1. Introduction
As a member of the superfamily of Transient Receptor Potential (TRP) ion channels, TRPA1 is expressed in sensory neurons, and co-localizes with TRPV1, calcitonin gene-related peptide (CGRP), substance P, and bradykinin receptors [19,30,36]. TRPA1 expression is increased in pain models and avulsion-injured human dorsal root ganglion (DRG) neurons [1,15,30,36]. It is activated by noxious cold, intracellular Ca2+, hypertonic solutions, and pharmacological agents [2,20,36,39,40]. Most notably, TRPA1 is covalently modified and activated by electrophilic compounds, including pungent natural products (eg, allyl isothiocyanate [AITC]), environmental irritants (acrolein), and endogenous molecules involved in pain, oxidative stress, and inflammation (eg, 4-hydroxynonenal [4-HNE]) [3,37]. Therefore, TRPA1 acts as a molecular integrator of various stimuli to mediate sensation, pain, and neurogenic inflammation.
One piece of compelling evidence for TRPA1 involvement in pain derives from a recent finding that a gain of function mutation in TRPA1 causes familial episodic pain syndrome in human beings [23]. In addition, TRPA1 agonists evoke acute pain, hyperalgesia, inflammation, and airway hypersensitivity [4,37]. Gene knockout impairs sensory functions and sensitivity to agonists [3,20,24,37]. TRPA1-sepcific antisense oligodeoxynucleotides attenuates cold hyperalgesia in rats with inflammation and neuropathy [21,30]. Furthermore, antagonists attenuate pain in several animal models [13,28,32,38]. Collectively, these studies suggest that TRPA1 may represent an important target for development of analgesic and anti-inflammatory drugs.
Despite recent progress, many questions remain concerning the physiological function and therapeutic utility of targeting TRPA1. The role of TRPA1 in noxious cold sensation is controversial [12,25]. It is not known whether TRPA1 is involved in regulating body temperature. The human gain-of-function mutation data support the role of TRPA1 in pain sensation [23], but they do not pinpoint the exact role of TRPA1 in chronic pain states and the analgesic profile of TRPA1 antagonists. Loss-of-function mutation data will be more informative but currently are not available. The major hurdle to defining the therapeutic utility of TRPA1 antagonists is the lack of potent, selective, and bioavailable compounds. To date, several antagonists have been reported, with IC50 values of 3.1 to 6.9μmol/L for AP18, 6.2 to 41.8μmol/L for HC-030031, and 14.3μmol/L for Chembridge-5861528 [13,28,32,38]. The selectivity and drug exposure of ChemBridge-5861528 have not been reported. AP18 is selective for TRPA1 versus several other TRP channels (tested at 50μmol/L); however it is not systematically bioavailable through oral or i.p. dosing. HC-030031 has been widely used in behavioral studies. Although it was reported to be selective against several other TRP channels (IC50 >10 or 20μmol/L), when tested at 10μmol/L in radioligand binding assays, HC-030031 inhibited several proteins involved in pain signaling including sodium channels (40%), sigma receptors (37%), monoamine transporters (37%), and cannabinoid CB2 receptor (33%) [13]. Here we report the discovery of A-967079, a potent, highly selective, and orally bioavailable TRPA1 antagonist. We investigate the potential role of TRPA1 in pathological pain, noxious cold sensation, body temperature regulation, and cardiovascular function.
2. Methods
2.1. Clones, cells, and in vitro assays
Full-length cDNAs for human, rat, mouse TRPA1, human TRPV1, rat TRPV2, human TRPV4, and human TRPM8 were cloned in the pcDNA3.1/V5-His Topo vector and transiently expressed in HEK-293F cells as described previously [10]. Rat DRG neurons were prepared as reported previously [36]. Human epithelial keratinocytes from Lonza (Basel, Switzerland) were used to evaluate endogenous TRPV3. Intracellular Ca2+ assays were performed using either FLIPR, or ratiometric Ca2+ imaging. Whole-cell currents were recorded using identical external and internal solutions containing (in mmol/L): 140 NaCl, 2 MgCl2, 5 EGTA, 10 HEPES (300mOsmo/L, pH 7.4). Currents were elicited from a holding potential of –60mV, or a 200-millisecond voltage ramp ranging from −80 to +80mV applied every second. CGRP release from rat DRG neurons was measured as in Supplemental Methods.
