Selective Transient Receptor Potential Melastatin 8 Antagonist Research Articles

KPR-5714, a selective transient receptor potential melastatin 8 antagonist, improves voiding dysfunction in rats with bladder overactivity but does not affect voiding behavior in normal rats.

Transient receptor potential melastatin 8 (TRPM8) has a role in the abnormal sensory transduction of the bladder and is involved in the pathophysiology of hyperactivity bladder disorders. The aim of this study is to examine the effects of KPR-5714, a novel and selective TRPM8 antagonist, on voiding dysfunction induced by bladder afferent hyperactivity via mechanosensitive C-fibers in rats. The effects of intragastric administration of KPR-5714 on bladder overactivity induced by intravesical instillation of 10 mM ATP were investigated using cystometry in conscious female rats. We examined the effects of oral administration of KPR-5714 on voiding behavior using a metabolic cage in normal male rats and rats with an intratesticular injection of 3% acetic acid. In cystometry measurements, the intercontraction interval was decreased by intravesical ATP instillation. KPR-5714 (0.1, 0.3, and 1 mg/kg) dose-dependently prolonged the shortened intercontraction interval provoked by ATP. In voiding behavior measurements, intratesticular injection of acetic acid decreased the mean voided volume and increased voiding frequency. KPR-5714 (0.1 and 0.3 mg/kg) dose-dependently increased the mean voided volume and decreased voiding frequency without affecting the total voided volume in these rats. However, KPR-5714 (1 and 10 mg/kg) did not influence the voiding behavior in normal rats. The present results suggest that KPR-5714 improves voiding dysfunction by inhibiting the enhanced activity of mechanosensitive bladder C-fibers in rats with bladder overactivity and shows no significant change in voiding behavior in normal rats.

Efficacy of the combination of KPR-5714, a novel transient receptor potential melastatin 8 (TRPM8) antagonist, and β3-adrenoceptor agonist or anticholinergic agent on bladder dysfunction in rats with bladder overactivity

Transient receptor potential melastatin 8 (TRPM8) channels may contribute to the pathophysiological bladder afferent hyperactivity, thus a TRPM8 antagonist would be a promising therapeutic target for the bladder hypersensitive disorders including urinary urgency in overactive bladder (OAB). We aimed to investigate a pharmacological effect of KPR-5714, a novel selective TRPM8 antagonist, on TRPM8 channels, M3 receptors and β3-adrenoceptors using the transfected cells of each gene at first. Then, combination effects of KPR-5714 and mirabegron, a β3-adrenoceptor agonist, or tolterodine tartrate, an anticholinergic agent, were studied on rhythmic bladder contractions (RBCs) in normal rats and bladder function in frequent-voiding rats. In vitro measurements showed that KPR-5714 acts on neither β3-adrenoceptor nor M3 receptor. In normal rats, KPR-5714 and mirabegron significantly reduced the frequency of RBCs, and a combined administration showed an additive effect. In rats with cerebral infarction, KPR-5714 and mirabegron significantly reduced the voiding frequency, and a combined administration showed an additive effect. In rats exposed to cold temperature, KPR-5714 and tolterodine tartrate significantly reduced the voiding frequency accompanied by the increased mean voided volume, and a combined administration showed additive effects. The present study demonstrated that the combined administration of KPR-5714 and mirabegron or tolterodine tartrate showed the additive effects on bladder dysfunction in different animal models, suggesting that the combination therapy of TRPM8 antagonist and β3-adrenoceptor agonist or anticholinergic agent can be the potential treatment option for obtaining additive effects in comparison with monotherapy for OAB.

Identification of N-acyl-N-indanyl-α-phenylglycinamides as selective TRPM8 antagonists designed to mitigate the risk of adverse effects

Transient receptor potential melastatin 8 (TRPM8), a temperature-sensitive ion channel responsible for detecting cold, is an attractive molecular target for the treatment of pain and other disorders. We have previously discovered a selective TRPM8 antagonist, KPR-2579, which inhibited bladder afferent hyperactivity induced by acetic acid instillation into the bladder. However, additional studies have revealed potential adverse effects with KPR-2579, such as the formation of a reactive metabolite, CYP3A4 induction, and convulsions. In this report, we describe the optimization of α-phenylglycinamide derivatives to mitigate the risk of these adverse effects. The optimal compound 13x exhibited potent inhibition against icilin-induced wet-dog shakes and cold-induced frequent voiding in rats, with a wide safety margin against the potential side effects.

