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Research ArticleDrug Discovery and Translational Medicine

DFL23448, A Novel Transient Receptor Potential Melastin 8–Selective Ion Channel Antagonist, Modifies Bladder Function and Reduces Bladder Overactivity in Awake Rats

Francesco A. Mistretta, Andrea Russo, Fabio Castiglione, Arianna Bettiga, Giorgia Colciago, Francesco Montorsi, Laura Brandolini, Andrea Aramini, Gianluca Bianchini, Marcello Allegretti, Silvia Bovolenta, Roberto Russo, Fabio Benigni and Petter Hedlund
Journal of Pharmacology and Experimental Therapeutics January 2016, 356 (1) 200-211; DOI: https://doi.org/10.1124/jpet.115.228684

Abstract

The transient receptor potential melastin 8 ion channel (TRPM8) is implicated in bladder sensing but limited information on TRPM8 antagonists in bladder overactivity is available. This study characterizes a new TRPM8-selective antagonist (DFL23448 [5-(2-ethyl-2H-tetrazol-5-yl)-2-(3-fluorophenyl)-1,3-thiazol-4-ol]) and evaluates it in cold-induced behavioral tests and tests on bladder function and experimental bladder overactivity in vivo in rats. DFL23448 displayed IC50 values of 10 and 21 nM in hTRPM8 human embryonic kidney 293 cells activated by Cooling Agent 10 or cold, but it had limited activity (IC50 > 10 μM) at transient receptor potential vanilloids TRPV1, TRPA1, or TRPV4 or at various G protein–coupled receptors. In rats, DFL23448 administered intravenously or orally had a half-life of 37 minutes or 4.9 hours, respectively. DLF23448 (10 mg/kg i.v.) reduced icilin-induced “wet dog–like” shakes in rats. Intravesical DFL23448 (10 mg/l), but not vehicle, increased micturition intervals, micturition volume, and bladder capacity. During bladder overactivity by intravesical prostaglandin E2 (PGE2), vehicle controls exhibited reductions in micturition intervals, micturition volumes, and bladder capacity by 37%–39%, whereas the same parameters only decreased by 12%–15% (P < 0.05–0.01 versus vehicle) in DFL23448-treated rats. In vehicle-treated rats, but not in DFL23448-treated rats, intravesical PGE2 increased bladder pressures. Intravenous DFL23448 at 10 mg/kg, but not 1 mg/kg DFL23448 or vehicle, increased micturition intervals, micturition volumes, and bladder capacity. During bladder overactivity by intravesical PGE2, micturition intervals, micturition volumes, and bladder capacity decreased in vehicle– and 1 mg/kg DFL23448–treated rats, but not in 10 mg/kg DFL23448–treated rats. Bladder pressures increased less in rats treated with DFL23448 10 mg/kg than in vehicle– or 1 mg/kg DFL23448–treated rats. DFL23448 (10 mg/kg i.v.), but not vehicle, prevented cold stress–induced bladder overactivity. Our results support a role for bladder TRPM8-mediated signals in experimental bladder overactivity.

Introduction

The introduction of urgency as a symptom of lower urinary tract dysfunction has impelled translational research to advance understanding of sensory functions of micturition pathways (Abrams et al., 2002; Birder and Wyndaele, 2013). Although the mechanisms of bladder sensation are not fully characterized, improved knowledge of its neurophysiology and pathophysiology has substantiated sensory pathways as central components of micturition and bladder disorders, and various afferent transmitter systems or receptors have been identified as putative targets for future drugs (Birder et al., 2010; Drake et al., 2010; Gonzalez et al., 2014). The transient receptor potential ion channel (TRP) superfamily is widely expressed in mammals and consists of six subfamilies of proteins, which may be considered as polymodal cell sensors that are involved in various biologic processes and diseases (Flockerzi and Nilius, 2014). Some TRPs that are expressed in the lower urinary tract have been correlated with sensory bladder function and dysfunction and are being explored as drug targets in bladder disease (Franken et al., 2014). A subset of TRP channels may be classified as thermosensors, including transient receptor potential vanilloid 1 (TRPV1; heat), transient receptor potential melastatin 8 (TRPM8; cold), and transient receptor potential ankyrin 1 (TRPA1; moderate cold); however, these TRP channels also respond to other noxious stimuli as well as various exogenous or endogenous agents (Andersson et al., 2010; Franken et al., 2014). TRPV1 is the channel best studied in the lower urinary tract, but the mechanism of action of TRPV1-active compounds is not completely established and their usefulness in lower urinary tract disorders is debated (Andersson et al., 2010; Franken et al., 2014). Previous studies in which TRPM8, together with TRPA1, was isolated from human prostate tissue suggest that both receptors may be involved in bladder responses to cold (Tsavaler et al., 2001; Franken et al., 2014). TRPM8 and TRPA1 are both expressed in mucosal cells and afferent nerves of the bladder and urethra, and both receptors have been implicated in bladder overactivity in experimental models or lower urinary tract symptoms in humans; however, their respective role in cold-induced bladder responses have not been clarified (Andersson et al., 2010; Franken et al., 2014). The bladder can react to thermal cold by at least two principally different neurophysiological doctrines: 1) by local activation of thermal receptors within its sphere, such as during the controversial ice-water test, which elicits a visceral sensorimotor reflex (the bladder-cooling reflex) (Hellström et al., 1991; Drake et al., 2010; Franken et al., 2014); or 2) by activation of somatosensory pathways, which is suggested to depend on segmental dichotomizing afferent axons of the skin and viscera (Shibata et al., 2011). However, the latter mechanism does not explain the occurrence of detrusor instability in response to everyday nonsegmental stimuli (e.g., hand-washing or cold weather urgency, or bladder contractions induced by cold exposure of cranial skin in rodents) (Choe et al., 1999; Ghei and Malone-Lee, 2005; Uvin et al., 2015). Less well understood processes or hypotheses such as plasticity in bladder sensory pathways (Chai et al., 1998), central nervous modulation of homeostatic afferent networks (Drake et al., 2010), or convergence of central nervous organization of somatosensory and central micturition pathways (Hedlund, 2015) have been suggested to construe acute cold-induced urgency (Uvin et al., 2015).

