The Inhibition of TREK-1 K+ Channels via Multiple Compounds Contained in the Six Kamikihito Components, Potentially Stimulating Oxytocin Neuron Pathways.

Oxytocin, a significant pleiotropic neuropeptide, regulates psychological stress adaptation and social communication, as well as peripheral actions, such as uterine contraction and milk ejection. Recently, a Japanese Kampo medicine called Kamikihito (KKT) has been reported to stimulate oxytocin neurons to induce oxytocin secretion. Two-pore-domain potassium channels (K2P) regulate the resting potential of excitable cells, and their inhibition results in accelerated depolarization that elicits neuronal and endocrine cell activation. We assessed the effects of KKT and 14 of its components on a specific K2P, the potassium channel subfamily K member 2 (TREK-1), which is predominantly expressed in oxytocin neurons in the central nervous system (CNS). KKT inhibited the activity of TREK-1 induced via the channel activator ML335. Six of the 14 components of KKT inhibited TREK-1 activity. Additionally, we identified that 22 of the 41 compounds in the six components exhibited TREK-1 inhibitory effects. In summary, several compounds included in KKT partially activated oxytocin neurons by inhibiting TREK-1. The pharmacological effects of KKT, including antistress effects, may be partially mediated through the oxytocin pathway.

A multi-centre, double-blind, randomized, placebo-controlled trial to evaluate the effectiveness and safety of ramelteon for the prevention of postoperative delirium in elderly cancer patients: a study protocol for JORTC-PON2/J-SUPPORT2103/NCCH2103.

Postoperative delirium is an important issue in cancer patients, affecting surgical outcomes and the quality of life. Ramelteon is a melatonin receptor agonist with high affinity for MT1 and MT2 receptors. Clinical trials and observational studies in Japan, including in surgical cancer patients, have shown efficacy of ramelteon in delirium prevention, with no serious safety concerns. However, clinical trials from the USA have reported conflicting results. A Japanese phase II study investigated the efficacy and safety of ramelteon for delirium prevention following gastrectomy in patients aged ≥75 years, with findings suggesting the feasibility of a phase III trial. The aim of this multi-centre, double-blind, randomized placebo-controlled phase III trial is to evaluate the effectiveness and safety of oral ramelteon for postoperative delirium prevention in cancer patients aged ≥65 years as advanced medical care. The trial protocol is described here.

Therapeutic Potential of Orally Administered Rubiscolin-6.

Rubiscolins are naturally occurring opioid peptides derived from the enzymatic digestion of the ribulose bisphosphate carboxylase/oxygenase protein in spinach leaves. They are classified into two subtypes based on amino acid sequence, namely rubiscolin-5 and rubiscolin-6. In vitro studies have determined rubiscolins as G protein-biased delta-opioid receptor agonists, and in vivo studies have demonstrated that they exert several beneficial effects via the central nervous system. The most unique and attractive advantage of rubiscolin-6 over other oligopeptides is its oral availability. Therefore, it can be considered a promising candidate for the development of a novel and safe drug. In this review, we show the therapeutic potential of rubiscolin-6, mainly focusing on its effects when orally administered based on available evidence. Additionally, we present a hypothesis for the pharmacokinetics of rubiscolin-6, focusing on its absorption in the intestinal tract and ability to cross the blood-brain barrier.

Distinct Profiles of Desensitization of µ-Opioid Receptors Caused by Remifentanil or Fentanyl: In Vitro Assay with Cells and Three-Dimensional Structural Analyses.

Remifentanil (REM) and fentanyl (FEN) are commonly used analgesics that act by activating a µ-opioid receptor (MOR). Although optimal concentrations of REM can be easily maintained during surgery, it is sometimes switched to FEN for optimal pain regulation. However, standards for this switching protocol remain unclear. Opioid anesthetic efficacy is decided in part by MOR desensitization; thus, in this study, we investigated the desensitization profiles of REM and FEN to MOR. The efficacy and potency during the 1st administration of REM or FEN in activating the MOR were almost equal. Similarly, in ß arrestin recruitment, which determines desensitization processes, they showed no significant differences. In contrast, the 2nd administration of FEN resulted in a stronger MOR desensitization potency than that of REM, whereas REM showed a higher internalization potency than FEN. These results suggest that different ß arrestin-mediated signaling caused by FEN or REM led to their distinct desensitization and internalization processes. Our three-dimensional analysis, with in silico binding of REM and FEN to MOR models, highlighted that REM and FEN bound to similar but distinct sites of MOR and led to distinct ß arrestin-mediated profiles, suggesting that distinct binding profiles to MOR may alter ß arrestin activity, which accounts for MOR desensitization and internalization.

