JPET# Stereoselective, High-Affinity Antagonist is a Useful Radioligand For The Human

Transcription

1 JPET This Fast article Forward. has not been Published copyedited and on formatted. July 27, The 2007 final as version DOI: /jpet may differ from this version. JPET# [ 3 H]A [1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea]: A Novel, Stereoselective, High-Affinity Antagonist is a Useful Radioligand For The Human TRPV1 Receptor Bruce R. Bianchi, Rachid El Kouhen, Torben R. Neelands, Chih-Hung Lee, Arthur Gomtsyan, Shirish N. Raja, Sriajan N. Vaidyanathan, Bruce Surber, Heath A. McDonald, Carol S. Surowy, Connie R. Faltynek, Robert B. Moreland, Michael F. Jarvis, Pamela S. Puttfarcken Departments of Neuroscience Research (B.R.B., R.E.K, T.R.N., C.-H.L, A.G., H.A.M., C.S.S., C.R.F., M.F.J., R.B.M., P.S.P.) and Radiochemistry (S.N.R., S.N.V., B.S.), Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics.

2 JPET# Running title: [ 3 H]A , A Novel TRPV1 Receptor Radioligand Author for correspondence: Pamela S. Puttfarcken Abbott Laboratories, R4PM, AP9A/2, 100 Abbott Park Rd, Abbott Park, IL address: pamela.puttfarcken@abbott.com Tel. (847) ; FAX (847) Number of text pages: 38 Number of tables: 3 Number of figures: 7 Number of words in Abstract: 179 Number of words in Introduction: 566 Number of words in Discussion: 1156 Number of references: 38 Abbreviations: TRPV1, transient receptor potential vanilloid-1; CHO cells, Chinese hamster ovary cells; DRG, dorsal root ganglion; D-PBS, Dulbecco s phosphate-buffered saline; HBSS, Hank s balanced salt solution; DMEM, Dulbecco s modified Eagle s medium; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene glycol-bis(2-aminoethylether)- N,N,N N -tetraacetic acid; HEPES, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; MES, 2-morpholinoethanesulfonic acid; ATP Mg, adenosine 5 -triphosphate magnesium salt; NGF, nerve growth factor; A , 1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-

3 JPET# yl-urea; A , 1-((S)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea; A , 1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea; A , 1-[3- (trifluoromethyl)pyridin-2-yl]-n-[4-(trifluoromethylsulfonyl)phenyl]-1,2,3,6- tetrahydropyridine-4-carboxamide; SB , 1-(2-bromo-phenyl)-3-[2-(ethyl-m-tolylamino)-ethyl]-urea; AMG6880, (E)-3-(2-(piperidin-1-yl)-6-(trifluoromethyl)pyridin-3- yl)-n-(quinolin-7-yl)acrylamide; JNJ compound, 1-[6-fluoro-1-(3-trifluoromethylbenzyl)-1,2,3,4-tetrahydro-naphthalen-2-yl]-3-isoquinolin-5-yl-urea; CPZ, capsazepine; CAP, capsaicin; RTX, resiniferatoxin; I-RTX, iodo-resiniferatoxin; NADA, N- arachidonoyl-dopamine; TFA, trifluoroacetic acid; FLIPR, fluorometric imaging plate reader; n H, Hill slope.

4 JPET# Abstract 1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea (A ) is a novel, stereoselective, competitive antagonist that potently blocks transient receptor potential vanilloid-1 (TRPV1) receptor-mediated changes in intracellular calcium concentrations (pic 50 = 8.31 ± 0.13). The (S)-stereoisomer, A , is 6.8-fold less potent (pic 50 = 7.47 ± 0.07). A also potently blocks capsaicin (CAP) and acid activation of native rat TRPV1 receptors in dorsal root ganglion (DRG) neurons. A was tritiated ([ 3 H]A ; 29.3 Ci/mmol) and utilized to study recombinant human TRPV1 (htrpv1) receptors expressed in CHO cells. [ 3 H]A labeled a single class of binding sites in htrpv1-expressing CHO cell membranes with high affinity (K D = 3.4 nm; B max = 4.0 pmol/mg protein). Specific binding of 2 nm [ 3 H]A to htrpv1-expressing CHO cell membranes was reversible. The rank order potency of TRPV1 receptor antagonists to inhibit binding of 2 nm [ 3 H]A correlated well with their functional potencies in blocking TRPV1 receptor activation. The present data demonstrate that A blocks polymodal activation of the TRPV1 receptor by binding to a single high-affinity binding site, and that [ 3 H]A possesses favorable binding properties to facilitate further studies of htrpv1 receptor pharmacology.

