P2X3 Receptor

Recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flow (Ki=22-92 nM) is potently blocked by A-317491 [1]. A concentration-dependent inhibition of dorsal root ganglion (DRG) currents is produced by A-317491 (1 nM-10 μM) with an IC50 of 15 nM[1].

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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 [1].

A-317491 (0.1-30 mg/kg; one single SC) reverses inflammatory mechanical hyperalgesia in rats in a dose-dependent manner[2]. Plasma half-life (7.38 h), clearance rate (1.83 L/h/kg), and volume of distribution (0.17 L/kg) are demonstrated by A-317491 (3-30 mg/kg; a single.v.)[2].

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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. [1]

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The effect of A-317491 (5-([(3-Phenoxybenzyl)[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid), a recently described selective P2X3 and P2X(2/3) receptor antagonist, on inflammatory mechanical hyperalgesia was examined. In the rat Freund's complete adjuvant model of inflammatory pain, s.c. administration of A-317491 dose-dependently reversed mechanical hyperalgesia. Maximum percent reversal (72%) was seen 3 h after administration at 10 mg/kg. Substantial plasma concentrations were measured for A-317491 after s.c. dosing 3, 10 and 30 mg/kg. However, the brain-to-plasma concentration ratio, determined 1 h after a 10 mg/kg s.c. dose, indicated limited penetration of A-317491 into the central nervous system. As revealed by neural activity recorded from single C-fiber nociceptive afferent in a Freund's complete adjuvant-inflamed rat skin-nerve preparation, topical application of A-317491 completely blocked afferent activation and mechanical sensitization induced by alpha,beta-methylene ATP, a P2X agonist. These results suggest that A-317491 is a peripherally acting P2X blocker. Its efficacy demonstrates the importance of peripheral P2X3/P2X(2/3) receptors in mediating ATP-associated mechanical hyperalgesia following inflammation, confirming previous suggestions of a significant role for P2X(2/3) [2].

Pharmacological Selectivity Studies. [1]
The activity of A-317491 (10 μM) was evaluated in a number of assays to assess pharmacological selectivity relative to 86 other cell-surface receptors, ion channels, transport sites, and enzymes including the opioid receptor subtypes and cycloxygenases 1 and 2, by use of standardized assay protocols as described.

Electrophysiology. [1]
Whole-cell patch-clamp recordings were obtained as described from stable cell lines or DRG neurons by using a modified extracellular saline consisting of 155 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 12 mM glucose, pH 7.4. The patch pipette solution consisted of 140 mM potassium aspartate, 20 mM NaCl, 10 mM EGTA, 5 mM Hepes. All cells were voltage-clamped at −60 mV, and series resistance was compensated 75–90% by using an Axopatch 200B amplifier.
Rat DRG neurons were prepared as described. Lumbar (L4–6) DRG were dissected and placed in DMEM containing 0.3% collagenase B for 60 min at 37°C. The collagenase was replaced with 0.25% trypsin (GIBCO/BRL) in Ca2+/Mg2+-free Dulbecco's PBS and further digested for 30 min at 37°C. Ganglia were washed in fresh DMEM, dissociated by trituration, and plated on polyethylenimine-treated coverslips. Cells were plated in 1 ml DMEM supplemented with 10% FBS, nerve growth factor (50 ng/ml), and 100 units/ml penicillin/streptomycin.
Drugs were applied to the cells by using a piezoelectric-driven glass theta tube positioned near the cell. During experiments, agonists were usually applied every 3 min. A-317491 was both preapplied and coapplied to cells during agonist application. Responses were acquired and digitized at 3 kHz, and analyzed by using pclamp software. Current amplitudes were measured at the peak of the response.

Animal/Disease Models: Male adult SD (Sprague-Dawley) rats received an intraplantar injection of Freund's complete adjuvant[2]
Doses: 0.1, 1, 3, 10, 30 mg/kg
Route of Administration: A single sc
Experimental Results: Produced a dose-dependent reduction in mechanical hyperalgesia 1 h, 3 h and 5 h post-administration.

