JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 JPET FastThis article Forward. has not beenPublished copyedited on and Augustformatted. 9,The 2007 final version as DOI:10.1124/jpet.107.127258 may differ from this version.
JPET # 127258
Pharmacology and antitussive efficacy of 4-(3-trifluoromethyl-pyridin-
2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-2-yl)- amide (JNJ17203212), a TRPV1 antagonist in guinea pigs
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Anindya Bhattacharya, Brian P. Scott, Nadia Nasser, Hong Ao, Michael P. Maher,
Adrienne E. Dubin, Devin M. Swanson, Nigel P. Shankley, Alan D. Wickenden, Sandra jpet.aspetjournals.org R. Chaplan
Department 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 and Johnson Pharmaceutical Research & Development LLC. at ASPET Journals on September 26, 2021 3210 Merryfield Row, San Diego, CA 92121
1
Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258
Running title: TRPV1 antagonism and cough
Corresponding author: Anindya Bhattacharya, Ph.D. Johnson and Johnson Pharmaceutical Research & Development 3210 Merryfield Row Downloaded from San Diego, CA 92121 [email protected] Phone: (858) 320-3429
Fax: (858) 450-2040 jpet.aspetjournals.org
Number of text pages: 36 at ASPET Journals on September 26, 2021 Number of tables: 2 Number of figures: 7 Number of references: 40 Number of Words in abstract: 240 Number of words in Introduction: 653 Number of words in Discussion: 1245
Nonstandard abbreviations: TRPV1 (transient receptor potential vanilloid 1); pKi (-log[Ki]); pKB (-log[KB]); pIC50 (-log[IC50]); pEC50 (-log[EC50]); FLIPR (fluorometric imaging plate reader); RTX (resiniferatoxin); CHO (chinese hamster ovary);
Section recommendation: Inflammation, Immunopharmacology and Asthma
2 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
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Abstract
TRPV1 plays an integral role in modulating the cough reflex and is an attractive antitussive drug target. The purpose of this study was to characterize a TRPV1 antagonist, JNJ17203212, against the guinea pig TRPV1 receptor in-vitro followed by a proof-of-principle study in an acid-induced model of cough. The affinity of JNJ17203212 for the recombinant guinea pig TRPV1 receptor was estimated by radioligand binding
and functionally characterized by antagonism of low pH and capsaicin-induced activation Downloaded from of the ion channel (FLIPR and electrophysiology). The nature of antagonism was further tested against the native channel in isolated guinea pig tracheal rings. Following jpet.aspetjournals.org pharmacokinetic characterization of JNJ17203212 in guinea pigs, pharmacodynamic and efficacy studies were undertaken to establish the antitussive efficacy of the TRPV1 antagonist. The pKi of JNJ17203212 for recombinant guinea pig TRPV1 was 7.14 ± 0.06. at ASPET Journals on September 26, 2021
JNJ17203212 inhibited both pH (pIC50 of 7.23 ± 0.05) and capsaicin (pIC50 of 6.32 ±
0.06)-induced channel activation. In whole-cell patch clamp the pIC50 for inhibition of guinea pig TRPV1 was 7.3 ± 0.01. JNJ17203212 demonstrated surmountable antagonism in isolated trachea with a pKB value of 6.2 ± 0.1. Intra-peritoneal administration of 20 mg/kg JNJ17203212 achieved a maximal plasma exposure of 8.0 ± 0.4 µM and attenuated capsaicin evoked coughs with similar efficacy to codeine (25 mg/kg). Lastly,
JNJ17203212 dose dependently produced antitussive efficacy in citric-acid induced experimental cough in guinea pigs. Our data provides preclinical support for developing
TRPV1 antagonists for the treatment of cough.
3 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
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Introduction
The transient receptor potential vanilloid, subtype I (TRPV1 or VR1) receptor is a ligand-gated cation channel that is activated or modulated by a variety of mediators believed to contribute to neuro-inflammation (Szallasi et al., 2007; Tominaga &
Tominaga, 2005). 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 (De Downloaded from
Petrocellis & Di Marzo, 2005; Ross, 2003), to mention a few. In addition, various endogenous mediators such as bradykinin, substance P, glutamate, prostaglandins and jpet.aspetjournals.org 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 neuro-inflammatory disorders (Jia et al., 2005;) and recent reviews on the at ASPET Journals on September 26, 2021 discovery of TRPV1 antagonists highlight efforts to develop novel therapeutics in this area (Correll & Palani, 2006; Kyle & 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 non-myelinated C- and myelinated
Aδ-fibres are drug targets for chronic cough, including TRPV1, acid-sensing ion channels and GPCRs 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 (Canning et al., 2006; Kollarik & Undem,
2006; Mazzone, 2004). TRPV1 is expressed on vagal afferents innervating the airway walls at the level of the guinea pig bronchi and trachea where it co-localizes with neuropeptides such as substance P and CGRP (Watanabe et al., 2006). In humans TRPV1
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JPET # 127258 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 Downloaded from activate TRPV1 receptors (Jia et al., 2002; Ricciardolo, 2001). Further validation for the therapeutic rationale of TRPV1 blockade in cough has been sought from pharmacological jpet.aspetjournals.org studies. Several studies have employed the TRPV1 antagonists, capsazepine and iodo-
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, were not effective at all at ASPET Journals on September 26, 2021
(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; Gill et al., 2004; Liu and Simon, 1997; McIntyre et al., 2001). Iodo-RTX may be partially de-iodinated 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 and colleagues (2006) reported that this compound was effective in a model of antigen-provoked cough in ovalbumin-sensitized guinea pigs. Nevertheless,
5 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258 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 4-
(3-trifluoromethylpyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-
2-yl)amide (JNJ17203212; Swanson et al., 2005) at the guinea pig TRPV1 homologue 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 Downloaded from the guinea pig TRPV1 receptor with in-vivo efficacy as an antitussive agent in a citric acid induced guinea-pig cough model. jpet.aspetjournals.org
at ASPET Journals on September 26, 2021
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Methods
Drugs: JNJ17203212, AMG517 and SB-705498 were synthesized in the laboratories of Johnson & Johnson Pharmaceutical Research & Development, LLC.