5 JPET# Introduction The transient receptor potential vanilloid-1 (TRPV1) receptor is a nonselective cation channel, characterized by six transmembrane domains and intracellular N- and C- termini (Benham et al., 2002). The distribution of the TRPV1 receptor is widespread throughout the nervous system, showing highest levels of expression in sensory neurons of the dorsal root and trigeminal ganglia (Mezey et al., 2000; Sanchez et al., 2001). It is believed that nociceptive signalling of acute and chronic inflammatory pain is mediated in part by activation of the TRPV1 receptor (Honore et al., 2005). Both native and recombinant TRPV1 receptors are activated by diverse pain-producing stimuli, such as noxious heat (> 43 o C), protons (< ph 6), capsaicin (CAP) and resiniferatoxin (RTX) (Szallasi and Blumberg, 1999; Caterina and Julius, 2001). Furthermore, several naturally occurring lipids have been described to have agonist activity at the TRPV1 receptor, including the endocannabinoid anandamide (Smart et al., 2000) and N-acyl-dopamine derivatives, such as N-arachidonoyl-dopamine (NADA) (Huang et al., 2002). Small molecule antagonists of the TRPV1 receptor have been identified from diverse structural classes (Correll and Palani, 2006) and reported to be effective in preclinical animal models of pain and hyperalgesia. These include: 1-(2-bromo-phenyl)-3-[2- (ethyl-m-tolyl-amino)-ethyl]-urea (SB ) (Rami et al., 2004); (E)-3-(2-piperindin- 1-yl)-6-(trifluoromethyl)pyridin-3-yl)-N-(quinolin-7-yl)acrylamide (AMG6880) (Doherty et al., 2005; Gavva et al., 2005); 1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4- (trifluoromethylsulfonyl)phenyl]-1,2,3,6-tetrahydropyridine-4-carboxamide (A ) (Cui et al., 2006); 1-[6-fluoro-1-(3-trifluoromethyl-benzyl)-1,2,3,4-tetrahydronaphthalen-2-yl]-3-isoquinolin-5-yl-urea (JNJ compound) (Jetter et al., 2004), and 1-

6 JPET# isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea (A ) (El Kouhen et al., 2005 ) (Fig. 1). A has been shown to reduce nociception in rat models of post-operative pain and chronic inflammatory pain, and also showed partial efficacy in a neuropathic pain model (Honore et al., 2005). A also potently blocked other modes of TRPV1 activation including anandamide, NADA, acid and heat with equivalent efficacy (El Kouhen et al., 2005). The relative efficacy of small molecule TRPV1 receptor antagonists to block these different modes of TRPV1 receptor activation could be an important factor in determining their antinociceptive activity in vivo (Gavva et al., 2005). In vitro binding assays have been developed previously for the TRPV1 receptor using the potent agonist radioligand [ 3 H]-resiniferatoxin (RTX) (Szallasi and Blumberg, 1990; Szallasi et al., 1999) and also the antagonist radioligand [ 125 I]-iodo-resiniferatoxin (I- RTX) (Wahl et al., 2001). A potential problem with [ 3 H]RTX is that it is an agonist radioligand, and may bind to sites on the TRPV1 protein that represent one mode of activation of the TRPV1 receptor only. This was later resolved upon the development of [ 125 I]I-RTX (an antagonist radioligand sharing the same pharmacophore as [ 3 H]RTX). [ 125 I]I-RTX has been reported to potently block activation of the TRPV1 receptor by CAP, acid and heat in in vitro functional studies (Seabrook et al., 2002). However, both [ 3 H]RTX and [ 125 I]I-RTX exhibit a high degree of nonspecific binding, and additional treatment of membranes with the vanilloid binding agent α1-acid glycoprotein at 4 o C is required to reduce the nonspecific binding (Szallasi et al., 1992; Szallasi and Blumberg, 1993; Wahl et al., 2001). A (1-((R)-5-tert-butyl-indan-1-yl)-3-isoquinolin-5-yl-urea, Fig. 1) is one of the first chiral non-vanilloid TRPV1 antagonists reported (Gomtsyan et al., 2005). It is

7 JPET# a competitive antagonist of CAP at the recombinant htrpv1 receptor, that shows stereospecific activity in blocking TRPV1 receptor mediated changes in intracellular calcium concentrations. A tritiated form of A was synthesized with high specific activity ([ 3 H]A ; 29.3 Ci/mmol) and found to be a useful radioligand to study the recombinant htrpv1 receptor in a heterologous expression system. The present study describes the synthesis, pharmacology, and binding properties of [ 3 H]A

8 JPET# Materials and Methods CAP and capsazepine(cpz) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Olvanil (N-9-Z-octadecenoyl-vanillamide) and NADA were purchased from Tocris Cookson, Inc. (Ellisville, MO). RTX and tinyatoxin were purchased from LKT Laboratories, Inc. (St. Paul, MN). A , its enantiomer A , 1((S)-5-tertbutyl-indan-1-yl)-3-isoquinolin-5-yl-urea, A , A , SB , AMG6880 and the JNJ compound (Jetter et al., 2004) were synthesized in-house (Abbott, Abbott Park, IL)(Fig. 1). N-[4-[6-[(acetyloxy)methoxy]-2,7-difluoro-3-oxo-3H-xanthen-9-yl]-2- [2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]phenyl]- N-[2-[(acetyloxy)methoxy]-2-oxoethyl]-glycine, (acetyloxy)methyl ester (fluo-4 AM) was purchased from Texas Fluorescence Labs, Inc. (Austin, TX). F-12 nutrient mixture (Ham), Dulbecco s phosphate-buffered saline (D-PBS)(with Ca 2+, Mg 2+ and 1 mg/ml D- glucose)(ph 7.4), phosphate-buffered saline (PBS)(without Ca 2+, Mg 2+ ), Hank s balanced salt solution (HBSS)(without Ca 2+, Mg 2+ ), 0.25% trypsin-edta, penicillin/streptomycin and Lipofectamine Plus transfection reagent were purchased from Invitrogen Corp. (Grand Island, NY). Dulbecco s modified Eagle s medium (DMEM)(with 4.5 mg/ml D-glucose), L-glutamine, fetal bovine serum, bovine serum albumin (fraction V), collagenase/dispase (EC ). ph 7.4 Trizma pre-set crystals, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) and 2- morpholinoethanesulfonic acid (MES) were purchased from Sigma-Aldrich Co. (St. Louis, MO). Collagenase B and nerve growth factor (NGF) were purchased from Roche (Indianapolis, IN.), and geneticin (G418 sulfate) was purchased from Calbiochem-

