Transient receptor potential vanilloid 1 (TRPV1) is activated by a variety of stimulations, such as endogenous ligands and low pH, and is believed to play a role in pain transmission. TRPV1 antagonists have been reported to be effective in several animal pain models; however, some compounds induce hyperthermia in animals and humans. We discovered the novel TRPV1 antagonist (R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-carboxamide (AS1928370) in our laboratory. AS1928370 bound to the resiniferatoxin-binding site on TRPV1 and inhibited capsaicin-mediated inward currents with an IC50 value of 32.5 nM. Although AS1928370 inhibited the capsaicin-induced Ca2+ flux in human and rat TRPV1-expressing cells, the inhibitory effect on proton-induced Ca2+ flux was extremely small. In addition, AS1928370 showed no inhibitory effects on transient receptor potential vanilloid 4, transient receptor potential ankyrin 1, and transient receptor potential melastatin 8 in concentrations up to 10 μM. AS1928370 improved capsaicin-induced secondary hyperalgesia and mechanical allodynia in an L5/L6 spinal nerve ligation model in rats with respective ED50 values of 0.17 and 0.26 mg/kg p.o. Furthermore, AS1928370 alleviated inflammatory pain in a complete Freund's adjuvant model at 10 mg/kg p.o. AS1928370 had no effect on rectal body temperature up to 10 mg/kg p.o., although a significant hypothermic effect was noted at 30 mg/kg p.o. In addition, AS1928370 showed no significant effect on motor coordination. These results suggest that blockage of the TRPV1 receptor without affecting the proton-mediated TRPV1 activation is a promising approach to treating neuropathic pain because of the potential wide safety margin against hyperthermic effects. As such, compounds such as ASP1928370 may have potential as new analgesic agents for treating neuropathic pain.
Transient receptor potential vanilloid 1 (TRPV1), which is recognized as the receptor for capsaicin, the pungent substance in red hot chili peppers, is a ligand-gated nonselective cation channel with high permeability for Ca2+ (Caterina et al., 1997). In addition to exogenous agonists such as capsaicin and resiniferatoxin (RTX), low pH and heat can activate TRPV1 (Tominaga et al., 1998). Several lipid derivatives have been proposed as endogenous ligands, including anandamide, N-arachidonoyl dopamine, N-oleoyldopamine, and products of lipoxygenases (Hwang et al., 2000; Smart et al., 2000; Huang et al., 2002; Chu et al., 2003).
Primarily expressed in both central and peripheral terminals of nonmyelinated C fibers, TRPV1 plays an important role in pain signal transmission (Sasamura and Kuraishi, 1999). Furthermore, under certain nerve injury or inflammatory disease conditions, TRPV1 is up-regulated in nonmyelinated C fibers as well as myelinated A fibers (Hudson et al., 2001; Amaya et al., 2003; Rashid et al., 2003; Luo et al., 2004). This change in TRPV1 expression induces sensitization of primary afferent neurons, thereby leading to the induction of abnormal pain sensation.
Topical application of capsaicin is clinically effective in treating a variety of chronic painful conditions, including postherpetic neuralgia (Backonja et al., 2008) and HIV-associated distal sensory polyneuropathy (Simpson et al., 2008), and its analgesic action is considered a consequence of inhibition of TRPV1 function by desensitization (Holzer, 1991). However, capsaicin is not administered systemically because of its adverse effects, such as a burning sensation and Bezold-Jarisch reflex with bradycardia and hypopnea in the initial phase. Therefore, inhibition of TRPV1 function via an orally active antagonist is an attractive therapeutic strategy for treating chronic intractable pain.
A considerable number of TRPV1 antagonists have been discovered to date that have shown efficacy in several models of inflammatory and neuropathic pain (Pomonis et al., 2003; Honore et al., 2005; Kanai et al., 2007; Lehto et al., 2008). Despite their promising analgesic effects, however, several TRPV1 antagonists have recently been found to cause hyperthermia in laboratory animals (Gavva et al., 2007a,b). Furthermore, phase I clinical trials of AMG517 showed that TRPV1 antagonist administration induced severe hyperthermia both in healthy volunteers and in patients who had undergone third molar extraction (Gavva et al., 2008). However, of note is that the hyperthermic potential of TRPV1 antagonists varies among compounds; for example, previous studies found that capsazepine and SB-366791 did not change body temperature (Gavva et al., 2007b; Garami et al., 2010). Given such variety, the analgesic spectrum of TRPV1 antagonists without hypothermia-inducing effects merits further characterization.