BCTC and AMG9810 were purchased from Biomol International and Tocris Biosciences, respectively. Codeine was purchased from Sigma-Aldrich.
Cell culture: Guinea pig TRPV1 cDNA (accession number AJ492922.2) was
generated by RT-PCR from guinea pig brain mRNA and stably expressed in Chinese Downloaded from
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 G418 for jpet.aspetjournals.org selection. Cells were sub-cultured at approximately 90% confluency from a T-150 cm2 flask using 0.05% trypsin.
Radioligand binding: Radioligand binding was carried out with membrane at ASPET Journals on September 26, 2021 fractions prepared from CHO cells expressing recombinant guinea pig TRPV1 receptors.
Briefly, the cells were spun down in a tabletop centrifuge (1500 rpm for 5 minutes at
4°C) and the cell pellet was homogenized with a homogenization buffer (50 mM Tris-
HCl, 5 mM EDTA; pH 7.4). The supernatant was then centrifuged at 32000 g for 30 minutes at 4°C to collect the membrane pellet. The membrane pellet was re-suspended in assay buffer (in mM: sucrose 320, MgCl2 2, NaCl 5.8, KCl 5.0, CaCl2 0.75 and HEPES
20; pH 7.4) at a final protein concentration of 100 µg/ml. [3H]-RTX (Perkin Elmer) was
3 3 used as the tracer for the study. Approximately 0.2 – 0.3 nM of [ H]-RTX (KD of [ H]-
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 hours by filtration (GF/B filters pre-soaked with 0.3% PEI) using a
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JPET # 127258 wash buffer with 50 mM Tris-HCl and 0.1% triton-X. The filter-bound radioactivity was counted by a beta scintillation counter (Perkin Elmer).
Fluorescence assays: Fluorescent assays were performed using FLIPRTM (MDS
Analytical Technologies). CHO cells expressing the guinea pig TRPV1 receptor were seeded in black-walled clear-bottom 96-well plates at a density of 50,000 cells per well
(complete media without the antibiotic) and cultured overnight at 37º C.
2+ TETRA Ca influx assay (FLIPR ): On the day of the experiment, cells were washed 3 times Downloaded from with HEPES buffered saline (in mM: NaCl 137, MgCl2 0.5, KCl 2, dextrose 5, CaCl2 2 and HEPES 10; pH 7.4). Fluo-3 AM (Molecular Devices, CA) was then added to the cells jpet.aspetjournals.org at a concentration of 4µM and incubated at room temperature in the dark for 60 minutes.
After incubation with dye, cells were washed and serial dilutions of test compounds were added. After a 30-minute incubation at room temperature, changes in fluorescence were at ASPET Journals on September 26, 2021 monitored for 3 minutes after the addition of agonist (1µM capsaicin).
Low pH assay (FLIPRTETRA): Cells were washed with assay buffer (in mM: NaCl 130,
KCl 2, CaCl2 2, MgCl2 1, glucose 5 and HEPES 10; pH 7.4) and loaded with 0.2 µM pH- sensitive fluorescent dye, BCECF-AM (Hellwig et al., 2004) for 30 minutes at 25°C in the dark, and washed again with assay buffer. Test compounds were then added to the cells and incubated for an additional 30 minutes 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) on-line and changes of intracellular fluorescence were monitored.
Electrophysiology: CHO cells stably expressing the guinea pig TRPV1 receptor were plated at low density onto glass coverslips 24 -72 hours prior to recording. On the
8 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258 day of the experiment, glass cover slips containing cells were placed in a bath on the stage of an inverted microscope and perfused (approximately 1 ml/minute) with
extracellular solution of the following composition (in mM): 137 NaCl, 2 CaCl2, 5.4 KCl,
1 MgCl2, 5 glucose, and 10 HEPES, pH 7.4. Pipettes were filled with an intracellular
solution of the following composition (in mM): 40 KCl, 100 KF, 2 MgCl2, 10 EGTA,
10 HEPES, pH 7.3 to 7.4, and had a resistance of 2 to 4 MΩ. All recordings were made at
room temperature (22-24°C) in a modified, nominally calcium-free recording solution in Downloaded from which calcium was replaced with Ba2+ (2 mM) using a Multiclamp 700A amplifier and pClamp 9 software (Axon Instruments). Inward TRPV1 currents were measured using the jpet.aspetjournals.org whole-cell configuration of the patch-clamp technique at a holding potential of –60 mV.
The liquid junction potential was calculated to be 7.1mV at 20°C and voltage commands
were not corrected. Current records were acquired at 5 KHz and filtered at 2 KHz. at ASPET Journals on September 26, 2021
Capsaicin concentration-response curves were constructed by exposing CHO-TRPV1 cells to capsaicin (0.03 µM – 10 µM) for 10s every 82s using a SF-77B Fast-Step
Perfusion device (Warner Instruments). 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 calculated for each capsaicin concentration.
To determine the IC50 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 applied to cells using a SF-77B Fast-Step Perfusion device (Warner Instruments).
Maximum final DMSO concentration was 0.01%. Up to 5 concentrations of
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JNJ17203212 (0.01 – 1 µM in half-log increments) were applied to each cell with washout in between each concentration.
Isolated trachea: Guinea pigs (Hartley, females) were obtained from Charles
River Laboratories (NY). All procedures and experiments were carried out in accordance with the internationally accepted guidelines for the care and the use of the laboratory animals in research and were approved by the local IACUC. Following euthanization, the trachea was quickly dissected and rings were hung in 10 ml organ baths containing Downloaded from
Krebs’ buffer (in mM: KCl 4.6; KH2PO4 1.2; MgSO4 1.2; NaCl 118.2; glucose 10;
NaHCO3 24.8; CaCl2-2H20 2.5), 37°C, pH 7.4 and gassed with 95% O2, 5% CO2. jpet.aspetjournals.org Isometric contraction was measured by a force transducer (AD Instruments, Colorado
Springs, CO) connected to an electronic data acquisition system (EMKA Technologies,
Falls Church, VA). Tissues were equilibrated with intermittent washes to maintain a at ASPET Journals on September 26, 2021 resting tension of ~ 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 hour. 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 5 and 8 for treatment groups.