Identification of a natural compound, sesamin, as a novel TRPM8 antagonist with inhibitory effects on prostate adenocarcinoma

Transient receptor potential melastatin 8 (TRPM8) is a calcium ion-permeable cation channel that is used as a prognostic marker and therapeutic target for different tumor types. To identify natural selective TRPM8 antagonists, we tested 158 traditional Chinese medicine (TCM) compounds for the ability to inhibit TRPM8. Calcium mobilization assays were used to evaluate the 158 TCM compound components in HEK293 cells stably expressing TRPM8. An identified putative TRPM8 antagonist, sesamin, was further evaluated. Publicly available cancer OMICS data were used to explore the expression of TRPM8, its gene regulatory network, and the survival of patients with prostate adenocarcinoma (PRAD). The cytotoxicity and specificity of sesamin to TRPM8 were tested in HEK293/TRPM8 cells. The effect of sesamin on cell proliferation in PRAD cell lines was assessed. Sesamin selectively inhibited TRPM8 in HEK293/TRPM8 cells (IC50: 9.78 μM), and a molecular docking study confirmed the binding of sesamin to TRPM8. TRPM8 was highly overexpressed in PRAD, and high TRPM8 expression was associated with poor survival of PRAD patients. Functional network analysis suggested that TRPM8 has key effects on proliferation, survival, and invasion of prostate cancer cells. Cell proliferation assays supported these findings and showed that sesamin inhibited the proliferation of PRAD cell lines DU145 and LNCaP cells. These data revealed that abnormal TRPM8 expression is associated with PRAD and that sesamin is a new anti-PRAD candidate drug, exerting inhibitory effects on TRPM8.

KPR-5714, a Novel Transient Receptor Potential Melastatin 8 Antagonist, Improves Overactive Bladder via Inhibition of Bladder Afferent Hyperactivity in Rats

Transient receptor potential (TRP) melastatin 8 (TRPM8) is a temperature-sensing ion channel mainly expressed in primary sensory neurons (Aδ-fibers and C-fibers in the dorsal root ganglion). In this report, we characterized KPR-5714 (N-[(R)-3,3-difluoro-4-hydroxy-1-(2H-1,2,3-triazol-2-yl)butan-2-yl]-3-fluoro-2-[5-(4-fluorophenyl)-1H-pyrazol-3-yl]benzamide), a novel and selective TRPM8 antagonist, to assess its therapeutic potential against frequent urination in rat models with overactive bladder (OAB). In calcium influx assays with HEK293T cells transiently expressing various TRP channels, KPR-5714 showed a potent TRPM8 antagonistic effect and high selectivity against other TRP channels. Intravenously administered KPR-5714 inhibited the hyperactivity of mechanosensitive C-fibers of bladder afferents and dose-dependently increased the intercontraction interval shortened by intravesical instillation of acetic acid in anesthetized rats. Furthermore, we examined the effects of KPR-5714 on voiding behavior in conscious rats with cerebral infarction and in those exposed to cold in metabolic cage experiments. Cerebral infarction and cold exposure induced a significant decrease in the mean voided volume and increase in voiding frequency in rats. Orally administered KPR-5714 dose-dependently increased the mean voided volume and decreased voiding frequency without affecting total voided volume in these models. This study demonstrates that KPR-5714 improves OAB in three different models by inhibiting exaggerated activity of mechanosensitive bladder C-fibers and suggests that KPR-5714 may provide a new and useful approach to the treatment of OAB. SIGNIFICANCE STATEMENT: TRPM8 is involved in bladder sensory transduction and plays a role in the abnormal activation in hypersensitive bladder disorders. KPR-5714, as a novel and selective TRPM8 antagonist, may provide a useful treatment for the disorders related to the hyperactivity of bladder afferent nerves, particularly in overactive bladder.

Vascular Endothelial Growth Factor (VEGF) Induced Downstream Responses to Transient Receptor Potential Vanilloid 1 (TRPV1) and 3-Iodothyronamine (3-T1AM) in Human Corneal Keratocytes.

This study was undertaken to determine if crosstalk among the transient receptor potential (TRP) melastatin 8 (TRPM8), TRP vanilloid 1 (TRPV1), and vascular endothelial growth factor (VEGF) receptor triad modulates VEGF-induced Ca2+ signaling in human corneal keratocytes. Using RT-PCR, qPCR and immunohistochemistry, we determined TRPV1 and TRPM8 gene and protein coexpression in a human corneal keratocyte cell line (HCK) and human corneal cross sections. Fluorescence Ca2+ imaging using both a photomultiplier and a single cell digital imaging system as well as planar patch-clamping measured relative intracellular Ca2+ levels and underlying whole-cell currents. The TRPV1 agonist capsaicin increased both intracellular Ca2+ levels and whole-cell currents, while the antagonist capsazepine (CPZ) inhibited them. VEGF-induced Ca2+ transients and rises in whole-cell currents were suppressed by CPZ, whereas a selective TRPM8 antagonist, AMTB, increased VEGF signaling. In contrast, an endogenous thyroid hormone-derived metabolite 3-Iodothyronamine (3-T1AM) suppressed increases in the VEGF-induced current. The TRPM8 agonist menthol increased the currents, while AMTB suppressed this response. The VEGF-induced increases in Ca2+ influx and their underlying ionic currents stem from crosstalk between VEGFR and TRPV1, which can be impeded by 3-T1AM-induced TRPM8 activation. Such suppression in turn blocks VEGF-induced TRPV1 activation. Therefore, crosstalk between TRPM8 and TRPV1 inhibits VEGFR-induced activation of TRPV1.

Antinociceptive effect of two novel transient receptor potential melastatin 8 antagonists in acute and chronic pain models in rat

Transient receptor potential (TRP) channels are a superfamily of non-selective cation permeable channels involved in peripheral sensory signalling. Animal studies have shown that several TRPs are important players in pain modulation. Among them, the TRP melastatin 8 (TRPM8) has elicited more interest for its controversial role in nociception. This channel, expressed by a subpopulation of sensory neurons in dorsal root ganglia (DRG) and trigeminal ganglia (TG), is activated by cold temperatures and cooling agents. In experimental neuropathic pain models, an up-regulation of this receptor in DRG and TG has been observed, suggesting a key role for TRPM8 in the development and maintenance of pain. Consistent with this hypothesis, TRPM8 knockout mice are less responsive to pain stimuli. In this study, the therapeutic potential and efficacy of two novel TRPM8 antagonists, DFL23693 and DFL23448, were tested. Two potent and selective TRPM8 antagonists with distinct pharmacokinetic profiles, DFL23693 and DFL23448, have been fully characterized in vitro. In vivo studies in well-established models, namely, the wet-dog shaking test and changes in body temperature, confirmed their ability to block the TRPM8 channel. Finally, TRPM8 blockage resulted in a significant antinociceptive effect in formalin-induced orofacial pain and in chronic constriction injury-induced neuropathic pain, confirming an important role for this channel in pain perception. Our findings, in agreement with previous literature, encourage further studies for a better comprehension of the therapeutic potential of TRPM8 blockers as novel agents for pain management.

KPR-2579, a novel TRPM8 antagonist, inhibits acetic acid-induced bladder afferent hyperactivity in rats.

Transient receptor potential melastatin 8 (TRPM8) is proposed to be a promising therapeutic target for hypersensitive bladder disorders. We examined the effects of KPR-2579, a novel selective TRPM8 antagonist, on body temperature and on mechanosensitive bladder single-unit afferent activities (SAAs) provoked by intravesical acetic acid (AA) instillation in rats. Female Sprague-Dawley rats were used. Effects of cumulative intravenous (i.v.) administrations of KPR-2579 (0.03-1 mg/kg) on deep body temperature were investigated (N = 18). In separate animals, effects of bolus administration of KPR-2579 (0.03 or 0.3 mg/kg, i.v.) on bladder hyperactivity induced by intravesical instillation of 0.1% AA were investigated using cystometry (N = 57) in a conscious free-moving condition or urethane-anesthetized condition, and SAA measurements (N = 41) were performed in a urethane-anesthetized condition. KPR-2579 at any doses tested did not affect body temperature. In cystometry measurements, a high dose (0.3 mg/kg) of KPR-2579 counteracted the shortened intercontraction interval provoked by AA instillation. In SAA measurements, 48 single afferent fibers (n = 24 in each fiber) were isolated. AA instillations significantly increased the SAAs of C fibers, but not of Aδ fibers, in the presence of KPR-2579's vehicle and a low dose (0.03 mg/kg) of KPR-2579. Pretreatment with a high dose (0.3 mg/kg) of KPR-2579 significantly inhibited the AA-induced activation of C-fiber SAAs. The present results suggest that TRPM8 channels play a role in the AA-induced pathological activation of mechanosensitive bladder C fibers in rats. KRP-2579 may be a promising drug for hypersensitive bladder disorders.

Downregulations of TRPM8 expression and membrane trafficking in dorsal root ganglion mediate the attenuation of cold hyperalgesia in CCI rats induced by GFRα3 knockdown.

Cold hyperalgesia is an intractable sensory abnormality commonly seen in peripheral neuropathies. Although glial cell line-derived neurotrophic factor family receptor alpha3 (GFRα3) is required for the formation of pathological cold pain has been revealed, potential transduction mechanism is poorly elucidated. We have previously demonstrated the contribution of enhanced activity of transient receptor potential melastatin 8 (TRPM8) to cold hyperalgesia in neuropathic pain using a rat model of chronic constriction injury (CCI) to the sciatic nerve. Recently, the enhancement of TRPM8 activity is attributed to the increased TRPM8 plasma membrane trafficking. In addition, TRPM8 can be sensitized by the activation of GFRα3, leading to increased cold responses in vivo. The aim of this study was to investigate whether GFRα3 could influence cold hyperalgesia of CCI rats via modulating TRPM8 expression and plasma membrane trafficking in dorsal root ganglion (DRG). Mechanical allodynia, cold and heat hyperalgesia were measured on 1day before CCI and the 1st, 4th, 7th, 10th and 14th day after CCI. TRPM8 total expression and membrane trafficking as well as GFRα3 expression in DRG were detected by immunofluorescence and western blot. Furthermore, GFRα3 small interfering RNA (siRNA) was intrathecally administrated to reduce GFRα3 expression in DRG, and the effects of GFRα3 knockdown on CCI-induced behavioral sensitization as well as TRPM8 total expression and membrane trafficking in both mRNA and protein levels were investigated, and the change in coexpression of TRPM8 with GFRα3 was also evaluated. Then, the effect of GFRα3 activation with artemin on pain behavior of CCI rats pretreated with the selective TRPM8 antagonist RQ-00203078 was observed. Here we found that TRPM8 total expression and plasma membrane trafficking as well as GFRα3 expression in DRG were initially increased on the 4th day after CCI, and maintained at the peak level from the 10th to the 14th day, which entirely conformed with the induction and maintenance of behavioral-reflex facilitation following CCI. The coexpression of TRPM8 with GFRα3, which was mainly located in peptidergic C-fibers DRG neurons, was also increased after CCI. Downregulation of GFRα3 protein in DRG attenuated CCI-induced cold hyperalgesia without affecting mechanical allodynia and heat hyperalgesia, and reduced the upregulations of TRPM8 total expression and plasma membrane trafficking as well as coexpression of TRPM8 with GFRα3 induced by CCI. Additionally, the inhibition of TRPM8 abolished the influence of GFRα3 activation on cold hyperalgesia after CCI. Our results demonstrate that GFRα3 knockdown specially inhibits cold hyperalgesia following CCI via decreasing the expression level and plasma membrane trafficking of TRPM8 in DRG. GFRα3 and its downstream mediator, TRPM8, represent a new analgesia axis which can be further exploited in sensitized cold reflex under the condition of chronic pain.

Synthesis and optimization of novel α-phenylglycinamides as selective TRPM8 antagonists

Transient receptor potential melastatin 8 (TRPM8) is activated by innocuous cold and chemical substances, and antagonists of this channel have been considered to be effective for pain and urinary diseases. N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N-(2-thienylmethyl)benzamide hydrochloride (AMTB), a TRPM8 antagonist, was proposed to be effective for overactive bladder and painful bladder syndrome; however, there is a potential risk of low blood pressure. We report herein the synthesis and structure–activity relationships of novel phenylglycine derivatives that led to the identification of KPR-2579 (20l), a TRPM8 selective antagonist. KPR-2579 reduced the number of icilin-induced wet-dog shakes and rhythmic bladder contraction in rats, with no negative cardiovascular effects at the effective dose.

Functional role of the transient receptor potential melastatin 8 (TRPM8) ion channel in the urinary bladder assessed by conscious cystometry and ex vivo measurements of single‐unit mechanosensitive bladder afferent activities in the rat

To evaluate the role of the transient receptor potential melastatin 8 (TRPM8) channel on bladder mechanosensory function by using L-menthol, a TRPM8 agonist, and RQ-00203078 (RQ), a selective TRPM8 antagonist. Female Sprague-Dawley rats were used. In conscious cystometry (CMG), the effects of intravesical instillation of L-menthol (3 mm) were recorded after intravenous (i.v.) pretreatment with RQ (3 mg/kg) or vehicle. The direct effects of RQ on conscious CMG and deep body temperature were evaluated with cumulative i.v. administrations of RQ at 0.3, 1, and 3mg/kg. Single-unit mechanosensitive bladder afferent activities (SAAs) were monitored in a newly established ex vivo rat bladder model to avoid systemic influences of the drugs. Recordings were performed after cumulative intra-aortic administration of RQ (0.3 and 3 mg/kg) with or without intra-vesical L-menthol instillation (3 mm). Intravesical L-menthol decreased bladder capacity and voided volume, which was counteracted by RQ-pretreatment. RQ itself increased bladder capacity and voided volume, and lowered deep body temperature in a dose-dependent manner. RQ decreased mechanosensitive SAAs of C-fibres, and inhibited the activation of SAAs induced by intravesical L-menthol. Our results suggest that TRPM8 channels have a role in activation of bladder afferent pathways during filling of the bladder in the normal rat. This effect seems, at least partly, to be mediated via mechanosensitive C-fibres.

ChemInform Abstract: The Design and Synthesis of Novel, Phosphonate‐Containing Transient Receptor Potential Melastatin 8 (TRPM8) Antagonists.

AbstractCompound (Ib) is identified as a highly selective and robust TRPM8 antagonist in vitro that inhibits icilin‐induced behaviors in a rat WDS model.

Design and Optimization of Benzimidazole-Containing Transient Receptor Potential Melastatin 8 (TRPM8) Antagonists

Transient receptor potential melastatin 8 (TRPM8) is a nonselective cation channel that is thermoresponsive to cool to cold temperatures (8-28 °C) and also may be activated by chemical agonists such as menthol and icilin. Antagonism of TRPM8 activation is currently under investigation for the treatment of painful conditions related to cold, such as cold allodynia and cold hyperalgesia. The design, synthesis, and optimization of a class of selective TRPM8 antagonists based on a benzimidazole scaffold is described, leading to the identification of compounds that exhibited potent antagonism of TRPM8 in cell-based functional assays for human, rat, and canine TRPM8 channels. Numerous compounds in the series demonstrated excellent in vivo activity in the TRPM8-selective "wet-dog shakes" (WDS) pharmacodynamic model and in the rat chronic constriction injury (CCI)-induced model of neuropathic pain. Taken together, the present results suggest that the in vivo antagonism of TRPM8 constitutes a viable new strategy for treating a variety of disorders associated with cold hypersensitivity, including certain types of neuropathic pain.