Using TRPM8 or TRPA1 knockout mice, Uvin et al. (2015) recently identified TRPM8 as the sole sensor that is responsible for skin-cooling bladder contractions. Previously, increased bladder expression of TRPM8 in patients with painful bladder syndrome and idiopathic detrusor overactivity was correlated with frequency and pain (Mukerji et al., 2006). Furthermore, the bladder-cooling reflex has been related to bladder outlet obstruction in non-neurogenic patients with lower urinary tract symptoms (Hirayama et al., 2005). In agreement, rats with bladder outlet obstruction exhibited more pronounced urodynamic responses to intravesical menthol than sham-operated controls (Hayashi et al., 2011). The bladder expression of TRPM8 in bladder outlet obstruction was higher than in controls, suggesting that neuronal afferent input through TRPM8-mediated signals is augmented in experimental bladder outlet obstruction (Jun et al., 2012).

Effects of TRPM8 inhibition on bladder contractions and skin-cooling bladder responses in rats have hinted this pharmacological principle as a new putative therapeutic approach for overactive bladder syndrome or urgency (Lashinger et al., 2008; Lei et al., 2013; Uvin et al., 2015). However, information on the effect of TRPM8 antagonists in other bladder overactivity models is lacking. This study aimed to characterize DFL23448 [5-(2-ethyl-2H-tetrazol-5-yl)-2-(3-fluorophenyl)-1,3-thiazol-4-ol], a new TRPM8-selective antagonist, and to evaluate it in tests for cold-induced responses and tests on bladder function and experimental bladder overactivity induced by intravesical prostaglandin E2 (PGE2) in rats.

Materials and Methods

Chemicals

DFL23448 (Fig. 1) was synthesized as described in patent number WO20130922711(A1).

Fig. 1.
Fig. 1.

Structure of DFL23448.

In Vitro TRPM8 Assay

TRPM8 antagonist activity by DFL23448 was determined by measuring changes in intracellular calcium levels using a Ca2+-sensitive fluorescent dye. The experiments were performed using human embryonic 293 (HEK-293) cells stably expressing human TRPM8. Cells were seeded 10,000 cells/well in 384-well plates coated with poly(d-lysine) (Matrix black/clear-bottom plates; Thermo Scientific, Waltham, MA) in complete medium and grown overnight at 37°C, 5% CO2. Twenty-four hours after seeding, cell culture medium was removed and cells were washed with Tyrode’s assay buffer and then loaded with the fluorescent Ca2+ indicator Fluo-4 NW dye (Molecular Probes, Life Technologies, Paisley, UK) supplemented with water-soluble probenecid (Molecular Probes). Dye-loaded cell plates were incubated for 1 hour at room temperature. DFL233448, N-(3-aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)-benzamide hydrochloride (1:1) hyclate (AMTB), or vehicle was added and the kinetic response was monitored by a fluorometric imaging plate (FLIPRTETRA; Molecular Devices, Sunnyvale, CA) over a period of 5 minutes (300 seconds). Five minutes later, an injection of the reference agonist Cooling Agent 10 at EC80 concentration was performed. The signal of the emitted fluorescence was recorded for an additional 3 minutes. Data were analyzed using Spotfire DecisionSite software (version 9.1.1; TIBCO Software Inc., Palo Alto, CA). The bioactivity exerted by DFL23448, AMTB, or vehicle was expressed as percent inhibition and IC50 values were then calculated. The percentage scale is defined by 100% inhibition, in which the relative fluorescence units of the test were identical to the MIN controls in the second injection (capsazepine at IC100, 50 µM), and 0% inhibition, in which the relative fluorescence units of the test were identical to the MAX controls in the second injection (Cooling Agent 10 at EC80, 20–30 µM), as described in the following section.

In Vitro Cold Stimulation Assay

The temperature activation cell-based assay described by Aneiros and Dabrowski (2009) was modified and used to assess the activity by DFL23448 to inhibit cold-induced TRPM8 stimulation. HEK-293 cells stably expressing human TRPM8 were seeded (1.5–1.8 × 106) in a T75 flask in complete medium. Three to four days after seeding (approximately 80% confluent cells), the medium was removed and cells were loaded with a solution of Screen Quest Fluo-8 NW dye (ABD Bioquest, Sunnyvale, CA) in the dark. Dye-loaded cell flasks were incubated for 45 minutes at room temperature in the dark, and the Fluo-8 NW solution was then removed and cells were seeded in 96-well assay plates (MicroAmp Optical 96-Well Reaction Plates; Applied Biosystems, Life Technologies, Carlsbad, CA) at 100,000 cells/well (20 µl/well). DFL23448 was added and incubated at room temperature for 5 minutes. The signal was recorded for 2 minutes at 25°C, and then the temperature was lowered to subphysiologic levels and the signal was recorded for 3 minutes by the ABI Prism 7900HT Sequence Detection System (Life Technologies). The fluorescence difference (ΔF = fluorescence525nm at 14°C – fluorescence525nm at 25°C) was assessed. The analysis was performed computing ΔF/F0, where F0 was the fluorescent signal at the starting temperature (25°C). ΔF/F0 was normalized versus maximum signals [buffer plus 0.5% dimethylsulfoxide (DMSO)] and inhibitor controls (minimum signals, IC100 of 4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (BCTC) to obtain percent activity according to the following formula: percent activity = −100 × (X – MAX/MIN − MAX), where X is the measured value of a well, MIN is the median minimum signal control, and MAX is the median maximum signal control. IC50 curves were generated by fitting the fluorescence data with a sigmoidal curve equation using GraphPad Prism software (version 5, GraphPad Software Inc., La Jolla, CA). All data point determinations were performed in duplicates and are presented as mean and standard error of the mean.