Pharmacokinetics: Guinea pigs (Hartley, male) were obtained from Charles River
Laboratories surgically prepared with carotid artery and jugular vein catheters. Three guinea pigs (mean body weight of 411 ± 5 grams) had both cannulae fitted with injection ports under isoflurane anesthesia and, upon recovery, were then administered a single 20 mg/kg dose of JNJ17203212 in a 15% solutol in 5% dextrose solution by intraperitoneal
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(i.p) injection. Blood (approximately 0.25 ml) was collected from either catheter at 15,
30, 45, 60, 75, 90, 120, 135, or 240 minutes after drug administration. Plasma was prepared by centrifugation of blood and was stored at -20o C. Plasma concentrations of
JNJ17203212 were determined by LC-MS/MS.
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. Downloaded from
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 hour prior to the capsaicin jpet.aspetjournals.org challenge. Individual guinea pigs were placed in an exposure chamber with airflow of 3
L/min to acclimatize 10 minutes prior to the capsaicin or citric acid challenge. Cough responses were induced by exposure to capsaicin aerosol (15µM) or citric acid aerosol at ASPET Journals on September 26, 2021
(1.0 M) generated by an ultrasonic nebulizer at a nebulization rate of 0.6ml/min for 4 minute and 10 minute, respectively. Coughs were counted manually for a total of 15 minutes starting from the initiation of the irritant challenge (4-minute capsaicin challenge or 10-minute citric acid challenge). Animals were immediately removed from the exposure chamber and euthanized. Terminal blood samples were taken, spun down, and the plasma aliquoted and stored at –80oC prior to analytical chemistry. All blood samples were taken within one minute of the animals’ removal from the exposure chamber.
Plasma concentrations of JNJ17203212 were determined by LC-MS/MS.
Data and statistical analysis: In-vitro data points for concentration-response were fitted by non-linear regression (Graph Pad Prism, version 4.0) using the following four parameter general logistic equation:
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(logEC – Log Agonist) Hill slope Response = Basal + (Max – Basal) / [1 + 10 50 ]
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. pA2 estimates were determined at two antagonist concentrations (1 and 3 µM) of JNJ17203212 from the
FLIPR assay as each concentration of the compound produced a dextral shift with no Downloaded from 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 jpet.aspetjournals.org tissue assay).
For electrophysiology, linear leak was corrected off-line and percent inhibition was
calculated from leak corrected records according to: at ASPET Journals on September 26, 2021
% Inhibition = (1-(current, drug/ [(current, pre-drug + current, post-drug)/2])*100
For the cough studies in conscious guinea pigs, a one-way analysis of variance (ANOVA) 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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JPET # 127258
Results
We determined the affinity of JNJ17203212 for recombinant guinea pig TRPV1 receptors using a tritiated-resiniferatoxin (RTX) binding assay. In addition, since the pharmacology of guinea pig TRPV1 has not been characterized in detail, especially for antagonists (see Savidge et al., 2002; McLoed et al., 2006; Gunthorpe et al., 2007), we compared the affinity of JNJ17203212 with a number of recently described TRPV1
antagonists (Figs. 1 & 2 and Table 1), namely SB-705498 (Rami et al., 2006), AMG9810 Downloaded from
(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 jpet.aspetjournals.org 3 Figure 1. The equilibrium dissociation constant (KD) of [ H]-RTX for the guinea pig
TRPV1 was ~ 0.3 nM (inset; Figure 2). JNJ17203212 displaced tritiated-RTX from membrane preparations of CHO cells expressing the guinea pig TRPV1 ion channel (Fig. at ASPET Journals on September 26, 2021
2) with potency (pIC50) of 7.03 ± 0.07 and an affinity (pKi) of 7.14 ± 0.06. For comparison, Figure 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
13 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258 measured against the guinea pig receptor are very similar to those measured against the human homologue but tend to be higher (up to 10 fold) than those measured against the rat receptor.
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 non-
physiological conditions. Therefore, we next evaluated the ability of JNJ17203212 to Downloaded from 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 ± jpet.aspetjournals.org 0.03. As shown in Figure 3A, JNJ17203212 inhibited capsaicin (1µM)-induced increases in calcium fluorescence with a pIC50 of 6.32 ± 0.06. SB-705498 and AMG9810 were
equi-potent with JNJ17203212, as was observed in the radioligand-binding assay. The at ASPET Journals on September 26, 2021 pIC50 of SB-705498 and AMG9810 was 6.26 ± 0.08 and 6.49 ± 0.11, respectively (Fig.
3A). In order 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). Since TRPV1 is also modulated and activated by protons, low pH (4.5) was also used to activate recombinant guinea pig TRPV1 in a
14 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258 functional fluorescence assay. JNJ17203212 inhibited proton activation of guinea pig
TRPV1 receptor (Fig. 3C) with a pIC50 of 7.23 ± 0.05, again comparable to 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.
In order to confirm TRPV1 antagonistic activity using a more direct approach, we
studied the activity of JNJ17203212 against guinea pig TRPV1 using the whole-cell Downloaded from voltage-clamp technique (Fig. 4). In these studies, capsaicin concentration dependently increased TRPV1 channel activity. Representative traces showing capsaicin-induced jpet.aspetjournals.org 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 at ASPET Journals on September 26, 2021 close to maximal at 10 µM and the pEC50 of capsaicin was 6.1 ± 0.01. As shown in the inset to Figure 4B, 1uM 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. 4B). More importantly, this experiment demonstrated that JNJ17203212 is rapidly reversible as is shown in the inset. Taken together, our data demonstrates that JNJ17203212 is a competitive and reversible antagonist at the recombinant guinea pig TRPV1 receptor.