2.2. In vivo pain and thermal sensation assays
Male Sprague–Dawley rats (weighing 200–300g; Charles River, Wilmington, MA) were used in most experiments, and CD1 mice (weighing 20–25g, Charles River) were used in cold plate tests. All in vivo models are well established and institutionally approved by Abbott Laboratories (detailed in Supplemental Methods). Briefly, in the AITC-induced nocifensive response model, 5% mustard oil in 50μL ethanol (∼0.48M AITC) was injected subcutaneously into the dorsal aspect of the right hind paw, and flinching responses were recorded during a 5-minute period. In the osteoarthritic rat pain model, unilateral knee joint osteoarthritis was induced by injecting MIA into the joint cavity of the right hind limb. After 3weeks, peak hind limb grip force (CFmax in gram) was measured by recording the maximum compressive force exerted on the hind limb and normalized by kilogram of body weight. In the chronic constriction injury model of neuropathic pain, acetone-mediated cold allodynia was assessed by spraying acetone (100μL) onto the plantar surface of the hind paw; and subsequent foot withdrawal response was graded in a four-point scale: 0, no response; 1, brisk withdrawal or flick of the paw; 2, repeated flicking of the paw; 3, repeated flicking and licking of the paw. Mechanical allodynia was evaluated in chronic construction, spinal nerve ligation and Complete Freund’s Adjuvant (CFA) models using calibrated von Frey filaments. Noxious heat sensation was evaluated as tail withdrawal latency after tail immersion in 55°C water. Noxious cold sensation was evaluated in the cold plate test; latency to first jump and cumulative forepaw shivering episodes in 4minutes were measured. Compounds were dosed orally 1hour before testing in 100% PEG-400 vehicle. Animals were randomized for dosing, and experiments were conducted in a blinded manner. Data were analyzed by using Origin 6.0 (OriginLab Corporation, Northampton, MA) or GraphPad Prism4 (GraphPad Software Inc., La Jolla, CA) and expressed as mean±SEM (except for cold allodynia, for which mean±interquartile is used). Significance between groups was derived from analysis of variance and post hoc comparison.
2.3. Radiotelemetry for core body temperature and cardiovascular function measurement
Conscious, freely moving, male Sprague–Dawley rats were instrumented with telemetry transmitters to allow monitoring of core body temperature, mean arterial pressure and heart rates, as detailed in Supplemental Methods. Compounds were dosed orally in a vehicle containing 80% oleic acid, 10% Cremaphore EL, and 10% PEG-400.
2.4. Chemical compounds
A-967079 (Fig. 1A) and A-995662 ((R)-8-[4-methyl-5-(4-trifluoromethyl-phenyl)-oxazol-2-ylamino]-1,2,3,4-tetrahydro-naphthalen-2-ol) were synthesized at Abbott Laboratories, with purity >98% as determined by 1H NMR and mass spectrometry. Other compounds were obtained from Sigma-Aldrich (St. Louis, MO) or Calbiochem.
;)
Fig. 1.:
A-967079 is a potent blocker of human and rat TRPA1 channels. (A) Chemical structure of A-967079. (B) A-967079 blocked AITC (30 μmol/L)-evoked Ca2+ increase in cells expressing hTRPA1, as represented by changes of fluorescence signals (RFU) in a FLIPR-based Ca2+ assay. Arrows indicate compound additions. (C and D) Concentration-effect relationships for inhibition of human and rat TRPA1 by A-967079, AP18, and HC030031; n = 4–8 (number of wells). (E) A-967079 blocked AITC (100 μmol/L)-evoked currents in a representative HEK-293F cell expressing hTRPA1 (held at −60 mV). (F) Concentration–effect relationships for A-967079 inhibition of human and rat TRPA1 currents; n = 5. (G) A-967079 blocked both inward (−80 mV) and outward currents (+80 mV) when assessed in a 200-ms voltage ramp protocol (from –80 mV to +80 mV) applied every second.
3. Results
From a high-throughput screen (>1,000,000 compounds) and synthetic efforts, we identified a family of TRPA1 antagonists belonging to the α,β-unsaturated oxime series [9,31]. This class of compounds had comparable pharmacology at human and rodent TRPA1, but many compounds exhibited poor pharmacokinetic profiles and insufficient drug exposure, including AP18, a previously disclosed structural analogue [32]. Iterative synthesis and screening led to identification of A-967079, an analogue with favorable drug-like properties (MW 207; cLogP 3.53; >300μmol/L aqueous solubility; Fig. 1A). In fluorescence based glutathione assay [10], A-967079 (0.2mmol/L), and glutathione (2mmol/L) were stirred in buffered aqueous tetrahydrofuran (pH 7.5) at ambient temperature. After 16 hours, >95% A-967079 remained intact as revealed by nuclear magnetic resonance spectroscopy and liquid chromatography–mass spectrometry analysis. In La antigen-based ALARM nuclear magnetic resonance spectroscopy and ALARM mass spectrometry [10], A-967079 did not modify La-antigen, indicating lack of chemical reactivity.
3.1. A-967079 is a potent TRPA1 antagonist
The effect of A-967079 on TRPA1 was first tested in a fluorometric imaging plate reader (FLIPR)-based Ca2+ assay. In cells expressing recombinant human TRPA1, AITC (30μmol/L, ∼EC80 concentration) evoked robust increase of intracellular Ca2+; A-967079 blocked AITC responses in a concentration-dependent manner, with an IC50 of 0.067±0.004μmol/L (n=8; Fig. 1B and C). In parallel experiments, AP18 and HC-030031 inhibited human TRPA1 with IC50 of 6.9±0.5μmol/L and 6.9 ± 0.4μmol/L, respectively (n=8; Fig. 1C). In cells expressing rat TRPA1, A-967079, AP18, and HC-030031 blocked 30μmol/L AITC-evoked Ca2+ influx with an IC50 of 0.289±0.017μmol/L, 8.8±0.6μmol/L, and 6.9±0.4μmol/L, respectively (n=4–8; Fig. 1D). Hence, A-967079 is ∼100-fold more potent on human TRPA1 and 24-fold more potent on rat TRPA1, compared with AP18 and HC030031. A-967079 also potently blocked mouse TRPA1 (IC50=0.296±0.026μmol/L, n=4).