In Vitro TRP Selectivity Assay

TRPA1-, TRPV1-, and TRPV4-expressing HEK-293 cells were analyzed for response to various compounds using a Ca2+ mobilization-dependent fluorescence signal in 384 MTP format (see above). Cells were seeded at 10,000 cells per well in 384 MTP (Matrix black/clear-bottom plates) in complete medium (25 μl/well). Twenty-four hours after seeding, the medium was removed and cells were loaded with 20 μl/well of the Fluo-8 NW dye solution. The dye-loaded cell plates were incubated for 1 hour at room temperature. DFL23448 was added to the wells of an assay plate in a 1.5% DMSO solution. Then a Tyrode's buffer (10 μl) was added to the wells (final DMSO concentration of 0.5%) by the FLIPRTETRA plate. The kinetic response was monitored by the instrument over a period of 3 minutes (200 seconds). A Hereafter, reference agonists (capsaicin, GSK1016790A [(N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide], and isothiocyanate) in assay buffer (10 μl) were added to the wells by the FLIPRTETRA plate. The signal of the emitted fluorescence was recorded for an additional 3 minutes.

Radioligand Binding Assay for Selectivity versus G Protein–Coupled Receptors

Evaluation of the affinity of DFL23448 for human adrenoceptors (α1A, α2A, β1, β2), histamine receptors (H1, H2, M2), muscarinic receptors (M2, M3), neurokinin-1 receptor, opioid receptors (δ, κ, µ), opioid receptor like-1, serotonin 5-HT1A receptor, cannabinoid CB2 receptor, dopamine receptors (D2, D3), and bradykinin BK1 receptor was determined by radioligand binding assays. Cell membrane homogenates (48 µg protein) were incubated for 60 minutes at 22°C with the respective reference compound in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 2 mM MgCl2, and 1 mM EDTA. After incubation, the samples were filtered rapidly under a vacuum through glass fiber filters (GF/B; Packard Instruments, Meriden, CT) presoaked with 0.3% polyethylenimine PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter; Packard Instruments). The filters were dried and they were then counted for radioactivity in a scintillation counter (Topcount; Packard Instruments) using a scintillation cocktail (Microscint 0; Packard Instruments). Results are expressed as the percent inhibition of the control radioligand-specific binding. The standard reference compounds were tested in each experiment at several concentrations to obtain a competition curve, from which the IC50 value was calculated.

Animals and Ethical Approval

A total of 40 male and 29 female Sprague-Dawley rats (200–230 g; Charles River Laboratories, Calco, Italy) were used for this study. The animals were caged individually under clean conditions at the animal facilities of the IRCCS Ospedale San Raffaele Urological Research Institute (Milan, Italy) or the University of Naples Department of Experimental Pharmacology (Naples, Italy). Rats had free access to pellets and water during a 12-hour/12-hour dark/light cycle (light from 6 AM to 6 PM). All of the environmental conditions, as well as all procedures adopted throughout the study for housing and handling of the animals, were in strict compliance with European Economic Community regulations and Italian Guidelines for Laboratory Animal Welfare. Animals were randomly distributed into the experimental groups (Quickcalcs; GraphPad Software Inc., La Jolla, CA). All animals were euthanized by CO2 asphyxiation; in some experiments, urinary bladders or dorsal root ganglia were extracted for real-time quantitative polymerase chain reaction (PCR). The protocols were approved by the Animal Ethics Committee of IRCCS Ospedale San Raffaele and the University of Naples.

Pharmacokinetic Studies

The pharmacokinetics of DFL23448 dissolved in 50% PEG400 (Sigma-Aldrich, Milan, Italy) and 50% 1× phosphate-buffered saline (PBS; pH 7.4) was investigated after single intravenous (3 mg/kg) and oral (10 mg/kg) administrations in male rats. DFL23448 was given as a single dose in three animals per group by intravenous (3 mg/kg; bolus) and oral (10 mg/kg by gavage) administrations to evaluate the pharmacokinetic parameters. In other experiments (three animals per group), DFL23448 (1 or 10 mg/kg) was also given intravenously to evaluate pharmacokinetic parameters.

Icilin-Induced “Wet Dog” Shaking in Rats

Icilin, a TRPM8 agonist, was used to induce shaking in male rats. Animals were first habituated to the testing room for 30 minutes, and they were then pretreated with intravenous vehicle or DFL23448 (10 mg/kg); 40 minutes later, they were injected with icilin (1 mg/kg i.p.). The number of wet dog–like shakes (WDSs) of the neck, head, and trunk in each animal was recorded for a period of 30 minutes after icilin administration.

Micturition and Bladder Overactivity Studies

Surgical Procedures.

Female rats were anesthetized with ketamine (10 mg/kg intraperitoneally, Ketalar Vet; Pfizer Inc., Rome, Italy) and xylazine (50 mg/kg intraperitoneally, Rompun Vet; Bayer Animal Health, Milano, Italy) and placed in a thermoregulated surgical area. Bladder and intravenous catheters were implanted as previously described (Hedlund et al., 2007). Briefly, the lower urinary tract of the animals was exposed through a lower midline abdominal incision. In each rat, the bladder orientation was assessed by checking its craniocaudal alignment and the position of the median ligament. A polyethylene collar-fitted PE-50 catheter (Clay-Adams, Parsippany, NJ) was inserted into the bladder dome and held in place with a purse-string suture. In some experiments, a second polyethylene PE-50 catheter (stretched at the tip) filled with a heparinized saline solution (100 IU/ml) was inserted into the left femoral vein. Both catheters were tunnelled subcutaneously and secured to the skin of the neck by a silk ligature, and the free ends (identified by a color code) were sealed.

