Efficacy and safety of the emedastine patch, a novel transdermal drug delivery system for allergic rhinitis: Phase III, multicenter, randomized, double-blinded, placebo-controlled, parallel-group comparative study in patients with seasonal allergic rhinitis.

BACKGROUND: The emedastine patch was developed in Japan as the first transdermal drug delivery system of emedastine difumarate for allergic rhinitis. METHODS: A multicenter, randomized, double-blind, placebo-controlled, parallel-group comparison was conducted in patients with seasonal allergic rhinitis. Patients were administered Emedastine patches (4 or 8 mg), placebo, or levocetirizine hydrochloride (5 mg tablet) once daily for 2 weeks (double-dummy technique). The primary objective was superiority to placebo by the change of the total nasal symptom score (sneezing, rhinorrhea, and nasal congestion) in Week 2. Levocetirizine was a reference drug and not a comparator in this study. RESULTS: A total of 1276 patients were randomized to receive the 4 mg emedastine patch (n = 384), 8 mg emedastine patch (n = 382), placebo (n = 384), or levocetirizine (n = 126). The least squares mean (LSM) of the change from baseline of the total nasal symptom score (TNSS) in Week 2 was significantly larger in both emedastine patch groups than in the placebo group (adjusted p < 0.0001). In secondary analysis, LSM of the change in the TNSS was -1.20, -1.49, -0.44, and -1.32 in the 4 mg emedastine patch, 8 mg patch, placebo, and levocetirizine, respectively. Reductions in the number of episodes and scores of individual nasal symptoms were all significantly larger throughout the day in the emedastine patch groups than the placebo group (all p < 0.05). No clinically significant safety problems occurred. CONCLUSIONS: The emedastine patch (4 and 8 mg) effectively and safely controlled symptoms of seasonal allergic rhinitis with sustained action throughout the day. STUDY REGISTRATION: JapicCTI-153092.

Validation of the Modified Fatigue Impact Scale and the relationships among fatigue, pain and serum interleukin-6 levels in patients with neuromyelitis optica spectrum disorder.

Fatigue and pain are disabling symptoms in patients with neuromyelitis optica spectrum disorder (NMOSD). The Modified Fatigue Impact Scale (MFIS) has not yet been validated in patients with NMOSD, and anti-interleukin-6 (IL-6) receptor antibody was reported to decrease pain and fatigue in patients with NMOSD. The aim of this study was to validate MFIS and to investigate the relationships among fatigue, pain and serum IL-6 levels in patients with NMOSD. MFIS and the Multidimensional Fatigue Inventory (MFI), an established scale for fatigue, were administered to patients with NMOSD and age- and sex-matched healthy controls (HCs). The Pain Effects Scale score and serum IL-6 levels were also measured in patients with NMOSD. Correlations among clinical characteristics, laboratory data and each score were investigated. To validate MFIS in patients with NMOSD, MFIS was administered twice within 4days from the first administration. Fifty-one patients answered the first MFIS, and 26 patients answered the second MFIS. There was no difference between the first and second MFIS scores. Patients with NMOSD had higher MFIS and MFI scores than HCs. No correlations were observed between serum IL-6 levels and either score. MFIS was validated in patients with NMOSD. Serum IL-6 levels may not be involved in the pathogenesis of fatigue and pain in patients with NMOSD.

Optimum Dose of Once-Daily Oxybutynin Patch in Japanese Patients with Overactive Bladder: A Randomized Double-Blind Trial Versus Placebo.