The effect of A-967079 was further evaluated using whole-cell electrophysiological recordings. HEK-293F cells were transiently transfected with human or rat TRPA1 plus green fluorescent protein. In cells expressing human TRPA1 (held at −60mV), AITC (100μmol/L) elicited robust inward currents, which were subsequently blocked by co-application of A-967079 in a concentration-dependent manner (Fig. 1E). The IC50 values were 0.051±0.001μmol/L on human TRPA1 and 0.101±0.008μmol/L on rat TRPA1 (n=5; Fig. 1F). When evaluated using a 200-ms voltage ramp protocol (from –80mV to +80mV), A-967079 completely blocked both inward at −80mV and outward currents at +80mV (n=4; Fig. 1G).
In addition to AITC, TRPA1 can also be activated by a variety of other stimuli, including reactive ligands (eg, formaldehyde, 4-HNE, H2O2), nonreactive ligands (eg, trinitrophenol, URB597,) and mechanical stimuli (eg, hypertonicity). A-967079 blocked all modes of TRPA1 activation (Supplemental Table 1), indicating that the inhibitory effect of A-967079 is independent of the nature of TRPA1 activators. We showed previously that TRPA1 undergoes pore dilation, a property manifested by changes in ion selectivity and increases in Yo-Pro uptake [8]. As expected, A-967079 potently blocked TRPA1-mediated Yo-Pro uptake (Supplemental Table 1).
3.2. A-967079 is highly selective for TRPA1
A-967079 (up to 100μmol/L) did not activate human TRPV1, TRPV3, TRPV4, or TRPM8 or rat TRPV2. It also did not significantly (<20%) inhibit activation of these channels by their respective agonists (Table 1). In radioligand binding assays for a panel of 75 enzymes, G-protein-coupled receptors, transporters, and ion channels (CEREP, Poitiers, France), 10μmol/L A-967079 exhibited modest inhibition on ligand binding to dopamine transporter (43%), norepinephrine transporter (32%), melatonin receptor MT1 (31%), with no significant effects (<30%) on other 72 proteins (Supplemental Table 2). Thus, A-967079 is >1000-fold selective over other TRP channels and >150-fold selective over 75 other tested proteins.
Table 1: Selectivity of A-967079.
3.3. A-967079 blocks natively expressed TRPA1 and CGRP release in DRG neurons
We evaluated the effect of A-967079 on endogenously expressed TRPA1 channels in rat DRG neurons. In ratiometric Ca2+ imaging experiments, 30.8% of DRG neurons (99 of 321) were TRPA1 positive, as judged by a >50% change in F340/F380 ratio in response to AITC (100μmol/L). In a representative optical field, 4 of 8 neurons produced robust, long-lasting responses to AITC (Fig. 2A); A-967079 (1μmol/L) reversibly blocked AITC responses, in accordance with a noncovalent interaction between A-967079 and TRPA1.
Fig. 2.:
A-967079 blocks endogenous expressed TRPA1 and CGRP releases in rat DRG neurons. (A) Repeated application of A-967079 (1 μmol/L) reversibly blocked AITC (100 μmol/L)-evoked Ca2+ signals in a representative ratiometric Ca2+ imaging experiment. Four of 8 neurons responded to AITC stimuli. (B) A-967079 (5 μmol/L) blocked CGRP release evoked by 200 μmol/L AITC and 10 μmol/L bradykinin (∗ P < .05, Student t-test), but not by 300 nmol/L capsaicin, n = 4 to 8.
CGRP is a vasoactive neuropeptide and a key mediator of neurogenic inflammation. TRPA1 has been shown to be co-localized with CGRP in a subset of sensory neurons, and its activation leads to CGRP release from central and peripheral nerve endings [36,37]. In our study, AITC (200μmol/L) stimulated CGRP release from DRG neurons, and the effect was blocked by preincubation with A-967079 (Fig. 2B). TRPA1 has also been shown to be indirectly activated by bradykinin through PLC signaling pathways [2]. Consistently, A-967079 blocked bradykinin (10μmol/L) stimulated CGRP release. In contrast, A-967079 did not block capsaicin (300nmol/L)-evoked CGRP release mediated through TRPV1 activation.
3.4. A-967079 is orally bioavailable in rodents
Upon oral administration (p.o.) at 2mg/kg in naive rats, A-967079 exhibited oral bioavailability (F=37%) with a maximum plasma concentration of 0.72μmol/L after 0.5hour and a plasma half-life of 0.81hour. A-967079 penetrated the blood–brain barrier, with a brain/plasma ratio of 0.75. Sixty minutes after oral dosing at 6.2, 20.7, and 62.1mg/kg in rats, plasma concentrations of A-967079 were 1.54±0.43, 6.22±1.29, and 11.8±1.7μmol/L, respectively (n=6). At 62.1mg/kg dosing, the free plasma concentration was 0.47±0.04μmol/L (n=6), 4.7-fold over IC50, or equivalent to IC85 in electrophysiology assay. The robust free plasma exposure and high degree of selectivity (Table 1) enabled us to use A-967079 as an in vivo tool to explore TRPA1 function.
3.5. A-967079 is efficacious in preclinical pain models
Subcutaneous injection of mustard oil (5%, or 0.48mol/L AITC in 50μL) into the rat hind paw resulted in robust flinching responses; oral dosing of A-967079 prevented AITC-induced flinching behaviors in a dose-dependent fashion, demonstrating an “on-target” effect (Fig. 3A). A-967079 had no effects on locomotor activity at these doses.