Study Design.

This first study was conducted in awake rats to assess effects by intravesical administration of vehicle (n = 8) or DFL23448 (10 mg/l; n = 6) on baseline urodynamic recordings and during experimental bladder overactivity induced by intravesical PGE2 administration (Lee et al., 2008). During bladder overactivity, vehicle or DFL23448 was administered in the PGE2 solution.

In another study, 1 mg/kg (n = 7) or 10 mg/kg (n = 8) DFL23448 or vehicle (n = 8) was administered intravenously during baseline urodynamic recordings and after PGE2-induced (intravesical) bladder overactivity in awake rats. In separate experiments using male rats, effects of DFL23448 (n = 8) or vehicle (n = 6) were evaluated during cold stress–induced bladder overactivity (Chen et al., 2010). After in vivo experiments, urinary bladders and dorsal root ganglia were harvested from the rats for real-time quantitative PCR investigations of isolated tissues.

Cystometry in Awake Rats.

Urodynamic investigations were performed in awake rats 3 days after the bladder catheterization (Hedlund et al., 2007). Rats were placed in metabolic cages without restraint and bladder catheters were connected via a T tube to a P23 DC pressure transducer (Statham Instruments, Oxnard, CA) and a CMA 100 microinjection pump (Carnegie Medicine AB, Solna, Sweden). Micturition volumes and bladder pressures were recorded continuously with AcqKnowledge 3.8.1 software and an MP100 data acquisition system (Biopac Systems, Santa Barbara, CA) connected to a Model 7E polygraph (Grass Technologies, Middleton, WI). Before the start of the cystometries and when solutions for bladder infusions were changed, the bladder was emptied via the bladder catheter.

To record the micturition volume, room temperature saline was infused into the bladder continuously at a rate of 10 ml/h; a fluid collector connected to an FT03 D force displacement transducer (Grass Technologies) registered the data acquired.

Recorded parameters included the following: basal pressure (the lowest bladder pressure during filling), threshold pressure (bladder pressure immediately before micturition), maximum pressure (maximum bladder pressure during a micturition cycle), micturition volume (volume of expelled urine), residual volume (registered volume of liquid in the bladder after micturition), and micturition intervals (time between two micturition maximum pressures). Bladder capacity was calculated as the sum of micturition volume and residual volume (Hedlund et al., 2007). Reproducible micturition cycles were recorded for a minimum of 30 minutes to serve as baseline values. Once baseline urodynamics and drug activity (intravesical or intravenous) had been registered on a normal pattern, a 50-μM PGE2 saline solution was infused intravesically to induce bladder overactivity and urodynamic parameters were recorded. For intravesical administration of DFL23448, total exposure of the drug to the bladder mucosa was calculated as follows: the mass of the compound in the infusion liquid (10 mg/l = 10 μg/ml) multiplied by the speed of the infusion (10 ml/h) and the duration of the infusion. In other animals, the hind legs were shaved the day before urodynamic investigations; during cystometry after baseline bladder function was established, a 50% menthol spray (100 μl once every 5 minutes for 30 minutes) was applied to the exposed skin as previously described (Chen et al., 2010).

Real-Time Quantitative PCR

Immediately after the rats were euthanized, sixth lumbar dorsal root ganglia and bladder tissues were harvested and snap-frozen in liquid nitrogen. Tissues were then homogenized to allow RNA recovery and retrotranscription and were further processed for real-time PCR as previously described (Streng et al., 2008). RNA extraction was performed using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA), according to the manufacturer’s indications. The retrotranscription reaction was carried out according to the specifications of the Transcriptor High-Fidelity cDNA synthesis kit (Roche, Milan, Italy), starting from 0.5 μg total RNA. The cDNA product was amplified by 35 PCR cycles (30 seconds at 95°C, annealing for 1 minute at 58°C, and extension for 30 seconds at 72°C) and final elongation at 72°C for 5 minutes for TRPM8 and actin. Each PCR product was then amplified by 35 PCR cycles and validated by size determination after separation on 2% agarose gel.

Drugs and Solutions

Icilin, AMTB, BCTC, capsaicin, capsazepine, menthol, allyl isothiocyanate, and GSK1016790A were purchased from Sigma-Aldrich (Milan, Italy), and Cooling Agent 10 was from Takasago (Milan, Italy). During the awake cystometries, DFL23448 (Dompe Pharmaceuticals, L’Aquila, Italy) was administered in 10% Solutol-HS15 (Sigma-Aldrich) and N-methylpyrrolidone (NMP) [2:1 Solutol/NMP (w/v); Sigma-Aldrich] and 90% PBS at 10 mg/l intravesically or at 1 or 10 mg/kg intravenously. Subsequent dilutions of the drug were made on the day of the experiment using a saline solution. Control animals received vehicle alone (0.5 ml i.v. per rat; 10% Solutol-NMP and 90% PBS). A saline solution containing 50 µM PGE2 (Sigma-Aldrich) was prepared on the day of experiments for intravesical administration to induce bladder overactivity. Icilin 1 mg/kg (0.5 mg/1 ml; 0.5 ml i.p. per rat) was dissolved in 1% Tween 80 and distillated water. A 99% menthol solution was dissolved at 40°C and diluted to a 50% menthol solution with distilled water.

Statistical Calculations

Values are given as means ± S.E.M. For multiple comparisons of means, an analysis of variance with Newman-Keuls post hoc test was used. For repeated measures, a one-way analysis of variance (Newman-Keuls) for repeated measures was used. Pairwise and nonpairwise comparisons were made by the t test. Significant differences were accepted when P < 0.05. All statistical calculations were based on the number of individual animals. All calculations were performed using SigmaPlot 11.0 software (Systat Software Inc., San Jose, CA).

