A-317491

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

重组人和大鼠 P2X3 和 P2X2/3 受体 (Ki=22-92 nM) 可有效阻断钙流[1]。 A-317491 (1 nM-10 μM) 对背根神经节 (DRG) 电流产生浓度依赖性抑制，IC50 为 15 nM[1]。

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P2X3和P2X2/3受体高度定位于感觉传入神经的外周和中枢过程，这些通道的激活有助于ATP的旋感作用。A-317491是一种新型的P2X3和P2X2/3受体激活的非核苷酸拮抗剂。A-317491能有效阻断重组人和大鼠P2X3和P2X2/3受体介导的钙通量（Ki=22-92 nM），对其他P2受体和其他神经递质受体、离子通道和酶具有高度选择性（IC50>10 microM）。A-317491还阻断了大鼠背根神经节神经元中的天然P2X3和P2X2/3受体。阻断含P2X3的通道具有立体特异性，因为A-317491的R-对映体（A-317344）在P2X3和P2X2/3受体上的活性明显降低[1]。

在大鼠中，A-317491（0.1-30 mg/kg；单次皮下注射）可剂量依赖性地逆转炎症性机械痛觉过敏[2]。 A-317491（3-30 mg/kg；单次皮下注射）的血浆半衰期为 7.38 小时，清除率为 1.83 L/h/kg，分布容积为 0.17 L/kg [2]。

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A-317491剂量依赖性地（ED50=30微千克皮下注射）减少了大鼠完全弗氏佐剂诱导的热痛觉过敏。A-317491在减轻慢性神经收缩损伤后的热痛觉过敏和机械性异常性疼痛方面最有效（ED50=10-15微千克皮下注射）。R-对映体A-317344在这些慢性疼痛模型中没有活性。尽管A-317491在慢性疼痛模型中具有活性，但在急性疼痛、术后疼痛和内脏疼痛的动物模型中，它在减少伤害感受方面无效（ED50>100微千克皮下注射）。目前的数据表明，P2X3和P2X2/3受体的强效选择性拮抗剂有效地减少了神经损伤和慢性炎症性伤害感，但P2X3和P2X2/3受体激活可能不是急性、急性炎症或内脏疼痛的主要介质。[1]

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研究了最近描述的选择性P2X3和P2X（2/3）受体拮抗剂A-317491（5-（[（3-苯氧基苄基）[（1S）-1,2,3,4-四氢-1-萘基]氨基]羰基）-1,2,4-苯三甲酸）对炎性机械性痛觉过敏的影响。在大鼠弗氏完全佐剂炎性疼痛模型中，皮下注射A-317491可剂量依赖性地逆转机械性痛觉过敏。以10mg/kg给药后3小时，观察到最大百分比的逆转（72%）。皮下注射3、10和30mg/kg后，测量了A-317491的实质性血浆浓度。然而，皮下注射10mg/kg后1小时测定的脑与血浆浓度比表明A-317491对中枢神经系统的渗透有限。正如弗氏完全佐剂性炎症大鼠皮肤神经制剂中单个C纤维伤害性传入记录的神经活动所揭示的那样，局部应用a-317491完全阻断了P2X激动剂α，β-亚甲基ATP诱导的传入激活和机械致敏。这些结果表明，A-317491是一种外周作用的P2X阻断剂。其疗效证明了外周P2X3/P2X（2/3）受体在介导炎症后ATP相关机械性痛觉过敏中的重要性，证实了之前关于P2X（2/3）发挥重要作用的建议[2]。

药理学选择性研究。[1]
通过使用所述的标准化测定方案，在多个测定中评估了A-317491（10μM）的活性，以评估相对于86个其他细胞表面受体、离子通道、转运位点和酶（包括阿片受体亚型和环氧化酶1和2）的药理学选择性。

电生理学。[1]
如所述，通过使用由155 mM NaCl、5 mM KCl、2 mM CaCl2、1 mM MgCl2、10 mM Hepes、12 mM葡萄糖组成的改良细胞外盐水，从稳定的细胞系或DRG神经元获得全细胞膜片钳记录，pH 7.4。贴片移液管溶液由140 mM天冬氨酸钾、20 mM NaCl、10 mM EGTA、5 mM Hepes组成。所有电池都被电压箝位在-60 mV，使用Axopatch 200B放大器对串联电阻进行了75-90%的补偿。
按所述制备大鼠DRG神经元。解剖腰椎（L4-6）DRG，并将其置于含有0.3%胶原酶B的DMEM中，在37°C下放置60分钟。在无Ca2+/Mg2+的Dulbecco's PBS中用0.25%胰蛋白酶（GIBCO/BRL）代替胶原酶，并在37°C下进一步消化30分钟。神经节在新鲜的DMEM中洗涤，通过研磨分离，并镀在聚乙烯亚胺处理的盖玻片上。将细胞接种在1ml DMEM中，DMEM补充了10%FBS、神经生长因子（50ng/ml）和100单位/ml青霉素/链霉素。
通过使用位于细胞附近的压电驱动玻璃θ管将药物施加到细胞上。在实验过程中，通常每3分钟施用一次激动剂。在施用激动剂期间，A-317491既可以预先施用，也可以联合施用到细胞上。在3 kHz下采集并数字化响应，并使用pclamp软件进行分析。在响应峰值处测量电流振幅。

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

141.44

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.42 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (4.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (4.42 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。