Fig. 3.:
A-967079 exhibits analgesic efficacy in rat pain models. (A) A-967079 (squares) dose dependently attenuated the nocifensive flinching evoked by 5% mustard oil relative to vehicle (triangle) (∗∗ P < .01), n = 6. (B) In the MIA-induced osteoarthritic (OA) pain model, A-967079 (squares) reversed the grip force deficit relative to vehicle (V, triangle) (∗∗ P < .01). Grip force in age-matched naive rats is also shown (N, circle). ED50 value was derived using least-squares linear regression; n = 12.
A-967079 was evaluated in a rat model of osteoarthritic (OA) pain induced by injection of monoiodoacetate (MIA) into the knee joint. Three weeks after MIA injection, OA pain was manifested as a decrease in hind limb grip force. A-967079 dose dependently and fully reversed the grip force deficit in OA rats with an ED50 of 23.2mg/kg, p.o. (95% confidence interval, 16.5–32.7mg/kg) (Fig. 3B).
3.6. A-967079 has distinct effects on cold sensation in naive and injured states
The effect of A-967079 on noxious cold sensation was examined in the cold plate test using CD1 mice. To avoid tissue injury and muscle stiffness associated with extreme cold (eg, −5°C or −10°C), 0°C and 5°C temperatures were chosen. Between vehicle- and A-967079 (62.1mg/kg, p.o.)-treated groups, no difference was observed in the latency to first jump (0°C cold plate, Fig. 4A), or in total forepaw shivering responses in 4min (5°C cold plate, Fig. 4B). On noxious heat sensation, we compared A-967079 with A-995662, a potent and selective TRPV1 antagonist (IC50: 15±3nmol/L, n=12). A-967079 did not affect tail withdrawal latency to hot water immersion at 55°C (Fig. 3C); in contrast, A-995662 (11.6mg/kg, p.o.) significantly increased the withdrawal latency in this assay (Fig. 3D), consistent with the role of TRPV1 in noxious heat sensation. Therefore, A-967079 does not alter acute sensation to noxious cold or noxious heat in uninjured animals.
Fig. 4.:
A-967079 does not affect noxious thermosensation in normal animals, but reverses cold allodynia after nerve injury. (A and B) In the noxious cold plate assay, A-967079 (62.1 mg/kg, p.o.) did not produce significant changes in the latency to first jump at 0°C (n = 10), or changes in the number of forepaw shivering episodes over a 4-minute time span at 5°C (n = 10). (C) A-967079 did not change withdraw latency in 55°C tail immersion test (n = 18). (D) A-995662 (TRPV1 antagonist, 11.6 mg/kg, p.o.) significantly increased withdrawal latency in the 55°C tail immersion test (∗ P < .05, n = 18). (E) In the chronic constriction injury model of neuropathic pain, A-967079 attenuated cold allodynia evoked by acetone spray (∗ P < .05). Groups are represented as Contra, contralateral (n = 48); Ipsi, ipsilateral (n = 48); Vehicle, vehicle-treated (n = 16); and A-967079-treated group (n = 16). Data are presented as mean ± SEM in A–D and as median ± interquartile range in E.
Next, we examined whether A-967079 affects cold allodynia under neuropathy. In the chronic constriction injury model, a brief application of acetone on the paw produced cold allodynia, as represented by a robust nocifensive response of the ipsilateral but not the contralateral paw (Fig. 4E). After baseline testing, animals were dosed with vehicle or A-967079 (62.1mg/kg, p.o.). A-967079 attenuated cold allodynia evoked by acetone spray by 46.4%±9.0% (n=16). In contrast, A-967079 failed to produce a significant attenuation of mechanical allodynia in chronic constriction injury model (Fig. 5A). Similarly, A-967079 did not attenuate mechanical allodynia in spinal nerve ligation model or CFA models (Fig. 5 B and C). Gabapentin (100mg/kg, p.o.) consistently exhibited 50% to 60% efficacy in attenuating mechanical allodynia in these models (data not shown).
Fig. 5.:
A-96709 does not attenuate mechanical allodynia in neuropathic and inflammatory pain models. For the neuropathic models (chronic constriction injury and spinal nerve ligation), mechanical allodynia was assessed 10 to 14 days after surgery. For the CFA model, mechanic allodynia was assessed 48 hours after intrapaw injection of CFA. The strength of the maximum filament used for von Frey testing was 15.0 g, which was considered the maximal possible response (dotted line on top). Compared with vehicle (triangle), A-967079 (square) did not exhibit significant effects on mechanical allodynia in CCI (A), SNL (B), or CFA (C) model (n = 8). Gabapentin (100 mg/kg, p.o.) exhibited 50% to 60% efficacy in these models (data not shown).
3.7. A-967079 does not alter body temperature regulation or cardiovascular functions
To determine whether TRPA1 is involved in regulating body temperature, core body temperature was continuously monitored in freely moving rats instrumented with telemetry transmitters. Treatment with the TRPV1 antagonist A-995662 immediately increased body temperature, by 0.6° and 0.9°C with the 1.16- and 3.88-mg/kg doses, respectively (Fig. 6A), confirming the previous finding that TRPV1 is involved in regulating body temperature [16,35]. In contrast, at doses producing analgesic efficacy (20.7 and 62.1mg/kg), A-967079 did not elevate or decrease core body temperature compared with responses in vehicle-treated rats (Fig. 6B). In addition, A-967079 had no effect on either mean arterial pressure or heart rate, suggesting a lack of effect on major cardiovascular functions (Supplemental Fig. 1 A and B).