Results

Effect of DFL23448 on [Ca2+]i by TRPM8 Agonists in HEK-293 Cells

In Vitro TRPM8 Assay.

DFL23448 (Fig. 2) or AMTB concentration-dependently (0.03 nM to 1 μM; each n = 4) inhibited changes in intracellular Ca2+ levels. The vehicle (n = 4) had no effect. The IC50 values for DFL23448 and AMTB were 10.1 ± 0.2 nM and 60 ± 0.3 nM, respectively.

Fig. 2.
Fig. 2.

In vitro TRPM8 and cold stimulation assays. (A) Concentration-dependent inhibitory effect of DFL23448 (n = 4) on Cooling Agent 10–activated HEK-293 cells expressing human TRPM8. (B) Concentration-dependent inhibitory effect of DFL23448 (n = 4) on cold-induced activation of HEK-293 cells expressing human TRPM8. Data are given as means ± S.E.M.

In Vitro Cold Assay.

Concentration-dependent (0.1 nM to 1 μM) effects on ΔF were achieved with DFL23448 (Fig. 2). The compound exhibited an IC50 value of 21.4 ± 0.3 nM (n = 4).

In Vitro Selectivity of DFL23448 Versus Other TRP Channels or G Protein–Coupled Receptors

DFL23448 did not inhibit the calcium efflux (IC50 values > 10 μM) triggered by capsaicin, GSK1016790A, or isothiocyanate, respectively (each n = 4). In a panel of G protein–coupled receptors, DFL23448 (each n = 4) exhibited no or limited inhibition of reference compound binding to targets (Table 1).

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TABLE 1

Radioligand binding assay

Values are given as means ± S.E.M. (n = 4 for each receptor).

Pharmacokinetics of DFL23448 in the Rat

Intravenous administration of DFL23448 (3 mg/kg; n = 3) showed a clearance of 2.7 ml/min per kilogram, a half-life of 38 minutes, and a distribution volume of 0.069 l/kg. The plasma concentrations of free DFL23448 (1 or 10 mg/kg; each n = 3) after intravenous administration are depicted in Fig. 3. At 1 mg/kg, the plasma concentration of DFL23448 declined below the compounds IC50 value at TRPM8 within 30 minutes after dosing, whereas a dose of 10 mg/kg sufficiently covered the IC50 value over time (Fig. 3).

Fig. 3.
Fig. 3.

Pharmacokinetics. Plasma concentrations (in micromoles) over time (minutes) of free DFL23448 after intravenous administration of 1 mg/kg (white circles; n = 3) or 10 mg/kg (black circles; n = 3). The arrow indicates intravenous injection. The dotted line indicates the in vitro IC50 value for DFL23448 at inhibiting Cooling Agent 10–activated HEK-293 cells expressing human TRPM8. Data are given as means ± S.E.M.

Oral administration of 10 mg/kg DFL23448 (n = 3) caused a maximum plasma concentration of 42 ng/ml and the tmax occurred at 4.3 hours after dosing. The oral half-life was 4.9 hours.

Icilin-Induced WDSs in Rats

Icilin induced WDSs in the vehicle group (n = 10; 174.1 ± 10.5) and in rats treated with 10 mg/kg DFL23448 (n = 10; 109.5 ± 21.6; P < 0.05).

Cystometry in Awake Rats

Intravenous DFL23448 in Cold Stress–Induced Bladder Overactivity.

Skin application of menthol (Fig. 4; Table 2) induced bladder overactivity in vehicle-treated rats (n = 6) but not in DFL23448-treated rats (10 mg/kg i.v.; n = 8). In DFL23448-treated rats, micturition intervals, micturition volumes, and bladder capacity increased by 13%–19% in response to menthol on the skin. By contrast, these parameters decreased by 30%–44% in vehicle-treated rats (P < 0.001 versus DFL23448). Residual volume was unchanged in DFL23448-treated rats before and after skin-menthol provocation, whereas it increased from 0.02 ± 0.01 to 0.06 ± 0.01 ml in vehicle-treated rats (P < 0.05 versus DFL23448). In DFL23448-treated rats, basal pressure and threshold pressure decreased (9%–10%) and maximum pressure increased (9%) after skin application of menthol. In vehicle-treated controls, reverse responses to menthol were recorded; basal pressure and threshold pressure increased by 31%–36% (P < 0.01 versus DFL23448) and maximum pressure decreased by 14% ± 3% (P < 0.001 versus DFL23448).

Fig. 4.
Fig. 4.

Cystometry in awake rats. Cold stress–induced bladder overactivity. (A and B) Representative tracings describing the finding that skin application of menthol induced bladder overactivity in vehicle-treated rats (A), but not in rats treated with 10 mg/kg (intravenous) DFL23448 (B). Note the increase in micturition frequency (left upper panel of bladder pressure and left lower panel of micturition volume) in vehicle-treated rats (A), which is not observed in DFL23448-treated rats (B) (right upper panel of pressure and right lower panel of volume).

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TABLE 2

In vivo cystometry in awake rats: effect of intravenous DFL23448 (10 mg/kg) or vehicle on cold stress–induced bladder overactivity by skin application of menthol (50% solution)

Values are given as means ± S.E.M. (numbers are individual animals).

Intravesical DFL23448 in PGE2-Induced Bladder Overactivity.

Compared with baseline, DFL23448 (n = 6; Table 3) prolonged micturition intervals by 54% ± 15% and increased micturition volumes and bladder capacity by 38% ± 12% and 42% ± 13%, respectively (all P < 0.05). Residual volume did not change in response to DFL23448.

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TABLE 3

In vivo cystometry in awake rats: effect of intravesical vehicle or DFL23448 on intravesical PGE2-induced bladder overactivity

Values are given as means ± S.E.M.

