The TRPA1 receptor‐channel is a therapeutic target for the discovery of novel pain drugs. In man, a single gain‐of‐function mutation in TRPA1, N855S, is known to be the cause of a familial episodic pain syndrome (FEPS). Multimodal activation by various stimuli is a fundamental feature of TRPA1 so that the precise manner in which TRPA1 and N855S‐TRPA1 is activated under pathophysiological conditions is not known, but is thought to be due to a combined effect of an array of inflammatory and nociceptive activators. Since the therapeutic potential of this class of drugs may depend on their efficacy to inhibit TRPA1 function by diverse activation mechanisms, we evaluated the activity of a group of novel TRPA1 antagonists, ALGX‐2542, 2567, 2582, 2586 and 2596 by multiple assay methods to determine mechanisms for inhibition and assess the therapeutic potential for this drug series. In the FLIPR‐calcium based pharmacological assays, the compounds demonstrated potent inhibition of either FFA or AITC induced activation of human TRPA1 (hTRPA1) expressed in HEK‐293 cells with IC50s ranging from 15–88 nM. The potent activity of these ALGX antagonists was confirmed in whole‐cell patch clamp electrophysiological assays. To demonstrate open channel block, hTRPA1 currents were first activated by AITC and upon reaching a pseudo‐steady state, each cell was exposed to a single concentration of an ALGX antagonist. All ALGX antagonists rapidly and completely blocked the open channel currents at 10‐fold their respective IC50 values. To compare open channel block and drug interactions with resting state channels, cells were pre‐incubated with ALGX antagonists for 5 min in the presence of external Ca2+, and then challenged with AITC. For this condition, all ALGX antagonists also completely blocked hTRPA1 activation, indicating that these compounds are effective inhibitors of both open and resting state hTRPA1 channels. Calcium imaging studies also showed that ALGX‐2542 potently inhibits activation by AITC, H202, and cold. The pharmacological activity of this series of antagonists was further defined for native human TRPA1 channels endogenously expressed in the WI‐38 human cell line. Utilizing the pre‐incubation FLIPR protocol, the series of ALGX antagonists was also found inhibit agonist‐induced increases in [Cai2+] with nearly equal IC50 values as found in the recombinant hTRPA1‐HEK assay system. The quantitative agreement (r2=0.95) demonstrates that the pharmacological activity defined in the recombinant assay systems directly translates to native hTRPA1 receptor‐channels. Furthermore, these antagonists also completely inhibited the AITC‐activation of the N855S hTRPA1 channels. Remarkably, the inhibitory effects of this series of antagonists is independent of the mode of TRPA1 activation, which includes structurally diverse agonists, H202, and cold temperatures with similar IC50s. This multimodal profile may be an optimal feature for producing maximal clinical efficacy for a pain drug targeting TRPA1. Our results identify a class of dr...

Currently, there are no approved disease‐modifying drugs for osteoarthritis (OA). OA patients depend upon analgesic drugs such as nonsteroidal anti‐inflammatory drugs (NSAIDs) and opioid analgesics for pain management. Despite existing therapies, pain relief in OA remains largely inadequate. Recently, the functional role for TRPA1 receptor‐channels which are expressed on nociceptor sensory nerves and other non‐neuronal cells within the joint has been identified, although studies of the role of TRPA1 in the pathogenesis of OA pain are limited. TRPA1 represents a promising target for the development of novel therapeutic agent for managing OA pain. This study investigated the effects of ALGX‐XC20, a highly selective and potent TRPA1 antagonist with a long in‐vivo half‐life on pain behaviors and cartilage damage in a rat model of monosodium iodoacetate (MIA)‐induced OA. The analgesic effect of a single dose (30 mg/kg; oral) of ALGX‐XC20 was evaluated for a period of 14 days post‐OA induction and compared to celecoxib (30 mg/kg; oral), a selective cyclooxygenase‐2 (COX‐2) inhibitor by using pain‐related behavior assessments, including hindlimb weight distribution and von Frey tests. The histological scoring of cartilage damage was also determined according to the Mankin grading system. The MIA‐induced OA rat group displayed a large reduction in the weight bearing distribution on the ipsilateral hindlimb (p<0.01–0.05) and hind paw mechanical withdrawal thresholds compared to saline control group, which was maintained for 14 days. The MIA‐evoked reduction in weight bearing distribution was significantly reversed (~50%) in the MIA‐induced group treated with ALGX‐XC20 compared to the MIA‐treated group for the 2‐week duration, while the antiallodynic effect of ALGX‐XC20 was less pronounced. In addition, histopathology studies showed that ALGX‐XC20 was also effective in reducing the severe cartilage damage in the MIA‐injected knee joint as shown by a significant improvement in the Mankin score in the ALGX‐XC20 treated group at 14 days (p<0.05). The magnitude of the effect on spontaneous weight bearing behavior due to ALGX‐XC20 treatment is closely comparable to the effects observed for the MIA‐celecoxib group and reported for TRPA1‐KO animals. The positive analgesic effects in this OA model due to pharmacological inhibition of TRPA1 by ALGX‐XC20 occur without any adverse behavioral effects. These in vivo data suggest that ALGX‐XC20 may be used as a novel therapeutic analgesic/anti‐inflammatory drug for the treatment of chronic arthritis, such as OA pain. This TRPA1 antagonist drug may also represent a new class of disease‐modifying drug by providing a chondroprotective effect on articular cartilage.