Transient receptor potential vanilloid 1 (TRPV1) activation in peripheral sensory nerve is known to be associated with various pain-related diseases, thus TRPV1 has been the focus as a target for drug discovery. In this study, we characterized the pharmacological profiles of (3S)-3-(hydroxymethyl)-4-(5-methylpyridin-2-yl)-N-[6-(2,2,2-trifluoroethoxy)pyridin-3-yl]-3,4-dihydro-2H-benzo[b][1,4]oxazine-8-carboxamide (JTS-653), a novel TRPV1 antagonist. JTS-653 displaced [3H]resiniferatoxin binding to human and rat TRPV1. JTS-653 competitively antagonized the capsaicin-induced activation of human TRPV1 with pA2 values of 10.1. JTS-653 also inhibited proton-induced activation of human and rat TRPV1 with IC50 values of 0.320 and 0.347 nM, respectively. Electrophysiological studies indicated that JTS-653 blocked heat-induced inward currents in rat TRPV1 with IC50 values of 1.4 nM. JTS-653 showed weak or no inhibitory effects on other TRP channels, receptors, and enzymes. JTS-653 significantly prevented capsaicin-induced mechanical hyperalgesia at 1 mg/kg p.o. and attenuated carrageenan-induced mechanical hyperalgesia at 0.3 mg/kg p.o. JTS-653 significantly attenuated carrageenan-induced thermal hyperalgesia at 0.1 mg/kg p.o. and fully reversed at 0.3 mg/kg p.o. without affecting the volume of the carrageenan-treated paw. JTS-653 showed a transient increase of body temperature at 0.3 mg/kg p.o. These results indicated that JTS-653 is a highly potent and selective TRPV1 antagonist in vitro and in vivo and suggested that JTS-653 is one of the most potent TRPV1 antagonists. The profiles of JTS-653, high potency in vivo and transient hyperthermia, seem to be associated with polymodal inhibition of TRPV1 activation.
TRP channels have been proposed as the detectors for a wide range of environmental stimuli. TRP channels play an important role in the detection of noxious stimuli and thus have been highlighted as potential targets for drug discovery (Moran et al., 2011). TRPV1 is a member of the TRP channel family and has been cloned as a receptor for capsaicin (Caterina et al., 1997). TRPV1 is a Ca2+-permeable, nonselective cation channel that is activated by chemical or heat stimuli. Capsaicin, resiniferatoxin, protons (acidification), and heat (>43°C) activate TRPV1. Some endogenous compounds, including anandamide (Smart et al., 2000), 12-hydroperoxyeicosatetraenoic acid (Hwang et al., 2000), N-arachidonoyl-dopamine (Huang et al., 2002), and N-oleoyldopamine (Chu et al., 2003), have been reported as the endogenous ligands of TRPV1. A subset of unmyelinated sensory fibers expresses TRPV1 on the nerve endings. TRPV1 activation elicited by chemical and thermal stimuli depolarizes the afferent sensory nerves and produces pain.
Many studies have suggested that the activation or sensitization of TRPV1 is associated with various pain-related diseases. In pathological conditions, TRPV1 expression is up-regulated in the affected organs, such as nerve, skin (Facer et al., 2007), the gastrointestinal tract (Yiangou et al., 2001), bladder (Apostolidis et al., 2005), and airways (Groneberg et al., 2004). Moreover, TRPV1 is sensitized by inflammatory mediators or growth factors produced in response to tissue injury. Prostaglandin E2, bradykinin, ATP, and nerve growth factor activate their receptors and modify TRPV1 via protein kinase C, protein kinase A, phosphatidylinositol-3-kinase, or phospholipase C, leading to TRPV1 sensitization (Patapoutian et al., 2009). Moreover, capsaicin has been clinically used to reduce chronic pain (Anand and Bley, 2011), because capsaicin deprives the function of TRPV1-sensitive sensory nerves. Therefore, TRPV1 is an appropriate target molecule for pain treatment.