DFL23448 per se did not have effects on basal pressure or threshold pressure, whereas maximum pressure was increased by 33% ± 6% (P < 0.05).

After intravesical administration of PGE2 in DFL23448-treated rats (n = 6; Table 3), micturition intervals decreased by only 15% ± 8% (P = 0.14; Fig. 5). In comparison, intravesical administration of PGE2 reduced micturition intervals by 38% ± 5% in nontreated control rats (n = 8; P < 0.05 versus DFL23448; Fig. 5). Micturition volume was not significantly reduced by PGE2 in DFL23448-treated rats (−13% ± 4%), although it was reduced from 0.66 ± 0.1 to 0.41 ± 0.08 ml in controls (−39% ± 5%; P < 0.05 versus DFL23448; Fig. 5). The residual volume was similar in both groups before and after intravesical administration of PGE2. bladder capacity was reduced by 12% ± 4% in DFL23448-treated rats and 37% ± 6% in controls (P < 0.05 versus DFL23448) after intravesical administration of PGE2 (Fig. 5; Table 3). For controls, significant (P < 0.05–0.01) increases in basal pressure (34% ± 8%), threshold pressure (17% ± 3%), and maximum pressure (33% ± 6%) were recorded (Fig. 5; Table 3) after intravesical administration of PGE2. On the contrary, smaller (nonsignificant) increases in basal pressure (13% ± 5%; P = 0.06 versus vehicle), threshold pressure (15.9% ± 9.2%), and maximum pressure (2% ± 6%; P < 0.01 versus vehicle) were noted in DFL23448-treated rats after intravesical administration of PGE2 (Fig. 5; Table 3).

Fig. 5.
Fig. 5.

Cystometry in awake rats. Bladder overactivity induced by intravesical infusion of PGE2 (50 μM). Effect by DFL23448 (10 mg/kg intravesically, n = 6) or vehicle (intravesically, n = 8) on PGE2-induced changes in micturition interval, micturition volume, bladder capacity, basal pressure, threshold pressure, and micturition pressure. Data are given as mean percentages ± S.E.M. of intravesical PGE2-induced changes from baseline. *P < 0.05; **P < 0.01 DFL23448 versus vehicle (nonpairwise comparisons by the t test based on the number of individual animals).

Intravenous DFL23448 in PGE2-Induced Bladder Overactivity.

At baseline, 1 mg/kg DFL23448 (n = 7; Table 4) did not influence micturition intervals, micturition volume, or bladder capacity. Compared with baseline, the higher dose of 10 mg/kg DFL23448 (n = 8; Table 4) increased micturition intervals by 13% ± 4% (P < 0.05), micturition volume by 19% ± 46% (P < 0.05), and bladder capacity by 17% ± 46% (P < 0.05). This effect was not registered for vehicle-treated rats (n = 8; Table 4). Bladder pressures were similar for drug or vehicle at baseline. No changes in the intravesical pressures from baseline were noted for rats treated with DFL23448 or vehicle.

View this table:
TABLE 4

In vivo cystometry in awake rats: effect of intravenous vehicle, 1 mg/kg DFL23448, or 10 mg/kg DFL23448 on intravesical PGE2-induced bladder overactivity

Values are given as means ± S.E.M.

After intravesical administration of PGE2 (Fig. 6; Table 4), micturition intervals, micturition volume, and bladder capacity decreased (P < 0.05–0.01) by 27%–34% in controls (Fig. 6). Similarly, in rats treated with 1 mg/kg DFL23448, decreases (P < 0.05–0.01) in micturition intervals, micturition volume, and bladder capacity amounted to 31%–35% in response to intravesical PGE2 (Fig. 6). In the 10 mg/kg DFL23448–treated group (Fig. 6; Table 4), micturition intervals, micturition volume, and bladder capacity were reduced only by 3%–6%. The residual volume increased in vehicle controls (P < 0.05) but did not change in rats treated with 1 or 10 mg/kg DFL23448 (Table 4).

Fig. 6.
Fig. 6.

Cystometry in awake rats. Bladder overactivity induced by intravesical infusion of PGE2; 50 μM). Effect by intravenous 1 mg/kg (n = 7) or 10 mg/kg (n = 8) DFL23448 or vehicle (n = 8) on PGE2-induced changes in micturition interval, micturition volume, bladder capacity, basal pressure, threshold pressure, and micturition pressure. Data are given as mean percentages ± S.E.M. of intravesical PGE2-induced changes from baseline. *P < 0.05 DFL23448 versus vehicle (Newman-Keuls analysis of variance for repeated measures based on the number of individual animals).

In vehicle-treated controls, basal pressure, threshold pressure, and maximum pressure increased (P < 0.01) by 41%–75% after intravesical administration of PGE2 compared with baseline (Fig. 6; Table 4). For rats treated with 1 mg/kg DFL23448, basal pressure increased by 37% ± 5% (P < 0.05), whereas smaller increases in threshold pressure (18%) and maximum pressure (21%) were recorded after intravesical administration of PGE2 (Fig. 6; Table 4). In rats treated with 10 mg/kg DFL23448, smaller increases in basal pressure (12%), threshold pressure (6%), and maximum pressure (20%) were recorded after intravesical administration of PGE2.

Real-Time Quantitative PCR

The highest TRPM8 expression was detected in rat sixth lumbar dorsal root ganglia. Nearly 10-fold less expression of TRPM8 was encountered in rat bladders. Neither exposure to intravesical PGE2 nor intravenous treatment with DFL23488 had effects on TRPM8 expression levels in the dorsal root ganglia (n = 5) or bladder tissue (Fig. 7; n = 4).

Fig. 7.
Fig. 7.