Fig. 6.:
Distinctive effects of A-995662 and A-967079 on core body temperature. (A) TRPV1 antagonist A-995662 elevates core body temperature. (B) A-967079 does not affect body temperature. Compound dosing is indicated by an arrow (time zero). The ∼1°C increase in body temperature (before compounds dosing at 0 min) represents change from dark to light circle.
4. Discussion
In the current study, we report the discovery of A-967079, a potent blocker of TRPA1 channels. It is an oxime analogue that emerged from high-throughput screen and extensive medicinal chemistry efforts [9,31]. Structurally A-967079 resembles AP18, with a methyl addition and a chlorine–fluorine substitution. However, compared with AP18 and HC-030031, A-967079 is significantly more potent (∼100-fold on human and 24-fold on rat TRPA1) and has a much improved pharmacokinetic profile. Furthermore, A-967079 is highly selective, with >1000-fold selectivity over other TRP channels and >150-fold selectivity over 75 other tested proteins. Using A-967079 as a tool to explore TRPA1 function, we have made the following findings.
First, A-967079 significantly attenuates AITC-induced nocifensive responses in a dose-related fashion (Fig. 3A). The lack of complete inhibition could be due to the very high concentration of AITC (0.48mol/L, local paw injection) and relatively low concentration of A-967079 in the paw. In addition, this could be due to effects independent of TRPA1. As shown previously, the nocifensive responses to high concentrations of AITC and formalin were reduced but not abolished in knockout mice [24,28], suggesting that AITC and formalin may cause tissue injury and engage other targets beyond TRPA1 [33]. The movement-evoked, MIA-induced osteoarthritis pain model has been well validated and may have predictive value for human osteoarthritis pain [7]. In this model, full analgesic efficacy was observed for tramadol, celecoxib, and diclofenac; moderate effects for indomethacin (41%), duloxetine (50%), and gabapentin (47%) were observed [7]. A-967079 fully attenuates grip force deficit in the MIA-induced osteoarthritis pain model, with ∼80% efficacy at 62.1mg/kg oral dosing. Interestingly, in human patients with osteoarthritis, levels of 4-HNE (an endogenous TRPA1 agonist) are significantly increased in synovial fluid, as evidenced by a 1.9-fold increase in HNE/protein adducts compared with normal synovial fluid [29]. Furthermore, the HNE/protein adducts in osteoarthritic chondrocytes are significantly increased by oxidative stress-related molecules including H2O2 and TNFα. The elevated 4-HNE could damage cartilage by transcriptional and post-translational modification of proteins such as type II collagen. In addition, 4-HNE could directly excite neurons expressing TRPA1, leading to chronic pain and inflammation. Osteoarthritis affects more than one third of the population more than 65years of age, and accounts for 25% of visits to primary care physicians. Given the high unmet medical need in osteoarthritic pain relief, it will be important to explore whether the efficacy observed in the preclinical osteoarthritis pain model can translate into human osteoarthritis.
Second, A-967079 does not affect noxious cold sensation in naive mice. The role of TRPA1 in cold sensation has been a subject of extensive debate [5,25]. Channel activation by noxious cold has been reported in several studies [20,36], but disputed, or attributed to an indirect, intracellular Ca2+-mediated mechanism [19,40]. Studies using knockout mice have also produced conflicting results. Some studies have shown that TRPA1-deficient mice were significantly less responsive to acetone-mediated evaporative cooling, cold plate, and tail immersion [20,24]. In contrast, no behavioral or cutaneous fiber sensitivity deficits to cold were observed in other studies [3,26]. Recently, it has been reported that TRPA1 may play a role in cold sensing in visceral sensory neurons [14] but not in cutaneous nociceptors [12]. In the current study, A-967079 does not produce deficits in the cold plate test (0° and 5°C) in naive mice, suggesting TRPA1 may not play a prominent role in acute noxious cold sensation in normal animals.
Third, A-967079 attenuates cold allodynia in the chronic constriction injury model of neuropathic pain, confirming the role of TRPA1 in cold allodynia suggested by previous studies using TRPA1-specific antisense [21,30]. The different effects on cold allodynia (pathological) and acute sensation to noxious cold (physiological) are apparently perplexing, but reminiscent of a previous finding that block of the voltage-gated sodium channel Nav1.8 affects heat hyperalgesia in injured rats but not acute heat sensation in naïve rats [18]. Cold sensation is a complex process and involves many other ion channels including Nav1.8, epithelial sodium channel DRASIC, K+ channel TREK1 and TRPM8. Under physiological conditions, these ion channels and their related signaling pathways may sufficiently confer noxious cold sensation independent of TRPA1. This notion is consistent with our present data and consistent with the robust cold sensitivity observed in TRPA1 knockout mice and in ruthenium red-treated cutaneous fibers [12,26]. However, upon nerve injury and development of cold allodynia, the relative contribution of TRPA1 could be significantly increased. Indeed, in several neuropathic and inflammatory pain models, TRPA1 expression and the proportion of cold- and AITC-responsive Aδ fibers are significantly increased [15,21,30]. The activity of TRPA1 can be further boosted by the release of proalgesic and proinflammatory agents (eg, bradykinin) [2]. Of note, both A-967079 and TRPA1 antisense exhibit significant, but not complete reversal of cold allodynia, suggesting the involvement of other TRPA1-independent mechanisms. In any case, our data suggest that TRPA1 plays distinctive role in sensing cold under physiological and pathological states, and antagonists may have utility in treating cold allodynia, a common symptom associated with neuropathic pain in human beings.
