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 M) 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 dosedependently (ED50 ‫؍‬ 30 mol͞kg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 ‫؍‬ 10 -15 mol͞kg 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 (ED 50 >100 mol͞kg 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 P2X 3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X 3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.T he cloning and characterization of the P2X 3 receptor, a specific ATP-sensitive ligand-gated ion channel that is selectively localized on peripheral and central processes of sensory afferent neurons (1-3), has generated much interest in the role of this receptor in nociceptive signaling (4). The discovery of the P2X 3 receptor has provided a putative mechanism for previous reports that ATP, released from sensory nerves (5), produces fast excitatory potentials in dorsal root ganglion (DRG) neurons (6). These actions appear to be physiologically relevant because iontophoretic application of ATP to human skin elicits pain (7) and exogenously applied ATP enhances pain sensations in a human blister base model (8).The P2X 3 receptor is natively expressed as a functional homomer and as a heteromultimeric combination with the P2X 2 (P2X 2/3 ) receptor (1, 2, 9). Both P2X 3 -containing channels are expressed on a high proportion of isolectin IB4-positive neurons in DRG (3, 10). These receptors share similar pharmacological profiles (11), but differ in their acute desensitization kinetics (10, 12). Immunohistochemical studies have shown that P2X 3 receptor expression is up-regulated in DRG neurons and ipsilateral spinal cord after chronic constriction injury (CCI) of the sciatic nerve (13). Additionally, CCI results in a specific ectopic sensitivity to ATP that is not observed on contralateral (uninjured) nerves (14).Recently, the phenotyp...

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Despite the increasing interest in TRPA1 channel as a pain target, its role in cold sensation and body temperature regulation is not clear; the efficacy and particularly side effects resulting from channel blockade remain poorly understood. Here we use a potent, selective, and bioavailable antagonist to address these issues. A-967079 potently blocks human (IC(50): 51 nmol/L, electrophysiology, 67 nmol/L, Ca(2+) assay) and rat TRPA1 (IC(50): 101 nmol/L, electrophysiology, 289 nmol/L, Ca(2+) assay). It is >1000-fold selective over other TRP channels, and is >150-fold selective over 75 other ion channels, enzymes, and G-protein-coupled receptors. Oral dosing of A-967079 produces robust drug exposure in rodents, and exhibits analgesic efficacy in allyl isothiocyanate-induced nocifensive response and osteoarthritic pain in rats (ED(50): 23.2 mg/kg, p.o.). A-967079 attenuates cold allodynia produced by nerve injury but does not alter noxious cold sensation in naive animals, suggesting distinct roles of TRPA1 in physiological and pathological states. Unlike TRPV1 antagonists, A-967079 does not alter body temperature. It also does not produce locomotor or cardiovascular side effects. Collectively, these data provide novel insights into TRPA1 function and suggest that the selective TRPA1 blockade may present a viable strategy for alleviating pain without untoward side effects.

Vanilloid receptor type 1 (TRPV1) is a ligand-gated nonselective cation channel that is considered to be an important integrator of various pain stimuli such as endogenous lipids, capsaicin, heat, and low pH. In addition to expression in primary afferents, TRPV1 is also expressed in the CNS. To test the hypothesis that the CNS plays a differential role in the effect of TRPV1 antagonists in various types of pain, the analgesic effects of two TRPV1 antagonists with similar in vitro potency but different CNS penetration were compared in vivo. In these models, the potency of the two compounds was similar after intrathecal administration. However, when administered orally, A-784168, with good CNS penetration, was much more potent than A-795614. Together, these results demonstrate that TRPV1 receptors in the CNS play an important role in pain mediated by central sensitization. In addition, these results demonstrate that significant CNS penetration is necessary for a TRPV1 antagonist to produce broad-spectrum analgesia.

Abundantly expressed in pain-sensing neurons, TRPV1, TRPA1 and TRPM8 are major cellular sensors of thermal, chemical and mechanical stimuli. The function of these ion channels has been attributed to their selective permeation of small cations (e.g., Ca 2+ , Na + and K + ), and the ion selectivity has been assumed to be an invariant fingerprint to a given channel. However, for TRPV1, the notion of invariant ion selectivity has been revised recently. When activated, TRPV1 undergoes time and agonist-dependent pore dilation, allowing permeation of large organic cations such as YoPro and NMDG + . The pore dilation is of physiological importance, and has been exploited to specifically silence TRPV1-positive sensory neurons. It is unknown whether TRPA1 and TRPM8 undergo pore dilation. Here we show that TRPA1 activation by reactive or non-reactive agonists induces Yo-Pro uptake, which can be blocked by TRPA1 antagonists. In outside-out patch recordings using NMDG + as the sole external cation and Na + as the internal cation, TRPA1 activation results in dynamic changes in permeability to NMDG + . In contrast, TRPM8 activation does not produce either Yo-Pro uptake or significant change in ion selectivity. Hence, pore dilation occurs in TRPA1, but not in TRPM8 channels.

The TRPV1 antagonist A-995662 demonstrates analgesic efficacy in monoiodoacetate-induced osteoarthritic (OA) pain in rat, and repeated dosing results in increased in vivo potency and a prolonged duration of action. To identify possible mechanism(s) underlying these observations, release of neuropeptides and the neurotransmitter glutamate from isolated spinal cord was measured. In OA rats, basal release of glutamate, bradykinin and calcitonin gene-related peptide (CGRP) was significantly elevated compared to naïve levels, whereas substance P (SP) levels were not changed. In vitro studies showed that capsaicin-evoked TRPV1-dependent CGRP release was 54.7+/-7.7% higher in OA, relative to levels measured for naïve rats, suggesting that TRPV1 activity was higher under OA conditions. The efficacy of A-995662 in OA corresponded with its ability to inhibit glutamate and CGRP release from the spinal cord. A single, fully efficacious dose of A-995662, 100 micromol/kg, reduced spinal glutamate and CGRP release, while a single sub-efficacious dose of A-995662 (25 micromol/kg) was ineffective. Multiple dosing with A-995662 increased the potency and duration of efficacy in OA rats. Changes in efficacy did not correlate with plasma concentrations of A-995662, but were accompanied with reductions in spinal glutamate release. These findings suggest that repeated dosing of TRPV1 antagonists enhances therapeutic potency and duration of action against OA pain, at least in part, by the sustained reduction in release of glutamate and CGRP from the spinal cord.

The vanilloid receptor transient receptor potential type V1 (TRPV1) integrates responses to multiple stimuli, such as capsaicin, acid, heat, and endovanilloids and plays an important role in the transmission of inflammatory pain. Here, we report the identification and in vitro characterization of A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel, potent, and selective TRPV1 antagonist. A-425619 was found to potently block capsaicin-evoked increases in intracellular calcium concentrations in HEK293 cells expressing recombinant human TRPV1 receptors (IC 50 ϭ 5 nM). A-425619 showed similar potency (IC 50 ϭ 3-4 nM) to block TRPV1 receptor activation by anandamide and N-arachidonoyl-dopamine. Electrophysiological experiments showed that A-425619 also potently blocked the activation of native TRPV1 channels in rat dorsal root ganglion neurons (IC 50 ϭ 9 nM). When compared with other known TRPV1 antagonists, A-425619 exhibited superior potency in blocking both naive and phorbol estersensitized TRPV1 receptors. Like capsazepine, A-425619 demonstrated competitive antagonism (pA 2 ϭ 2.5 nM) of capsaicinevoked calcium flux. Moreover, A-425619 was 25-to 50-fold more potent than capsazepine in blocking TRPV1 activation. A-425619 showed no significant interaction with a wide range of receptors, enzymes, and ion channels, indicating a high degree of selectivity for TRPV1 receptors. These data show that A-425619 is a structurally novel, potent, and selective TRPV1 antagonist.

Exogenous ATP has been shown to be algogenic in both animal and humans. Research has focused on the P2X3 ligand-gated ion channel, as it is preferentially expressed on nociceptive C-fibers. In addition, P2X3 receptor gene disrupted mice show decreased responses to somatic painful stimuli. However, the potential role of P2X receptor activation in visceral pain has not yet been evaluated. In the present study, the systemic administration of suramin, and pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid, PPADS, both non-selective P2X receptor antagonists, dose-dependently reduced acetic acid-induced abdominal constrictions in mice (ED(50)=34.5 micromol/kg and ED50=70 micromol/kg, respectively). Furthermore, 2'-(or-3')-O-(trinitrophenyl)adenosine 5'- tri-phosphate (TNP-ATP) potently (IC50=10 nM) blocked the functional activation of P2X3 receptors in vitro and attenuated acetic acid-induced visceral pain. In the abdominal constriction assay, TNP-ATP (ED(50)=6.35 micromol/kg, i.p.) was 6-10 fold more potent than suramin and PPADS to reduce nociceptive behavior. In addition, TNP-ATP was 10 fold more potent than TNP-AMP (2'-(or-3')-O-(trinitrophenyl)adenosine 5'-mono-phosphate) (ED50=63.5 micromol/kg, i.p.) at reducing acetic acid-induced nociception. At the highest dose, TNP-ATP completely abolished nociceptive behavior, as did morphine (ED50=3 micromol/kg, i.p.). While TNP-ATP is also a potent antagonist of P2X1 receptors, P2X1 receptor mediated responses have not been shown in dorsal root ganglia and diinosine pentaphosphate, IP5I, a potent and selective P2X1 receptor antagonist, was ineffective at reducing abdominal constrictions. Thus, the antinociceptive effects of TNP-ATP appear to be mediated through activation of homomeric P2X3and/or heteromeric P2X2/3 receptors. Together, these results show that activation of P2X3 containing receptors plays a role in the transmission of inflammatory visceral pain.