Real-time quantitative PCR. Expression of TRPM8 in the sixth lumbar dorsal root ganglion (L6 DRG; each n = 5) and urinary bladder (each n = 4) of rats treated with intravenous DFL23448 (10 mg/kg) or vehicle that were exposed to intravesical (i.ves.) infusion of PGE2 (50 μM) or saline (PGE2 vehicle). Data are given as means ± S.E.M. Values are normalized to the internal control gene hypoxanthine ribosyltransferase (HPRT).

Discussion

Population-based surveys show that lower urinary tract symptoms are highly prevalent, occurring in 62% and 67% of men and women, and the prevalence increases with age (Irwin et al., 2006). Storage symptoms, including urgency (a sudden compelling desire to pass urine that is difficult to defer), are the most common complaints and occur in more than one-half of the survey populations (Abrams et al., 2002; Irwin et al., 2006). Interestingly, patients with overactive bladder report that cold weather aggravates urgency symptoms and these patients also experience acute cold-induced urgency (Kondo et al., 1992; Choe et al., 1999; Ghei and Malone-Lee, 2005; Uvin et al., 2014). It is proposed that imbalances of sensory functions within the urothelium and the suburothelial space, along with potential abnormalities of central function in processing of afferent signals, significantly contribute to the generation of storage lower urinary tract symptoms (Roosen et al., 2009). Advances in the understanding of TRPs in bladder sensing suggest that TRPM8 has important functions in bladder physiology and pathophysiology and that this receptor may be an emerging drug target in lower urinary tract symptoms (Franken et al., 2014; Uvin et al., 2015).

Our study characterizes DFL23448 as a functionally active TRPM8 antagonist that inhibits cold-induced responses in vitro and counteracts in vivo cold-provoked reactions and bladder overactivity in rats. Using an in vitro assay, the functional inhibitory activity of DFL23448 on human TRPM8 receptors was evaluated. In accordance with previous findings, Cooling Agent 10, an efficient TRPM8 agonist, induced [Ca2+]i changes in TRPM8-expressing HEK-293 cells (Behrendt et al., 2004). In a similar manner as AMTB (a selective TRPM8 antagonist that exhibited inhibitory activity around its expected IC50 value for TRPM8 in our investigation), DFL23448 also counteracted [Ca2+]i changes in response to Cooling Agent 10 (Journigan and Zaveri, 2013). Furthermore, human TRPM8-expressing cells displayed increased [Ca2+]i responses to decreasing temperature and these changes were concentration-dependently attenuated by DFL23448. In addition, the currently reported pharmacological properties of DFL23448 suggest that the compound exhibits an approximately 200-fold functional selectivity for TRPM8 versus TRPA1, TRPV1, and TRPV4 and it does not inhibit radioligand binding to a large number of G protein–coupled receptors. However, it cannot be excluded that DFL23448 may act at other receptors that are not accounted for in this investigation.

In agreement with previous findings, icilin (a potent and efficacious TRPM8 agonist) induced reproducible responses during the in vivo pharmacodynamics WDS test in rats (Behrendt et al., 2004; Horne et al., 2014). These responses were inhibited by DFL23448 by approximately 40%. Because the icilin WDS test reflects somatomotor cold behaviors, we also assessed effects of DFL23448 on visceral in vivo responses to cold (Werkheiser et al., 2009). TRPM8 was recently identified as the main mediator for the skin-cooling bladder reflex in rats and this model is proposed as relevant to simulate human acute cold-induced urgency (Uvin et al., 2015). Similar to cutaneous cold-sensitive C-fibers in humans, type 1 cold-sensitive C-fibers of the rat hind limb skin are reported to be sensitive to TRPM8 agonists (Campero et al., 2009; Teliban et al., 2014). Corresponding to results by Chen et al. (2010), cutaneous application of the TRPM8 agonist menthol on vehicle-treated rats caused reproducible bladder overactivity that was characterized by increased micturition frequency and deranged intravesical pressures (Behrendt et al., 2004; Chen et al., 2010). Supporting a role for TRPM8 in the skin-cooling bladder reflex in vivo in rats, the bladder overactivity responses recorded in this study were abolished by DFL23448. Urodynamic parameters that may be considered to reflect bladder storage and sensory micturition functions (e.g., micturition interval, micturition volume, bladder capacity, and threshold pressure) were reversely affected by DFL23448 compared with vehicle-treated rats during menthol provocation. This is supported by previous reports that environmental cold induces bladder overactivity via resiniferatoxin-sensitive nerves in conscious rats (Imamura et al., 2008). In contrast with other studies that reported no or nonsignificant increases in intravesical pressure in response to skin application of menthol, rats in this study did exhibit increased basal and maximal intravesical pressures under the same experimental procedures (Chen et al., 2010; Lei et al., 2013). These effects were not observed in DFL23448-treated rats. Whether TRPM8 signals also influence efferent functions of micturition may also require further research.

The in vivo functional role for TRPM8 in bladder function has previously been studied mainly in rats exposed to menthol, or cold stimuli by acetone or air, and in knockout mice (Nomoto et al., 2008; Jun et al., 2012; Lei et al., 2013; Uvin et al., 2015). Less is known from other bladder overactivity models. In humans, peripheral TRPM8 plasticity has been suggested to be associated with overactive and painful bladder syndromes in humans (Mukerji et al., 2006). Various models are available to simulate these conditions; of these, acute administrations of agents into the bladder are common and conveniently performed to stimulate specific receptors or to produce inflammation/hypersensitivity (Andersson et al., 2011). In this study, we used intravesical infusion of PGE2, which causes bladder overactivity in rodents and in humans (Andersson et al., 2011). Levels of PGE2 have also been reported to increase in urine from patients with overactive bladders and PGE2 has been proposed as a biomarker for the condition (Kim et al., 2005, 2006; Cho et al., 2013). Furthermore, intravesical PGE2 has been shown to activate afferent C-fibers from the urinary bladder (Aizawa et al., 2010). Because TRPM8 has been located in the urothelium, sensory nerve fibers, and suburothelial varicose terminals of the lower urinary tract (Hayashi et al., 2009; Andersson et al., 2011), we first assessed the effect of intravesical DFL23448 on urodynamic parameters to understand whether the compound has a local site of action in the bladder. At baseline in naive rats, intravesical DFL23448 had already reduced micturition frequency and concomitantly increased codependent bladder volumes. This may be expected since stimulatory effects on micturition have been reported after intravesical administration of menthol using the same rat model for cystometry (Nomoto et al., 2008).