(R)-N-(1-Methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-carboxamid (AS1928370) is a novel and orally active TRPV1 antagonist that we identified in our research. Here, we characterized the inhibition mode of AS1928370 against TRPV1 receptors in in vitro studies. We then evaluated the analgesic effects of orally administered AS1928370 in a capsaicin-induced secondary hyperalgesia model, a neuropathic pain model, and an inflammatory pain model. We then assessed the effects of AS1928370 on body temperature and motor coordination to characterize the potential of TRPV1 receptor blockade as a novel analgesic agent. The results were compared with those of the conventional TRPV1 antagonist N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC; Pomonis et al., 2003).
Materials and Methods
AS1928370 (see Fig. 1), BCTC, and 1-(aminomethyl)-cyclohexane-acetic acid (gabapentin) were synthesized at Astellas Pharma Inc. (Ibaraki, Japan). A monohydrochloride salt of AS1928370 and BCTC were used as AS1928370 and BCTC, respectively. Diclofenac sodium, RTX, and capsaicin were purchased from Sigma-Aldrich (St. Louis, MO). For in vivo studies, AS1928370 and gabapentin were suspended in 0.5% methylcellulose aqueous solution. Diclofenac sodium was dissolved in distilled water. These compounds were administered orally at an administration volume of 5 ml/kg. BCTC was dissolved in 10% dimethyl sulfoxide and 10% Cremophor and was administered intraperitoneally at an administration volume of 5 ml/kg. Drug concentrations were calculated in terms of free base.
Male Sprague-Dawley rats (weight range, 165–310 g; Japan SLC, Hamamatsu, Japan) were used for all in vivo experiments. Animals were group-housed and kept on a 12-h light/dark cycle (lights on from 7:30 AM to 7:30 PM) with free access to food and water. All animal experimental procedures were approved by the Committee for Animal Experiments of Astellas Pharma Inc. and conformed to the International Guiding Principles for Biomedical Research Involving Animals (CIOMS) and Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, 2006). All efforts were made to minimize the number of animals used and their suffering.
Human embryonic kidney (HEK) 293 cells (American Type Culture Collection, Manassas, VA) were cultured on a collagen I-coated dish in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in 5% CO2. HEK293 cells stably expressing human or rat TRPV1 (human or rat TRPV1-HEK293) were generated by transfection with a pcDNA 3.1(+) expression vector encoding the appropriate TRPV1 cDNA. Lipofectamine 2000 transfection regent (Invitrogen, Carlsbad, CA) was used for stable transfections, and 800 μg/ml G-418 was used as a selection agent. The positive clones that showed high responses in a capsaicin-induced Ca2+ flux assay were used in all in vitro studies. HEK293 cells stably expressing other transient receptor potential channels (TRPs) were constructed using similar procedures. For electrical recording, cells were plated on poly-d-lysine-coated glass coverslips at a density of approximately 1.5 × 104 cells/cm2. For the Ca2+ flux assay, cells were plated on multiwell plates at a density of 1.5 × 105 cells/cm2.
[3H]RTX Binding Assay.
The binding assay was conducted using a method modified from that of Szallasi et al. (1999). In brief, human TRPV1-HEK293 cell membrane (30 μg of protein), [3H]RTX (2.2 nM; PerkinElmer Life and Analytical Sciences, Waltham, MA), and competitors were incubated at 37°C for 60 min in 0.3 ml of 25 mM Tris-HCl, pH 7.4, containing 0.025% bovine serum albumin. To determine nonspecific binding, 1 μM RTX was used. Membrane-bound [3H]RTX was separated from the free [3H]RTX by adding 50 μl of α1-acid glycoprotein and subjecting the solution to 5-min incubation and rapid filtration through GF/B filters (Whatman, Maidstone, UK). The radioactivity was determined by scintillation counting. Specific binding was defined as a portion of total binding, which was replaced by 1 μM RTX. Data were analyzed using a modified Hill equation as in the study by Acs et al. (1994) with the computer program Origin7.5 (OriginLab Corp, Northampton, MA).