9 JPET# Novabiochem Corp. (San Diego, CA). CHO cells were obtained from American Type Culture Collection (Manassas, VA). [ 3 H]A Synthesis and Purification A mixture of 6,8-diiodo-isoquinolin-5-ylamine (8.72 mg, mmol), 10% PD/C (7.12 mg), triethylamine (75 µl) and methanol (2 ml) was attached to a tritiation manifold and degassed by freeze-pump-thaw. Tritium gas (0.063 mmol, 3654 mci) was introduced, and then the mixture was vigorously stirred for 2.5 hr at o C. Excess of gas was removed to a charcoal trap cooled to 196 o C. The catalyst was filtered, and the labile tritium from the filtrate was removed by three evaporations of methanol to obtain 505 mci of crude [6,8-3 H]-isoquinolin-5-ylamine (1)(47% product formation with R f ~ 0.45)(Fig. 2). Crude 1 was purified by solid phase extraction using silica gel and 5% methanol in methylene chloride (> 98% radiochemically pure). To synthesize [ 3 H]A , a mixture of purified 1 (100 mci), 5-tert-butyl-1-isocyanato-indan (7.01 mg, mmol) and 10 µl of toluene was stirred for 24 hr at o C. Solvent was removed on a rotary evaporator to obtain crude product (54% product formation with R f ~ 0.3)(Fig. 2). The crude product was then applied to two preparative silica gel thin layer chromatography plates (1500 µ, 20 x 20 cm, 5% methanol in methylene chloride with 0.1% ammonium hydroxide), and bands corresponding to the product were scraped and extracted with 5% methanol in methylene chloride with 0.1% ammonium hydroxide (4 x 25 ml). Solvent was removed on a rotary evaporator to yield 36 mci of [ 3 H]A (> 90% radiochemically pure). Additional and final purification of [ 3 H]A was achieved with high performance liquid chromatography. The residue was dissolved in acetonitrile (1 ml) and water (1 ml) with 0.1% trifluoroacetic acid (TFA), and then a 400

10 JPET# µl sample was injected onto a Phenomenex Luna C18 column (5 µ, 4.6 x 250 mm). [ 3 H]A was eluted off at a flow rate of approximately 4 ml/min, increasing the gradient mobile phase B from 5% to 95% over a 20 min period (mobile phase A = 0.1% TFA/water and mobile phase B = 0.1% TFA/acetonitrile) and then held at 95% of B for 5 min. Peak elution was detected with an Agilent variable wavelength UV detector set at 215 nm and chemstation software (Agilent Labs, Palo Alto, CA). The fractions containing [ 3 H]A were collected at approximately 14.5 min. The above purification procedure was repeated four more times to process all of the [ 3 H]A , then the fractions were combined and solvents were evaporated under vacuum. The end product was dissolved in 5 ml ethanol (> 97% radiochemically pure). The specific activity was determined to be 29.3 Ci/mmol based on mass spectrometry by measuring the isotopic ratios compared with authentic A Cell Transfection and Culture Human TRPV1 was cloned from ileum as described by Witte et al. (2002). Sequence analysis showed that the cdna coded an amino acid sequence identical to AL The pcineo expression vector containing cdna for the wild type htrpv1 receptor was introduced into CHO cells using the Lipofectamine Plus transfection protocol (Invitrogen Corp., Grand Island, NY). Single colonies surviving selection by G418 sulfate were screened for functional expression of the htrpv1 receptor in response to CAP stimulation (100 nm) using the Ca 2+ flux assay. CHO cells were grown in F-12 nutrient mixture (Ham) containing 2 mm L-glutamine and 10% (v/v) fetal bovine serum and maintained in a 37 o C incubator under a humidified 5% CO 2 atmosphere. CHO cells

11 JPET# stably expressing the htrpv1 receptor were grown in the same medium supplemented with 300 µg/ml G418 sulfate. TRPV1 function remained stable over many cell passages. Ca 2+ Flux Assay Cellular flux of Ca 2+ was measured in htrpv1-expressing CHO cells using the fluorescent Ca 2+ chelating dye fluo-4 AM. Cells were grown as a monolayer in black 96- well tissue culture plates (with clear bottoms)(costar, Corning, NY). Prior to start of the assay, the growth medium was removed and cells were preincubated with 2 µm fluo-4 AM (in D-PBS, ph 7.4, containing Ca 2+, Mg 2+, and 1 mg/ml D-glucose) for 2 hr at 25 o C. To remove extracellular dye, cells were then washed five times with 200 µl of assay buffer (D-PBS, ph 7.4, containing Ca 2+, Mg 2+, and 1 mg/ml D-glucose) using a MultiWash Advantage multiplate washer (Model )(Tricontinent, Inc., Suffolk, UK). All compounds were dissolved in DMSO (10 mm). Test compound plates were prepared using a Biomek 2000 robotic workstation, programmed to change pipet tips following each dilution. Compounds, 50 µl, were added to the cells at a delivery rate of 50 µl/s. For determination of agonist activity, a single addition of the test compounds was made at the 10 s time point of the experimental run. For determination of antagonist activity, a second addition of the TRPV1 receptor agonist CAP (50 nm final concentration) was made 5 min after addition of the test compounds to challenge the TRPV1 receptor. Schild analyses of A were also double addition experiments where half-log concentration-effect curves of CAP were generated in the presence of five different concentrations of A (5, 20, 40, 320 and 1280 nm). Final assay volume was 200 µl. Length of the experimental run was 5 min for single addition experiments and 10 min for double addition experiments. Changes in fluorescence were recorded in a

