P2X3 Receptor (Ki = 22, 22, 9, and 92 nM for hP2X3, rP2X3, hP2X2/3, and rP2X2/3, respectively)

Recombinant human and rat P2X3 and P2X2/3 receptors (Ki=22-92 nM) efficiently block calcium flux[1]. The 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].

In rats, inflammatory mechanical hyperalgesia is dose-dependently reversed by A-317491 (0.1–30 mg/kg; single subcutaneous injection) [2]. The plasma half-life of A-317491 (3–30 mg/kg; single subcutaneous injection) is 7.38 hours, while its clearance is 1.83 L/h/kg and its volume of distribution is 0.17 L/kg [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 demonstrate that A-317491 is a potent and selective antagonist of P2X3 and P2X2/3 receptors. Like the nucleotide-based antagonist 2′,3′-O-2,4,6-trinitrophenyl (TNP)-ATP, A-317491 is a competitive antagonist of P2X2/3 receptors. However, unlike TNP-ATP, which also has high affinity for P2X1 receptors, A-317491 exhibits >100-fold selectivity for P2X3 and P2X2/3 receptors compared with its activity at other P2X receptor subtypes. A-317491 shows only very weak or no affinity for a large selection of other cell surface receptors, ion channels, and enzymes. A-317491, at concentrations up to 100 μM, also did not inhibit ectonucleotidase activity as measured by [32P]ATP degradation (unpublished observations). The specificity of the antagonist actions of A-317491 for P2X3 and P2X2/3 receptor blockade is further supported by the significantly weaker activity of the R-enantiomer, A-317344, as a P2X3 receptor antagonist. Electrophysiological data from both recombinant and native P2X3 receptor-mediated responses demonstrate that receptor block is rapid in onset, reversible, and devoid of nonspecific effects. A-317491 is also not susceptible to metabolic dephosphorylation like TNP-ATP. Thus, A-317491 represents the first non-nucleotide, potent and selective, antagonist of P2X3-containing channels. [1]
A-317491 effectively reduced nociception in the CFA-induced model of chronic inflammatory pain and was particularly potent in reducing both thermal hyperalgesia and tactile allodynia in the CCI neuropathic pain model. The enhanced antinociceptive efficacy of A-317491 in the CCI model is consistent with the previously documented up-regulation of P2X3-containing channels in rat DRG and spinal dorsal horn in this model. Although less active, A-317491 also significantly reduced tactile allodynia thresholds in the L5/L6 nerve injury model. After L5/L6 nerve ligation, there is a significant decrease in the density of IB4-positive small diameter neurons in the L5/L6 DRG and a corresponding reduction in P2X3 immunoreactivity. However, a subpopulation of IB4 negative larger diameter neurons in the L5/L6 DRG remain intact, show P2X3 immunoreactivity, and demonstrate both fast (P2X3-like) and slowly (P2X2/3-like) desensitizing responses to ATP. Taken together, these data provide neurochemical and functional evidence that activation of P2X3 and P2X2/3 receptors is modulated during chronic pain and blockade of these receptors can reduce nociception mediated by both small and larger diameter sensory neurons in chronic pain states.

C33H27NO8

565.57

565.174

C, 70.08; H, 4.81; N, 2.48; O, 22.63

475205-49-3

A-317491 sodium salt hydrate

9829395

Off-white to pink solid powder

6.293

3

8

9

42

979

1

C1C[C@@H](C2=CC=CC=C2C1)N(CC3=CC(=CC=C3)OC4=CC=CC=C4)C(=O)C5=CC(=C(C=C5C(=O)O)C(=O)O)C(=O)O

VQGBOYBIENNKMI-LJAQVGFWSA-N

InChI=1S/C33H27NO8/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)/t29-/m0/s1

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (4.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.42 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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