Whole-cell patch-clamp recordings were obtained from human TRPV1-HEK293 cells using fire-polished glass electrodes (resistance, 2–5 MΩ) filled with a solution containing 120 mM CsCl, 2 mM MgCl2, 10 mM HEPES, 10 mM BAPTA, 4 mM MgATP, 0.4 mM Na2GTP, and 4 mM Na2 creatine phosphate, pH 7.2, with CsOH. Cells were continuously perfused at room temperature (20–25°C) with a bath solution containing 145 mM NaCl, 5 mM CsCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4, with NaOH. All cells were voltage-clamped at −60 mV, and series resistance was compensated 75 to 90% using an Axopatch 1D amplifier (Molecular Devices, Sunnyvale, CA). Capsaicin (1 μM) was delivered to the cells using a U-tube positioned near the cell, and during experiments, capsaicin was usually applied for 10 s every 3 min. AS1928370 (3–100 nM) was applied to the cells via a bath solution. Capsaicin-induced current amplitudes were measured at the response peak. IC50 values were calculated using nonlinear regression analysis with the SAS package version 8.2 (SAS Institute, Cary, NC).
Capsaicin- and Proton-Induced Ca2+ Flux Assay.
Hanks' balanced salt solution without phenol red with 20 mM HEPES added was used as the assay buffer for the capsaicin-induced Ca2+ flux assay. To avoid proton buffering, Hanks' citrate buffer containing 107 mM NaCl, 5.4 mM KCl, 1.26 mM CaCl2·2H2O, 0.8 mM MgSO4, 0.44 mM KH2PO4, 0.34 mM Na2HPO4, 10 mM sodium citrate, and 5.6 mM d-glucose, pH 7.4, was used as assay buffer for proton-induced Ca2+ flux assay. Component A of a calcium 3 assay kit (Molecular Devices) was dissolved in the assay buffer with 0.5 mM probenecid and used as the dye-loading buffer. Human or rat TRPV1-HEK293 cells were loaded with Ca2+ reporter dye and left to sit for 2 h at room temperature. Intracellular Ca2+ concentration was measured using a fluorometric imaging plate reader (Molecular Devices). AS1928370 solution was added 5 min before adding either the submaximal concentration of capsaicin solution (80 nM) or low pH solution, pH 6.0. IC50 values were calculated using nonlinear regression analysis with SAS package version 8.2.
Selectivity for Other TRPs and Ion Channels.
Inhibitory effects for other TRPs were evaluated in HEK293 cells stably expressing TRPs using the Ca2+ flux assay described above. Hypotonic solution (distillated water), allyl isothiocyanate (2 nM; Wako Pure Chemicals, Osaka, Japan), and menthol (400 μM; Sigma-Aldrich) were used as agonists for TRPV4, TRPA1, and TRPM8, respectively. Investigations of binding affinity to other ion channels were performed at Sekisui Medical Co., Ltd. (Ibaraki, Japan) using published protocols. Veratridine-induced [14C]guanidinium uptake assay in rat cortical cell cultures for the investigation of sodium channels was performed using the method of Bönisch et al. (1993).
Plasma and brain samples were collected at 0.5, 1, 2, and 4 h after drug administration in rats and stored at −20°C until use. Brain samples were homogenized with 100 mM Na-K phosphate buffer, pH 7.4. After the addition of diazepam (1 μg/ml; Wako Pure Chemicals) as the internal standard, plasma and brain samples were treated by protein precipitation with acetonitrile. After centrifugation (1500g at 4°C for 10 min), the supernatant was separated and used for analysis. The concentration of AS1928370 in the samples was quantified using high-performance liquid chromatography coupled with a triple quadruple mass spectrometer (API2000; Applied Biosystems, Foster City, CA).
Capsaicin-Induced Secondary Hyperalgesia Model.