12 JPET# fluorometric imaging plate reader (FLIPR)(Molecular Devices, Sunnyvale, CA)(λ EX = 488 nm, λ EM = 540 nm). The peak increase in fluorescence over baseline was calculated, and expressed as a percentage of the maximal or control response to CAP. A fourparameter logistic Hill equation was then used to curve-fit the concentration-effect data, and derive EC 50 and IC 50 values (GraphPad Software, Inc., San Diego, CA). DRG Neuronal Cultures All experiments were carried out in accordance with the guidelines and the approval of the Institutional Animal Care and Use Committee (IACUC). DRG cultures were prepared according to previous studies (El Kouhen et al., 2005), with minor modifications. Six to eight day-old Sprague-Dawley rats (Charles River Laboratories International, Inc., Wilmington, MA.) were deeply anesthetized with CO 2 and euthanized by decapitation. DRGs were rapidly removed and collected in HBSS. DRGs were transferred to a tube containing 0.1% collagenase/dispase and 0.1% collagenase B and allowed to incubate at 37 o C for one hour. Following the incubation, the tissue was centrifuged at 600 rpm for 5 min, the supernatant removed, and replaced with 0.25% trypsin-edta, and allowed to incubate at 37 o C for an additional 30 min. The tissue was centrifuged and dissociated by trituration in DMEM, with sequential use of a plastic Pasteur pipette. Undisrupted tissue fragments were allowed to settle and the supernatant was transferred to a new tube and centrifuged. The tissue pellet was resuspended in HBSS, triturated, layered over HBSS containing 2% fetal bovine serum and centrifuged at 600 rpm for 5 min. The resulting pellet was resuspended in DMEM containing 100 units/ml penicillin and 100 µg/ml streptomycin, 10% fetal bovine serum and 100 ng/ml

13 JPET# NGF. Cells were plated onto Biocoat poly-d-lysine coverslips (BD Biosciences, Bedford, MA). All experiments were conducted 24 to 48 hours following plating. Whole-Cell Patch Clamp Electrophysiology DRG neurons plated on poly-d-lysine-coated coverslips were maintained at room temperature in an extracellular recording solution (ph 7.4, 325 mosm) consisting of (in mm): 155 NaCl, 5 KCl, 2 CaCl 2, 1 MgCl 2, 10 HEPES, 12 glucose. For experiments involving application of acidic solution (ph 5.5), HEPES was replaced with MES in the external solution. Patch-pipettes composed of borosilicate glass (1B150F-3; World Precision Instruments, Inc, Sarasota, FL), were pulled and fire-polished using a DMZ- Universal micropipette puller (Zeitz-Instruments, Martinsried, Germany). Pipettes (2-6 MΩ) were filled with an internal solution (ph 7.3, 295 mosm) consisting of (in mm): K-aspartate, 20 KCl, 1 MgCl 2, 10 EGTA, 5 HEPES, 2 ATP Mg. Standard wholecell recording techniques were utilized for voltage-clamp studies using an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Coverslips plated with DRG neurons (20-48 hours after dissociation) were placed in a perfusion chamber and following establishment of whole-cell recording conditions bath perfusion (~2 ml/min) was initiated. Application of control bath solution through a MPRE8 multi-barrel application device with a common 360 µm polyimide tip (Cell Microcontrols, Norfolk, VA), positioned ~100 µm from the cell, was continued throughout the recording except during drug application. Each drug reservoir was connected to solenoid teflon valves that were controlled by a ValveLink16 system (AutoMate Scientific, San Francisco, CA). Drugs were applied using rapid valve switching of the ValveLink system controlled by the data acquisition software

14 JPET# pclamp9.0 (Axon instruments). Activators (1 µm CAP or ph 5.5) were applied for 5 seconds at 2-minute intervals to individual cells until subsequent responses produced responses with similar amplitudes. At this point, A was pre-applied for seconds prior to co-application with each activator of TRPV1. Peak amplitudes were measured, and expressed as a percentage of the control response to activator alone. A nonlinear regression sigmoidal function (GraphPad Prism Software, Inc., San Diego, CA) was then used to curve-fit the concentration-effect data and derive an IC 50 value (maximum values were constrained to not exceed 100% and minimal values were constrained not to go below 0%). Membrane Preparation htrpv1-expressing and untransfected (null) CHO cells were rinsed with ice-cold PBS and harvested from 150 cm 2 flasks by manual scraping. The cells were pelleted by lowspeed centrifugation (1000 x g for 10 min) at 4 o C, and then resuspended in ice-cold 10 mm HEPES buffer, ph 7.8, containing 0.32 M sucrose (1.5 ml per 150 cm 2 flask). The cells were homogenized in a glass-teflon vessel (motor-driven, 10 up-and-down strokes), and the homogenate was centrifuged at low-speed (1,000 x g for 10 min) at 4 o C. The resulting pellet (P1) was then re-homogenized (in ½ volume) and centrifuged again at low-speed. The supernatants from the two low-speed spins were pooled together and centrifuged at 20,000 x g for 60 min to pellet the membranes (P2). The resulting P2 membrane fraction was resuspended in ice-cold 50 mm Tris HCl buffer, ph 7.4 (0.25 ml per 150 cm 2 flask) by homogenization. Protein concentration was determined with a Bio- Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Volume was adjusted with