Fourth, at robust free plasma exposure (0.47μmol/L, or ∼IC85), A-967079 did not attenuate mechanical hypersensitivity in chronic constriction injury, spinal nerve ligation, and CFA models. This data are consistent with the previous report that TRPA1 specific antisense oligodeoxynucleotides reduced cold, but not heat or mechanical hypersensitivity [30]. In addition, TRPA knockout mice did not differ in baseline mechanical thresholds from wild-type mice [3]. Moreover, knockout mice were able to developed normal mechanical allodynia but not cold allodynia [11,32]. Collectively, these findings suggest that TRPA1 may not play a significant role in mechanical hypersensitivity. On the other hand, HC-030031 and AP-18 were reported to attenuate mechanical hypersensitivity in spinal nerve ligation (by 41%) and in CFA model (by ∼30%), respectively [13,32]. HC-030031 (at 100μmol/L) was also shown to reduce von Frey evoked cutaneous nerve firing (by ∼50%) [22]. Nonetheless, these effects could not be conclusively attributed to TRPA1 blockade because of the relative weak potency of tool compounds, the lack of high level selectivity and the absence of free drug exposure data (see Introduction). Therefore the exact role of TRPA1 in mechanical hypersensitivity is not settled and should be addressed by future studies.
Fifth, at doses producing analgesic effects, A-967079 does not affect body temperature, cardiovascular function, or locomotor function. The TRPV1 antagonist A-995662 increases body temperature, consistent with the role of TRPV1 in regulating body temperature [6,16,34,35]. Given its close association with TRPA1 (co-localization, functional synergy, etc), it is important to investigate whether TRPA1 possesses similar function. Previous studies have shown that intraperitoneal administration of cinnamaldehyde cause hypothermia in mice [17], whereas intragastric administration of AITC and cinnamaldehyde cause hyperthermia [27]. Besides the inconsistency of these results, the 2 studies may have been confounded by the promiscuous nature of AITC and cinnamaldehyde (ie, covalently modifying many other proteins) and their tendency to evoke events not directly linked to temperature regulation (eg, pain, inflammation and tissue injury). In contrast, A-967079 is nonreactive, highly selective, and highly bioavailable. At doses producing robust analgesic efficacy, A-967079 does not alter body temperature in naïve animals, suggesting that TRPA1 may not play a role in thermoregulation under normal physiological conditions. Alternatively, TRPA1 may still play a role; but in the absence of endogenous basal activity, blockade of TRPA1 produces no effects. Future studies are required to determine whether TRPA1 regulates body temperature under pathological conditions, especially those involving fever and/or production of endogenous TRPA1 ligands.
The present findings have significant implications for drug discovery efforts targeting TRPA1. The analgesic efficacy demonstrated by A-967079 supports the potential utility of TRPA1 antagonists for treating osteoarthritic pain and alleviating cold allodynia, a common symptom associated with neuropathic pain in human beings. Equally important, the lack of side effects (eg, temperature sensation, temperature regulation, and cardiovascular and locomotive functions) suggests a favorable safety profile associated with TRPA1 blockade. It will be of high interest to determine whether the lack of severe side effects can be extended to other TRPA1 antagonists. In addition, it is important to determine whether TRPA1 block produces detrimental effects under pathological conditions (eg, heat or endotoxic challenge). Nonetheless, our data provide the first indication of a favorable safety profile and should stimulate future drug discovery efforts targeting TRPA1.
In summary, we report A-967079 as a potent, highly selective, and orally bioavailable antagonist of TRPA1 channel. We demonstrate that selective block of TRPA1 attenuates osteoarthritic pain- and neuropathy-induced cold allodynia, but does not affect noxious cold sensation in naive animals. In addition, blockade of TRPA1 does not alter body temperature regulation, locomotor activity, or cardiovascular function. Although more work is required, these data reveal novel insights into TRPA1 function and suggest that selective blockade of TRPA1 may present an attractive strategy for alleviating pain.
Conflict of interest statement
All authors are employed by Abbott Laboratories and may own stock options of Abbott Laboratories.
Acknowledgements
We thank Dr. Michael Jarvis for comments on the manuscript. This work was supported by Abbott Laboratories.
References
[1]. Anand U, Otto WR, Facer P, Zebda N, Selmer I, Gunthorpe MJ, Chessell IP, Sinisi M, Birch R, Anand P. TRPA1 receptor localisation in the human peripheral nervous system and functional studies in cultured human and rat sensory neurons.
Neurosci Lett. 2008;438:221-227.
[2]. Bandell M, Story GM, Hwang SW, Viswanath V, Eid SR, Petrus MJ, Earley TJ, Patapoutian A. Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin.
Neuron. 2004;41:849-857.
[3]. Bautista DM, Jordt SE, Nikai T, Tsuruda PR, Read AJ, Poblete J, Yamoah EN, Basbaum AI, Julius D. TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents.
Cell. 2006;124:1269-1282.
