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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Departments of Pain and Related Disorders (A.B., B.P.S., N.N., H.A., M.P.M., A.E.D., A.D.W., S.R.C.), Neuroscience (D.M.S.), and Physiological Systems (N.P.S.), Johnson & Johnson Pharmaceutical Research & Development, LLC, San Diego, California
Received June 18, 2007; accepted July 25, 2007.
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Abstract |
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; Szallasi et al., 2007). This ion channel is a polymodal nociceptor that is activated by vanilloids such as capsaicin (Caterina et al., 1997), noxious heat (Cesare et al., 1999), low pH, polyamines (Ahern et al., 2006), and lipid mediators such as anandamide (Ross, 2003; De Petrocellis and Di Marzo, 2005), to mention a few. In addition, various endogenous mediators such as bradykinin, substance P, glutamate, prostaglandins, and ATP sensitize TRPV1 (Szallasi et al., 2007). As an integrator and mediator of nociceptive and/or inflammatory stimuli, TRPV1 is an attractive therapeutic target for the treatment of various neuroinflammatory disorders (Jia et al., 2005), and recent reviews on the discovery of TRPV1 antagonists highlight efforts to develop novel therapeutics in this area (Correll and Palani, 2006; Kyle and Tafesse, 2006; Szallasi et al., 2007).
Chronic cough is a symptomatic manifestation of airway hyperactivity. Receptors present on sensory nerve endings and in cell bodies of nonmyelinated C- and myelinated A
-fibers are drug targets for chronic cough, including TRPV1, acid-sensing ion channels, and G protein-coupled receptors for bradykinin, neurokinin, and cannabinoids (Geppetti et al., 2006; Kollarik et al., 2007). TRPV1 plays a critical role in the sensory regulation and/or sensitization of the cough reflex in animals (Mazzone, 2004; Canning et al., 2006; Kollarik and Undem, 2006). TRPV1 is expressed on vagal afferents innervating the airway walls at the level of the guinea pig bronchi and trachea where it colocalizes with neuropeptides such as substance P and calcitonin gene-related peptide (Watanabe et al., 2006). In humans TRPV1 is up-regulated in patients with chronic cough (Groneberg et al., 2004), and capsaicin-induced cough responses are increased in patients with inflammatory lung diseases such as asthma, bronchitis, chronic obstructive pulmonary disease, and upper respiratory tract infection, probably as a result of TRPV1 sensitization. In animal models, TRPV1-mediated cough is sensitized by inflammatory pathways activated by protease-activated and bradykinin receptors (Carr et al., 2003; Gatti et al., 2006). In guinea pigs, experimental cough can be induced by citric acid, capsaicin, and anandamide, all of which activate TRPV1 receptors (Ricciardolo, 2001; Jia et al., 2002). Further validation for the therapeutic rationale of TRPV1 blockade in cough has been sought from pharmacological studies. Several studies have used the TRPV1 antagonists capsazepine and iodo-resiniferatoxin (RTX). Although these studies are generally supportive (Lalloo et al., 1995; Trevisani et al., 2004), these agents are not fully efficacious, and in one study, they were not effective at all (Lewis et al., 2007). Unfortunately, capsazepine and iodo-RTX are not ideal in vivo pharmacological tools. For example, capsazepine is a relatively weak antagonist, with species and modality-specific activity and limited TRPV1 selectivity (Docherty et al., 1997; Liu and Simon, 1997; McIntyre et al., 2001; Gill et al., 2004). Iodo-RTX may be partially deiodinated in vivo and/or may possess partial agonist activity (Shimizu et al., 2005). Thus, in vivo data generated with these agents should be interpreted with a degree of caution. Perhaps the strongest pharmacological evidence for a role of TRPV1 receptors in cough comes from a recent study with the "second generation" TRPV1 antagonist BCTC. McLeod et al. (2006) reported that this compound was effective in a model of antigen-provoked cough in ovalbumin-sensitized guinea pigs. Nevertheless, additional studies, using different compounds and different cough models are required to further strengthen the therapeutic rationale of TRPV1 antagonism in cough.