Capsaicin-induced secondary hyperalgesia model was performed using a method modified from that of Kim et al. (2001). Capsaicin (10 μg) dissolved in 10 μl of 20% 2-hydroxypropyl-β-cyclodextrin was injected intradermally into the center of the plantar surface of the left hindpaw. Thirty minutes after capsaicin injection, the mechanical threshold in unrestrained rats was assessed using the von Frey hair test. The proximal half of the third or fourth toes of the left hindpaw was stimulated with von Frey hairs (0.41–15.14 g), and the 50% paw withdrawal threshold was determined using up-down paradigms (Chaplan et al., 1994). Before drug evaluation, the rats underwent withdrawal threshold measurement and were allocated to groups to minimize the differences in average threshold among the groups. Behavioral testing was performed in a blind fashion. In the calculation of ED50 values, the thresholds in control and vehicle-treated groups were designated as 100 and 0%, respectively.
Spinal Nerve Ligation Model.
Spinal nerve ligation (SNL) surgery was conducted based on the method of Kim and Chung (1992). In brief, the left L5 and L6 spinal nerves were tightly ligated using 6-0 silk thread, and the wound was sutured under pentobarbital sodium anesthesia. The mechanical threshold in unrestrained rats was then measured using the von Frey hair test as follows: the plantar surface of the hindpaw was stimulated with von Frey hairs (0.41–15.14 g) with ascending force until a paw withdrawal response was elicited. The lowest amount of force required to elicit a response was designated as the withdrawal threshold. On postsurgery day 6, the rats underwent withdrawal threshold measurement and were allocated to groups to minimize the differences in average threshold among the groups. Drug evaluation was performed on postsurgery day 7. Behavioral testing was performed in a blind fashion. In the calculation of ED50 values, the thresholds of contralateral and ipsilateral paws in the vehicle-treated group were designated as 100 and 0%, respectively.
Complete Freund's Adjuvant Model.
The analgesic effect in a complete Freund's adjuvant (CFA)-induced inflammatory pain model was evaluated according to the method of Kanai et al. (2007). In brief, inflammation was induced via an injection of Mycobacterium tuberculosis H37Ra strain (0.3 mg; Difco, Detroit, MI) in liquid paraffin (0.1 ml) into the left hind pad. The thermal withdrawal latency was assessed using a plantar test (Ugo Basile, Comerio, Italy). On the second day after CFA injection, the rats underwent latency measurement and were allocated to groups to minimize the differences in average latency among the groups; drug evaluation was performed on the same day. Behavioral testing was performed in a blind fashion.
Rectal Body Temperature.
Before the drug evaluation, handling treatments were done twice on the study day and the previous day. Rats were then placed individually into stainless steel cages (20 × 30 × 20 cm) and allowed to acclimate for 1 h. Rectal temperature was measured using a microprobe thermometer (BAT-12; Muromachi Kikai Co., Ltd., Tokyo, Japan) before dosing and 0.5, 1, 1.5, 2, 3, and 4 h after dosing.
Rotarod Performance Assessment.
Motor coordination was measured using an accelerating rotarod apparatus (model LE8500; Panlab, Barcelona, Spain) that was set to accelerate from 4 to 40 rpm over 5 min. Each rat was subjected to three training sessions. Rats that remained on the rod for over 90 s were used for the test session and were allocated to four groups to minimize the differences in average maximum performance times in the training sessions among groups. In the test session, retention time was measured twice, and the mean was adopted as a data point for each animal.
Data were expressed as the mean ± S.E.M. Significance of differences between two groups was assessed using Student's t test, whereas that among more than two groups was assessed using Dunnett's multiple comparison tests. In time-course measurement studies, analyses were conducted using two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni's post tests. P < 0.05 was considered significant.
[3H]RTX Binding Assay.
The specific [3H]RTX binding to human TRPV1-HEK293 cell membrane showed saturable binding, with a Kd value of 1.4 ± 0.18 nM and a Bmax value of 2.2 ± 0.021 pmol/mg protein in a positive cooperativity. Insertion into the Hill equation yielded a Hill coefficient of 1.3 ± 0.067. In competition experiments, ASP1928370 displaced [3H]RTX binding with a Ki value of 131 ± 8.0 nM and a Hill coefficient value of 1.53 ± 0.25 (Fig. 2A).