OBJECTIVE: To evaluate the efficacy, safety, and optimum dose of once-daily oxybutynin patch for overactive bladder. METHODS: A randomized double-blind trial was conducted in patients with overactive bladder symptoms for ≥24 weeks, who received an oxybutynin patch (73.5 or 105 mg) or placebo once daily for 8 weeks. The primary endpoint was the change in the daily frequency of micturition from baseline to the end of study. RESULTS: A total of 579 patients were randomized to the placebo group (n = 164), 73.5 mg oxybutynin patch group (n = 166), and 105 mg oxybutynin patch group (n = 165). The frequency of micturition (mean ± standard deviation) decreased by 1.19 ± 1.80 in the placebo group, 1.87 ± 1.93 in the 73.5 mg group, and 1.80 ± 1.76 in the 105 mg group. Compared with the placebo group, micturition decreased significantly in the 73.5 mg and 105 mg groups (t-test: P = 0.0025 and 0.0039, respectively), while the decrease was similar in both oxybutynin groups. The oxybutynin groups showed significant improvement of urgency, urge incontinence, incontinence, nocturia, mean voided volume, and five or six domains of the King's Health Questionnaire. Dry mouth was noted in 12.1% of the 73.5 mg group and 13.3% of the 105 mg group. Constipation was comparable between the oxybutynin groups and the placebo group. Application site reactions were less frequent in the 73.5 mg group than the 105 mg group. CONCLUSION: The efficacy of oxybutynin patch was confirmed, and the optimum dose for Japanese patients with overactive bladder was 73.5 mg.

Temperature receptors in cutaneous nerve endings are thermostat molecules that induce thermoregulatory behaviors against thermal load.

When skin temperature falls below a set-point, mammals experience "cold in the skin" and exhibit heat-seeking behaviors for error correction. Physiological thermostats should perform the behavioral thermoregulation, and it is important to identify the thermostats. A classical model of the sensory system states that thermoreceptors (e.g., thermoTRPs) in skin nerve endings are sensors that transform temperature into the firing rate codes that are sent to the brain, where the codes are decoded as "cold" by a labeled line theory. However, the view that the temperature code is transformed into "cold" (not temperature) is conflicting. Another model states that a thermostat exists in the brain based on the view that a skin thermo-receptor is a sensor. However, because animals have no knowledge of the principle of temperature measurement, the brain is unable to measure skin temperature with a thermometer calibrated based on a code table of each sensor in the skin. Thus, these old models cannot identify the thermostats. We have proposed a new model in which temperature receptors in a nerve ending are molecules of the thermostats. When skin temperature falls below a set-point, these molecules as a whole induce impulses as command signals sent to the brain, where these impulses activate their target neurons for "cold" and heat-seeking behaviors for error correction. Our study challenges the famous models that sensory receptor is a sensor and the brain is a code processor.

Prostaglandin transporter in the rat brain: its localization and induction by lipopolysaccharide.

Prostaglandin E2 (PGE2) is produced in the brain during infectious/inflammatory diseases, and it mediates acute-phase responses including fever. In the recovery phase of such diseases, PGE2 disappears from the brain through yet unidentified mechanisms. Rat prostaglandin transporter (PGT), which facilitates transmembrane transport of PGE2, might be involved in the clearance of PGE2 from the brain. Here, we examined the cellular localization of PGT mRNA and its protein in the brains of untreated rats and those injected intraperitoneally with a pyrogen lipopolysaccharide (LPS) or saline. PGT mRNA was weakly expressed in the arachnoid membrane of untreated rats and saline-injected ones, but was induced in blood vessels of the subarachnoidal space and choroid plexus and in arachnoid membrane at 5 h and 12 h after LPS injection. In the same type of cells, PGT-like immunoreactivity was found in the cytosol and cell membrane even under nonstimulated conditions, and its level was also elevated after LPS injection. PGT-positive cells in blood vessels were identified as endothelial cells. In most cases, PGT was not colocalized with cyclooxygenase-2, a marker of prostaglandin-producing cells. The PGE2 level in the cerebrospinal fluid reached its peak at 3 h after LPS, and then dropped over 50% by 5 h, which time point coincides with the maximum PGT mRNA expression and enhanced level of PGT protein. These results suggest that PGT is involved in the clearance of PGE2 from the brain during the recovery phase of LPS-induced acute-phase responses.

Efficacy and safety of once-daily oxybutynin patch versus placebo and propiverine in Japanese patients with overactive bladder: A randomized double-blind trial.