In the present study we characterized the in vitro and in vivo pharmacology of JNJ17203212 (Swanson et al., 2005) at the guinea pig TRPV1 homolog, with the ultimate goal of testing the efficacy of JNJ17203212 in a guinea model of cough. Our data clearly demonstrate that JNJ17203212 is a reversible competitive antagonist at the guinea pig TRPV1 receptor, with in vivo efficacy as an antitussive agent in a citric acid-induced guinea pig cough model.
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Materials and Methods |
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Cell Culture
Guinea pig TRPV1 cDNA (accession no. AJ492922
[GenBank]
.2) was generated by reverse transcription-polymerase chain reaction from guinea pig brain mRNA and stably expressed in Chinese hamster ovary (CHO) cells. Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 500 µg/ml of the antibiotic Geneticin (G-418; Invitrogen, Carlsbad, CA) for selection. Cells were subcultured at approximately 90% confluence from a T-150-cm2 flask using 0.05% trypsin.
Radioligand Binding
Radioligand binding was carried out with membrane fractions prepared from CHO cells expressing recombinant guinea pig TRPV1 receptors. In brief, the cells were spun down in a tabletop centrifuge (1500 rpm for 5 min at 4°C), and the cell pellet was homogenized with a homogenization buffer (50 mM Tris-HCl and 5 mM EDTA, pH 7.4). The supernatant was then centrifuged at 32,000g for 30 min at 4°C to collect the membrane pellet. The membrane pellet was resuspended in assay buffer (320 mM sucrose, 2 mM MgCl2, 5.8 mM NaCl, 5.0 mM KCl, 0.75 mM CaCl2, and 20 mM HEPES, pH 7.4) at a final protein concentration of 100 µg/ml. [3H]RTX (PerkinElmer Life and Analytical Sciences, Boston, MA) was used as the tracer for the study. Approximately 0.2 to 0.3 nM [3H]RTX (KD of [3H]RTX for the guinea pig TRPV1 is 
0.3 nM) was used as the tracer concentration, which was then displaced by increasing concentrations of the compounds tested. The incubation was terminated after 2 h by filtration [GF/B filters (Whatman, Maidstone, UK) presoaked with 0.3% polyethyleneimine] using a wash buffer with 50 mM Tris-HCl and 0.1% Triton-X. The filter-bound radioactivity was counted by a beta scintillation counter (PerkinElmer Life and Analytical Sciences).
Fluorescence Assays
Fluorescent assays were performed using FLIPR (MDS Analytical Technologies, Concord, ON, Canada). CHO cells expressing the guinea pig TRPV1 receptor were seeded in black-walled clear-bottomed 96-well plates at a density of 50,000 cells per well (complete media without the antibiotic), and they were cultured overnight at 37°C.
Ca2+ Influx Assay (FLIPRTETRA). On the day of the experiment, cells were washed three times with HEPES-buffered saline (137 mM NaCl, 0.5 mM MgCl2, 2 mM KCl, 5 mM dextrose, 2 mM CaCl2, and 10 mM HEPES, pH 7.4). Fluo-3 acetoxymethyl ester (Molecular Devices, Sunnyvale, CA) was then added to the cells at a concentration of 4 µM, and then cells were incubated at room temperature in the dark for 60 min. After incubation with dye, cells were washed, and serial dilutions of test compounds were added. After a 30-min incubation at room temperature, changes in fluorescence were monitored for 3 min after the addition of agonist (1 µM capsaicin).