Next, we assessed the pharmacology of JNJ17203212 in a native guinea pig system to understand whether any differences exist between recombinant and native guinea pig TRPV1 prior to in-vivo experiments. We used isolated guinea pig trachea as a
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JPET # 127258 native test system for JNJ17203212 (Fig. 5). Capsaicin produced a concentration- dependent contraction of isolated guinea pig tracheal rings with a potency (pEC50) of 6.5
± 0.07. Increasing concentrations of JNJ17203212 produced a dextral shift of the capsaicin concentration response curve with no apparent suppression of the maximal tissue contraction, suggesting competitive antagonism, as was seen previously in the recombinant system. 1, 3 and 10 µM JNJ17203212 shifted the potency of capsaicin to 6.2
± 0.07, 5.9 ± 0.08 and 5.2 ± 0.07, respectively. When the agonist concentration ratios Downloaded from were plotted against the negative logarithm of JNJ17203212 (Schild analysis), the estimate of affinity for JNJ17203212 (pKB) as measured by the x-intercept was 6.2 ± 0.1 jpet.aspetjournals.org (inset), with a slope not significantly different from unity.
A prerequisite for in-vivo studies is good exposure following drug administration
via a suitable route. We therefore, measured plasma concentrations of JNJ17203212 (20 at ASPET Journals on September 26, 2021 mg/kg) in guinea pigs following administration by the intraperitoneal (i.p) route. Plasma exposures of JNJ17203212 increased rapidly after a single i.p dose, reaching a maximum concentration (Cp) of 8.0 ± 0.4 µM at 50 ± 10 min post-dose. Plasma concentrations were maintained above the lowest estimate of TRPV1 affinity (pKB = 6.2 in the isolated trachea assay) for the duration of the pharmacokinetic study (4 hours). Having demonstrated good systemic exposure we tested the pharmacodynamic efficacy of
JNJ17203212 in guinea pigs where the coughing reflex was initiated by aerosolized capsaicin with a local target concentration of 15 µM (Fig. 7A). The mean number of capsaicin-induced cough responses recorded in vehicle pre-treated guinea pigs was 3.0 ±
0.5. This level of response was reduced significantly to 1.0 ± 0.21 coughs in
JNJ17203212 (20 mg/kg; i.p) pre-treated guinea pigs (p <0.001). For comparison,
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JPET # 127258 codeine (25 mg/kg; i.p), included as a positive control antitussive agent, reduced the number of coughs to 0.58 ± 0.15 (p <0.001). These data indicate that JNJ17203212 is capable of reaching the site of capsaicin action (presumably sensory neurons innervating the upper airway) and exerting a TRPV1 antagonist effect in-vivo. This model then, represents an excellent pharmacodynamic assay for JNJ17203212, but is of questionable pathophysiological relevance. Therefore, we wanted to test the efficacy of the compound in a more disease relevant model of experimental cough. The citric acid-induced cough Downloaded from model was used for that purpose (see Methods) where 1 M citric acid aerosol produced
17.7 ± 8.9 (n = 6) coughs (Fig. 7B). JNJ17203212 (n = 6 per group) dosed 1 hour prior to jpet.aspetjournals.org the citric acid challenge reduced the number of coughs dose dependently and at a dose of
20mg/kg, the number of coughs was reduced to 4.5 ± 3.6 (n = 6). The pharmacodynamic
efficacy correlated well with the plasma exposure of JNJ17203212, and antitussive at ASPET Journals on September 26, 2021 efficacy was seen only at plasma concentrations greater than 1 µM (inset). The number of coughs in the codeine-treated animals was 7.8 ± 5.5 (n = 6). Statistical analysis revealed an overall effect of JNJ17203212 (p < 0.05; ANOVA), although post hoc analysis using the Tukey-Kramer multiple comparisons test indicated that no single dose of
JNJ17203212 exerted a significant reduction in coughing. Given the clear trend toward dose-dependent antitussive efficacy of JNJ17203212, we postulated that the inability to demonstrate statistical significance was a reflection of the inadequate power of the study, rather than proof of the null hypothesis. In order to increase the power of the study, we increased the number of animals in the vehicle, high dose JN17203212 (20mg/kg) and codeine groups to n = 12 per group. As illustrated in Fig. 7C, the antitussive effects of
JNJ17203212 (20 mg/kg) and codeine (25mg/kg) were statistically significant (p <0.01)
17 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258 in the powered study. The antitussive efficacy of JNJ17203212 was comparable to codeine, where citric acid-induce coughs were reduced from 25.2 ± 3.8 coughs in vehicle treated guinea pigs to 9.1 ± 2.2 and 10.1 ± 2.4 coughs in JNJ17203212 and codeine- treated animals, respectively.
Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021
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Discussion
In this study we thoroughly characterized JNJ17203212 at both the recombinant and native guinea pig TRPV1 receptor and provided proof-of-concept for the efficacy of
TRPV1 antagonists in experimental cough. JNJ17203212, along with four other previously described TRPV1 antagonists, competed with RTX for the binding site at the recombinant guinea pig TRPV1 receptor. Based on the binding data, it seems that the
pharmacology of the guinea pig TRPV1 channel is more predictive or closely related to Downloaded from the human homologue, as previously suggested by Savidge et al. (2002) on the basis of capsazepine activity. Likewise, the compounds tested seem to have a lower affinity at the jpet.aspetjournals.org rat compared to 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 at ASPET Journals on September 26, 2021 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 appears that the guinea pig may represent a good model species for the study of TRPV1 antagonists, as previously suggested by Walker at 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: 6.12 ± 0.05) and the native (tissue
19 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
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contraction: 6.2 ± 0.1) guinea pig TRPV1 receptor. Interestingly though, 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 Downloaded from potency of antagonism of JNJ17203212 in whole-cell electrophysiology was 7.3 ± 0.01,
2+ significantly different from inhibition of capsaicin-induced Ca mobilization (pIC50 of jpet.aspetjournals.org 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) at ASPET Journals on September 26, 2021 and Gunthorpe et al., (2007) recently published similar data for SB-705498. While 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
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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 modality (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 equi-effective against both capsaicin- and low-pH-induced activation of rat
and human TRPV1. The pharmacology of TRPV1 then appears to be both species and Downloaded from modality specific, pointing to the value of studying TRPV1 antagonists against different activation modalities and also in different animal species. jpet.aspetjournals.org 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, at ASPET Journals on September 26, 2021 codeine. Although it is possible that the in-vivo effects of JNJ17203212 may be mediated via a non-TRPV1 mechanism, we believe this is unlikely for the following reasons.