OBJECTIVES: To evaluate the efficacy and safety of once-daily oxybutynin patch therapy for overactive bladder. METHODS: A randomized double-blind trial was carried out in patients with overactive bladder syndrome, who received an oxybutynin patch, propiverine (20 mg) or placebo once daily for 12 weeks. The primary efficacy end-point was the change of the mean daily number of micturitions in week 12. RESULTS: A total of 1530 patients were randomized to receive the oxybutynin patch (573), propiverine (576) or placebo (381). The change of the mean daily frequency of micturition from baseline in the full analysis set was -1.89 ± 2.04 with the oxybutynin patch, which was significantly higher than with placebo (-1.44 ± 2.23) (P = 0.0015). The difference of the mean change in the mean daily number of micturitions between the oxybutynin patch and propiverine groups showed a 95% confidence interval of -0.28 to 0.21, and the upper limit of this interval was below the predefined non-inferiority margin of 0.37, showing non-inferiority of the oxybutynin patch to propiverine. The incidence of dry mouth and constipation was higher with propiverine than with the oxybutynin patch or placebo. Application site dermatitis was more frequent with the oxybutynin patch (31.8%) than with propiverine (5.9%) or placebo (5.2%), but the dermatitis was generally mild. CONCLUSION: This trial shows the efficacy of the new once-daily oxybutynin patch for overactive bladder. Despite a higher rate of dermatitis with the oxybutynin patch, dry mouth and constipation occurs less often than during treatment with propiverine.

Sensory stimuli induce nuclear translocation and phosphorylation of nuclear factor κ B in primary sensory neurons of mice.

Nuclear factor kappa B (NF-κB) is a transcription factor, which is translocated to the nucleus when activated. Herein, we demonstrate immunohistochemically that electrical, chemical, and thermal stimuli, applied to the skin of mice, all induced nuclear translocation and phosphorylation of NF-κB in dorsal root ganglia (DRG) neurons. The latency of this response was short, with effects observable in as little as 3min following stimulation. Few nuclear phospho-NF-κB-positive neurons were observed in DRG innervating unstimulated regions. These results suggest somatosensory stimuli quickly induce NF-κB-mediated gene transcription in DRG, and phospho-NF-κB could be a suitable histological marker for activated DRG neurons.

Cooling-sensitive TRPM8 is thermostat of skin temperature against cooling.

We have shown that cutaneous cooling-sensitive receptors can work as thermostats of skin temperature against cooling. However, molecule of the thermostat is not known. Here, we studied whether cooling-sensitive TRPM8 channels act as thermostats. TRPM8 in HEK293 cells generated output (y) when temperature (T) was below threshold of 28.4°C. Output (y) is given by two equations: At T >28.4°C, y = 0; At T <28.4°C, y  =  -k(T - 28.4°C). These equations show that TRPM8 is directional comparator to elicits output (y) depending on negative value of thermal difference (ΔT  =  T - 28.4°C). If negative ΔT-dependent output of TRPM8 in the skin induces responses to warm the skin for minimizing ΔT recursively, TRPM8 acts as thermostats against cooling. With TRPM8-deficient mice, we explored whether TRPM8 induces responses to warm the skin against cooling. In behavioral regulation, when room temperature was 10°C, TRPM8 induced behavior to move to heated floor (35°C) for warming the sole skin. In autonomic regulation, TRPM8 induced activities of thermogenic brown adipose tissue (BAT) against cooling. When menthol was applied to the whole trunk skin at neutral room temperature (27°C), TRPM8 induced a rise in core temperature, which warmed the trunk skin slightly. In contrast, when room was cooled from 27 to 10°C, TRPM8 induced a small rise in core temperature, but skin temperature was severely reduced in both TRPM8-deficient and wild-type mice by a large heat leak to the surroundings. This shows that TRPM8-driven endothermic system is less effective for maintenance of skin temperature against cooling. In conclusion, we found that TRPM8 is molecule of thermostat of skin temperature against cooling.

Intragastric administration of allyl isothiocyanate increases carbohydrate oxidation via TRPV1 but not TRPA1 in mice.