Low-pH Assay (FLIPRTETRA). Cells were washed with assay buffer (130 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2,5mM glucose, and 10 mM HEPES, pH 7.4), and then they were loaded with 0.2 µM pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6')-carboxyfluorescein-acetoxymethyl ester (Hellwig et al., 2004) for 30 min at 25°C in the dark, and then they were washed again with assay buffer. Test compounds were then added to the cells and incubated for an additional 30 min at room temperature. Intracellular acidification was initiated by adding the low-pH buffer (assay buffer with 20 mM 2-[N-morpholino]ethanesulfonic acid, pH 4.5) online, and changes of intracellular fluorescence were monitored.
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. All recordings were made at room temperature (22–24°C) in a modified, nominally calcium-free recording solution in which calcium was replaced with 2 mM Ba2+ using a Multiclamp 700A amplifier and pClamp 9 software (Molecular Devices). Inward TRPV1 currents were measured using the whole-cell configuration of the patch-clamp technique at a holding potential of –60 mV. The liquid junction potential was calculated to be 7.1 mV at 20°C, and voltage commands were not corrected. Current records were acquired at 5 KHz and filtered at 2 KHz. Capsaicin concentration-response curves were constructed by exposing CHO-TRPV1 cells to capsaicin (0.03–10 µM) for 10 s every 82 s using an SF-77B Fast-Step Perfusion device (Warner Instruments, Hamden, CT). Complete concentration-response curves were constructed in both ascending and descending order in each cell. Linear leak was corrected off-line, and the average response was calculated for each capsaicin concentration. To determine the IC50 value for JNJ17203212, TRPV1 currents were activated using 1 µM capsaicin (approximately EC50 concentration). JNJ17203212 was diluted from a 10 mM DMSO stock solution into calcium-free extracellular solution containing 1 µM capsaicin, and it was applied to cells using an SF-77B Fast-Step Perfusion device (Warner Instruments). Maximum final DMSO concentration was 0.01%. Up to five concentrations of JNJ17203212 (0.01–1 µM in half-log increments) were applied to each cell, with washout in between each concentration.
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1 g. Tissues were then challenged with 70 mM KCl to assess tissue viability and for normalization of tissue contraction to test substances. Tissues were then returned to baseline tone by washing and then incubated with JNJ17203212 or vehicle (0.1% DMSO) for 1 h. A concentration-response curve was then generated by cumulative addition of capsaicin (from 10 nM to 10 µM) in half-log increments. Animal numbers varied between five and eight for treatment groups.
Pharmacokinetics
Male Hartley guinea pigs were obtained from Charles River Laboratories surgically prepared with carotid artery and jugular vein catheters. Three guinea pigs (mean body weight of 411 ± 5 g) had both cannulae fitted with injection ports under isoflurane anesthesia, and, upon recovery, they were administered a single 20 mg/kg dose of JNJ17203212 in a 15% Solutol in 5% dextrose solution by i.p. injection. Blood (approximately 0.25 ml) was collected from either catheter at 15, 30, 45, 60, 75, 90, 120, 135, or 240 min after drug administration. Plasma was prepared by centrifugation of blood, and it was stored at –20°C. Plasma concentrations of JNJ17203212 were determined by liquid chromatography-tandem mass spectrometry.
Experimental Cough in Guinea Pigs
Guinea pigs were randomly assigned to various test groups, and the experimenter was blinded to the treatments. The blinding code was not revealed to the experimenter until coughs from all animals had been tallied. Guinea pigs were dosed with JNJ17203212, codeine, or vehicle (15% Solutol in 5% dextrose solution; n = 6–12 per group) via the i.p. route 1 h before the capsaicin challenge. Individual guinea pigs were placed in an exposure chamber with airflow of 3 l/min to acclimatize 10 min before the capsaicin or citric acid challenge. Cough responses were induced by exposure to capsaicin aerosol (15 µM) or citric acid aerosol (1.0 M) generated by an ultrasonic nebulizer at a nebulization rate of 0.6 ml/min for 4 and 10 min, respectively. Coughs were counted manually for a total of 15 min starting from the initiation of the irritant challenge (4-min capsaicin challenge or 10-min citric acid challenge). Animals were immediately removed from the exposure chamber and euthanized. Terminal blood samples were taken, spun down, and the plasma was aliquoted and stored at –80°C before analytical chemistry. All blood samples were taken within 1 min of the animals' removal from the exposure chamber. Plasma concentrations of JNJ17203212 were determined by liquid chromatography-tandem mass spectrometry.