Firstly, in our hands, JNJ17203212 behaves as a highly selective TRPV1 antagonist. 1
µM JNJ17203212 did not significantly displace radioligands binding to a panel of receptors and transporters (CEREP; Table 2A), nor did it inhibit related TRP channels such as TRPV2, TRPV4 or TRPA1 (Table 2B). Although JNJ17203212 (1 µM) exhibited some weak TRPM8 inhibition, it is unlikely that TRPM8 occupancy by JNJ17203212 produced antitussive efficacy, as 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. These data also indicate that at the doses of JNJ17203212 used in the present study, the compound is
21 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258 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, tissue distribution etc. Thus, we believe that the Downloaded from 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 jpet.aspetjournals.org 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 at ASPET Journals on September 26, 2021
(Swanson et al., 2005). Hence, our data supports 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
22 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258 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 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 provides preclinical support for developing TRPV1 antagonists
for the treatment of cough and suggests that TRPV1 antagonists may hold promise as Downloaded from broad-spectrum therapeutics for the treatment of a variety of human disorders. jpet.aspetjournals.org at ASPET Journals on September 26, 2021
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Acknowledgements
The authors acknowledge Stefan Masure and Marc Merckens (Johnson and Johnson
PRD, Beerse) for providing the guinea pig TRPV1 cell line and Ruggero Galici (Johnson and Johnson PRD, San Diego) for helpful comments on statistical analyses. The guinea pig cough studies were performed by Pneumolabs (UK). Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021
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References
Ahern GP, Wang X and Miyares RL (2006) Polyamines are potent ligands for the
capsaicin receptor TRPV1. J Biol Chem 281:8991-8995.
Cannning BJ, Mori N and Mazzone SB (2006) Vagal afferent nerves regulating the cough
reflex. Respir Physiol Neurobiol 152:223-242.
Carr MJ, Kollarik M, Meeker SN and Undem BJ (2003) A role of TRPV1 in bradykinin-
induced excitation of vagal airway afferent nerve terminals. J Pharmacol Exp Ther Downloaded from
304:1275-1279.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD and Julius D (1997) jpet.aspetjournals.org The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature
389:816-824.
Cesare P, Moriondo A, Vellani V and McNaughton PA (1999) Ion channels gated by at ASPET Journals on September 26, 2021
heat. Proc Natl Acad Sci USA 96:7658-7663.
Correll CC and Palani A (2006) Advances in the development of TRPV1 antagonists.
Expert Opin Ther Patents 16:783-795.
De Petrocellis L and Di Marzo V (2005) Lipids as regulators of the activity of transient
receptor potential type V1 (TRPV1) channels. Life Sci 77:1651-1666.
Docherty RJ, Yeats JC and Piper AS (1997) Capsazepine block of voltage-activated
calcium channels in adult rat dorsal root ganglion neurones in culture. Br J
Pharmacol 121:1461-1467.
Gatti R, Andre E, Amadesi S, Dinh TQ, Fischer A, Bunnett NW, Harrison S, Geppetti P
and Trevisani M (2006) Protease-activated receptor 2 activation exaggerates TRPV1-
mediated cough in guinea pigs. J Appl Physiol 101:506-511.
25 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258
Gavva NR, Bannon AW, Immke DC, Tamir R, Klinosky L, Hever G, Wang J, Kuang R,
Wang W, Davis J, Zajic G, Arik L, Shi L, Bak A, Surapaneni S, Hovland D, Louis J,
Norman MH, Magal E and Treanor JS (2006) Preclinical pharmacology of AMG517:
a novel TRPV1 antagonist in the clinic. Society for Neuroscience, Atlanta, GA.
Gavva NR, Kionsky L, Qu Y, Shi L, Tamir R, Edenson S, Zhang TJ, Viswanadhan VN,
Toth A, Pearce LV, Vanderah TW, Porreca F, Blumberg PM, Lile J, Sun Y, Wild K,
Louis J-C and Treanor JS (2004) Molecular Determinants of Vanilloid Sensitivity in Downloaded from
TRPV1. J Biol Chem 279:20283-20295.
Gavva NR, Tamir R, Qu Y, Klionsky L, Zhang TJ, Immke D, Wang J, Zhu D, Vanderah jpet.aspetjournals.org TW, Porreca F, Doherty EM, Norman MH, Wild KD, Bannon AW, Louis J-C and
Treanor JS (2005) AMG 9810 [(E)-3-(4-t-butylphenyl)-N-(2,3-
dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide], a novel vanilloid receptor 1 (TRPV1) at ASPET Journals on September 26, 2021
antagonist with antihyperalgesic properties. J Pharmacol Exp Ther 313:474-484.
Geppetti P, Materazzi S and Nicoletti P (2006) The transient receptor potential vanilloid
1: role in airway inflammation and disease. Eur J Pharmacol 533:207-214.
Gill CH, Randall A, Bates SA, Hill K, Owen D, Larkman PM, Cairns W, Yusaf SP,
Murdock PR, Strijbos PJ, Powell AJ, Benham CD and Davies CH (2004)
Characterization of the human HCN1 channel and its inhibition by capsazepine. Br J
Pharmacol 143:411-421.
Groneberg DA, Niimi A, Dinh QT, Cosio B, Hew M, Fischer A and Chung KF (2004)
Increased expression of transient receptor potential vanilloid-1 in airway nerves of
chronic cough. Am J Respir Crit Care Med 170:1276-1280.
26 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258
Gunthorpe MJ, Hannan SL, Smart D, Jerman JC, Arpino S, Smith GD, Brough S, Wright
J, Egerton J, Lappin SC, Holland, VA, Winborn K, Thompson M, Rami HK, Randall
A and Davis JB (2007) Characterization of SB-705498, a potent and selective TRPV1
antagonist which inhibits capsaicin-, acid-, and heat-mediated activation of the
vanilloid receptor. J Pharmacol Exp Ther 321:1183-1192.
Hellwig N, Plant TD, Janson W, Schafer M, Schultz G and Schaefer M (2004) TRPV1
acts as a proton channel to induce acidification in nociceptive neurons. J Biol Chem Downloaded from
279:34553-34561.