In our electrophysiological assay using a whole-cell patch-clamp technique, application of 1 μM capsaicin to human TRPV1-HEK293 cells produced inward currents that recovered slowly to the baseline level after agonist removal (Fig. 2B). AS1928370 inhibited the capsaicin-induced currents in a concentration-dependent manner with an IC50 value of 32.5 ± 6.2 nM.
Capsaicin- and Proton-Induced Ca2+ Flux Assay.
In the Ca2+ flux assay using the fluorometric imaging plate reader system, ASP1928370 concentration-dependently inhibited capsaicin-induced Ca2+ flux in human and rat TRPV1-HEK293 cells, with IC50 values of 301 ± 21 and 884 ± 146 nM, respectively (Fig. 3A). In contrast, AS1928370 showed a weak or null effect on proton-induced Ca2+ flux in human and rat TRPV1-HEK293 cells (Fig. 3B). It is noteworthy that BCTC inhibited both capsaicin- and proton-induced Ca2+ flux in rat TRPV1-HEK293 cells, with IC50 values of 4.8 ± 1.7 and 4.7 ± 2.7 nM, respectively (data not shown).
Selectivity for Other TRPs and Ion Channels.
AS1928730 showed neither agonistic nor antagonistic activity on TRPV4, TRPA1, and TRPM8 in concentrations up to 10 μM (Table 1). In addition, 10 μM AS1928370 did not inhibit radioligand binding by more than 50% at calcium channel type L, calcium channel type N, potassium channel KATP, and potassium channel SKCa, whereas AS1928730 at the same concentration showed 91% inhibition on sodium channel site 2. However, AS1928370 did not inhibit veratridine-induced [14C]guanidinium uptake assay (sodium channel) in concentrations up to 10 μM (data not shown).
The plasma and brain concentrations of AS1928370 reached peak levels of 31 ng/ml (68 nM) and 161 ng/g tissue (355 nM) 1 h after oral administration (3 mg/kg) in rats (Fig. 4). The plasma peak levels of AS1928370 after administration of 10 and 30 mg/kg p.o. were 100 and 310 ng/ml, respectively (data not shown).
Capsaicin-Induced Secondary Hyperalgesia Model.
Intraplantar injection of capsaicin significantly reduced paw withdrawal thresholds 30 min after injection. In turn, AS1928370 administration resulted in dose-dependent recovery of the threshold, with an ED50 value of 0.17 mg/kg p.o. [95% confidence interval (CI), 0.036–0.31] (Fig. 5A). The effects at 0.3 and 1 mg/kg p.o. were statistically significant, and the threshold recovered to nearly the normal level. In addition, BCTC significantly recovered the threshold at 10 and 30 mg/kg i.p. (Fig. 5B).
In an SNL model, withdrawal thresholds of paws that had been operated on were significantly lower than in contralateral nonoperated paws. AS1928370 dose-dependently recovered the threshold, with an ED50 value of 0.26 mg/kg p.o. (95% CI, 0.0010–0.83) (Fig. 6A). The effects at 1 and 3 mg/kg p.o. were statistically significant. In addition, BCTC (30 and 100 mg/kg i.p.) and gabapentin (300 mg/kg p.o.) also significantly improved mechanical allodynia in this model, and BCTC (100 mg/kg i.p.) increased the threshold over the level in the control group (Fig. 6, B and C).
Intraplantar injection of CFA significantly reduced paw withdrawal latency, and AS1928370 exerted a significant effect on thermal hyperalgegia at 10 mg/kg p.o. (Fig. 7A). Furthermore, BCTC (30 and 100 mg/kg i.p.) and diclofenac sodium (1 and 3 mg/kg p.o.) both significantly recovered thermal hyperalgesia in this model (Fig. 7, B and C).
Rectal Body Temperature.
AS1928370 induced no significant change in body temperature at doses up to 10 mg/kg p.o. (Fig. 8A). Although the greatest relative increase was observed at 0.3 mg/kg p.o., the temperature differed from that of the vehicle-treated group by only approximately 0.3°C. However, AS1928370 significantly reduced body temperatures at 30 mg/kg p.o. In contrast, BCTC significantly increased rectal body temperatures at 10 to 100 mg/kg i.p., and the greatest difference from the vehicle-treated group was approximately 1.0°C (Fig. 8B).