Data and Statistical Analysis
In vitro data points for concentration-response were fitted by nonlinear regression (GraphPad Prism, version 4.0; GraphPad Software Inc., San Diego, CA) using the following four-parameter general logistic equation: response = basal + (max – basal)/[1 + 10(logEC50 – log agonist)Hill slope].
Potency (pEC50 or pIC50) was estimated for the concentration that produced half-maximal effect. For Schild analysis, the shift in potency of the agonist in the presence of the antagonist was used to calculate the concentration ratio (CR) to determine pA2 or pKB estimates, according to the methods of Arunlakshana and Schild (1959). pA2 estimates were determined at two antagonist concentrations (1 and 3 µM) of JNJ17203212 from the FLIPR assay, because each concentration of the compound produced a dextral shift with no apparent suppression of the maxima. Schild estimates of pKB were obtained by plotting log (CR – 1) against log concentration of JNJ17203212 (for both the FLIPR and isolated tissue assay).
For electrophysiology, linear leak was corrected off-line, and percentage of inhibition was calculated from leak corrected records according to the following equation: % inhibition = (1 – (current, drug/[(current, predrug + current, postdrug)/2]) x 100.
For the cough studies in conscious guinea pigs, a one-way analysis of variance was assessed to determine whether the treatment groups (JNJ17203212 and codeine-treated) demonstrated statistical significance from vehicle treatment, followed by the Tukey-Kramer multiple comparisons post hoc test.
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; McLoed et al., 2006; Gunthorpe et al., 2007), we compared the affinity of JNJ17203212 with a number of recently described TRPV1 antagonists (Figs. 1 and 2; Table 1), namely, SB-705498 (Rami et al., 2006), AMG9810 (Gavva et al., 2005), BCTC (Valenzano et al., 2003), and AMG517 (Gavva et al., 2006). The chemical structures of the TRPV1 antagonists used in the present study are shown in Fig. 1. The equilibrium dissociation constant (KD) of [3H]RTX for the guinea pig TRPV1 was 0.3 nM (Fig. 2, inset). JNJ17203212 displaced tritiated-RTX from membrane preparations of CHO cells expressing the guinea pig TRPV1 ion channel (Fig. 2), with potency (pIC50) of 7.03 ± 0.07 and an affinity (pKi) of 7.14 ± 0.06. For comparison, Fig. 2 also shows displacement curves for SB-705498 and AMG9810. SB-705498 and AMG9810 exhibited similar potency to JNJ17203212, with pIC50 values of 6.95 ± 0.05 and 7.1 ± 0.10, respectively. In addition to these compounds, binding affinities for two other TRPV1 ligands, BCTC and AMG517, are shown in Table 1. The rank order of antagonist affinity for guinea pig TRPV1 was AMG517 > BCTC > AMG9810 > JNJ17203212 = SB-705498. These data show that JNJ17203212 displaces RTX binding from guinea pig TRPV1 receptor with modest affinity. Table 1 also compares the binding affinity for of these five TRPV1 antagonists with their respective binding affinity at recombinant rat and human TRPV1. In general, the rank order of antagonist affinity is similar across the three species. Interestingly, the affinities measured against the guinea pig receptor are very similar to those measured against the human homolog, but they tend to be higher (up to 10-fold) than those measured against the rat receptor.
