antagonism may offer benefit in sensory disorders. Recently, drug-like P2X3 antagonists, active in a many inflammatory and visceral pain models, have emerged. Significantly, these compounds have no overt CNS action and are inactive versus acute nociception. Selectively targeting ATP sensitization of PANs may lead to therapies that block inappropriate chronic signals at their source, decreasing drivers of peripheral and central wind-up, yet leaving defensive nociceptive and brain functions unperturbed. This article reviews this evidence, focusing on how ATP sensitization of PANs in visceral "hollow" organs primes them to chronic discomfort, irritation and pain (symptoms) as well as exacerbated autonomic reflexes (signs), and how the use of isolated organ-nerve preparations has revealed this mechanism. Urinary and airways systems share many features: dependence on continuous afferent traffic to brainstem centers to coordinate efferent autonomic outflow; loss of descending inhibitory influence in functional and sensory disorders; dependence on ATP in mediating sensory responses to diverse mechanical and chemical stimuli; a mechanistically overlapping array of existing medicines for pathological conditions. These similarities may also play out in terms of future treatment of signs and symptoms, in the potential for benefit of P2X3 antagonists.
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