15 JPET# additional ice-cold 50 mm Tris HCl buffer, ph 7.4, to obtain 50 µg protein per 90 µl. Membranes were stored at 80 o C until use. Radioligand Binding Membranes were incubated with [ 3 H]A and test compounds in a final reaction volume of 200 µl in 12 x 75 mm polypropylene round-base tubes (Sarstedt, Inc., Newton, NC). Working concentrations (2.22X) of [ 3 H]A were prepared in 50 mm Tris HCl buffer, ph 7.4, containing 2.22 mg/ml BSA. All compounds were dissolved in DMSO (10 mm), and diluted to 10X final concentrations in distilled water. Assay incubations were initiated with the addition of membrane suspension (50 µg protein in 50 mm Tris HCl buffer, ph 7.4). Final concentration of BSA was 1 mg/ml. For both ligand-competition and saturation binding experiments, incubations were carried out for 60 min at 25 o C, and nonspecific binding was defined with 3 µm A Saturationbinding isotherms were generated at concentrations of [ 3 H]A between 0.05 and 20 nm. The final radioligand concentration was 2 nm in association, dissociation and ligand-competition binding experiments. To ascertain association kinetics, incubations were carried out at 25 o C, and terminated at different times (1, 2.5, 5, 7.5, 10, 20, 30, 45 and 60 min). To ascertain dissociation kinetics, equilibrium was established (60 min incubation at 25 o C), and then 3 µm A was added to inhibit binding over different times (1, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 min). All incubations were terminated by rapid filtration over Whatman glass fiber filter paper (GF/B)(fired) using a 48-well Brandel cell harvester (Model M-48)(Brandel, Inc., Gaithersburg, MD). Filters were washed three times with ice-cold 50 mm Tris HCl buffer, ph 7.4, and

16 JPET# transferred to scintillation vials (mini Poly-Q vials, Beckman Coulter, Fullerton, CA). Ecolume LSC (MP Biomedicals, Inc., Aurora, OH) was added to each vial, and bound tritium radioactivity (dpm) was counted in a Beckman LS6500 scintillation counter. Data analysis was accomplished using GraphPad Prism (GraphPad Software, Inc., San Diego, CA). The specific bound counts (dpm) from the saturation binding experiments were converted to pmol/mg protein, and then concentration-effect data was curve-fit to a one-site binding (hyperbola) equation to derive the dissociation constant of the radioligand (K D ) and number of binding sites (B max ). For ligand-competition binding experiments, the specific bound counts (dpm) were expressed as a percentage of the maximal binding observed in absence of test compound, and then the concentration-effect data was curve-fit to a four-parameter logistic Hill equation to derive the potency (IC 50 ) of the test compound. The equilibrium dissociation constant (K i ) of the test compound was calculated by the Cheng-Prusoff equation: K i = IC 50 /(1 + ([L]/K D ))(Cheng and Prusoff, 1973). For association binding experiments, the specific bound counts (dpm) were expressed as a percentage of the maximal binding observed at equilibrium, and then the time course was curve-fit to a one-phase exponential association equation to derive the observed on-rate (k ob ). For dissociation binding experiments, the specific bound counts (dpm) were expressed as a percentage of the maximal binding observed at equilibrium (before addition of 3 µm A ), and then the time course was curve-fit to a one-phase exponential decay equation to derive the off-rate (k off ). The on-rate (k on ) was calculated from the equation k on = (k ob - k off )/[L].

17 JPET# Results A is a Potent Antagonist at the Recombinant Human TRPV1 Receptor The potency of A , as well as a number of TRPV1 receptor agonists and antagonists was determined in htrpv1-expressing CHO cells using a FLIPR-based assay to measure changes in intracellular calcium concentrations (El Kouhen et al., 2005) (Table 1). All of the TRPV1 receptor agonists increased intracellular calcium concentrations in a concentration-dependent fashion. CAP was 50-fold more potent than NADA, a putative endogenous agonist (Huang et al., 2002), in activating TRPV1 receptors (pec 50 = 7.54 ± 0.11 and 5.83 ± 0.10, respectively). CAP did not evoke a Ca 2+ influx response in untransfected CHO cells (data not shown). A fully blocked the ability of 50 nm CAP to activate TRPV1 receptors (pic 50 = 8.31 ± 0.13)(Fig. 3A). A was approximately 6.8-fold more potent than its enantiomer A (pic 50 = 7.47 ± 0.07), and 58-fold more potent than the prototypical TRPV1 receptor antagonist CPZ (pic 50 = 6.55 ± 0.04) (Table 2). The nature of TRPV1 antagonism was determined by Schild analysis. The concentration-effect curve of CAP was shifted to the right in the presence of increasing concentrations of A (Fig. 3B), without affecting the maximum response, indicating that A is a competitive antagonist of CAP at the htrpv1 receptor. A Schild plot with linear regression of dose-ratios gave a straight line with a slope of 1.39 ± 0.20, and goodness of fit r 2 equal to 0.94 (Fig. 3C). A is a Potent Antagonist at the Native Rat TRPV1 Receptor Whole-cell patch-clamp recordings were made from rat DRG neurons voltageclamped at 60 mv. Application of 1 µm CAP, the EC 50 value for CAP at recombinant