The effects by PGE2 in the bladder are suggested to be mediated by sensitization of afferent nerves and by modulation of efferent neurotransmission (Andersson and Wein, 2004; Aizawa et al., 2010); in agreement with our previous experience, infusion of PGE2 into bladders of vehicle controls caused bladder overactivity with increased frequency, decreased micturition volume, and increased pressures (Gandaglia et al., 2014). These effects were not observed in rats treated with intravesical DFL23448. Thus, under this experimental condition, it seems likely that DFL23448 may act at structures in the bladder. We cannot exclude that some absorption may have occurred from the lower urinary tract. However, compared with the current pharmacokinetic data based on 3 mg/kg i.v. and 10 mg/kg p.o. administration of DFL23448 in rats, total bladder mucosa exposure of 50–150 μg (from the end of baseline registration to the end of cystometry) of the compound per rat seems unlikely to yield relevant systemic drug levels.

Systemic (intravenous) administration of 10 mg/kg DFL23448 was efficacious to reduce PGE2-induced (intravesical) bladder overactivity and exhibited similar effects on urodynamic parameters as when the compound was infused locally into the bladder. At this dose, DFL23448 per se also decreased baseline-voiding frequency, although this effect was less pronounced compared with that after intravesical administration of the compound. By contrast, systemic administration of BCTC (another TRPM8 antagonist) did not influence urodynamic parameters in naive rats but it exhibited dose-dependent effects on menthol or cold stress–induced bladder overactivity (Lei et al., 2013).

In this study, a dose-dependent effect of DFL23448 on urodynamic parameters during PGE2-induced bladder overactivity may be considered. Intravenous DFL23448 at 1 mg/kg neither prolonged micturition intervals during baseline nor inhibited a PGE2-induced increase in voiding frequency, but it counteracted changes in bladder pressures in response to intravesical PGE2. These findings are supported by the pharmacokinetic data showing that 10 mg/kg (but not 1 mg/kg) DFL23448 reached sufficient temporal plasma concentrations that cover the in vitro IC50 value of the compound at TRPM8 and correspond to the duration of the in vivo experimental situations.

Systemic exposure of DFL23448 to rats did not influence the expression patterns of TRPM8 in peripheral lower urinary tract sensory pathways or in the urinary bladder. However, from a putative therapeutic perspective, additional investigations on effects on micturition pathways by regular chronic dosing of drugs that target this receptor are necessary.

In conclusion, we report that DFL23448 is a selective antagonist of the TRPM8 that is systemically active to counteract behaviors and visceral responses to skin application of TRPM8 agonists and cooling mimetic agents. The compound prolongs the storage phase of micturition and effectively inhibits experimental bladder overactivity in rats. Our results support previous findings that implicate a role for TRPM8 in bladder function and dysfunction. In view of recent human proof-of-concept studies with a TRPM8 antagonist for cold pressor pain (Winchester et al., 2014), our data contribute information to further explore TRPM8 as a possible drug target for human lower urinary tract symptoms.

Authorship Contributions

Participated in research design: Mistretta, Brandolini, Aramini, Benigni, Hedlund.

Conducted experiments: Mistretta, A. Russo, Castiglione, Bovolenta, Bettiga, Colciago, R. Russo, Benigni, Hedlund.

Contributed new reagents or analytic tools: Brandolini, Aramini, Bianchini.

Performed data analysis: Mistretta, A. Russo, Bettiga, R. Russo, Benigni, Hedlund.

Wrote or contributed to the writing of the manuscript: Mistretta, Castiglione, Montorsi, Brandolini, Aramini, Bianchini, Allegretti, Benigni, Hedlund.

Footnotes

    • Received August 14, 2015.
    • Accepted November 5, 2015.

  • This research was supported by an unrestricted grant from Dompe Pharmaceuticals and the Gester Foundation.

  • dx.doi.org/10.1124/jpet.115.228684.

Abbreviations

DFL23448
5-(2-ethyl-2H-tetrazol-5-yl)-2-(3-fluorophenyl)-1,3-thiazol-4-ol
DMSO
dimethylsulfoxide
GSK1016790A
N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide
HEK-293
human embryonic kidney 293
NMP
N-methylpyrrolidone
PBS
phosphate-buffered saline
PCR
polymerase chain reaction
PGE2
prostaglandin E2
TRP
transient receptor potential ion channel
TRPA
transient receptor potential ankyrin
TRPM8
transient receptor potential melastin 8 ion channel
TRPV
transient receptor potential vanilloid
WDS
wet dog–like shake

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Research ArticleDrug Discovery and Translational Medicine

TRPM8 as a Drug Target in Models for Bladder Overactivity

Francesco A. Mistretta, Andrea Russo, Fabio Castiglione, Arianna Bettiga, Giorgia Colciago, Francesco Montorsi, Laura Brandolini, Andrea Aramini, Gianluca Bianchini, Marcello Allegretti, Silvia Bovolenta, Roberto Russo, Fabio Benigni and Petter Hedlund
Journal of Pharmacology and Experimental Therapeutics January 1, 2016, 356 (1) 200-211; DOI: https://doi.org/10.1124/jpet.115.228684
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