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Although radioligand binding is a powerful technique for measuring receptor affinity under equilibrium conditions, it does not necessarily discriminate between agonists and antagonists, and it involves the study of TRPV1 channels under nonphysiological conditions. Therefore, we next evaluated the ability of JNJ17203212 to inhibit capsaicin-induced activation of guinea pig TRPV1 in a functional calcium mobilization assay (FLIPR). Capsaicin activated guinea pig TRPV1, with a pEC50 of 6.4 ± 0.03. As shown in Fig. 3A, JNJ17203212 inhibited 1 µM capsaicin-induced increases in calcium fluorescence, with a pIC50 of 6.32 ± 0.06. SB-705498 and AMG9810 were equipotent with JNJ17203212, as was observed in the radioligand-binding assay. The pIC50 of SB-705498 and AMG9810 was 6.26 ± 0.08 and 6.49 ± 0.11, respectively (Fig. 3A). To study the mechanism of antagonism more thoroughly for JNJ17203212, we used increasing concentrations of the compound to produce dextral shift of the capsaicin-induced calcium response. JNJ17203212 produced a concentration dependent dextral shift of the capsaicin concentration-response curve with no apparent suppression of the maxima, indicating competitive and surmountable antagonism (Fig. 3B). The pA2 estimate of JNJ17203212 at 1 and 3 µM was 6.24 ± 0.06 and 6.50 ± 0.07, respectively. In line with the pA2 estimates, a linear regression of the dextral shifts against each concentration of JNJ17203212 tested (Schild analysis) yielded a straight line, with a pKB of 6.12 ± 0.05 (data not shown). Because TRPV1 is also modulated and activated by protons, low pH (4.5) was used to activate recombinant guinea pig TRPV1 in a functional fluorescence assay. JNJ17203212 inhibited proton activation of guinea pig TRPV1 receptor (Fig. 3C), with a pIC50 of 7.23 ± 0.05, again comparable with AMG9810 (7.44 ± 0.05). Surprisingly, SB-705498 was less potent (pIC50 of 6.23 ± 0.05) than JNJ17203212 or AMG9810 in the low-pH assay, pointing to differences in pharmacology based on the modality of receptor activation.
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To confirm TRPV1 antagonistic activity using a more direct approach, we studied the activity of JNJ17203212 against guinea pig TRPV1 using the whole-cell voltage-clamp technique (Fig. 4). In these studies, capsaicin concentration-dependently increased TRPV1 channel activity. Representative traces showing capsaicin-induced guinea pig TRPV1 inward currents are shown in Fig. 4A. As shown in this figure, capsaicin induced a slow and sustained inward current that was rapidly reversed on removal of capsaicin. On average, the activation of recombinant guinea pig TRPV1 was close to maximal at 10 µM, and the pEC50 of capsaicin was 6.1 ± 0.01. As shown in Fig. 4B, 1 µM capsaicin induced a robust inward current that exhibited only limited desensitization over a period of several minutes. JNJ17203212 produced a potent, rapid, concentration-dependent attenuation of this capsaicin response. On average, the pIC50 for inhibition of the established capsaicin response was 7.3 ± 0.01 (Fig. 4C). More importantly, this experiment demonstrated that JNJ17203212 is rapidly reversible. Together, our data demonstrate that JNJ17203212 is a competitive and reversible antagonist at the recombinant guinea pig TRPV1 receptor.
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) on the basis of capsazepine activity. Likewise, the compounds tested seem to have a lower affinity at the rat compared with human and guinea pig TRPV1. Guinea pig and human TRPV1 contain a leucine residue at position 547 (rat TRPV1 contains a methionine) that is suggested to be important for inhibition of proton activation (Gavva et al., 2004). The similarity of guinea pig and human TRPV1 with respect to antagonist affinity might suggest that this leucine residue is also involved in high-affinity antagonist binding, although the contribution of other residues common to guinea pig and human TRPV1 cannot be discounted. Regardless of the molecular basis of these findings, it seems that the guinea pig may represent a good model species for the study of TRPV1 antagonists, as suggested previously by Walker et al. (2003).