First, we demonstrate that AS1928370 competitively inhibited binding of [3H]RTX to human TRPV1 and blocked capsaicin-induced inward currents. Although the compound also inhibited capsaicin-mediated Ca2+ flux, it showed minimal inhibitory effect on proton-mediated Ca2+ flux. A large number of TRPV1 antagonists have been reported with a range of differing antagonistic profiles against several types of stimulations; for example, some compounds, such as BCTC and AMG517, prevent both capsaicin- and proton-evoked activation, whereas others, such as capsazepine and SB-366791, inhibit only capsaicin-induced activation, working species-dependently (Garami et al., 2010). One previous study suggested that TRPV1 antagonists that inhibited both types of activation bind to the intramembrane RTX-binding pocket and either stabilize or lock the channel conformation so that protons cannot activate TRPV1. In contrast, antagonists that inhibit only capsaicin-induced activation bind to the same pocket but do not stabilize or lock channel confirmation (Gavva et al., 2005). Our data obtained here suggest that our novel compound AS1928370 belongs to this latter group of TRPV1 antagonists.
Next, we demonstrated AS1928370 was capable of improving mechanical allodynia in an SNL neuropathic pain model and that its analgesic efficacy was comparable with that of gabapentin, a widely used agent for treating neuropathic pain. Although TRPV1 antagonists that prevent both capsaicin- and proton-evoked activation, such as BCTC and A-425619, have already been reported to show analgesic effects in neuropathic pain models such as an SNL model or a chronic constriction injury (CCI) model (Pomonis et al., 2003; Honore et al., 2005), analgesic effects of TRPV1 antagonists that exert no inhibitory effect on proton-evoked activation have not been characterized in a SNL or CCI model. Capsazepine was previously reported to have no significant effect in a partial sciatic nerve ligation model; however, the doses used in that study (1–30 mg/kg s.c.) were lower than the dose (100 mg/kg s.c.) that perfectly recovered capsaicin-induced secondary hyperalgesia (Walker et al., 2003). In the present study, AS1928370, as well as BCTC, showed a significant effect in SNL model at the dose that exerted full recovery in a capsaicin-induced secondary hyperalgesia model, indicating that the analgesic effect of AS1928370 in the SNL model is mediated by TRPV1 blockage. This interpretation is supported by our pharmacokinetics data that show that the brain concentration of AS1928370 at 3 mg/kg p.o. sufficiently covered the IC50 value obtained in our electrophysiological assay (355 versus 32.5 nM). In addition, we demonstrated the selectivity of AS1928370 against several transient receptor potential channels and other relevant ion channels; however, further selectivity study is needed to exclude a possibility of an off-target mechanism.
TRPV1 is up-regulated on nociceptive Aδ and C fibers and newly expressed on tactile Aβ fibers under neuropathic pain conditions (Hudson et al., 2001; Rashid et al., 2003). Several putative endogenous ligands, such as anandamide and N-arachidonoyl dopamine, are likely to bind to RTX-binding sites on these up-regulated TRPV1 molecules, subsequently inducing release of calcitonin gene-related peptide, substance P, and glutamate from primary afferent endings (Huang et al., 2002; Immke and Gavva, 2006; Medvedeva et al., 2008). As a result, nociceptive neurons in the dorsal horns of the spinal cord become sensitized because of repeated stimulations of these neurotransmitters. Given that AS1928370 is expected to antagonize the endogenous ligands as well as capsaicin at RTX-binding sites, AS1928370 likely attenuates neuropathic pain primarily by blocking endogenous ligand-mediated neurotransmitter release, not by blockage of proton-evoked TRPV1 activation. In the present study, analgesic efficacy of AS1928370 looked weaker than that of BCTC; however, it is not obvious whether this difference is due to the lack of ability to block proton-evoked TRPV1 activation. Even TRPV1 antagonists with the same inhibition-mode showed different analgesic efficacy in each report; for example, BCTC fully recovered allodynia in the CCI model, whereas A-425619 partially recovered allodynia in the SNL and CCI models (Pomonis et al., 2003; Honore et al., 2005). Therefore, further evaluations of several TRPV1 antagonists are needed under the same experimental condition.