Discovery of 7-Azanorbornane-Based Dual Agonists for the Delta and Kappa Opioid Receptors through an In Situ Screening Protocol.

In medicinal chemistry, the copper-catalyzed click reaction is used to prepare ligand candidates. This reaction is so clean that the bioactivities of the products can be determined without purification. Despite the advantages of this in situ screening protocol, the applicability of this method for transmembrane proteins has not been validated due to the incompatibility with copper catalysts. To address this point, we performed ligand screening for the µ, δ, and κ opioid receptors using this protocol. As we had previously reported the 7-azanorbornane skeleton as a privileged scaffold for the G protein-coupled receptors, we performed the click reactions between various 7-substituted 2-ethynyl-7-azanorbornanes and azides. Screening assays were performed without purification using the CellKeyTM system, and the putative hit compounds were re-synthesized and re-evaluated. Although the "hit" compounds for the µ and the δ receptors were totally inactive after purifications, three of the four "hits" for the κ receptor were true agonists for this receptor and also showed activities for the δ receptor. Although false positive/negative results exist as in other screening projects for soluble proteins, this in situ method is effective in identifying novel ligands for transmembrane proteins.

Effects of an oral mucosa protective formulation on chemotherapy- and/or radiotherapy-induced oral mucositis: a prospective study.

BACKGROUND: Oral mucositis (OM) associated with cancer treatment not only impairs patients' quality of life but also causes treatment delays or changes. This prospective exploratory study was conducted to evaluate the efficacy of Episil® oral liquid, which is an approved protective formulation for the oral mucosa in patients with OM. The extent of the pain-relieving effect, feeling during use, and adverse events or problems were evaluated. METHODS: In total, 10 Japanese cancer patients with OM receiving chemotherapy, pretreatment therapy for hematopoietic stem cell transplantation, or radiation therapy for head and neck cancer were enrolled. RESULTS: A numerical rating scale (NRS) was used to assess oral pain intensity due to OM. Compared to baseline, the mean NRS began to decrease at 5 min after using Episil® (7.1 ± 1.4 to 4.6 ± 2.87; p = 0.264). A significant decrease was observed in the pain score after using Episil® compared with that before using Episil®, and this effect lasted up to 120 min. The protective effects of Episil® were observed 3-5 min after application. Some patients felt slight soreness or discomfort when applying Episil®. However, this discomfort due to Episil®'s stimulation was within the allowable range and transient. No adverse events were observed in any of the cases. CONCLUSIONS: The results of this prospective study showed that Episil® could be an effective treatment to relieve oral pain in Japanese patients with moderate to severe OM, and this newly approved product might adequately support patients' oral intake. TRIAL REGISTRATION: University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) ( UMIN000031921 ).

Involvement of spinal G-protein inwardly rectifying potassium (GIRK) channels in the enhanced antinociceptive effects of the activation of both µ-opioid and cannabinoid CB1 receptors.

Neuropathic pain is refractory to opioid analgesics. Since there are functional linkages between µ-opioid receptors (MOR) and cannabinoid receptors (CBR), the present study was designed to investigate the interactions between MOR and CB1R based on antinociceptive effects for neuropathic pain mediated through G protein-coupled inwardly-rectifying potassium channels (GIRKs). The antinociceptive effects against pseudonociceptive response or neuropathic pain of MOR and CBR agonists were assessed in mice with or without partial sciatic nerve ligation. To investigate the functional interaction between MOR and CB1R, electrophysiological recording through GIRK was performed using the two-electrode voltage-clamp method in oocytes along with Western blotting in the spinal cord of mice. Co-administration of the MOR agonist DAMGO and the CB1R agonist CP55,940 augmented inwardly rectifying K+ currents in Xenopus oocytes co-expressing MOR, CB1R and GIRK1/2. Further, combination of morphine and the CBR agonist WIN-55,212-2 produced prominent antinociceptive effects in an i.t. GIRK1 inhibitor-reversible manner. Furthermore, CB1R was upregulated under neuropathic pain in the spinal cord, and such upregulation and antinociceptive effects were not altered by repeated treatment with morphine plus WIN-55,212-2. Our findings suggest that co-administration of MOR and CBR agonists could enhance their antinociceptive effects through GIRK1 in the spinal cord of mice.

Prevention of delirium with agitation by yokukansan in older adults after cancer surgery.

OBJECTIVE: Preventing postoperative delirium with agitation is vital in the older population. We examined the preventive effect of yokukansan on postoperative delirium with agitation in older adult patients undergoing highly invasive cancer resection. METHODS: We performed a secondary per-protocol analysis of 149 patients' data from a previous clinical trial. Patients underwent scheduled yokukansan or placebo intervention 4-8 days presurgery and delirium assessment postoperatively. Delirium with agitation in patients aged ≥75 years was assessed using the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition, and the Japanese version of the Delirium Rating Scale-Revised-98. We assessed odds ratios for yokukansan (TJ-54) compared with placebo for the manifestation of postoperative delirium with agitation across patients of all ages (n = 149) and those aged ≥65 years (n = 82) and ≥ 75 years (n = 21) using logistic regression. RESULTS: Delirium with agitation manifested in 3/14 and 5/7 patients in the TJ-54 and placebo groups, respectively, among those aged ≥75 years. The odds ratio for yokukansan vs. placebo was 0.11 (95% confidence interval: 0.01-0.87). An age and TJ-54 interaction effect was detected in patients with delirium with agitation. No intergroup differences were observed in patients aged ≥65 years or across all ages for delirium with agitation. CONCLUSIONS: This is the first study investigating the preventive effect of yokukansan on postoperative delirium with agitation in older adults. Yokukansan may alleviate workforce burdens in older adults caused by postoperative delirium with agitation following highly invasive cancer resection.

Novel Opioid Analgesics for the Development of Transdermal Opioid Patches That Possess Morphine-Like Pharmacological Profiles Rather Than Fentanyl: Possible Opioid Switching Alternatives Among Patch Formula.

BACKGROUND: Transdermal fentanyl is widely used in the treatment of severe pain because of convenience, safety, and stable blood concentrations. Nevertheless, patients often develop tolerance to fentanyl, necessitating the use of other opioids; transdermal buprenorphine patch is widely used as an analgesic agent, though available formulation does not provide comparable analgesic effect as transdermal fentanyl patch. Opioids bind to the opioid receptor (OR) to activate both G protein-mediated and ß-arrestin-mediated pathways. We synthesized morphine-related compounds with high transdermal absorbability (N1 and N2) and evaluated their OR activities pharmacologically in comparison with fentanyl and morphine. METHODS: In cells stably expressing µ-opioid receptor (MOR), δ-opioid receptor (DOR), and κ-opioid receptor (KOR), G protein-mediated pathways were assessed using the CellKey and an intracellular cyclic adenosine monophosphate (cAMP) assay, while ß-arrestin-mediated pathways were analyzed with ß-arrestin recruitment and receptor internalization assays. Furthermore, analgesic effects were evaluated using a tail-flick test in mice, and the analgesic effect on fentanyl-tolerant mice was evaluated. RESULTS: In the CellKey and cAMP assays, both N1 and N2 showed the highest affinity for MOR and acted as full agonists as well as partial agonists for DOR and KOR. In the ß-arrestin and internalization assays, only fentanyl acted as a full agonist; N1 and N2 acted as partial agonists of MOR. In the mouse tail-flick test, N1 and N2 showed analgesic effects equivalent to those of fentanyl and morphine. In fentanyl-tolerant mice, fentanyl showed a diminished analgesic effect, whereas N1 and N2 as well as morphine retained their analgesic effects. CONCLUSIONS: While N1 and N2 have higher transdermal absorbability than fentanyl, they also have analgesic effects comparable to those of morphine, suggesting that they may be attractive compounds for the development of novel opioid patches for transitioning from fentanyl patches.