In line with the RTX competition data, JNJ17203212 was also a competitive antagonist of capsaicin-induced activation of both the recombinant (intracellular Ca2+ mobilization and electrophysiology) and native (tissue contraction) TRPV1 receptor. The functional estimate of affinity (pKB) for JNJ17203212 was strikingly similar when measured at the recombinant (Ca2+ response of 6.12 ± 0.05) and the native (tissue contraction of 6.2 ± 0.1) guinea pig TRPV1 receptor. Interestingly, functional pKB estimates were significantly different from the measured binding affinity (pKi). It is possible that RTX (for binding) and capsaicin (for function) may present the guinea pig TRPV1 in a high- and a low-affinity state for JNJ17203212, which may then indicate that the binding site of JNJ17203212 does not completely overlap with that of capsaicin and RTX. In this context, it will be very interesting to perform a functional affinity estimate for JNJ17203212 using RTX as the stimulus. Another apparent paradox was that the potency of antagonism of JNJ17203212 in whole-cell electrophysiology was 7.3 ± 0.01, significantly different from inhibition of capsaicin-induced Ca2+ mobilization (pIC50 of 6.32 ± 0.06). It is possible that JNJ17203212 displays a higher affinity against the "open-state" of the channel or displays some degree of voltage dependence, as was observed in a preliminary set of experiments with JNJ17203212 (A. D. Wickenden, unpublished data), and Gunthorpe et al. (2007) recently published similar data for SB-705498. Although the exact reasons for these apparent discrepancies are not clearly understood, it is likely that experimental differences in potency/affinity estimates can be accounted for by small differences in the assay conditions (e.g., method of channel activation, intact cells or membranes, extent of equilibration).
The TRPV1 receptor is endogenously gated by low pH, and protons are potentially involved in the activation of TRPV1 at the site of tissue injury, inflammation, and ischemia. Furthermore, our aim was to test the efficacy of JNJ17203212 in an acid-induced experimental cough model. Therefore, we measured the antagonist potency of JNJ17203212 using low pH as the stimulus for TRPV1 activation. Our results show that JNJ17203212 was a potent (pIC50 of 7.23 ± 0.05) inhibitor of proton-induced guinea pig TRPV1 activation, as was AMG9810. Surprisingly, SB-705498 exhibited a very different pharmacology against low pH. In fact, between the three assays (binding, capsaicin, and low pH), SB-705498 exhibited weak antagonism in only the low pH assay (pIC50 of 6.23 ± 0.05), whereas AMG9810 behaved quite similarly to JNJ17203212 in all three types of assays. Interestingly, Gunthorpe et al. (2007) recently published data suggesting SB-705498 is equieffective against both capsaicin- and low-pH-induced activation of rat and human TRPV1. The pharmacology of TRPV1 thus seems to be both species- and modality-specific, pointing to the value of studying TRPV1 antagonists against different activation modalities and also in different animal species.