Pharmaceutical companies have focused on the development of TRPV1 antagonists. Selective TRPV1 antagonists have been reported to date, and those studies have indicated that TRPV1 antagonists are effective in animal models of inflammatory pain, neuropathic pain (Pomonis et al., 2003; Honore et al., 2005), cystitis (Charrua et al., 2009), and cough (Maher et al., 2011). Several compounds have advanced into studies evaluating pain-related diseases, including pain, osteoarthritis, and gastroesophageal reflux disease (Moran et al., 2011). However, the clinical efficacy of these compounds has not yet been proven. In addition, previous studies have reported that hyperthermia is a common on-target effect of TRPV1 antagonists. TRPV1 antagonists produce a transient increase in the body temperature in multiple species (Gavva et al., 2007b), including humans (Gavva et al., 2008; Othman et al., 2011). Most of these TRPV1 antagonists have been reported to equally inhibit TRPV1 activation by diverse stimuli, such as capsaicin, proton, and heat (Gavva et al., 2007a; Surowy et al., 2008).
Some TRPV1 antagonists, such as (R,E)-N-(2-hydroxy-2,3-dihydro-1H-inden-4-yl)-3-(2-(piperidin-1-yl)-4-(trifluoromethyl)phenyl)acrylamide (AMG8562) and (R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-carboxamide (AS1928370), have been reported not to cause hyperthermia in rodents (Lehto et al., 2008; Watabiki et al., 2011). However, these compounds showed weak efficacy in models of inflammatory pain. In vitro studies showed that AMG8562 and AS1928370 inhibited capsaicin-induced activation of TRPV1 but not proton-induced activation. These observations indicated the possibility that the in vivo pharmacological profile of a TRPV1 antagonist could be determined by its in vitro inhibitory profile against TRPV1 activation induced by diverse stimuli.
In this study, we characterized the in vitro and in vivo pharmacological profiles of a novel and selective TRPV1 antagonist, (3S)-3-(hydroxymethyl)-4-(5-methylpyridin-2-yl)-N-[6-(2,2,2-trifluoroethoxy)pyridin-3-yl]-3,4-dihydro-2H-benzo[b][1,4]oxazine-8-carboxamide (JTS-653) (Fig. 1). In in vitro experiments, we investigated the effects of JTS-653 on capsaicin-, proton-, and heat-induced activation of TRPV1 and examined the effects of JTS-653 on other TRP channels, receptors, ion channels, and enzymes. In in vivo experiments, we investigated the effects of JTS-653 on capsaicin- or carrageenan-induced hyperalgesia in rats and assessed the effect on body temperature in normal rats.
Materials and Methods
JTS-653 was synthesized at the Central Pharmaceutical Research Institute, Japan Tobacco Inc. (Osaka, Japan). Capsaicin, RTX, indomethacin, 2-aminoethyl diphenylborinate (2-APB), 4α-phorbol-12,13-didecanoate (4α-PDD), and (−)-menthol were purchased from Sigma-Aldrich (St. Louis, MO). Allyl isothiocyanate was purchased from Nacalai Tesque Inc. (Kyoto, Japan). In the in vitro experiments, JTS-653 and the TRP channel agonists were dissolved in dimethyl sulfoxide and diluted with the buffer used in each experiment. In the in vivo experiments, JTS-653 and indomethacin were suspended in 0.5% (w/v) methyl cellulose solution.
Male 6-week-old Sprague-Dawley rats (Japan SLC, Shizuoka, Japan) were used in most of the experiments. Evaluation of the body temperature was conducted with male 5-week-old Sprague-Dawley rats (Charles River Japan, Yokohama, Japan). Animals were group-housed and kept in a 12-h light/dark cycle with free access to food and water. These studies complied with the guidelines for animal experimentation at our laboratories.
Human embryonic kidney (HEK) 293 cells were cultured in basal medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin) at 37°C under the 5% CO2 in air. The cells stably expressing TRPV1 were cultured in basal medium containing 500 μg/ml G418.
Stable Transfection of TRPV1 cDNA.
The pcDNA 3.1-expression vector (Invitrogen, Carlsbad, CA) encoding human or rat TRPV1 cDNA was transfected into HEK 293 cells with Lipofectamine 2000 (Invitrogen). These cells were cultured in basal medium containing 500 μg/ml G418 for 2 weeks. The positive colonies were picked up and expanded. These clones were screened based on the response to capsaicin (1 μM) or low pH solution, pH 5.5, by the Ca2+ influx assay described below. The clones responsive to both stimuli were selected and used for the experiments.
Transient Transfection of TRP Channel cDNA.
HEK 293 cells were seeded into the collagen I-coated flask and cultured in basal medium. These cells were transiently transfected with a pcDNA 3.1-expression vector encoding human TRP channels (TRPV1, TRPV3, TRPV4, TRPM8, and TRPA1) or rat TRPV1 gene. The transfection was performed with Lipofectamine 2000 on the day before the experiments.
HEK 293 cells were transiently transfected with human or rat TRPV1 cDNA and cultured overnight. The cells were collected and resuspended in an ice-cold 25 mM HEPES buffer supplemented with protease inhibitors (1 tablet; 50 ml; Roche Diagnostics, Basel, Switzerland). These cells were homogenized with a Polytron homogenizer (Polytron PT10-35; Kinematica, Lucerne, Switzerland). The homogenate was centrifuged at 1,000g for 10 min, and the resulting supernatant was further centrifuged at 150,000g for 30 min. The pellet was resuspended in ice-cold 20 mM Tris-HCl buffer, pH 7.5, and stored at −80°C.
Radioligand Binding Assay.
Membranes were incubated with 0.5 nM [3H]RTX (39.8 Ci/mmol; PerkinElmer Life and Analytical Sciences, Waltham, MA) and JTS-653 for 60 min at 37°C in a final volume of 0.5 ml of 10 mM Tris-HCl buffer. The incubation was initiated with the addition of the membrane suspension (human TRPV1, 20 μg of protein; rat TRPV1, 2 μg of protein), and terminated by rapid filtration through glass microfiber filters (GF/C; Whatman, Clifton, NJ) presoaked in 10 mM Tris-HCl buffer containing 0.5% polyethyleneimine. The radioactivity was measured with a liquid scintillation counter (TRI-CARB 2500TR and 1900TR; PerkinElmer Life and Analytical Sciences). Nonspecific binding of [3H]RTX was defined with 1 μM RTX, and specific binding was calculated by subtracting the nonspecific binding from the total binding. Data were analyzed by using the four-parameter logistic-Hill equation with Prism version 4.03 (GraphPad Software Inc., San Diego, CA). The equilibrium dissociation constant (Ki) of JTS-653 was calculated by the modified Cheng-Prusoff equation (Cheng, 2004).
Ca2+ Influx Assay for TRPV1.
These experiments were performed with HEK 293 cells stably expressing human and rat TRPV1. Changes in the intracellular calcium levels by TRPV1 activation were measured according to the method described by El Kouhen et al. (2005) with slight modifications. In brief, the cells (5 × 104 cells/well) were seeded onto a black-walled, clear-bottom, 96-well plate coated with poly-d-lysine (BD Biosciences, San Jose, CA) and cultured overnight. Then the medium was removed, and Ca2+ indicator dye was loaded into the cells with a Fluo-4 NW Calcium Assay Kit (Invitrogen). The dye was dissolved in Hanks' balanced saline solution (Invitrogen) containing 20 mM HEPES, pH 7.4. The cells were incubated for 30 min at 37°C and 30 min at room temperature. Fluorescence was measured with a fluorometric imaging plate reader (Molecular Devices, Sunnyvale, CA) as relative light units (RLU; λEX = 488 nm, λEM = 540 nm). JTS-653 was added to the plate, then capsaicin or proton stimulus was applied, and the RLU value was recorded for 190 s. Proton stimuli were applied with a low pH solution, pH 5.5, containing 20 mM MES. The basal RLU value was defined as the RLU obtained at 10 s after adding JTS-653. The changes in the RLU were calculated by subtracting the basal RLU value from the maximal RLU value during stimulation and used for data analysis. Data analysis was performed with Prism version 4.03 (GraphPad Software Inc.) and SAS System release 8.2 (SAS Institute Japan Co., Ltd., Tokyo, Japan).
HEK 293 cells stably expressing rat TRPV1 were plated onto glass coverslips. These coverslips were incubated for 24 to 72 h at 33°C before being used for the experiments. Whole-cell patch-clamp recordings were conducted as described previously (Tominaga et al., 2001). In brief, coverslips plated with cells were placed in a recording chamber filled with bath solution consisting of 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM EGTA, 10 mM glucose, and 10 mM HEPES, pH 7.4 (adjusted with NaOH). Recording pipettes were filled with pipette solution consisting of 140 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH 7.4 (adjusted with KOH) All recordings were performed on well isolated, single-phase bright cells at room temperature by using an Axopatch 200B amplifier controlled via pClamp10.2 software (Molecular Devices). The cells were voltage-clamped at −60 mV. Drug applications were controlled by using perfusion valve control systems (VC-6; Warner Instruments, Hamden, CT), and temperature jumps were controlled by using a temperature controller (TC-344B; Warner Instruments) with an in-line heater. A preheated bath solution (approximately 50°C) was applied to the cells at 3-min intervals until the subsequent current was stable. At that point, typically after initial current rundown, JTS-653 was applied for 2 min. Then the cells were stimulated by coapplication of JTS-653 and the preheated bath solution. For data analysis, relative current were calculated as a percentage of the peak current observed before the application of JTS-653 or vehicle [0.1% (v/v) dimethyl sulfoxide]. Each relative current was normalized with the mean of the relative currents obtained from the vehicle-treated cells.
Ca2+ Influx Study for the Other TRP Channels.
These experiments were performed with HEK 293 cells transiently expressing the human TRP channel (TRPV3, TRPV4, TRPM8, or TRPA1). The cells expressing TRPV3, TRPM8, and TRPA1 were suspended in Hanks' balanced saline solution containing 20 mM HEPES, pH 7.4. The cells expressing TRPV4 were suspended in the solution reported by Xu et al. (2003) containing 88 mM NaCl, 5 mM KCl, 1 mM CaCl2, 5.5 mM glucose, 10 mM HEPES, and 100 mM mannitol, pH 7.4. The cells were treated with 5 μM Fura2-AM (Dojindo Laboratories, Kumamoto, Japan) and 0.01% Pluronic F-127 (Invitrogen) to load Fura-2AM into the cells. The cells were incubated for 30 min at 37°C and washed by centrifugation, and then the cell debris was removed. The ratio of the fluorescence intensity (F340/F380; excitation at 340 nm and 380 nm; emission 500 nm) was measured with a CAF-110 fluorescence spectrofluorometer (Jasco, Tokyo, Japan). The blank value was defined as the F340/F380 value obtained from the mock cells stimulated with vehicle, and the F340/F380 values for the transfected cells were corrected by subtraction of the blank value. We used 2-APB, 4α-PDD, (−)-menthol, and allyl isothiocyanate as agonists for TRPV3, TRPV4, TRPM8, and TRPA1, respectively. The activation of TRPV3, TRPV4, and TRPA1 was conducted at 37°C. The activation of TRPM8 was conducted at 25°C. The cells (2.5 × 105 cells) and JTS-653 were incubated in the cuvette for 10 min, and the basal F340/F380 value was acquired. The agonists were added, and the F340/F380 was recorded between 1 and 2 min after adding agonists. The change in F340/F380 was calculated as the difference between the maximal value and the basal value and was used for data analysis.
Selectivity for Other Receptors and Enzymes.
The binding affinities of JTS-653 to 34 receptors (including ion channels) and inhibitory effects on five enzyme reactions were investigated at a concentration of 10 μM. Investigation was performed at Daiichi Pure Chemicals Co., Ltd. (Ibaraki, Japan).
Capsaicin-Induced Mechanical Hyperalgesia.
JTS-653 was orally administered 1.5 h before the capsaicin treatment. Capsaicin (10 μg/site; 50 μl) was injected into the left hindpaw, whereas sham animals were injected with olive oil. Mechanical hyperalgesia was assessed by measuring the paw withdrawal threshold (PWT) with a dynamic plantar aesthesiometer (Ugo Basile, Comerio, Italy). Continuously increasing pressure was applied to the hindpaw through a filament, and the pressure that caused paw withdrawal was recorded as the PWT. Measurements of the PWT were conducted in a blind fashion at 2 h after administration of JTS-653.
Carrageenan (1% solution; 50 μl) was injected into the left hindpaw at 1 h before oral administration of the test compounds. Mechanical hyperalgesia was assessed by measuring the PWT by the method of Randall and Selitto (1957) with a pressure analgesymeter (Unicom, Chiba, Japan). Thermal hyperalgesia was assessed by measuring paw withdrawal latency (PWL) as described by Hargreaves et al. (1988) with a plantar thermal stimulator (Ugo Basile). An infrared heat stimulus was applied to the hindpaw, and the time required for the paw withdrawal was recorded as the PWL. Measurements of the PWT or PWL were conducted at 2 h after administration of the test compounds. After measurement of the PWL, the hindpaw volume was measured with a plethysmometer (Unicom). These measurements were performed in a blind fashion.
Body Temperature Evaluation.
Rectal body temperatures were measured with a digital thermometer (KN-91; Natsume Seisakusho Co., Ltd., Tokyo, Japan) before and 1, 2, 4, 8, and 24 h after oral administration of JTS-653.
Data were expressed as the mean ± S.E. Statistical analysis was performed with SAS System release 8.2. Comparison between three or more groups was conducted by Dunnett's multiple comparison test. Comparison between two groups was conducted by Student's t test. In the time-course measurement study, analysis was conducted by a two-way repeated-measures analysis of variance followed by Dunnett's multiple comparison test. p < 0.05 was considered statistically significant.
Effect of JTS-653 on [3H]RTX Binding.
The effect of JTS-653 on [3H]RTX binding to TRPV1 was investigated with a radioligand binding assay. [3H]RTX showed saturable and specific binding to human and rat TRPV1 in a positive cooperativity. The Kd values for human and rat TRPV1 were 0.834 ± 0.050 and 0.334 ± 0.070 nM, respectively. The Bmax values for human and rat TRPV1 were 4.16 ± 0.70 and 11.6 ± 0.75 pmol/mg protein, respectively. The Hill coefficients for human and rat TRPV1 were 1.60 ± 0.087 and 1.52 ± 0.18, respectively. In the competition experiments, JTS-653 displaced [3H]RTX binding to human and rat TRPV1 in a concentration-related manner with Ki values of 11.44 ± 6.4 and 4.40 ± 1.3 nM, respectively (Fig. 2). The Hill coefficients for human and rat TRPV1 were 1.23 ± 0.20 and 0.99 ± 0.11, respectively.
Effect of JTS-653 on TRPV1 Activation.
The effects of JTS-653 on the activation of TRPV1 were investigated by a Ca2+ influx assay. Capsaicin-induced TRPV1 activation was attenuated in the presence of JTS-653. JTS-653 produced a parallel and rightward shift of the concentration response curve of capsaicin and Schild analysis provided linear Schild plots (Fig. 3; representative data). The pA2 value for JTS-653 obtained from three independent experiments was 10.1 ± 0.02. The slope of the Schild plots was 0.89 ± 0.06, indicating that JTS-653 showed competitive inhibition. JTS-653 inhibited capsaicin- and proton-induced activation of human and rat TRPV1 in a concentration-related manner (Fig. 4). JTS-653 inhibited the 30 nM capsaicin-induced activation of human and rat TRPV1 with IC50 values of 0.236 ± 0.089 and 0.247 ± 0.063 nM, respectively. JTS-653 also inhibited proton-induced activation of human and rat TRPV1 with IC50 of 0.320 ± 0.059 and 0.347 ± 0.026 nM, respectively.
The effects of JTS-653 on the heat-induced activation of rat TRPV1 were investigated by whole-cell patch-clamp recording. JTS-653 decreased the heat-evoked inward currents (Fig. 5A, representative current traces) and showed its inhibitory effect in a concentration-related manner (Fig. 5B) with IC50 values of 1.4 nM.
Effect of JTS-653 on Other TRP Channels.
The effects of JTS-653 on other human TRP channels were investigated with Ca2+ influx assay. JTS-653 inhibited agonist-induced activation of each TRP channel (Table 1), and the IC50 value for each TRP channel was more than 1000-fold higher than that for TRPV1 obtained from Ca2+ influx assay.
Effect of JTS-653 on Other Receptors and Enzymes.
The selectivity of JTS-653 was investigated based on binding affinities to 34 receptors and inhibitory effects on five enzyme reactions at 10 μM. JTS-653 showed weak or no inhibitory effects on these molecules. JTS-653 showed 57% inhibition on the [3H]batrachotoxin binding to the rat sodium channel and less than 20% inhibition on the other receptors, ion channels, and enzymes.
Effect of JTS-653 on Capsaicin-Induced Mechanical Hyperalgesia.
The effect of JTS-653 on capsaicin-induced mechanical hyperalgesia was investigated. Olive oil slightly, but not significantly, decreased the PWT of the ipsilateral hindpaw. Capsaicin significantly decreased the PWT of the ipsilateral hindpaw in the vehicle group, indicating that capsaicin induced mechanical hyperalgesia. JTS-653 prevented the decrease of PWT in a concentration-related manner. JTS-653 significantly blocked mechanical hyperalgesia at 1 mg/kg or above and showed its maximum effect at 1 mg/kg. JTS-653 did not change the PWT of the contralateral hindpaw at the highest dose (Fig. 6).
Effect of JTS-653 on Carrageenan-Induced Hyperalgesia.
The effect of JTS-653 on carrageenan-induced hyperalgesia was investigated. Carrageenan significantly decreased PWT of the ipsilateral hindpaw in the vehicle group, indicating that carrageenan induced mechanical hyperalgesia. JTS-653 inhibited the decrease of PWT in a concentration-related manner. JTS-653 significantly attenuated thermal hyperalgesia at 0.3 mg/kg or above and showed its maximum effect at 1 mg/kg. Indomethacin also significantly inhibited the decrease of PWT. JTS-653 and indomethacin did not change the PWT of the contralateral hindpaw at the highest dose (Fig. 7).
Carrageenan significantly decreased PWL of the ipsilateral hindpaw in the vehicle group, indicating that carrageenan induced thermal hyperalgesia. JTS-653 inhibited the decrease of PWL in a concentration-related manner. JTS-653 significantly attenuated thermal hyperalgesia at 0.1 mg/kg or above. JTS-653 and indomethacin fully reversed thermal hyperalgesia. JTS-653 and indomethacin did not change the PWL of the contralateral hindpaw at the highest dose (Fig. 8A).
JTS-653 did not affect the volume of the ipsilateral hindpaw at the highest dose, whereas indomethacin produced a significant decrease of the volume of the ipsilateral hindpaw (Fig. 8B).
Effect of JTS-653 on Body Temperature.
The effect of JTS-653 on body temperature was investigated in normal rats. JTS-653, at doses of 0.3 and 3 mg/kg, transiently increased body temperature and showed a maximum increase in body temperature 1 h after dosing (Fig. 9). The hyperthermia was significant up to 4 h after dosing, and the body temperature returned to the level in the vehicle group by 8 h after dosing.
In this study, we characterized the pharmacological profiles of a novel TRPV1 antagonist, JTS-653. JTS-653 has been shown to be a potent and selective TRPV1 antagonist. JTS-653 inhibited the activation of TRPV1 induced by capsaicin, proton, and heat. Orally administered JTS-653 exerted a potent antihyperalgesic effect and caused transient hyperthermia in rats, indicating that JTS-653 antagonizes TRPV1 activation in vivo.
The present study demonstrates that JTS-653 is a potent TRPV1 antagonist. JTS-653 inhibited [3H]RTX binding to human and rat TRPV1. JTS-653 competitively inhibits capsaicin-induced activation of human TRPV1 with a pA2 value of 10.1. In addition, JTS-653 antagonized capsaicin- and proton-induced activation of human and rat TRPV1. Many studies have reported the potency of TRPV1 antagonists against capsaicin-induced TRPV1 activation. The pA2 values for (R)-(5-tert-butyl-2,3-dihydro-1H-inden-1-yl)-3-(1H-indazol-4-yl)-urea (ABT-102) and N-(2-bromophenyl)-N′-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolidin-3-yl)]urea (SB-705498), which are in clinical studies, against human TRPV1 activation have been reported as 8.3 (Surowy et al., 2008), 8.2 (Gavva et al., 2007a), and 7.4 (Gunthorpe et al., 2007), respectively. Antagonists, such as 1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea (A-425619), N-(4-[6-(4-trifluoromethyl-phenyl)-pyrimidin-4-yloxy]-benzothiazol-2-yl)-acetamide (AM517), AMG8562, (2-(2,6-dichlorobenzyl)thiazolo(5,4-d)pyrimidin-7-yl)-(4-trifluoromethylphenyl)amine (JNJ-39729209), and AS1928370 have been shown to inhibit human TRPV1 activation with similar or less potency compared with those compounds (El Kouhen et al., 2005; Gavva et al., 2007a; Lehto et al., 2008; Maher et al., 2011; Watabiki et al., 2011). Therefore, our results suggest that JTS-653 has higher potency against capsaicin-induced TRPV1 activation than other previously reported TRPV1 antagonists. We also showed that JTS-653 potently blocked proton-induced activation of human TRPV1 with an IC50 value of 0.32 nM. The other TRPV1 antagonists described above have been shown to have similar or less inhibitory potency for proton-induced TRPV1 activation compared with JTS-653 (El Kouhen et al., 2005; Gavva et al., 2007a; Gunthorpe et al., 2007; Lehto et al., 2008; Surowy et al., 2008; Maher et al., 2011; Watabiki et al., 2011). Taken together, our study suggests that JTS-653 can be classified as one of the most potent TRPV1 antagonists in vitro.
Our electrophysiological studies revealed that JTS-653 blocked heat-induced inward currents in HEK 293 cells expressing rat TRPV1. Considering the results of the Ca2+ influx assay, it is suggested that JTS-653 inhibits multiple modes of TRPV1 activation. Several TRPV1 antagonists, such as ABT-102 (Surowy et al., 2008) and A-425619 (Gavva et al., 2007b) have been reported to have an inhibitory effect on the activation of rat TRPV1 elicited by capsaicin, proton, and heat. Thus our results demonstrated that JTS-653 seems to exhibit a similar inhibitory profile as those compounds. Many TRPV1 antagonists, including A-425619, have been reported to equally block the heat-induced activation of human and rat TRPV1 (Gavva et al., 2007b; Papakosta et al., 2011). Meanwhile, modality-selective antagonists for human TRPV1, which had low inhibitory effects on TRPV1 activation induced by specific stimuli, have not yet been reported. Thus the pharmacological significance of modality-selective TRPV1 inhibition in humans is unknown. Recent studies have shown the key domains in TPRV1 for modality-selective TRPV1 inhibition (Aneiros et al., 2011; Papakosta et al., 2011; Yao et al., 2011). For example, Papakosta et al. have reported the key domain related to heat-induced TRPV1 activation. N-(4-tert-butylbenzyl)-N′-[3-fluoro-4-(methylsulfonylamino)benzyl] thiourea (JYL-1421), which potently inhibits the capsaicin-induced TRPV1 activation of human and rat TRPV1, potently blocked the heat-induced activation of human TRPV1 but weakly blocked that of rat TRPV1. They demonstrated that the key domain related to this species difference was the transmembrane domains 5/6 by using the chimeric approach. Such observations would be helpful for finding the modality-selective TRPV1 antagonist.
JTS-653 prevented capsaicin-induced mechanical hyperalgesia without affecting the paw withdrawal threshold in the nontreated paw. These results suggest that JTS-653 specifically antagonized TRPV1 activation in vivo and did not affect the sensitivity of the sensory nerves to mechanical stimuli in the normal tissue. Although JTS-653 inhibited the TRP channels and sodium channels, the IC50 values for these molecules were more than 1000-fold higher than that for TRPV1 obtained from the Ca2+ influx assay. These findings support the notion that JTS-653 showed antihyperalgesic effects through TRPV1 inhibition. Topically applied capsaicin promotes the release of neuropeptides, such as calcitonin gene-related peptide and substance P, from the central terminals of capsaicin-sensitive nerves, resulting in sensitization of the neurons in the spinal dorsal horns and mechanical hyperalgesia (Sun et al., 2004). Our study demonstrated that JTS-653 potently inhibits capsaicin-induced TRPV1 activation and has selectivity toward TRPV1 in vitro, suggesting that JTS-653 prevents capsaicin-induced hyperalgesia through TRPV1 inhibition at the nerve endings.
JTS-653 blocked carrageenan-induced thermal and mechanical hyperalgesia without affecting the volume of the inflamed paw. These results suggest that an anti-inflammatory effect was not involved in the antihyperalgesic effect of JTS-653. Carrageenan promoted inflammation in the paw and decreased the mechanical and thermal thresholds. It has been known that tissue acidification during inflammation causes the sensitization of the peripheral sensory nerves. Steen et al. (1992) have shown that the threshold to mechanical stimuli in the skin was decreased by perfusion with acidic solution to the ending of capsaicin-sensitive nerves. This observation suggested that mechanical hyperalgesia in inflammation probably is associated with proton-sensitive molecules, including TRPV1. Furthermore, inflammatory mediators and growth factors are produced in the inflammatory tissues, and these substances sensitize TRPV1. Prostaglandin E2, bradykinin, ATP, and nerve growth factor activate their receptors and promote TRPV1 sensitization by the modification of TRPV1 by protein kinase C, protein kinase A, phosphatidylinositol-3-kinase, or phospholipase C (Patapoutian et al., 2009). Tominaga et al. (2001) have shown that ATP increased the TRPV1 currents evoked by capsaicin and proton and decreased the threshold of TRPV1 to thermal stimuli below the normal body temperature. These studies suggest that sensitized TRPV1 in inflammation decreases the thresholds to diverse stimuli and causes mechanical and thermal hyperalgesia. Our in vitro studies indicated that JTS-653 potentially inhibited TRPV1 activation induced by multiple stimuli. These results suggest that the high potency and efficacy of JTS-653 on inflammatory pain are caused by the inhibitory effect of JTS-653 on multiple modes of TRPV1 activation. AMG8562 and AS1928370, which inhibited capsaicin-induced activation but did not inhibit proton-induced TRPV1 activation, showed weak efficacy in models of inflammatory pain (Lehto et al., 2008; Watabiki et al., 2011). These observations support the notion that the ability to inhibit multiple modes of TRPV1 activation may be required for adequate analgesic effect on inflammatory pain.