Evaluation of the Intracellular Signaling Activities of κ-Opioid Receptor Agonists, Nalfurafine Analogs; Focusing on the Selectivity of G-Protein- and ß-Arrestin-Mediated Pathways.

Opioid receptors (ORs) are classified into three types (µ, δ, and κ), and opioid analgesics are mainly mediated by µOR activation; however, their use is sometimes restricted by unfavorable effects. The selective κOR agonist nalfurafine was initially developed as an analgesic, but its indication was changed because of the narrow safety margin. The activation of ORs mainly induces two intracellular signaling pathways: a G-protein-mediated pathway and a ß-arrestin-mediated pathway. Recently, the expectations for κOR analgesics that selectively activate these pathways have increased; however, the structural properties required for the selectivity of nalfurafine are still unknown. Therefore, we evaluated the partial structures of nalfurafine that are necessary for the selectivity of these two pathways. We assayed the properties of nalfurafine and six nalfurafine analogs (SYKs) using cells stably expressing κORs. The SYKs activated κORs in a concentration-dependent manner with higher EC50 values than nalfurafine. Upon bias factor assessment, only SYK-309 (possessing the 3S-hydroxy group) showed higher selectivity of G-protein-mediated signaling activities than nalfurafine, suggesting the direction of the 3S-hydroxy group may affect the ß-arrestin-mediated pathway. In conclusion, nalfurafine analogs having a 3S-hydroxy group, such as SYK-309, could be considered G-protein-biased κOR agonists.

Variants That Affect Function of Calcium Channel TRPV6 Are Associated With Early-Onset Chronic Pancreatitis.

BACKGROUND & AIMS: Changes in pancreatic calcium levels affect secretion and might be involved in development of chronic pancreatitis (CP). We investigated the association of CP with the transient receptor potential cation channel subfamily V member 6 gene (TRPV6), which encodes a Ca2+-selective ion channel, in an international cohort of patients and in mice. METHODS: We performed whole-exome DNA sequencing from a patient with idiopathic CP and from his parents, who did not have CP. We validated our findings by sequencing DNA from 300 patients with CP (not associated with alcohol consumption) and 1070 persons from the general population in Japan (control individuals). In replication studies, we sequenced DNA from patients with early-onset CP (20 years or younger) not associated with alcohol consumption from France (n = 470) and Germany (n = 410). We expressed TRPV6 variants in HEK293 cells and measured their activity using Ca2+ imaging assays. CP was induced by repeated injections of cerulein in TRPV6mut/mut mice. RESULTS: We identified the variants c.629C>T (p.A210V) and c.970G>A (p.D324N) in TRPV6 in the index patient. Variants that affected function of the TRPV6 product were found in 13 of 300 patients (4.3%) and 1 of 1070 control individuals (0.1%) from Japan (odds ratio [OR], 48.4; 95% confidence interval [CI], 6.3-371.7; P = 2.4 × 10-8). Twelve of 124 patients (9.7%) with early-onset CP had such variants. In the replication set from Europe, 18 patients with CP (2.0%) carried variants that affected the function of the TRPV6 product compared with 0 control individuals (P = 6.2 × 10-8). Variants that did not affect the function of the TRPV6 product (p.I223T and p.D324N) were overrepresented in Japanese patients vs control individuals (OR, 10.9; 95% CI, 4.5-25.9; P = 7.4 × 10-9 for p.I223T and P = .01 for p.D324N), whereas the p.L299Q was overrepresented in European patients vs control individuals (OR, 3.0; 95% CI, 1.9-4.8; P = 1.2 × 10-5). TRPV6mut/mut mice given cerulein developed more severe pancreatitis than control mice, as shown by increased levels of pancreatic enzymes, histologic alterations, and pancreatic fibrosis. CONCLUSIONS: We found that patients with early-onset CP not associated with alcohol consumption carry variants in TRPV6 that affect the function of its product, perhaps by altering Ca2+ balance in pancreatic cells. TRPV6 regulates Ca2+ homeostasis and pancreatic inflammation.

Further investigation of the rapid-onset and short-duration action of the G protein-biased µ-ligand oliceridine.

TRV130 (oliceridine), a G protein-biased ligand for µ-opioid receptor, has recently been synthesized. It is considered to have strong antinociceptive effects and only minor adverse effects. However, whether or not oliceridine actually exhibits an ideal pharmacological profile as an analgesic has not yet been fully clarified in animal studies. This study examined the pharmacological profile of oliceridine in cells and animals. Oliceridine (10 µM) did not produce any µ-opioid receptor internalization in cells even though it increased impedance, which reflects the activation of Gi protein using the CellKey™ system, and inhibited the formation of cAMP. In mice, oliceridine (0.3-10 mg/kg) produced a dose-dependent antinociceptive effect with a rapid-onset and short-duration action in the hot-plate test, as well as antihyperalgesia after sciatic nerve ligation without the development of antinociceptive tolerance using the thermal hyperalgesia test. On the other hand, oliceridine inhibited gastrointestinal transit. Furthermore, oliceridine produced rapid-onset hyperlocomotion at antinociceptive doses; sensitization developed in mice and an emetic effect was observed in ferrets. These results indicate that, although oliceridine may produce dopamine-related behaviors even through selective stimulation of the G-protein-biased µ-opioid receptor pathway, it still offers advantages for breakthrough pain without antinociceptive tolerance with adequate doses.

Local Anesthetics Inhibit Transient Receptor Potential Vanilloid Subtype 3 Channel Function in Xenopus Oocytes.

BACKGROUND: The transient receptor potential vanilloid subtype 3 (TRPV3) channel is activated by innocuous temperature and several chemical stimuli. It is proposed to be involved in pathological pain development and is therefore considered a potential target for treating pain. Local anesthetics have been used for patients with both acute and chronic pain. Although blockage of the voltage-gated sodium channel is the primary mechanism by which local anesthetics exert their effects, they cannot be explained by this mechanism alone, especially in pathologic states such as chronic pain. Indeed, the effects of local anesthetics on multiple targets involved in the pain pathway have been reported. It has also been suggested that modulating the function of transient receptor potential (TRP) channels (eg, TRPV1 and transient receptor potential ankyrin 1 [TRPA1]) is one of the mechanisms of action of local anesthetics. However, the effects of local anesthetics on TRPV3 have not been reported. METHODS: We expressed TRPV3 in Xenopus oocytes and investigated the effects of local anesthetics on 2-aminoethoxydiphenyl borate (2APB)-induced currents using 2-electrode voltage-clamp techniques. RESULTS: Clinically used local anesthetics inhibited the 2APB-activated currents from the TRPV3 channel in a concentration-dependent manner at pharmacologically relevant concentrations with half maximal inhibitory concentration (IC50) values of 2.5 (lidocaine), 1.4 (mepivacaine), 0.28 (ropivacaine), and 0.17 (bupivacaine) mmol/L, respectively. Conversely, these local anesthetics also directly induced currents at higher concentrations, although these currents were quite small compared to the 2APB-induced currents. We found that the inhibition of TRPV3 by lidocaine is noncompetitive and independent of intracellular signaling cascades. 2APB-induced TRPV3 currents were reduced by extracellular N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) but not by intracellular QX-314 nor benzocaine. Moreover, lidocaine showed a use-dependent block in TRPV3 inhibition. Finally, QX-314 appeared to slightly permeate the activated TRPV3 channel pore based on examination of oocytes coexpressing TRPV3 and a sodium channel. These results suggest that local anesthetics could inhibit TRPV3 channel function by extracellular interactions of their charged forms with the channel pore. CONCLUSIONS: Local anesthetics inhibited TRPV3 2APB-induced currents at pharmacologically relevant concentrations when TRPV3 was expressed in Xenopus oocytes. These effects seem to occur via an extracellular interaction between the charged form of the anesthetic with the TRPV3 channel pore. These results help to elucidate the mechanisms of action of local anesthetics.

In Vitro Analyses of Spinach-Derived Opioid Peptides, Rubiscolins: Receptor Selectivity and Intracellular Activities through G Protein- and ß-Arrestin-Mediated Pathways.

Activated opioid receptors transmit internal signals through two major pathways: the G-protein-mediated pathway, which exerts analgesia, and the ß-arrestin-mediated pathway, which leads to unfavorable side effects. Hence, G-protein-biased opioid agonists are preferable as opioid analgesics. Rubiscolins, the spinach-derived naturally occurring opioid peptides, are selective δ opioid receptor agonists, and their p.o. administration exhibits antinociceptive effects. Although the potency and effect of rubiscolins as G-protein-biased molecules are partially confirmed, their in vitro profiles remain unclear. We, therefore, evaluated the properties of rubiscolins, in detail, through several analyses, including the CellKeyTM assay, cADDis® cAMP assay, and PathHunter® ß-arrestin recruitment assay, using cells stably expressing µ, δ, κ, or µ/δ heteromer opioid receptors. In the CellKeyTM assay, rubiscolins showed selective agonistic effects for δ opioid receptor and little agonistic or antagonistic effects for µ and κ opioid receptors. Furthermore, rubiscolins were found to be G-protein-biased δ opioid receptor agonists based on the results obtained in cADDis® cAMP and PathHunter® ß-arrestin recruitment assays. Finally, we found, for the first time, that they are also partially agonistic for the µ/δ dimers. In conclusion, rubiscolins could serve as attractive seeds, as δ opioid receptor-specific agonists, for the development of novel opioid analgesics with reduced side effects.

Prion protein interacts with the metabotropic glutamate receptor 1 and regulates the organization of Ca2+ signaling.

Cellular prion protein (PrP) is a membrane protein that is highly conserved among mammals and mainly expressed on the cell surface of neurons. Despite its reported interactions with various membrane proteins, no functional studies have so far been carried out on it, and its physiological functions remain unclear. Neuronal cell death has been observed in a PrP-knockout mouse model expressing Doppel protein, suggesting that PrP might be involved in Ca2+ signaling. In this study, we evaluated the binding of PrP to metabotropic glutamate receptor 1 (mGluR1) and found that wild-type PrP (PrP-wt) and mGluR1 co-immunoprecipitated in dual-transfected Neuro-2a (N2a) cells. Fluorescence resonance energy transfer analysis revealed an energy transfer between mGluR1-Cerulean and PrP-Venus. In order to determine whether PrP can modulate mGluR1 signaling, we performed Ca2+ imaging analyses following repetitive exposure to an mGluR1 agonist. Agonist stimulation induced synchronized Ca2+ oscillations in cells coexpressing PrP-wt and mGluR1. In contrast, N2a cells expressing PrP-ΔN failed to show ligand-dependent regulation of mGluR1-Ca2+ signaling, indicating that PrP can bind to mGluR1 and modulate its function to prevent irregular Ca2+ signaling and that its N-terminal region functions as a molecular switch during Ca2+ signaling.

Impact of chemotherapy on the association between fear of cancer recurrence and the gut microbiota in breast cancer survivors.

BACKGROUND: Dysfunctional processing of fear memory may be involved in the pathophysiology of fear of cancer recurrence (FCR), which is cited as the major unmet psychological need of cancer survivors. Emerging evidence has shown that the microbiota-gut-brain (MGB) axis affects depressive and anxiety disorders, and chemotherapy-associated psychological distress. We therefore hypothesized that the gut microbiota is associated with FCR in cancer survivors. METHODS: This cross-sectional study enrolled women diagnosed with invasive breast cancer who were not currently undergoing chemotherapy. Fecal samples were obtained to assess the gut microbiota. FCR grade was assessed using the Concerns About Recurrence Scale (CARS). RESULTS: Mean age of the participants (n = 126) was 58 years; 47% had stage I disease. Multiple regression analysis with adjustment for possible confounders showed that the relative abundance of the Bacteroides genus (beta = 0.180, p = 0.03) was significantly and directly associated with FCR. In the 57 participants with a history of chemotherapy, higher FCR was associated with lower microbial diversity (p = 0.04), lower relative abundance of Firmicutes (p = 0.03) and higher relative abundance of Bacteroidetes (p = 0.04) at the phylum level, and higher relative abundance of Bacteroides (p < 0.01) and lower relative abundance of Lachnospiraceae.g (p = 0.03) and Ruminococcus (p = 0.02) at the genus level. CONCLUSION: Our findings provide the first evidence of an association between the gut microbiota and FCR and suggest that chemotherapy-induced changes in gut microbiota can influence FCR. Further studies should examine the effects of the gut microbiota on FCR using a prospective design.