[4]. Bessac BF, Sivula M, von Hehn CA, Escalera J, Cohn L, Jordt SE. TRPA1 is a major oxidant sensor in murine airway sensory neurons.
J Clin Invest. 2008;118:1899-1910.
[5]. Caspani O, Heppenstall PA. TRPA1 and cold transduction: an unresolved issue?
J General Physiol. 2009;133:245-249.
[6]. Caterina MJ. On the thermoregulatory perils of TRPV1 antagonism.
Pain. 2008;136:3-4.
[7]. Chandran P, Pai M, Blomme EA, Hsieh GC, Decker MW, Honore P. Pharmacological modulation of movement-evoked pain in a rat model of osteoarthritis.
Eur J Pharmacol. 2009;613:39-45.
[8]. Chen J, Kim D, Bianchi BR, Cavanaugh EJ, Faltynek CR, Kym PR, Reilly RM. Pore dilation occurs in TRPA1 but not in TRPM8 channels.
Mol Pain. 2009;5:3.
[9]. Chen J, Lake MR, Sabet RS, Niforatos W, Pratt SD, Cassar SC, Xu J, Gopalakrishnan S, Pereda-Lopez A, Gopalakrishnan M, Holzman TF, Moreland RB, Walter KA, Faltynek CR, Warrior U, Scott VE. Utility of large-scale transiently transfected cells for cell-based high-throughput screens to identify transient receptor potential channel A1 (TRPA1) antagonists.
J Biomol Screen. 2007;12:61-69.
[10]. Chen J, Zhang XF, Kort ME, Huth JR, Sun C, Miesbauer LJ, Cassar SC, Neelands T, Scott VE, Moreland RB, Reilly RM, Hajduk PJ, Kym PR, Hutchins CW, Faltynek CR. Molecular determinants of species-specific activation or blockade of TRPA1 channels.
J Neurosci. 2008;28:5063-5071.
[11]. del Camino D, Murphy S, Heiry M, Barrett LB, Earley TJ, Cook CA, Petrus MJ, Zhao M, D’Amours M, Deering N, Brenner GJ, Costigan M, Hayward NJ, Chong JA, Fanger CM, Woolf CJ, Patapoutian A, Moran MM. TRPA1 contributes to cold hypersensitivity.
J Neurosci. 2010;30:15165-15174.
[12]. Dunham JP, Leith JL, Lumb BM, Donaldson LF. Transient receptor potential channel A1 and noxious cold responses in rat cutaneous nociceptors.
Neuroscience. 2010;164:1412-1419.
[13]. Eid SR, Crown ED, Moore EL, Liang HA, Choong KC, Dima S, Henze DA, Kane SA, Urban MO. HC-030031, a TRPA1 selective antagonist, attenuates inflammatory- and neuropathy-induced mechanical hypersensitivity.
Mol Pain. 2008;4:48.
[14]. Fajardo O, Meseguer V, Belmonte C, Viana F. TRPA1 channels mediate cold temperature sensing in mammalian vagal sensory neurons: pharmacological and genetic evidence.
J Neurosci. 2008;28:7863-7875.
[15]. Frederick J, Buck ME, Matson DJ, Cortright DN. Increased TRPA1, TRPM8, and TRPV2 expression in dorsal root ganglia by nerve injury.
Biochem Biophys Res Commun. 2007;358:1058-1064.
[16]. Gavva NR, Bannon AW, Surapaneni S, Hovland DN Jr, Lehto SG, Gore A, Juan T, Deng H, Han B, Klionsky L, Kuang R, Le A, Tamir R, Wang J, Youngblood B, Zhu D, Norman MH, Magal E, Treanor JJ, Louis JC. The vanilloid receptor TRPV1 is tonically activated in vivo and involved in body temperature regulation.
J Neurosci. 2007;27:3366-3374.
[17]. Harada M, Ozaki Y. Pharmacological studies on Chinese cinnamon. I. Central effects of cinnamaldehyde.
Yakugaku Zasshi. 1972;92:135-140.
[18]. Jarvis MF, Honore P, Shieh CC, Chapman M, Joshi S, Zhang XF, Kort M, Carroll W, Marron B, Atkinson R, Thomas J, Liu D, Krambis M, Liu Y, McGaraughty S, Chu K, Roeloffs R, Zhong C, Mikusa JP, Hernandez G, Gauvin D, Wade C, Zhu C, Pai M, Scanio M, Shi L, Drizin I, Gregg R, Matulenko M, Hakeem A, Gross M, Johnson M, Marsh K, Wagoner PK, Sullivan JP, Faltynek CR, Krafte DS. A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat.
Proc Natl Acad Sci USA. 2007;104:8520-8525.
[19]. Jordt SE, Bautista DM, Chuang HH, McKemy DD, Zygmunt PM, Hogestatt ED, Meng ID, Julius D. Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1.
Nature. 2004;427:260-265.
[20]. Karashima Y, Talavera K, Everaerts W, Janssens A, Kwan KY, Vennekens R, Nilius B, Voets T. TRPA1 acts as a cold sensor in vitro and in vivo.
Proc Natl Acad Sci USA. 2009;106:1273-1278.
[21]. Katsura H, Obata K, Mizushima T, Yamanaka H, Kobayashi K, Dai Y, Fukuoka T, Tokunaga A, Sakagami M, Noguchi K. Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats.
Exp Neurol. 2006;200:112-123.