18 JPET# TRPV1 using patch-clamp (Welch et al., 2000), to small to medium diameter DRG neurons (between µm) evoked an inward current in a majority (~70%) of the neurons tested. Pre-incubation of the cells with increasing concentrations of A ( nm) resulted in a concentration-dependent reduction in the peak CAP response (pic 50 = 8.92, n = 5-9)(Fig. 4A and 4B). Application of 10 nm A reduced the peak current by 89.9 ± 1.6 % (n = 9), and was completely reversible within 5-7 minutes following washout of the antagonist with control external solution (Fig. 4C and 4D). In addition, application of 10 nm A blocked 91.5 ± 4.0% (n = 6) of the peak current activated by application of an acidic solution (ph 5.5)(Fig. 4E and 4F). A is a Highly Selective TRPV1 Antagonist The selectivity of A was determined for more than 70 different neurotransmitter receptors and ion channels. A showed very weak or no affinity for a large selection of other cell surface receptors, ion channels, and enzymes (CEREP, Poiters, France; Supplementary Table). A also exhibited selectivity for TRPV1 compared to its activity at other TRP channels (TRPM8, IC 50 > 10 µm; TRPA1, IC 50 > 10 µm; TRPV3, IC 50 > 10 µm; and TRPV4, IC 50 > 10 µm). [ 3 H]A Labels the Recombinant htrpv1 Receptor with High Affinity Preliminary binding studies with 2 nm [ 3 H]A indicated a high percentage of nonspecific binding (~ 60%) relative to total binding in a crude membrane preparation of htrpv1-expressing CHO cells. Nonspecific binding was reduced significantly by (1) adding 1 mg/ml bovine serum albumin to the assay mixture and (2) replacing the crude membrane preparation with a P2 membrane fraction (50 µg protein per assay tube). Under these assay conditions, specific binding of 2 nm [ 3 H]A to the membranes

19 JPET# comprised about 90% of the total binding. Manipulation of other assay parameters including increasing the temperature to 37 o C, raising the ph to 7.8, or adding Ca 2+ /Mg 2+ to the assay mixture did not significantly increase the percentage of specifically bound radioligand (data not shown), so subsequent assay parameters were kept at 25 o C, ph 7.4, and Ca 2+ /Mg 2+ -free. Specific binding of 2 nm [ 3 H]A reached equilibrium within 30 min at 25 o C (Fig. 5A). Analysis of the association kinetics indicated that the t 1/2 for association was 4.94 ± 0.54 min (n = 3). Dissociation binding experiments revealed that the specific binding of [ 3 H]A was reversible and completely displaced by 3 µm A over a 60 min period at 25 o C (Fig. 5B). The t 1/2 for dissociation was 14.3 ± 2.5 min (n = 3). The k on and k off values were ± nm -1 min -1 and ± min -1, respectively (n = 3), yielding a K D value (k off /k on ) of 1.10 nm. Saturation binding experiments indicated that specific binding of [ 3 H]A was saturable over a 12- point concentration range between 0.05 and 20 nm when conducted at binding equilibrium (60 min at 25 o C)(Fig. 6A). An apparent K D value of 3.39 ± 0.34 nm (n = 3) was derived from a curve-fit of the concentration-effect data to a one-site binding model. The apparent B max was 4.02 ± 0.63 pmol/mg protein (n = 3). A Scatchard plot of the ratio of bound/free versus bound counts (expressed as pmol/mg protein) fit a straight line with a goodness of fit r 2 = (Fig. 6B), indicating that [ 3 H]A bound to a single class of binding sites. No specific [ 3 H]A binding was detected in untransfected CHO cell membranes (data not shown). Both TRPV1 receptor agonists and antagonists inhibited the specific binding of 2 nm [ 3 H]A in a concentration-dependent fashion (Fig. 7A and 7B; Table 3). The most

20 JPET# potent inhibitor was A (pk i = 8.13 ± 0.02; n H = 0.881). Its functionally weaker enantiomer A was approximately 15-fold less potent than A (pk i = 6.95 ± 0.08; n H = 1.08). The rank order potency of the TRPV1 receptor antagonists was A > AMG6880 > A JNJ Compound > A > A > SB > CPZ. The binding potencies of the TRPV1 receptor antagonists (expressed as pk i ) (Table 2) were positively correlated with their functional potencies in the Ca 2+ flux assay (expressed as pic 50 ). A linear regression curve-fit of the data (Fig. 7C) had a slope less than 1 (0.67 ± 0.14) and goodness of fit r 2 equal to 0.79, P < The rank order potency of the TRPV1 receptor agonists was RTX > tinyatoxin > olvanil > CAP > NADA. RTX inhibited binding of [ 3 H]A potently (pk i = 7.19 ± 0.23). The putative endovanilloid NADA was a weak inhibitor of binding. Poor aqueous solubility of NADA precluded testing at concentrations greater than 100 µm, so that only an estimate of its potency could be derived in the binding assay (pk i < 5.2). Nevertheless, these data agree with its low micromolar potency in the Ca 2+ flux assay (pec 50 = 5.83 ± 0.10)(Table 1). In contrast, CAP was also found to be a weak inhibitor of [ 3 H]A binding (pk i = 4.70 ± 0.33) (Table 3). Discussion TRPV1 is a ligand-gated, nonselective cation channel. The vanilloid, CPZ, was the first small molecule competitive antagonist to be reported (Walpole et al., 1994). However, CPZ exhibits weak potency at the rat (Szallasi et al., 1999) and human (El Kouhen et al., 2005) TRPV1 receptors, and is limited as a tool by its actions at other receptors, beside TRPV1 (Liu and Simon, 1997; Docherty et al., 1997). More recently, a number of structurally novel non-vanilloid TRPV1 antagonists have been described

21 JPET# (Gomtsyan et al., 2005, Rami et al., 2004, Doherty et al., 2005; Gavva et al., 2005, Jetter et al., 2004). A is one such compound that potently blocks TRPV1 receptor activation by capsaicin, acid, and heat (Gomtsyan et al., 2005; El Kouhen et al., 2005) and effectively reduces inflammatory pain in animal models (Honore et al., 2005). Further structure-activity studies of A led to the preparation of the (R) and (S) stereosiomers, A and A The (R) stereoisomer (A ) was 6.8-fold more potent than the (S) enantiomer in blocking TRPV1 receptor mediated changes in intracellular calcium concentrations. Furthermore, A demonstrates a high degree of specificity compared to its activity at other cell surface receptors and ion channels. Based on the properties described above, we developed a radioligand binding assay. For a radioligand to have widespread application, it should be competitive with a number of different structural types of receptor ligands. [ 3 H]A competed with two other related isoquinolinyl urea series, i.e., A (El Kouhen et al., 2005), a JNJ compound (Jetter et al., 2004), as well as a 2-bromophenyl urea (SB , Rami et al., 2004); an N-aryl cinnamide (AMG6880, Doherty et al., 2005; Gavva et al., 2005) and a highly lipophilic ligand from a series of tetrahydopyridines (A , Cui et al., 2006). All were found to completely inhibit binding at or near the highest concentrations tested and a linear correlation was observed between functional inhibition of the calcium flux assay, and competition binding across the different structural classes. Evaluation of a number of TRPV1 agonists and antagonists revealed that with the exception of CAP, agonists and antagonists of the TRPV1 receptor inhibited [ 3 H]A binding to htrpv1-expressing CHO cell membranes with potencies in general agreement with those observed in a Ca 2+ flux assay for the TRPV1 receptor. In contrast,

22 JPET# CAP showed significant potency differences in the radioligand binding (pki = 4.7 ± 0.33) and functional (pec 50 = 7.54 ± 0.11) assays. This observation is consistent with previous studies that have reported discrepancies between binding and functional ( 45 Ca uptake) potencies for CAP in both rat DRG neurons and htrpv1-expressing CHO and HEK293 cells (Szallasi et al., 1999; Acs et al., 1996). While the mechanistic basis for this discrepancy remains unclear, several possibilities may contribute to these findings. While A , A , and other TRPV1 antagonists (Gavva et al., 2005) are functionally competitive blockers of capsaicin s ability to activate TRPV1, these ligands may not share overlapping binding sites. This hypothesis is also supported by data showing that these antagonists can also fully block TRPV1 receptor activation by acid and heat (El Kouhen et al., 2005; Gavva et al., 2005; Neelands et al., 2005). It is also likely that cell preparation and type of assay may also account for differences between binding and functional data. In this regard, a previous study from our laboratory has demonstrated that the host cell expression system influences the pharmacology of TRPV1 (Bianchi et al., 2006). Additionally, the functional potency of CAP for activating TRPV1 is assay-dependent with high potency (pec 50 = 7.54 ± 0.11) in the Ca 2+ flux assay and much lower affinity in whole-cell patch-clamp studies (EC 50 = 640 nm) (Neelands et al, 2005). TRPV1 can be activated by vanilloids and bioactive lipids, protons, or heat (Ferrer- Montiel et al., 2004). Although protons and heat are thought to act at different sites in the protein, antagonists competitive with CAP can block all three modes of activation by an as yet undefined mechanism, presumably allosteric in nature (Bianchi et al., 2006, El Kouhen et al., 2005). This site has yet to be resolved by conventional means using either

23 JPET# a high affinity cross-linkable ligand and subsequent peptide mapping or the resolution of a TRPV1 x-ray crystallographic structure. Consequently, molecular biological studies using domain swaps and site directed mutagenesis comprise our current models. Using domain swaps between human and rat TRPV1, the region encompassed by the second through fourth transmembrane spanning domains were demonstrated to be key in capsaicin binding (Jordt and Julius, 2002). Mutagenesis studies have further defined the importance of tyrosine 511 and serine 512 in the third intracellular loop as well as isoleucine 514 and valine 518 in the third transmembrane spanning domain and methionine 547 in the fourth transmembrane spanning domain (Gavva et al., 2005, Johnson et al., 2006; Jordt and Julius, 2002; Phillips et al, 2004, Sutton et al., 2005). In this report, CAP was less potent in competition binding assays for tritiated A than olvanil. The structural difference between olvanil and CAP is an eighteen versus a ten carbon lipid tail. Consistent with a role for a lipid component, a recent report of ultrapotent capsaicinoid TRPV1 agonists shows that the most potent agonists had more lipophillic tails, for example, phenylacetylrinvanil (EC 50 = 90 pm, Appendino et al., 2005). Since A competes with capsaicin in functional calcium flux assays (Figure 3), it is possible that the actual binding site of the ligand is deeper in the transmembrane domains as suggested for RTX (Chou et al., 2004). Development of a useful, equilibrium binding assay for TRPV1 has been confounded by availability of potent and selective antagonists as well as the propensity for lipophillic toxins such as RTX and I-RTX to exhibit high nonspecific binding and nonsaturable binding properties (Szallasi et al., 1992; Wahl et al., 2001). Radiolabeled A offers several advantages over the use of either [ 3 H]RTX or [ 125 I]I-RTX. The binding

24 JPET# protocol is relatively simple, using a rapid filtration step, and is accompanied by lower nonspecific binding levels than previously described binding assays (Szallasi et al., 1992; Szallasi and Blumberg, 1993; Wahl et al., 2001). A represents one of the first in a new class of TRPV1 radioligands. While the competition with other TRPV1 antagonists and agonists was not examined, the 3- methylisoquinoline derivative of A was tritiated and used as a probe of the ligand binding site (compound A in Sutton et al., 2005; Johnson et al. 2006). This compound also binds potently to human TRPV1 (K d = 6.2nM) and was sensitive to the methionine 547 to leucine change near the extracellular side of fourth transmembrane spanning domain (Johnson et al., 2006). The present data demonstrate that A is a stereoselective high-affinity antagonist radioligand for the TRPV1 receptor. [ 3 H]A proved to be a useful radioligand to study the recombinant htrpv1 receptor in a heterologous expression system, possessing high affinity for the htrpv1 receptor with minimal nonspecific binding. These qualities may provide a unique tool to further investigate the biology of TRPV1.

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28 JPET# Honore P, Wismer CT, Mikusa J, Zhu CZ, Zhong C, Gauvin DM, Gomtsyan A, El Kouhen R, Lee CH, Marsh K, Sullivan JP, Faltynek CR and Jarvis MF (2005) A [1-isoquinolin-5-yl-3-(4-trifluoromethyl-benzyl)-urea], a novel transient receptor potential type V1 receptor antagonist, relieves pathophysiological pain associated with inflammation and tissue injury in rats. J Pharmacol Exp Ther 314: Huang SM, Bisogno T, Trevisani M, Al-Hayani A, De Petrocellis L, Fezza F, Tognetto M, Krey TF, Chu CJ, Miller JD, Davies SN, Walker JM, and Di Marzo V (2002) An endogenous capsaicin-like substance with high potency at recombinant and native vanilloid VR1 receptors. Proc. Natl. Acad. Sci. 99(12): Jetter MC, Youngman MA, McNally JJ, McDonnell M, Dax, SL, Codd EE, Colburn RW, Stone DJ, Zhang SP, Flores CM, Nasser N and Dubin A. β-amino ureas as novel VR1 antagonists: synthesis, in vitro and in vivo activity. Program No. MEDI 88. The 228 th ACS National Meeting, in Philadelphia, PA, August 22-25, Johnson DM, Garrett EM, Rutter R, Bonnert TP, Gao Y-D, Middleton RE, and Sutton KG (2006). Functional mapping of the transient receptor potential vanilloid 1 intracellular binding site. Mol. Pharmacol. 70: Jordt S-E and Julius D (2002) Molecular basis for species-specific sensitivity to hot chili pepers (2002) Cell 108:

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33 JPET# Legends for Figures Figure 1. Chemical structures of A , A , A , A , SB , AMG6880, JNJ Compound and CPZ. Figure 2. Synthesis of [ 3 H]A Figure 3. Antagonist profile of A in recombinant htrpv1-expressing CHO cells. A, inhibition of CAP (50 nm)-evoked Ca 2+ flux by A , A and CPZ. Data are expressed as a percentage of the control response to CAP, and are shown as means ± S.E.M. of 3 determinations. B, Concentration-effect curves of CAP alone and together with 5, 20, 40, 320 and 1280 nm A Data are expressed as a percentage of the maximal response to CAP (in absence of antagonist). C, Schild plot of A with linear regression of dose-ratios gave a straight line with a slope of 1.39 ± 0.20, and goodness of fit r 2 equal to All data are shown as means ± S.E.M. of 3 determinations. Figure 4. Antagonist profile of A in cultured rat DRG neurons. A, representative traces illustrating concentration-dependent inhibition by A (1 and 10 nm) of inward currents elicited by application of 1 µm CAP to small-diameter rat DRG neurons that were voltage-clamped at -60 mv. B, CAP-evoked currents were reduced in the presence of A in a concentration-dependent manner (pic 50 = 8.92; n = 5-9). C, representative trace showing that 10 nm A was completely

34 JPET# reversible. D, time course illustrating that peak current amplitudes return to control levels within 5-7 minutes following the application of 10 nm A (n = 3). E, representative traces of inward currents evoked by application of an acidic solution (ph 5.5), in the presence (10 nm) and absence (control) of A F, average peak current evoked by application of ph 5.5-solution alone ( pa, n = 6) and following inhibition by 10 nm A ( pa, n = 6) in rat DRG neurons. Figure 5. Binding kinetics of [ 3 H]A in membranes prepared from recombinant htrpv1-expressing CHO cells. A, representative association time course for 2 nm [ 3 H]A Nonspecific binding was defined with 3 µm A Specific bound counts (dpm) were calculated by subtracting nonspecific from total bound counts, and expressed as a percentage of the maximal specific bound counts (% bound) observed over 60 min at 25 o C. B, representative dissociation time course. Dissociation of [ 3 H]A was initiated by addition of 3 µm A , and carried out over a 60 min period at 25 o C. The binding observed at the 60 min time point was taken to represent nonspecific binding. Specific bound counts (dpm) were calculated by subtracting nonspecific from total bound counts, and expressed as a percentage of the maximal specific bound counts (% bound). Figure 6. Specific binding of [ 3 H]A in membranes prepared from recombinant htrpv1-expressing CHO cells was saturable. A, representative saturation binding curves. Total bound counts (dpm)(filled circles) and nonspecific bound counts (dpm)(open circles) are shown for 12 different radioligand concentrations (between 0.05