Carvacrol inhibits the neuronal voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.3, Nav1.7, and Nav1.8 expressed in Xenopus oocytes with different potencies.

Carvacrol is the predominant monoterpene in essential oils from many aromatic plants. Several animal studies showing analgesic effects of carvacrol indicate potential of carvacrol as a new medication for patients with refractory pain. Voltage-gated sodium channels (Nav) are thought to have crucial roles in the development of inflammatory and neuropathic pain, but there is limited information about whether the analgesic mechanism of carvacrol involves Nav. We used whole-cell, two-electrode, voltage-clamp techniques to examine the effects of carvacrol on sodium currents in Xenopus oocytes expressing α subunits of Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8. Carvacrol dose-dependently suppressed sodium currents at a holding potential that induced half-maximal current. The half-maximal inhibitory concentration values for Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 were 233, 526, 215, 367, and 824 µmol/L, respectively, indicating that carvacrol had more potent inhibitory effects towards Nav1.2 and Nav1.6 than Nav1.3, Nav1.7, and Nav1.8. Gating analysis showed a depolarizing shift of the activation curve and a hyperpolarizing shift of the inactivation curve in all five α subunits following carvacrol treatment. Furthermore, carvacrol exhibits a use-dependent block for all five α Nav subunits. These findings provide a better understanding of the mechanisms associated with the analgesic effect of carvacrol.

A novel method for evaluating activity of transient receptor potential channels using a cellular dielectric spectroscopy.

Cellular dielectric spectroscopy (CDS) is a novel technology enabling pharmacological evaluation of multiple receptor types with a label-free cell-based assay. We evaluated activities of a family of ligand-gated channels, transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels by an electrical impedance-based biosensor (CellKey™ system) using CDS. Measures of both potency (EC50) and efficacy (Emax) of these agonists with CellKey™ were almost identical to those made using the traditional Ca2+ influx assay in TRPV1- or TRPA1-expressing cells, suggesting that CellKey™ is a simpler and easier means of evaluating TRP activities.

A novel strategy for treatment of cancer cachexia targeting xanthine oxidase in the brain.

Cancer cachexia is a systemic wasting syndrome characterized by anorexia and loss of body weight. The xanthine oxidase (XO) inhibitor febuxostat is one of the promising candidates for cancer cachexia treatment. However, cachexic symptoms were not alleviated by oral administration of febuxostat in our cancer cachexia model. Metabolomic analysis with brains of our cachexic model showed that purine metabolism was activated and XO activity was increased, and thus suggested that febuxostat would not reach the brain. Accordingly, targeting XO in the brain, which controls appetite, may be an effective strategy for treatment of cancer cachexia.

Possible biased analgesic of hydromorphone through the G protein-over ß-arrestin-mediated pathway: cAMP, CellKey™, and receptor internalization analyses.

Morphine, fentanyl, and oxycodone are widely used as analgesics, and recently hydromorphone has been approved in Japan. Although all of these are selective for µ-opioid receptors (MORs) and have similar structures, their analgesic potencies and adverse effects (AEs) are diverse. Recent molecular analyses of MOR signaling revealed that the G protein-mediated signaling pathway causes analgesic effects and the ß-arrestin-mediated signaling pathway is responsible for AEs. We used several cell-based analyses that selectively measure cellular responses activated by either G protein- or ß-arrestin-mediated pathways. GloSensor™ cAMP, CellKey™, and receptor internalization assays were performed with four different types of cells stably expressing differentially labelled MOR. EC50 values measured by cAMP and CellKey™ assays had potencies in the order fentanyl ≤ hydromorphone < morphine ≤ oxycodone, all also exhibiting full agonist responses. However, in the internalization assay, only fentanyl elicited a full agonist response. Hydromorphone had the strongest potency next to fentanyl; however, contribution of the ß-arrestin-mediated pathway was small, suggesting that its effect could be biased toward the G protein-mediated pathway. Based on these properties, hydromorphone could be chosen as an effective analgesic.