In addition, AS1928370 exerted a significant effect in the CFA-induced inflammatory pain model. However, the effective dose of AS1928370 in the CFA model was much higher than that in capsaicin and neuropathic pain models. Capsazepine was reported to have no significant effect in the CFA model even at 100 mg/kg s.c. (Walker et al., 2003). Taken together, off-target mechanisms may be involved in the analgesic effect of AS1928370 in the CFA model. In contrast, TRPV1 antagonists that prevent both capsaicin- and proton-evoked activation showed significant effects in both models at similar doses (Pomonis et al., 2003; Honore et al., 2005). These results indicate that the inhibition of proton-evoked TRPV1 activation contributes to the efficacy in inflammatory pain models. Protons are actually elevated in inflammatory tissues and induce pain sensations by activating TRPV1 and acid-sensing ion channels (Leffler et al., 2006), implicating that tissue acidosis is an important potential source of inflammatory pain. AS1928370 is incapable of inhibiting this proton-evoked activation of rat TRPV1, which may explain the need for increased exposure to attenuate inflammatory pain.
We also evaluated the potential hyperthermic effect of AS1928370 on body temperature in rats. Of particular note is the fact that whereas BCTC induced a significant elevation in rectal body temperature, AS1928370 did not produce such significant increase. TRPV1 receptors have been reported to be involved in thermoregulation, and several TRPV1 antagonists, including BCTC, are known to cause hyperthermia in laboratory animals (Gavva et al., 2007a,b). However, a recent study showed that the hyperthermic effects of TRPV1 antagonists are strongly correlated with the compounds' inhibitory effects on proton-induced TRPV1 activation (Garami et al., 2010). They suggest that proton-mediated TRPV1 activation, particularly in abdominal viscera, likely plays an important role in thermoregulation, and therefore any disruption of this homeostatic system may lead to hyperthermia. We noted no change in body temperature in vivo with AS1928370 administration, compatible with these previous findings. However, AS1928370 did cause a significant reduction in rectal body temperature at the highest dose tested. Although AMG8562, which reversely potentiates proton-mediated TRPV1 activation in vitro, has been reported to cause hypothermia in rats (Lehto et al., 2008), AS1928370 does not potentiate a proton response in vitro. In addition, the dose of AS1928370 that induced hypothermic effect was much higher, and thus the mechanism remains unclear.
Finally, we performed a rotarod test in rats and detected no significant effects with AS1928370. Present first-line agents for neuropathic pain, such as gabapentin, frequently cause central side effects such as somnolence and dizziness in human patients and actually induce deficits in motor coordination in rats at analgesic doses (Patel et al., 2001). Taken together with these previous findings, our results suggest that AS1928370 has less liability to cause these side effects at analgesic doses.
In summary, AS1928370 is a novel TRPV1 antagonist with oral absorbability and brain penetrability, which has little inhibitory effect on proton-evoked TRPV1 activation. The compound's ameliorative effects on neuropathic pain are likely caused by the prevention of endogenous ligand-mediated TRPV1 activation. In addition, the compound did not cause hyperthermia or deficit in motor coordination at analgesic doses. These findings strongly suggest that blockage of TRPV1 receptors without affecting proton-mediated activation is a promising approach to treating neuropathic pain, and compounds such as AS1928370 therefore have potential as new analgesic agents for neuropathic pain.
Participated in research design: Watabiki, Kiso, Aoki, and Matsuoka.
Conducted experiments: Watabiki, Kiso, Tsuji, Kohara, and Kakimoto.
Contributed new reagents or analytic tools: Kuramochi and Yonezawa.
Performed data analysis: Watabiki, Kiso, Tsuji, Kohara, and Kakimoto.
Wrote or contributed to the writing of the manuscript: Watabiki, Kiso, Aoki, and Matsuoka.
We thank Kenichi Kakefuda and Yukari Suzuki for technical assistance in the selectivity studies.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- transient receptor potential vanilloid 1
- human embryonic kidney
- transient receptor potential channel
- transient receptor potential vanilloid 4
- transient receptor potential ankyrin 1
- transient receptor potential melastatin 8
- spinal nerve ligation
- complete Freund's adjuvant
- analysis of variance
- confidence interval
- chronic constriction injury
- bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid.
- Received September 26, 2010.
- Accepted November 19, 2010.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics