Efficacy of the combination of KPR-5714, a novel transient receptor potential melastatin 8 (TRPM8) antagonist, and ß3-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 ß3-adrenoceptors using the transfected cells of each gene at first. Then, combination effects of KPR-5714 and mirabegron, a ß3-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 ß3-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 ß3-adrenoceptor agonist or anticholinergic agent can be the potential treatment option for obtaining additive effects in comparison with monotherapy for OAB.

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.

Longitudinal analysis of motor symptoms and histopathology in woozy mice, a model of cerebellar ataxia.

Woozy (wz) mice develop ataxia and carry a mutation in the Sil1 gene. Homozygous wz mice have been characterized histopathologically, but no details of their motor function have been reported. In the present study, to comprehensively understand the relationship between symptomatic progression and pathological feature, we evaluated motor function and neurodegeneration with age from presymptomatic to terminal stages. We evaluated the motor function of homozygous and heterozygous wz mice aged from 5 to 71 weeks. Motor function was evaluated using the rotarod test, the footprint test, and the parallel rod floor test. Furthermore, we carried out a histopathological analysis of the mice at several ages. Impairment of motor function in homozygous wz mice began at around 11 weeks of age and became markedly worse until around 14 weeks. Heterozygous wz mice did not show motor dysfunction until 71 weeks of age. Features of cerebellar ataxia were evaluated using the footprint test and the parallel rod floor test. In addition to the observation of ubiquitin-positive aggregates at 6 weeks of age, Purkinje cell loss at 9 weeks of age and cerebellar atrophy were confirmed by histopathology. Apart from the cerebellar changes, we detected no other pathology that could contribute toward ataxia. In heterozygous wz mice, only minimal formation of ubiquitin-positive aggregates was observed. Homozygous wz mice showed adult-onset ataxia with progressive neurodegeneration of the cerebellum. Homozygous wz mice might be useful as an animal model of diseases showing adult-onset ataxia because of cerebellar neurodegeneration.

Identification of cell adhesive sequences in the N-terminal region of the laminin α2 chain.

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin ß1 and anti-integrin α2ß1 antibodies. These results suggest that A2-8 promotes an integrin α2ß1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2ß1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2ß1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2ß1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.