[22]. Kerstein PC, del Camino D, Moran MM, Stucky CL. Pharmacological blockade of TRPA1 inhibits mechanical firing in nociceptors.
Mol Pain. 2009;5:19.
[23]. Kremeyer B, Lopera F, Cox JJ, Momin A, Rugiero F, Marsh S, Woods CG, Jones NG, Paterson KJ, Fricker FR, Villegas A, Acosta N, Pineda-Trujillo NG, Ramirez JD, Zea J, Burley MW, Bedoya G, Bennett DL, Wood JN, Ruiz-Linares A. A gain-of-function mutation in TRPA1 causes familial episodic pain syndrome.
Neuron. 2010;66:671-680.
[24]. Kwan KY, Allchorne AJ, Vollrath MA, Christensen AP, Zhang DS, Woolf CJ, Corey DP. TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction.
Neuron. 2006;50:277-289.
[25]. Kwan KY, Corey DP. Burning cold: involvement of TRPA1 in noxious cold sensation.
J Gen Physiol. 2009;133:251-256.
[26]. Kwan KY, Glazer JM, Corey DP, Rice FL, Stucky CL. TRPA1 modulates mechanotransduction in cutaneous sensory neurons.
J Neurosci. 2009;29:4808-4819.
[27]. Masamoto Y, Kawabata F, Fushiki T. Intragastric administration of TRPV1, TRPV3, TRPM8, and TRPA1 agonists modulates autonomic thermoregulation in different manners in mice.
Biosci Biotechnol Biochem. 2009;73:1021-1027.
[28]. McNamara CR, Mandel-Brehm J, Bautista DM, Siemens J, Deranian KL, Zhao M, Hayward NJ, Chong JA, Julius D, Moran MM, Fanger CM. TRPA1 mediates formalin-induced pain.
Proc Natl Acad Sci USA. 2007;104:13525-13530.
[29]. Morquette B, Shi Q, Lavigne P, Ranger P, Fernandes JC, Benderdour M. Production of lipid peroxidation products in osteoarthritic tissues: new evidence linking 4-hydroxynonenal to cartilage degradation.
Arthritis Rheum. 2006;54:271-281.
[30]. Obata K, Katsura H, Mizushima T, Yamanaka H, Kobayashi K, Dai Y, Fukuoka T, Tokunaga A, Tominaga M, Noguchi K. TRPA1 induced in sensory neurons contributes to cold hyperalgesia after inflammation and nerve injury.
J Clin Invest. 2005;115:2393-2401.
[31]. Perner R, Kort ME, Didomenico SJ, Chen J, Vasudevan A. United States patent application US2009/0176883 A1. Priority date: January 4, 2008. TRPA1 antagonists.
[32]. Petrus M, Peier AM, Bandell M, Hwang SW, Huynh T, Olney N, Jegla T, Patapoutian A. A role of TRPA1 in mechanical hyperalgesia is revealed by pharmacological inhibition.
Mol Pain. 2007;3:40.
[33]. Reeh P. TRPA1-mediated nociception: response to letter by Fischer et al.
Neuroscience. 2008;155:339. [author reply 340].
[34]. Romanovsky AA, Almeida MC, Garami A, Steiner AA, Norman MH, Morrison SF, Nakamura K, Burmeister JJ, Nucci TB. The transient receptor potential vanilloid-1 channel in thermoregulation: a thermosensor it is not.
Pharmacol Reviews. 2009;61:228-261.
[35]. Steiner AA, Turek VF, Almeida MC, Burmeister JJ, Oliveira DL, Roberts JL, Bannon AW, Norman MH, Louis JC, Treanor JJ, Gavva NR, Romanovsky AA. Nonthermal activation of transient receptor potential vanilloid-1 channels in abdominal viscera tonically inhibits autonomic cold-defense effectors.
J Neurosci. 2007;27:7459-7468.
[36]. Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures.
Cell. 2003;112:819-829.
[37]. Trevisani M, Siemens J, Materazzi S, Bautista DM, Nassini R, Campi B, Imamachi N, Andre E, Patacchini R, Cottrell GS, Gatti R, Basbaum AI, Bunnett NW, Julius D, Geppetti P. 4-Hydroxynonenal, an endogenous aldehyde, causes pain and neurogenic inflammation through activation of the irritant receptor TRPA1.
Proc Natl Acad Sci USA. 2007;104:13519-13524.
[38]. Wei H, Hamalainen MM, Saarnilehto M, Koivisto A, Pertovaara A. Attenuation of mechanical hypersensitivity by an antagonist of the TRPA1 ion channel in diabetic animals.
Anesthesiology. 2009;111:147-154.
[39]. Zhang XF, Chen J, Faltynek CR, Moreland RB, Neelands TR. Transient receptor potential A1 mediates an osmotically activated ion channel.
Eur J Neurosci. 2008;27:605-611.
[40]. Zurborg S, Yurgionas B, Jira JA, Caspani O, Heppenstall PA. Direct activation of the ion channel TRPA1 by Ca
2+.
Nat Neurosci. 2007;10:277-279.
Appendix A Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.pain.2011.01.049.
Appendix A Supplementary data
Figure: Supplementary data: Supplementary material.
Figure: No caption availbel
Figure: No caption availbel
Figure: No caption availbel
Figure: No caption availbel
Figure: No caption availbel
Figure: No caption availbel