The transient receptor potential (TRP) channel family is composed of a wide variety of cation-permeable channels activated polymodally by various stimuli and is implicated in a variety of cellular functions. Recent investigations have revealed that activation of TRP channels is involved not only in nociception and thermosensation but also in thermoregulation and energy metabolism. We investigated the effect of intragastric administration of TRP channel agonists on changes in energy substrate utilization of mice. Intragastric administration of allyl isothiocyanate (AITC; a typical TRPA1 agonist) markedly increased carbohydrate oxidation but did not affect oxygen consumption. To examine whether TRP channels mediate this increase in carbohydrate oxidation, we used TRPA1 and TRPV1 knockout (KO) mice. Intragastric administration of AITC increased carbohydrate oxidation in TRPA1 KO mice but not in TRPV1 KO mice. Furthermore, AITC dose-dependently increased intracellular calcium ion concentration in cells expressing TRPV1. These findings suggest that AITC might activate TRPV1 and that AITC increased carbohydrate oxidation via TRPV1.

Activation of transient receptor potential ankyrin 1 by hydrogen peroxide.

Hydrogen peroxide (H(2)O(2)), which is contained in industrial products, is also generated within cells. H(2)O(2) causes pain but it has not been elucidated how it activates sensory neurons in the pain pathway. Here we show that transient receptor potential ankyrin 1 (TRPA1), expressed by sensory neurons in the pain pathway, is a receptor for H(2)O(2). H(2)O(2) activated mouse TRPA1 to induce Ca(2+) influx and elicit non-selective cation currents. These effects of H(2)O(2) were mimicked by both reactive oxygen species and reactive nitrogen species. Cysteine-reducing agents suppressed H(2)O(2)-induced TRPA1 activation, whereas cysteine-oxidizing agents activated TRPA1. H(2)O(2) caused Ca(2+) influx in a subset of dorsal root ganglia neurons, which responded to allyl isothiocyanate, a TRPA1 ligand. These results suggest that TRPA1 might be involved in the sensation of pain caused by H(2)O(2).

Application of menthol to the skin of whole trunk in mice induces autonomic and behavioral heat-gain responses.

When ambient temperature is decreased in mammals, autonomic and behavioral heat-gain responses occur to maintain their core temperatures. However, what molecules in cutaneous sensory nerve endings mediate cooling-induced responses is unclear. Recently, transient receptor potential melastatin-8 (TRPM8) has been identified in cell bodies of sensory neurons as low-temperature and menthol-activated cation channel. We hypothesized that TRPM8 mediates cooling-induced autonomic and behavioral heat-gain responses. To activate TRPM8 specifically, we applied 1-10% menthol to the skin of whole trunk in mice instead of cooling and measured core temperatures and autonomic and behavioral heat-gain responses. Solvent of menthol (100% ethanol) was used as control. Significant elevation of core temperatures was observed between 20 and 120 min after menthol application. Pretreatment with diclofenac sodium, an antipyretic drug, did not affect this hyperthermia, indicating that the menthol-induced hyperthermia is not fever. Menthol application induced a rise in oxygen consumption, shivering-like muscle activity, tail skin vasoconstriction (autonomic responses), and heat-seeking behavior. All of them are typical heat-gain responses. These results support the hypothesis that TRPM8 mediates cooling-induced autonomic and behavioral heat-gain responses.

Cold sensitivity of recombinant TRPA1 channels.

TRPM8 and TRPA1, members of the transient receptor potential (TRP) channel family, are candidates for cooling-activated receptors. It is accepted that TRPM8 responds to moderate cooling, although it is controversial whether TRPA1 responds to deep cooling. Here, using Ca(2+) imaging and/or patch-clamp recordings, we examined the thermal sensitivity of primary cultured dorsal root ganglion (DRG) neurons and mouse TRPA1-expressing human embryonic kidney (HEK) 293 cells. In a subset of cultured mouse DRG neurons, deep cooling (5-18 degrees C) and allyl isothiocyanate (AITC, agonist of TRPA1) induced increases in intracellular Ca(2+) level. Most AITC-sensitive (TRPA1-expressing) neurons responded to deep cooling. In TRPA1-expressing HEK293 cells, deep cooling and AITC-induced Ca(2+) responses and whole-cell currents. In inside-out patches excised from TRPA1-expressing HEK293 cells, deep cooling, and AITC activated the same channels, which were inhibited by camphor (antagonist for TRPA1). When temperature was decreased below 18 degrees C, unit conductance of the channel decreased but open probability of it increased. Deep cooling-induced increase of the open probability of TRPA1 may underlie the increase in whole-cell currents induced by deep cooling. It is concluded that TRPA1 is a deep cooling-activated channel, which supports the previous findings that TRPA1 responds to deep cooling.