The TRPV1 antagonist JNJ17203212 demonstrated antitussive efficacy in the citric acid-sensitized model of experimental cough in guinea pigs. Importantly, the maximal efficacy in this model was similar to that seen with the antitussive agent codeine. Although it is possible that the in vivo effects of JNJ17203212 may be mediated via a non-TRPV1 mechanism, we think this is unlikely for the following reasons. First, in our hands, JNJ17203212 behaves as a highly selective TRPV1 antagonist. At 1 µM, JNJ17203212 did not significantly displace radioligands binding to a panel of receptors and transporters (CEREP, Paris, France; Table 2), nor did it inhibit related TRP channels such as TRPV2, TRPV4, or TRPA1 (Table 3). Although 1 µM JNJ17203212 exhibited some weak TRPM8 inhibition, it is unlikely that TRPM8 occupancy by JNJ17203212 produced antitussive efficacy, because TRPM8 is not activated by capsaicin and acid, both of which induced cough in guinea pigs. Therefore, it is scientifically reasonable to infer that the antitussive efficacy of JNJ17203212 is due to TRPV1 blockade. Our data also indicate that at the doses of JNJ17203212 used in the present study, the compound is capable of reaching the site of capsaicin action (presumably sensory neurons innervating the upper airway) and exerting a TRPV1 antagonist effect in vivo. Finally, the plasma concentrations required for effect in our study were consistent with functional affinity estimates (pKB) measured against the native guinea pig TRPV1 receptor (6.2 ± 0.1). Indeed, this level of agreement between in vitro and in vivo estimates of potency is very respectable given the numerous confounding factors that can influence in vivo potency, such as plasma protein binding and tissue distribution. Thus, we think that the antitussive effects of JNJ17203212 seen in the present study are a consequence of TRPV1 antagonism. It is also worth mentioning that there were no obvious behavioral abnormalities at the highest dose (20 mg/kg) of JNJ17203212, both in the pharmacokinetic and in the efficacy studies. We did not measure body temperature in any of the guinea pig in vivo studies, although JNJ17203212 caused hyperthermia in rats (Swanson et al., 2005). Hence, our data support and extend previous findings on the antitussive effects of TRPV1 antagonists. Capsazepine and iodo-resiniferatoxin, both TRPV1 antagonists, have previously been shown to exhibit significant antitussive efficacy in the guinea pig citric acid model (Lalloo et al., 1995; Trevisani et al., 2004), although recently, Lewis et al. (2007) reported otherwise, albeit in a modified (smoking) model of citric acid-induced cough. In addition, the TRPV1 antagonist BCTC was efficacious in both the capsaicin pharmacodynamic model and in an ovalbumin-sensitized model (McLeod et al., 2006). These previously published data, together with our new data, provide preclinical support for developing TRPV1 antagonists for the treatment of cough associated with upper respiratory tract hyperactivity. Whether therapeutic intervention of TRPV1 results in an improved quality of life in patients suffering from chronic idiopathic cough remains to be tested in the clinic. In conclusion, JNJ17203212 is a competitive reversible antagonist of both recombinant and native guinea pig TRPV1. JNJ17203212 is bioavailable in guinea pigs by the intraperitoneal route, and it demonstrates antitussive efficacy in a citric acid-induced guinea pig cough model. To date, much emphasis has been placed on the development of TRPV1 antagonists for the treatment of pain. However, our data provide preclinical support for developing TRPV1 antagonists for the treatment of cough, and they suggest that TRPV1 antagonists may hold promise as broad-spectrum therapeutics for the treatment of a variety of human disorders.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: TRPV1, transient receptor potential vanilloid 1; RTX, resiniferatoxin; JNJ17203212, 4-(3-trifluoromethyl-pyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-2-yl)-amide; BCTC, 4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide; AMG517, N-(4-(6-(4-trifluoromethyl-phenyl)-pyrimidin-4-yloxy)-benzothiazol-2-yl)-acetamide; SB-705498, 1-(2-bromo-phenyl)-3-[1-(5-trifluoromethyl-pyridin-2-yl)-pyrrolidin-3-yl]-urea; AMG9810, 3-(4-tert-butyl-phenyl)-N-(2,3-dihydro-benzo[1,4]dioxin-6-yl)-acrylamide; CHO, Chinese hamster ovary; FLIPR, fluorometric imaging plate reader; DMSO, dimethyl sulfoxide; CR, concentration ratio; TRP, transient receptor potential; HEK, human embryonic kidney; RX821002, 2-(2-methoxy-1,4-benzodioxan-2yl)-2-imidazoline; CGS 21680, 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxami-doadenosine; CGP-12177, 4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one; SCH23390, R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; U-69593, (+)-(5
,7,8
)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide.
Address correspondence to: Dr. Anindya Bhattacharya, Johnson & Johnson Pharmaceutical Research and Development, LLC, 3210 Merryfield Row, San Diego, CA 92121. E-mail: abhatta2{at}prdus.jnj.com
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