In the present study, an orally bioavailable TRPA1 receptor antagonist, HC-030031, was used to further evaluate the role of TRPA1 receptors in models of inflammatory and neuropathic pain, and in tests of acute heat sensitivity and motor coordination. HC-030031 is a selective small molecule TRPA1 receptor antagonist (human IC50 = 4.9 μM). This compound previously was shown to inhibit AITC and formalin-evoked TRPA1 currents in vitro, and inhibit AITC and formalin-induced nociceptive behaviors in vivo following intraperitoneal dosing. Moreover, HC-030031 did not display any significant binding to 41 other receptors, ion channels, and transporters nor functional modulation of 7 enzymes that are known to modulate pain signaling in a target screen (< 40% effect at 10 μM). To confirm in vivo TRPA1 receptor engagement by HC-030031, effects of this compound on AITC-induced nociceptive behaviors were determined. When administered prior to application of AITC, HC-030031 (100 and 300 mg/kg, p.o.) was found to significantly inhibit AITC-induced nociceptive behaviors, suggesting that these doses effectively block TRPA1 receptor activation. Moreover, the fact that plasma exposures of 5.1 and 9.7 μM were obtained following oral dosing of 100 and 300 mg/kg, respectively, supports good oral bioavailability of this compound in rats.
To examine the role of TRPA1 receptors in inflammatory pain states, the effect of HC-030031 was evaluated in the CFA model of inflammatory pain. CFA is a commonly used rodent model of subchronic inflammatory pain , and in the present study oral administration of HC-030031 was found to reverse CFA-induced mechanical hypersensitivity, although efficacy was somewhat less than that of the positive comparator naproxen. These results are consistent with a number of previous reports that have supported a role for TRPA1 receptors in inflammatory pain. Using a small molecule TRPA1 receptor antagonist designated AP18 (mouse IC50 = 4.5 μM), Petrus et al. (2007) found that intra-paw injection of this compound completely reversed CFA-induced mechanical hypersensitivity in wild-type, but not TRPA1 knock-out mice. Additionally, this compound was found to reverse partially CFA-induced cold hypersensitivity. In a separate study using TRPA1 antisense oligodeoxynucleotides, Obata et al. (2005) found that intrathecal TRPA1 antisense administration inhibited CFA-induced cold allodynia, but not heat or mechanical hypersensitivity. Although it is unclear why effects on mechanical hypersensitivity were not observed, differences in experimental design may account for the differing results. For example, Obata et al. (2005) reported that antisense treatment only reduced the increased TRPA1 receptor expression following CFA injection, and thus greater receptor blockade/inactivation may be required to inhibit mechanical hypersensitivity. Additionally, differences in the mechanical stimuli used in these studies, and time of treatment relative to CFA injection (i.e. development vs maintenance) may account for the differing results. The contribution of TRPA1 receptors to inflammatory pain has been suggested to occur as a consequence of receptor sensitization and/or activation by a variety of proinflammatory mediators including bradykinin [5, 6, 22], protease activated receptor 2 (PAR2) agonists, and prostaglandins [16, 29, 30]. The variety of inflammatory mediators that have been found to directly activate or indirectly sensitize TRPA1 is consistent with the results from the present study supporting a role for TRPA1 in inflammatory pain.
In addition to effects on inflammatory pain, we found that oral administration of HC-030031 significantly reversed tactile hypersensitivity in the SNL model of neuropathic pain. The efficacy observed at the highest dose tested was comparable to the positive comparator pregabalin, which is considered by many to be the first-line treatment for neuropathic pain  (for recent review see Gajraj 2007). TRPA1 has been previously implicated in neuropathic pain based on the observation that TRPA1 mRNA expression was found to be increased in uninjured small diameter TrkA+ neurons in the L4 DRG following L5 SNL, and the time course of this upregulation was correlated with the development of cold hypersensitivity . More direct evidence for a role of TRPA1 in the maintenance of cold hypersensitivity in this study was provided by the observation that acute knock-down of TRPA1 following intrathecal administration of antisense oligodeoxynucleotides inhibited cold hypersensitivity. Although these data suggest an important role for TRPA1 in the maintenance of pain following neuropathy, the endogenous mediators and/or mechanisms responsible for TRPA1 activation in this case are less clear. In contrast to the efficacy achieved in the SNL model with pharmacological blockade of TRPA1, both mice lacking the function of TRPA1 and their wildtype littermates developed significant mechanical hypersensitivity in the spared nerve injury (SNI) model. The reason for this discrepancy is unclear, but one possibility is the upregulation of a protein that could compensate for the function of TRPA1. Another possibility is the potential embryonic rewiring of the neuronal circuitries expressing TRPA1 possibly masking the phenotype. Finally, while both SNL and SNI models of neuropathic pain are characterized by heightened sensitivity to mechanical stimulation, they differ in the nature of the injury.
In the present study, the plasma concentrations (i.e. ~5.0–10 μM) that produced antinociceptive efficacy were in a similar range compared to the in vitro potency values on TRPA1. This finding was somewhat unusual, since plasma exposures required to produce antinociceptive efficacy are typically significantly greater than the in vitro potency values for analgesic targets. Nevertheless, the fact that HC-030031 did not display off-target activity in a target screen, and was found to inhibit AITC-induced nociceptive behaviors at antinociceptive doses, suggests that the mechanism of action likely involves TRPA1 blockade, although other mechanisms cannot be ruled out.
While HC-030031 was effective in reversing mechanical hypersensitivity in models of inflammatory and neuropathic pain, it remains unclear how TRPA1 activation contributes to mechanosensation. Invertebrate and mammalian TRPA1 receptors were shown to be involved in mechanoreception [2, 26]. However, no data supporting a direct activation of this channel by mechanical stimulation is present to date. One possibility is that in response to mechanical stimulation, damaged/inflamed tissues release endogenous reactive mediators or respond by an increase in intracellular calcium, both known to directly activate TRPA1. Alternatively, inflammatory mediators may sensitize and/or lower TRPA1 threshold to mechanical stimulation leading to the development of mechanical hypersensitivity. Following injury, TRPA1 might enhance the mechanoresponsiveness of other proteins through direct or indirect mechanisms.
The observed efficacy of HC-030031 in inflammatory and neuropathic pain appears to not involve any coincident changes in acute heat sensitivity or motor coordination. Oral administration of HC-030031 failed to have any effect on hot plate response latencies or rotarod performance at doses which produced efficacy in the inflammatory and neuropathic pain models. Plasma exposures from these experiments were comparable to exposures obtained in the persistent pain model experiments, confirming lack of effect at doses that produced efficacy against inflammatory and neuropathic pain. The ineffectiveness of HC-030031 in the hot plate test is consistent with previous data showing a lack of noxious heat phenotype in TRPA1 knock-out mice and supports the notion that TRPA1 is not a heat sensor [6, 22]. Additionally, the results from the hot plate experiment are consistent with the lack of effect of HC-030031 on contralateral, uninjured hind paw withdrawal thresholds, in response to mechanical stimulation, in the CFA model suggesting that the antinociceptive effects are specific to injury-induced hypersensitivity. That HC-030031 had no effect on motor coordination at antinociceptive doses is consistent with a previous report demonstrating that the efficacy of this compound is likely specific to effects on sensory transmission and is not related to motor dysfunction .