Transient receptor potential channel TRPM8 agonists stimulate calcium influx and neurotensin secretion in neuroendocrine tumor cells.

TRPM8 is a member of the melastatin-type transient receptor potential ion channel family. Activation by cold or by agonists (menthol, icilin) induces a transient rise in intracellular free calcium concentration ([Ca(2+)](i)). Our previous study demonstrated that Ca(2+)-permeable cation channels play a role in IGF-1-induced secretion of chromogranin A in human neuroendocrine tumor (NET) cell line BON [Mergler et al.: Neuroendocrinology 2006;82:87-102]. Here, we extend our earlier study by investigating the expression of TRPM8 and characterizing its impact on [Ca(2+)](i) and the secretion of neurotensin (NT). We identified TRPM8 expression in NET BON cells by RT-PCR, Western blotting and immunofluorescence staining. Icilin increased [Ca(2+)](i) in TRPM8-transfected human embryonic kidney cells (HEK293) but not in mock-transfected cells. Icilin and menthol induced Ca(2+) transients in BON cells as well as in primary NET cell cultures of two different pancreatic NETs as detected by single cell fluorescence imaging. Icilin increased non-selective cation channel currents in BON cells as detected by patch-clamp recordings. This activation was associated with increased NT secretion. Taken together, this study demonstrates for the first time the expression TRPM8 in NET cells and its role in regulating [Ca(2+)](i) and NT secretion. The regulation of NT secretion in NETs by TRPM8 may have a potential clinical implication in diagnosis or therapy.

Cellular and molecular bases of the initiation of fever.

All phases of lipopolysaccharide (LPS)-induced fever are mediated by prostaglandin (PG) E2. It is known that the second febrile phase (which starts at approximately 1.5 h post-LPS) and subsequent phases are mediated by PGE2 that originated in endotheliocytes and perivascular cells of the brain. However, the location and phenotypes of the cells that produce PGE2 triggering the first febrile phase (which starts at approximately 0.5 h) remain unknown. By studying PGE2 synthesis at the enzymatic level, we found that it was activated in the lung and liver, but not in the brain, at the onset of the first phase of LPS fever in rats. This activation involved phosphorylation of cytosolic phospholipase A2 (cPLA2) and transcriptional up-regulation of cyclooxygenase (COX)-2. The number of cells displaying COX-2 immunoreactivity surged in the lung and liver (but not in the brain) at the onset of fever, and the majority of these cells were identified as macrophages. When PGE2 synthesis in the periphery was activated, the concentration of PGE2 increased both in the venous blood (which collects PGE2 from tissues) and arterial blood (which delivers PGE2 to the brain). Most importantly, neutralization of circulating PGE2 with an anti-PGE2 antibody both delayed and attenuated LPS fever. It is concluded that fever is initiated by circulating PGE2 synthesized by macrophages of the LPS-processing organs (lung and liver) via phosphorylation of cPLA2 and transcriptional up-regulation of COX-2. Whether PGE2 produced at the level of the blood-brain barrier also contributes to the development of the first phase remains to be clarified.

Ca2+-dependent PKC activation mediates menthol-induced desensitization of transient receptor potential M8.

In 1950, Hensel and Zotterman reported cooling-induced desensitization of cold receptors by extracellular discharge recordings of cold fibers. Since then, however, its intracellular mechanism has remained unresolved. We studied menthol-induced desensitization of cold/menthol receptors (TRPM8, transient receptor potential M8) expressed in HEK cells. TRPM8 desensitization depended on extracellular Ca2+ ions, indicating that Ca2+ influx-induced [Ca2+]i elevation caused the desensitization. We studied whether Ca2+-dependent kinase, PKC, mediated TRPM8 desensitization. PMA, a PKC activator, desensitized TRPM8. Inhibitor of Ca2+-dependent PKC isozymes specifically abolished PMA-induced TRPM8 desensitization. PMA similarly desensitized wild type TRPM8 and mutant TRPM8, in which serine or threonine residues in some putative PKC phosphorylation sites were replaced by alanine. PMA treatment did not induce internalization of TRPM8. As the basis of cooling-induced desensitization of cold receptors, we conclude that cooling-activated TRPM8 causes Ca2+-dependent PKC isozymes to desensitize TRPM8 itself.

Direct pyrogenic input from prostaglandin EP3 receptor-expressing preoptic neurons to the dorsomedial hypothalamus.

Fever is induced by a neuronal mechanism in the brain. Prostaglandin (PG) E2 acts as a pyrogenic mediator in the preoptic area (POA) probably through the EP3 subtype of PGE receptor expressed on GABAergic neurons, and this PGE2 action triggers neuronal pathways for sympathetic thermogenesis in peripheral effector organs including brown adipose tissue (BAT). To explore pyrogenic efferent pathways from the POA, we determined projection targets of EP3 receptor-expressing POA neurons with a special focus on rat hypothalamic regions including the dorsomedial hypothalamic nucleus (DMH), which is known as a center for autonomic responses to stress. Among injections of cholera toxin b-subunit (CTb), a retrograde tracer, into hypothalamic regions at the rostrocaudal level of the DMH, injections into the DMH, lateral hypothalamic area (LH) and dorsal hypothalamic area (DH) resulted in EP3 receptor immunolabelling in substantial populations of CTb-labeled neurons in the POA. Bilateral microinjections of muscimol, a GABA(A) receptor agonist, into the DMH and a ventral region of the DH, but not those into the LH, inhibited thermogenic (BAT sympathetic nerve activity, BAT temperature, core body temperature and expired CO2) and cardiovascular (arterial pressure and heart rate) responses to an intra-POA PGE2 microinjection. Further immunohistochemical observations revealed a close association of POA-derived GABAergic axon swellings with DMH neurons projecting to the medullary raphe regions where sympathetic premotor neurons for febrile and thermoregulatory responses are localized. These results suggest that a direct projection of EP3 receptor-expressing POA neurons to the DMH/DH region mediates febrile responses via a GABAergic mechanism.

TRPM8 protein localization in trigeminal ganglion and taste papillae.

TRPM8 is a TRP family cation channel which can be activated by cold stimuli or l-menthol. However, TRPM8 protein localization of nerve terminals in sensory organs remains unknown. Here we generated an antibody against TRPM8 and analyzed TRPM8 protein localization in trigeminal ganglia (TG) and in sensory nerve fibers in the tongue. TRPM8 immunoreactivity was detected in a subset of neurons with a small diameter in TG and in nerve fibers in the tongue. TRPM8-immunoreactive nerve fibers were rich in fungiform papillae, but sparse in foliate and circumvallate papillae. The TRPM8-immunoreactive nerve fibers reached the outer epithelial layer in each papilla, while no TRPM8-immunoreactive nerve fibers penetrated into taste buds. Double labeling analysis revealed that TRPM8 immunoreactivity was co-expressed with a part of TRPV1 or CGRP-immunoreactive neurons in TG. However, TRPM8 immunoreactivity was not observed in TRPV1- or CGRP-positive nerve fibers in fungiform, foliate, and circumvallate papillae. These results suggest that TRPM8 protein is present in sensory lingual nerve fibers mainly projected from TG and might work as cold and l-menthol receptors on tongue.

Visualization of synaptic Ca2+ /calmodulin-dependent protein kinase II activity in living neurons.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the CNS and critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, because of a lack of specific methods. We tried to address this by optically detecting the conformational change in CaMKII during activation using fluorescence resonance energy transfer (FRET). The engineered FRET probe Camuialpha detects calmodulin binding and autophosphorylation at threonine 286 that renders the enzyme constitutively active. In combination with two-photon microscopy, we demonstrate that Camuialpha can be used to observe temporal and spatial regulation of CaMKII activity in living neurons.