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Analgesia and Side-Effect Assays. [1]
A-317491 and A-317334 were evaluated in a number of well-characterized in vivo models to assess acute (noxious thermal, mechanical, and chemical stimulation), inflammatory (intraplantar formalin, carrageenan, and CFA), and neuropathic (CCI and L5/L6 nerve ligation) pain, as well as models of visceral (acetic acid-induced abdominal constriction, and normal and inflamed colonic distention) and postoperative pain. The specific methodologies for these nociceptive assays and the assessment of rat motor performance, hemodynamics, and general CNS function are described in detail in the Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org. Unless otherwise noted, all experimental and control groups contained at least six animals each, and data are expressed as mean ± SEM. Data analysis was conducted by using ANOVA and appropriate post hoc comparisons (P < 0.05) as described.

[1]. 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. 2002 Dec 24;99(26):17179-84.

[2]. A-317491, a selective P2X3/P2X2/3 receptor antagonist, reverses inflammatory mechanical hyperalgesia through action at peripheral receptors in rats. Eur J Pharmacol. 2004 Nov 3;504(1-2):45-53.

These data indicate that A-317491 is a potent and selective antagonist of the P2X3 and P2X2/3 receptors. Similar to the nucleotide antagonist 2′,3′-O-2,4,6-trinitrophenyl (TNP)-ATP, A-317491 is also a competitive antagonist of the P2X2/3 receptor. However, unlike TNP-ATP, which also has a high affinity for the P2X1 receptor, A-317491 exhibits selectivity for the P2X3 and P2X2/3 receptors that is more than 100-fold higher than its activity against other P2X receptor subtypes. A-317491 has weak or no affinity for many other cell surface receptors, ion channels, and enzymes. Even concentrations of A-317491 up to 100 μM did not inhibit extracellular nucleotidase activity (as determined by [32P]ATP degradation assays) (unpublished observations). The specific antagonistic effect of A-317491 on P2X3 and P2X2/3 receptor blockade is further supported by the significantly reduced activity of its R-enantiomer A-317344 as a P2X3 receptor antagonist. Electrophysiological data from recombinant and native P2X3 receptor-mediated responses indicate that the receptor blocking effect is rapid, reversible and without nonspecific effects. A-317491 is also not as readily dephosphorylated as TNP-ATP. Therefore, A-317491 is the first nonnucleotide, highly effective and selective P2X3 channel antagonist. [1]
A-317491 can effectively reduce nociceptive sensation in CFA-induced chronic inflammatory pain models, especially significantly reducing thermal hyperalgesia and tactile paresthesia in CCI neuropathic pain models. The enhanced analgesic effect of A-317491 in the CCI model is consistent with previously reported upregulation of P2X3 channels in the rat dorsal root ganglion (DRG) and dorsal horn of the spinal cord in this model. Although the activity was slightly weaker, A-317491 also significantly reduced the tactile abnormal pain threshold in the L5/L6 nerve injury model. After L5/L6 nerve ligation, the density of IB4-positive small-diameter neurons in the L5/L6 DRG was significantly reduced, and P2X3 immunoreactivity was correspondingly reduced. However, a portion of IB4-negative large-diameter neurons in the L5/L6 dorsal root ganglion remained intact, exhibiting P2X3 immunoreactivity and showing rapid (P2X3-like) and slow (P2X2/3-like) desensitization responses to ATP. In summary, these data provide neurochemical and functional evidence that activation of P2X3 and P2X2/3 receptors is modulated during chronic pain, and that blocking these receptors can reduce nociceptive sensation mediated by small- and large-diameter sensory neurons in chronic pain states.

C33H29NNAO9

606.57

606.17400

A-317491;475205-49-3

71312259

White to off-white solid powder

4

9

9

44

979

1

AMRIJNPVFCBGSV-UJXPALLWSA-N

InChI=1S/C33H27NO8.Na.H2O/c35-30(25-17-27(32(38)39)28(33(40)41)18-26(25)31(36)37)34(29-15-7-10-21-9-4-5-14-24(21)29)19-20-8-6-13-23(16-20)42-22-11-2-1-3-12-22;;/h1-6,8-9,11-14,16-18,29H,7,10,15,19H2,(H,36,37)(H,38,39)(H,40,41);;1H2/t29-;;/m0../s1

5-[(3-phenoxyphenyl)methyl-(1,2,3,4-tetrahydronaphthalen-1-yl)carbamoyl]benzene-1,2,4-tricarboxylic acid sodium salt hydrate

A-317491 sodium salt hydrate; PD078388; A-317491 sodium salt hydrate, >=98% (HPLC), powder

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O :~100 mg/mL (~164.86 mM)

Solubility in Formulation 1: 50 mg/mL (82.43 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)