Jia Y, McLeod RL and Hey JA (2005) TRPV1 receptor: a target for the treatment of pain, jpet.aspetjournals.org cough, airway disease and urinary incontinence. Drug News Perspect 18:165-171.
Jia Y, McLeod RL, Wang X, Parra LE, Egan RW and Hey JA (2002) Anandamide
induces cough in conscious guinea pigs through VR1 receptors. Br J Pharmacol at ASPET Journals on September 26, 2021
137:831-836.
Kollarik M, Ru F and Undem BJ (2007) Acid-sensitive vagal sensory pathways and
cough. Pulm Pharmacol Ther 20:402-411.
Kollarik M and Undem BJ (2006) Sensory transduction in cough-associated nerves.
Respir Physiol Neurobiol 152:243-254.
Kyle DJ and Tafesse L (2006) TRPV1 antagonists: a survey of the patent literature.
Expert Opin Ther Patents 16:977-996.
Lalloo UG, Fox AJ, Belvisi MG and Barnes PJ (1995) Capsazepine inhibits cough
induced by capsaicin and citric acid but not by hypertonic saline in guinea pigs. J
Appl Physiol 79:1082-1092.
27 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258
Lewis CA, Ambrose C, Banner K, Battram C, Butler K, Giddings J, Mok J, Nasra J,
Winny C and Poll C (2007) Animal models of cough: Literature review and
presentation of a novel cigaratte smoke-enhanced cough model in the guinea pig.
Pulm Pharmacol Ther 20:325-333.
Liu L and Simon SA (1997) Capsazepine, a vanilloid receptor antagonist, inhibits
nicotinic acetylcholine receptors in rat trigeminal ganglia. Neurosci Lett 228:29-32.
Mazzone SB (2004) Sensory regulation of the cough reflex. Pulm Pharmacol Ther Downloaded from
17:361-368.
McIntyre P, McLatchie LM, Chambers A, Phillips E, Clarke M, Savidge J, Toms C, jpet.aspetjournals.org Peacock M, Shah K, Winter J, Weerasakera N, Webb M, Rang HP, Bevan S and
James IF (2001) Pharmacological differences between the human and rat vanilloid
receptor 1 (VR1). Br J Pharmacol 132:1084-1094. at ASPET Journals on September 26, 2021
McLeod RL, Fernandez X, Correll CC, Phelps PT, Jia Y, Wang X and Hey JA (2006)
TRPV1 antagonists attenuate antigen-provoked cough in ovalbumin sensitized guinea
pigs. Cough doi:10.1186/1745-9974-2-10.
Rami HK, Thompson M, Stemp G, Fell S, Jerman JC, Stevens AJ, Smart D, Sargent B,
Sanderson D, Randall AD, Gunthorpe MJ and Davis JB (2006) Discovery of SB-
705498: a potent, selective and orally bioavailable TRPV1 antagonist suitable for
clinical development. Bioorg Med Chem Lett 16:3287-3291.
Ricciardolo FLM (2001) Mechanism of citric acid-induced bronchoconstriction. Am J
Med 111:18S-24S.
Ross RA (2003) Anandamide and vanilloid TRPV1 receptors. Br J Pharmacol 140:790-
801.
28 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258
Savidge J, Davis C, Shah K, Colley S, Phillips E, Ranasinghe S, Winter J, Kotsonis P,
Rang H and McIntyre P (2002) Cloning and functional characterization of the guinea
pig vanilloid receptor 1. Neuropharmacology 43:450-456.
Shimizu I, Iida T, Horiuchi N and Caterina MJ (2005) 5-Iodoresiniferatoxin evokes
hypothermia in mice and is a partial transient receptor potential vanilloid 1 agonist in
vitro. J Pharmacol Exp Ther 314:1378-1385.
Swanson DM, Dubin AE, Shah C, Nasser N, Chang L, Dax SL, Jetter M, Breitenbucher Downloaded from
JG, Liu C, Mazur C, Lord B, Gonzales L, Hoey K, Rizzolio M, Bongenstaetter M,
Codd EE, Lee DH, Zhang SP, Chaplan SR and Carruthers NI (2005) Identification jpet.aspetjournals.org and biological evaluation of 4-(3-trifluoromethylpyridin-2-yl)piperazine-1-carboxylic
acid (5-trifluoromethylpyridin-2-yl)amide, a high affinity TRPV1 (VR1) vanilloid
receptor antagonist. J Med Chem 48:1857-1872. at ASPET Journals on September 26, 2021
Szallasi A, Cortright DN, Blum CA and Eid SR (2007) The vanilloid receptor TRPV1: 10
years from channel cloning to antagonist proof-of-concept. Nat Rev Drug Discov
6:357-372.
Tominaga M and Tominaga T (2005) Structure and function of TRPV1. Pflugers Arch
451:143-150.
Trevisani M, Milan A, Gatti R, Zanasi A, Harrison S, Fontana G, Morice AH and
Geppetti P (2004) Antitussive activity of iodo-resiniferatoxin in guinea pigs. Thorax
59:769-772.
Valenzano KJ, Grant ER, Wu G, Hachicha M, Schmid L, Tafesse L, Sun Q, Rotshteyn Y,
Francis J, Limberis J, Malik S, Whittemore ER and Hodges D (2003) N-(4-
tertiarybutylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine -1(2H)-carbox-amide
29 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258
(BCTC), a novel, orally effective vanilloid receptor 1 antagonist with analgesic
properties: I. in vitro characterization and pharmacokinetic properties. J Pharmacol
Exp Ther 306:377-386.
Walker KM, Urban L, Medhurst SJ, Patel S, Panesar M, Fox AJ and McIntyre P (2003)
The VR1 Antagonist Capsazepine Reverses Mechanical Hyperalgesia in Models of
Inflammatory and Neuropathic Pain. J Pharmacol Exp Ther 304:56-62.
Watanabe N, Horie S, Michael GJ, Keir S, Spina D, Page CP and Priestley JV (2006) Downloaded from
Immunohistochemical co-localization of transient receptor potential vanilloid (TRPV)
1 and sensory neuropeptides in the guinea pig respiratory system. Neuroscience jpet.aspetjournals.org 141:1533-1543. at ASPET Journals on September 26, 2021
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Legends for Figures
Figure 1: Chemical structures of TRPV1 antagonists used in the study: JNJ17203212 (4-
(3-trifluoromethyl-pyridin-2-yl)-piperazine-1-carboxylic acid (5-trifluoromethyl-pyridin-
2-yl)-amide), 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), BCTC (4-(3-chloro-pyridin-2-yl)-piperazine-1-
carboxylic acid (4-tert-butyl-phenyl)-amide) and AMG517 (N-(4-(6-(4-trifluoromethyl- Downloaded from phenyl)-pyrimidin-4-yloxy)-benzothiazol-2-yl)-acetamide).
Figure 2: Concentration dependent displacement of [3H]-RTX binding to recombinant jpet.aspetjournals.org guinea pig TRPV1 by JNJ17203212, SB-705498 and AMG9810. Binding data are expressed as percentage of specific binding (determined by total binding minus binding in presence of 1 µM BCTC). Symbols represent mean ± standard error of the means from at ASPET Journals on September 26, 2021
3-4 independent experiments. Saturation binding of [3H]-RTX is shown as an inset to the figure.
Figure 3: (A) Concentration dependent attenuation of capsaicin (1 µM)-induced Ca2+ mobilization in recombinant guinea pig TRPV1-CHO cells. Data are expressed as percentage of the control response determined by capsaicin (1 µM) in the absence of
TRPV1 antagonists. (B) Dextral shift of the capsaicin concentration response curve based on the method of Arunlakshana and Schild. The concentration ratio was determined by dividing the EC50 of capsaicin in the presence of the JNJ17203212 with the EC50 in vehicle treated group. In this study, the capsaicin response is normalized to ionomycin (5
µM) added at the end of the run (3 minutes post capsaicin). (C) Concentration dependent attenuation of proton (pH 4.5) activation of TRPV1. Data is expressed as percentage of
31 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258 the control response determined by pH 4.5 in the absence of any TRPV1 antagonists.
Symbols represent mean ± standard error of the means from 3-4 independent experiments.
Figure 4: (A) Representative trace of capsaicin-induced inward Ba2+ current in guinea pig
TRPV1-CHO cells under whole-cell voltage clamp mode. Horizontal bar represents the time period of capsaicin application. (B) JNJ17203212 concentration dependently
inhibited 1 µM capsaicin-induced guinea pig TRPV1 activity. Symbols represent mean Downloaded from data from various independent experiments. A representative trace for such an experiment is shown as an inset. jpet.aspetjournals.org Figure 5: JNJ17203212 concentration dependently attenuated cumulative capsaicin- induced contraction of the isolated guinea pig trachea. Data is expressed as percentage of
KCl (70 mM)-induced contraction. Symbols represent mean contractility ± standard error at ASPET Journals on September 26, 2021 of means from 4-5 independent experiments. The Schild plot of log (CR-1) versus log of molar concentration of the antagonist is shown as an inset.
Figure 6: Plasma concentration (Cp) and time plot of JNJ17203212 dosed intra- peritonealy in guinea pigs. Symbols represent mean exposure from 3 independent animals.
Figure 7: (A) Pharmacodynamic effect of JNJ17203212 (20 mg/kg) and codeine (25 mg/kg) in capsaicin-induced experimental cough model in guinea pigs. Vertical bars represent mean number of coughs during the observation period. (B) Antitussive efficacy of JNJ17203212 in a citric acid-induced model of cough. The bars represent mean number of coughs from 6 animals. Plasma exposure at the end of the study (~ 75 minutes post-dose) is plotted against the mean number of experimental cough (inset). (C)
32 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
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Antitussive efficacy of JNJ17203212 (20 mg/kg) in a citric acid-induced model of cough.
The bars represent mean number of coughs from 12 animals. Asterisks indicate significant statistical difference (one * = p < 0.01; two * = p < 0.001) from vehicle treatment by the Tukey-Kramer post hoc analysis. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 26, 2021
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Table 1
Equilibrium binding affinities (pKi + standard error of means) of JNJ17203212
compared to TRPV1 antagonists; number of independent replicates shown in
parenthesis. Downloaded from
Rat TRPV1 Guinea pig TRPV1 Human TRPV1
JNJ17203212 6.5 ± 0.1 (n=5) 7.1 ± 0.06 (n=3) 7.3 ± 0.2 (n=3) jpet.aspetjournals.org SB-705498 6.1 ± 0.1 (n=3) 7.1 ± 0.04 (n=3) 6.9 ± 0.1 (n=3)
BCTC 8.5 ± 0.08 (n>11) 8.9 ± 0.08 (n=3) 8.7 ± 0.2 (n=3)
AMG9810 6.5 ± 0.04 (n=5) 7.6 ± 0.1 (n=3) 7.7 ± 0.1 (n=3) at ASPET Journals on September 26, 2021
AMG517 7.7 ± 0.03 (n=3) 9.1 ± 0.04 (n=3) 8.5 (n=3)
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Table 2
(A) Pharmacological selectivity of JNJ17203212 was ascertained at 1 µM using radioligand-binding assays at CEREP (France)
Target Species/tissue/cells Radioligand Percent displacement alpha 1 (non-selective) rat/cerebral cortex [3H] prazosin < 10 alpha 2 (non-selective) rat/cerebral cortex [3H] RX821002 < 10 adenosine 1 human/CHO [3H] DPCPX < 10 Downloaded from adenosine 2A human/HEK-293 [3H] CGS21680 < 10 adenosine 3 human/HEK-293 [125I] AB-MECA < 10 angiotensin 1 human/HEK-293 [125I][Sar1Ile8]-ATII < 10 benzodiazepine rat/cerebral cortex [3H] flunitrazepam < 10 jpet.aspetjournals.org beta 1 human/HEK-293 [3H](-)CGP12177 < 10 bradykinin 2 human/CHO [3H] bradykinin < 10 calcium channel, L-type rat/cerebral cortex [3H](-)D-888 < 10 cholecystokinin 1 human/CHO [125I] CCK-8s < 10 at ASPET Journals on September 26, 2021 chemokine 1 human/HEK-293 [125I] MIP-1α < 10 chloride channel rat/cerebral cortex [35S] BPS < 10 dopamine 1 human/CHO [3H] SCH23390 10 dopamine 2 human/HEK-293 [3H] spiperone < 10 dopamine transporter human/CHO [3H] BTCP < 10 delta opioid human/CHO [3H] DADLE < 10 endothelin A human/CHO [125I] endothelin-1 < 10 GABA rat/cerebral cortex [3H] GABA 19 galanin 2 human/CHO [125I] galanin < 10 histamine 1 guinea pig/cerebellum [3H] pyrilamine < 10 histamine 2 guinea pig/striatum [125I] APT < 10 serotonin 1A human/HEK-293 [3H] 8-OH-DPAT < 10 serotonin 1A rat/cerebral cortex [125I] CYP 11 serotonin 3 human/CHO [3H] BRL-43694 < 10 serotonin 5A human/CHO [3H] LSD < 10 serotonin 6 human/CHO [3H] LSD < 10 serotonin 7 human/CHO [3H] LSD < 10 potassium channel (V) rat/cerebral cortex [125I] α−dendrotoxin < 10
35 JPET Fast Forward. Published on August 9, 2007 as DOI: 10.1124/jpet.107.127258 This article has not been copyedited and formatted. The final version may differ from this version.
JPET # 127258
potassium channel (Ca) rat/cerebral cortex [125I] apamin < 10 kappa opioid rat/CHO [3H] U69593 14 muscarinic 1 human/CHO [3H] pirenzepine < 10 muscarinic 2 human/CHO [3H] AF-DX384 < 10 muscarinic 3 human/CHO [3H] 4-DAMP < 10 melanocortin 4 human/CHO [125I] NDP-α-MSH < 10 melatonin 1 human/CHO [125I] 2-iodomelatonin < 10 mu opioid human/HEK-293 [3H] diprenorphine < 10 sodium channel, site 2 rat/cerebral cortex [3H] batrachotoxin < 10 3 norepinephrine transporter human/CHO [ H] nisoxetine 12 Downloaded from neurokinin 2 human/CHO [125I] NKA < 10 neurokinin 3 human/CHO [3H] SR142801 < 10 neurotensin 1 human/CHO [125I] Tyr1-neurotensin < 10
somatostatin receptors mouse/AtT-20 [125I] Tyr11-somatostatin-14 < 10 jpet.aspetjournals.org vasopressin 1A human/CHO [3H] AVP < 10 VPAC1 human/CHO [125I] VIP < 10 neuropeptide Y1 human/SK-N-MC [125I] peptide YY < 10 125
neuropeptide Y2 human/KAN-TS [ I] peptide YY < 10 at ASPET Journals on September 26, 2021
(B) Pharmacological selectivity of JNJ17203212 was ascertained at 1 µM using functional assays.
TRP channels Species Agonist (conc) Percent inhibition TRPV2 human 2-APB (250 µM) < 10 TRPV4 human Hypo-osmolality (225 mOs) < 10 TRPA1 human AITC (12 µM) < 10 TRPM8 dog Icilin (600 nM) 33
2-APB: 2-aminoethoxydiphenyl borate; AITC: allylisothiocyanate
36 Figure 1
CF 3 O NN N
N HN CF3 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.
JNJ17203212 JPET FastForward.PublishedonAugust9,2007asDOI:10.1124/jpet.107.127258
F3C N O N N N H H Br SB-705498 O O
N O H
AMG9810
Cl O NN N HN
BCTC
NN
S O O N CF NH 3
AMG517
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at Figure 2 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonAugust9,2007asDOI:10.1124/jpet.107.127258
100 [3H]-RTX binding JNJ17203212 75 SB-705498 AMG9810 2500 50 2000 1500
cpm 1000 Specific binding
25 500 KD 0.03 ± 0.03
0 0.15 0.45 0.75 1.05 0 [3H-RTX] nM Specific binding (%) binding Specific -9 -8 -7 -6 -5
Log [Compound] M
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at A 100 capsaicin antagonism Figure 3 JNJ17203212 75 SB-705498 AMG9810
50 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.
25 JPET FastForward.PublishedonAugust9,2007asDOI:10.1124/jpet.107.127258 Capsaicin (%) 0 -9 -8 -7 -6 -5 Log [Compound] M 100 B capsaicin antagonism Vehicle 75 300 nM JNJ17203212 1 µM 50 3 µM
25 Ionomycin (%) Ionomycin 0 -8 -7 -6 -5 C Log [Capsaicin] M 100 low pH antagonism 75 50
pH 4.5 (%) 25 0 -9 -8 -7 -6 -5
Log [Compound] M
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JPET FastForward.PublishedonAugust9,2007asDOI:10.1124/jpet.107.127258
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at Figure 5 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonAugust9,2007asDOI:10.1124/jpet.107.127258
Vehicle 1 µM JNJ17203212 2.0 50 3 µM JNJ17203212 1.5 10 µM JNJ17203212 1.0
Log (CR-1) Log 0.5
0.0 25 -6.5 -6.0 -5.5 -5.0 -4.5 Log [JNJ17203212] M Contraction (%) 0 -9 -8 -7 -6 -5 -4
Log [Capsaicin] M
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10 8 6 4 Cp (µM) 2 0 0 60 120 180 240 300
Time (min)
Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on September 26, 2021 26, September on Journals ASPET at Figure 7 Citric acid-induced cough A 4 Capsaicin-induced cough B 30
3 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. 20 JPET FastForward.PublishedonAugust9,2007asDOI:10.1124/jpet.107.127258 2 JNJ17203212* * 1 Codeine 10 No. of Coughs of No. No. of Coughs of No. 0 e 0 icl ip) h ( (ip) e kg g .3 1 3 0 e V 0 1 20 in hicle de mg/ e JNJ17203212 (mg/kg; ip) o 0 5 mg/k V 2 2 C
Citric acid-induced cough C 30
20 ** ** Codeine JNJ-17203212 10 No. ofCoughs 0
le ) ip hic (ip) ( e g g V /k k g / m mg
5
20 2
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