JTS-653 transiently increased body temperature in normal rats at the dose that showed an antihyperalgesic effect. Hyperthermia had been known as on-target adverse effects of TRPV1 antagonists in various animals (Gavva et al., 2007b) and humans (Gavva et al., 2008; Othman et al., 2011). These compounds have been reported to equally inhibit TRPV1 activation evoked by capsaicin, proton, or heat. On the other hand, AMG8562 and AS1928370, which inhibited a specific mode of TRPV1 activation, have been reported to show no effect on body temperature in rats (Lehto et al., 2008; Watabiki et al., 2011). However, the effects of these compounds on human body temperature have not yet been shown. Garami et al. (2010) have examined the relative contribution of the blockade of capsaicin-, proton-, and heat-induced TRPV1 activation to the development of hyperthermia with mathematical modeling and proposed that the inhibition of proton- and/or capsaicin-induced activation was related to hyperthermia. On the contrary, Gavva et al. (2007b) showed that TRPV1 antagonists that were ineffective against proton-induced TRPV1 activation also caused hyperthermia and suggested that capsaicin and heat activation of TRPV1 were responsible for hyperthermia. Taken together, the critical factor determining TRPV1-mediated hyperthermia remains unclear. The studies of AMG8562 and AS1928370 suggest that the inhibition of specific mode of TRPV1 activation would cause an insufficient analgesic effect. It is likely that the important factor for the development of a hyperthermia-free TRPV1 antagonist could be independent of the inhibition of specific mode of TRPV1.
In addition to the high potency demonstrated in vitro, JTS-653 showed high potency in the in vivo studies. JTS-653 showed its maximal effects at 0.3 mg/kg (carrageenan-induced thermal hyperalgesia and hyperthermia). Previous TRPV1 antagonists showed their maximal effects at 0.3 mg/kg or higher (Honore et al., 2005; Gavva et al., 2007a; Maher et al., 2011). Thus our results demonstrated that JTS-653 is categorized in the most potent group of TRPV1 antagonists in vivo. Topical application of capsaicin was clinically used to reduce chronic pain, such as postherpetic neuralgia, because capsaicin blocks the function of TRPV1-sensitive sensory nerves and sensory nerve activation (Anand and Bley, 2011). Therefore, TRPV1 antagonists, including JTS-653, would be effective for TRPV1-mediated pain with systemic and topical application.
In summary, our findings demonstrated that JTS-653 is a highly potent TRPV1 antagonist in vitro and vivo. JTS-653 showed a sufficient efficacy on inflammatory pain and caused hyperthermia in rats. These profiles seem to be associated with polymodal inhibition of TRPV1 activation. Additional studies would be required for the clinical use of JTS-653.
Participated in research design: Kitagawa, Miyai, Usui, Hamada, Deai, Wada, Sakata, Hayashi, Tominaga, and Matsushita.
Conducted experiments: Miyai, Usui, Hamada, Deai, Wada, and Sakata.
Contributed new reagents or analytic tools: Koga.
Performed data analysis: Kitagawa, Miyai, Usui, Hamada, Deai, Wada, and Sakata.
Wrote or contributed to the writing of the manuscript: Kitagawa, Usui, and Sakata.
We thank Wataru Kondo, Shinji Yata, Yukihisa Wada, Norihisa Mera, and Akira Matsuo for helpful suggestions and support.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- transient receptor potential
- TRP vanilloid
- 2-aminoethyl diphenylborinate
- 4α-phorbol-12,13-didecanoate, RTX, resiniferatoxin
- human embryonic kidney
- relative light unit
- transient receptor potential melastatin 8
- transient receptor potential ankyrin 1
- 4-morpholineethanesulfonic acid
- paw withdrawal threshold
- paw withdrawal latency
- Received March 5, 2012.
- Accepted May 14, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics