Activation of tetrodotoxin-resistant sodium channels contributes to action potential electrogenesis in neurons. Antisense oligonucleotide studies directed against Nav1.8 have shown that this channel contributes to experimental inflammatory and neuropathic pain. We report here the discovery of A-803467, a sodium channel blocker that potently blocks tetrodotoxin-resistant currents (IC50 ‫؍‬ 140 nM) and the generation of spontaneous and electrically evoked action potentials in vitro in rat dorsal root ganglion neurons. In recombinant cell lines, A-803467 potently blocked human Nav1.8 (IC50 ‫؍‬ 8 nM) and was >100-fold selective vs. human Nav1.2, Nav1.3, Nav1.5, and Nav1.

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...

ATP-sensitive P2X 7 receptors are localized on cells of immunological origin including glial cells in the central nervous system. Activation of P2X 7 receptors leads to rapid changes in intracellular calcium concentrations, release of the proinflammatory cytokine interleukin-1␤ (IL-1␤), and following prolonged agonist exposure, cytolytic plasma membrane pore formation. P2X 7 knockout mice show reduced inflammation as well as decreased nociceptive sensitivity following peripheral nerve injury. A-740003 (N-(1-{[(cyanoimino)(5-quinolinylamino) methyl] amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide) is a novel competitive antagonist of P2X 7 receptors (IC 50 values ϭ 40 nM for human and 18 nM for rat) as measured by agonist-stimulated changes in intracellular calcium concentrations. A-740003 showed weak or no activity (IC 50 Ͼ 10 M) at other P2 receptors and an array of other neurotransmitter and peptide receptors, ion channels, reuptake sites, and enzymes.A-740003 potently blocked agonist-evoked IL-1␤ release (IC 50 ϭ 156 nM) and pore formation (IC 50 ϭ 92 nM) in differentiated human THP-1 cells. Systemic administration of A-740003 produced dose-dependent antinociception in a spinal nerve ligation model (ED 50 ϭ 19 mg/kg i.p.) in the rat. A-740003 also attenuated tactile allodynia in two other models of neuropathic pain, chronic constriction injury of the sciatic nerve and vincristine-induced neuropathy. In addition, A-740003 effectively reduced thermal hyperalgesia observed following intraplantar administration of carrageenan or complete Freund's adjuvant (ED 50 ϭ 38 -54 mg/kg i.p.). A-740003 was ineffective in attenuating acute thermal nociception in normal rats and did not alter motor performance at analgesic doses. These data demonstrate that selective blockade of P2X 7 receptors in vivo produces significant antinociception in animal models of neuropathic and inflammatory pain.

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The vanilloid receptor 1 (VR1, TRPV1), which is a member of the transient receptor potential (TRP) superfamily, is highly localized on peripheral and central processes of nociceptive afferent fibers. Activation of TRPV1 contributes to the pronociceptive effects of capsaicin, protons, heat, and various endogenous lipid agonists such as anandamide and N-arachidonoyl-dopamine. A-425619 [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)urea] is a novel potent and selective antagonist at both human and rat TRPV1 receptors. In vivo, A-425619 dose dependently reduced capsaicininduced mechanical hyperalgesia (ED 50 ϭ 45 mol/kg p.o.). A-425619 was also effective in models of inflammatory pain and postoperative pain. A-425619 potently reduced complete Freund's adjuvant-induced chronic inflammatory pain after oral administration (ED 50 ϭ 40 mol/kg p.o.) and was also effective after either i.t. administration or local injection into the inflamed paw. Furthermore, A-425619 maintained efficacy in the postoperative pain model after twice daily dosing p.o. for 5 days. A-425619 also showed partial efficacy in models of neuropathic pain. A-425619 did not alter motor performance at the highest dose tested (300 mol/kg p.o.). Taken together, the present data indicate that A-425619, a potent and selective antagonist of TRPV1 receptors, effectively relieves acute and chronic inflammatory pain and postoperative pain.The vanilloid receptor VR1 or TRPV1 is a nonselective cation channel that is activated by exogenous vanilloid compounds such as capsaicin (Caterina and Julius, 2001). Anatomical and functional studies have shown that TRPV1 receptors are expressed on peripheral nociceptors (for review, see Cortright and Szallasi, 2004). Recently, the analgesic potential of TRPV1 receptor blockade has been demonstrated by various approaches including gene disruption, neutralizing antibodies, or receptor antagonism (Caterina et al., 2000;Davis et al., 2000;Kamei et al., 2001;Walker et al., 2003). Although TRPV1 gene-disrupted mice showed mostly normal behavioral responses to noxious heat, they did not develop thermal hyperalgesia to mustard oil or complete Freund's adjuvant (CFA;Caterina et al., 2000). These results suggest that TRPV1 receptors are required for responses to noxious thermal stimuli under inflammatory conditions but that other mechanisms are in part responsible for normal sensation of noxious heat. Consistent with this conclusion, Davis et al. (2000) showed that TRPV1 knockout mice did not develop thermal hyperalgesia in response to carrageenan but showed normal responses to noxious heat. However, TRPV1 knockout mice did develop mechanical allodynia in response to CFA and mustard oil, showed normal responses to formalin, and developed both thermal hyperalgesia and mechanical allodynia after partial nerve injury (Caterina et al., 2000). A role for TRPV1 in thermal hypersensitivity has also been described in diabetic mice. Following i.t. administration of a TRPV1-neutralizing antibody, a partial reduction in thermal hypersen...

1 We have recently reported that systemic delivery of A-317491, the first non-nucleotide antagonist that has high affinity and selectivity for blocking P2X 3 homomeric and P2X 2/3 heteromeric channels, is antinociceptive in rat models of chronic inflammatory and neuropathic pain. In an effort to further evaluate the role of P2X 3 /P2X 2/3 receptors in nociceptive transmission, A-317491 was administered either intrathecally or into the hindpaw of a rat in several models of acute and chronic nociception. 2 Intraplantar (ED 50 ¼ 300 nmol) and intrathecal (ED 50 ¼ 30 nmol) injections of A-317491 produced dose-related antinociception in the CFA model of chronic thermal hyperalgesia. Administration of A-317491 by either route was much less effective to reduce thermal hyperalgesia in the carrageenan model of acute inflammatory hyperalgesia. 3 Intrathecal, but not intraplantar, delivery of A-317491 attenuated mechanical allodynia in both the chronic constriction injury and L5-L6 nerve ligation models of neuropathy (ED 50 ¼ 10 nmol for both models). Intrathecal injections of A-317491 did not impede locomotor performance. 4 Both routes of injection were effective in reducing the number of nocifensive events triggered by the injection of formalin into a hindpaw. Nocifensive behaviors were significantly reduced in both the first and second phases of the formalin assay (intrathecal ED 50 ¼ 10 nmol, intraplantar ED 50 4300 nmol). Nocifensive behaviors induced by the P2X receptor agonist a,b-meATP were also significantly reduced by intraplantar injection of A-317491. 5 These data indicate that both spinal and peripheral P2X 3 /P2X 2/3 receptors have significant contributions to nociception in several animal models of nerve or tissue injury. Intrathecal administration of A-317491 appears to be more effective than intraplantar administration to reduce tactile allodynia following peripheral nerve injury.

Several 3-acylindoles with high affinity for the CB(2) cannabinoid receptor and selectivity over the CB(1) receptor have been prepared. A variety of 3-acyl substituents were investigated, and the tetramethylcyclopropyl group was found to lead to high affinity CB(2) agonists (5, 16). Substitution at the N1-indole position was then examined. A series of aminoalkylindoles was prepared and several substituted aminoethyl derivatives were active (23-27, 5) at the CB(2) receptor. A study of N1 nonaromatic side chain variants provided potent agonists at the CB(2) receptor (16, 35-41, 44-47, 49-54, and 57-58). Several polar side chains (alcohols, oxazolidinone) were well-tolerated for CB(2) receptor activity (41, 50), while others (amide, acid) led to weaker or inactive compounds (55 and 56). N1 aromatic side chains also afforded several high affinity CB(2) receptor agonists (61, 63, 65, and 69) but were generally less potent in an in vitro CB(2) functional assay than were nonaromatic side chain analogues.

BACKGROUND AND PURPOSE Cannabinoid CB2 receptor activation by selective agonists has been shown to produce analgesic effects in preclinical models of inflammatory and neuropathic pain. However, mechanisms underlying CB2‐mediated analgesic effects remain largely unknown. The present study was conducted to elucidate the CB2 receptor expression in ‘pain relevant’ tissues and the potential sites of action of CB2 agonism in rats. EXPERIMENTAL APPROACH Expression of cannabinoid receptor mRNA was evaluated by quantitative RT‐PCR in dorsal root ganglia (DRGs), spinal cords, paws and several brain regions of sham, chronic inflammatory pain (CFA) and neuropathic pain (spinal nerve ligation, SNL) rats. The sites of CB2 mediated antinociception were evaluated in vivo following intra‐DRG, intrathecal (i.t.) or intraplantar (i.paw) administration of potent CB2‐selective agonists A‐836339 and AM1241. KEY RESULTS CB2 receptor gene expression was significantly up‐regulated in DRGs (SNL and CFA), spinal cords (SNL) or paws (CFA) ipsilateral to injury under inflammatory and neuropathic pain conditions. Systemic A‐836339 and AM1241 produced dose‐dependent efficacy in both inflammatory and neuropathic pain models. Local administration of CB2 agonists also produced significant analgesic effects in SNL (intra‐DRG and i.t.) and CFA (intra‐DRG) pain models. In contrast to A‐836339, i.paw administration of AM‐1241 dose‐relatedly reversed the CFA‐induced thermal hyperalgesia, suggesting that different mechanisms may be contributing to its in vivo properties. CONCLUSIONS AND IMPLICATIONS These results demonstrate that both DRG and spinal cord are important sites contributing to CB2 receptor‐mediated analgesia and that the changes in CB2 receptor expression play a crucial role for the sites of action in regulating pain perception.