Κ-opioid Receptor Antagonist Research Articles

Dexamethasone attenuates neuropathic pain through spinal microglial expression of dynorphin A via the cAMP/PKA/p38 MAPK/CREB signaling pathway

This study aimed to elucidate the opioid mechanisms underlying dexamethasone-induced pain antihypersensitive effects in neuropathic rats. Dexamethasone (subcutaneous and intrathecal) and membrane-impermeable Dex-BSA (intrathecal) administration dose-dependently inhibited mechanical allodynia and thermal hyperalgesia in neuropathic rats. Dexamethasone and Dex-BSA treatments increased expression of dynorphin A in the spinal cords and primary cultured microglia. Dexamethasone specifically enhanced dynorphin A expression in microglia but not astrocytes or neurons. Intrathecal injection of the microglial metabolic inhibitor minocycline blocked dexamethasone-stimulated spinal dynorphin A expression; intrathecal minocycline, the glucocorticoid receptor antagonist Dex-21-mesylate, dynorphin A antiserum, and κ-opioid receptor antagonist GNTI completely blocked dexamethasone-induced mechanical antiallodynia and thermal antihyperalgesia. Additionally, dexamethasone elevated spinal intracellular cAMP levels, leading to enhanced phosphorylation of PKA, p38 MAPK and CREB. The specific adenylate cyclase inhibitor DDA, PKA inhibitor H89, p38 MAPK inhibitor SB203580 and CREB inhibitor KG-501 completely blocked dexamethasone-induced anti-neuropathic pain and increased microglial dynorphin A exprression. In conclusion, this study reveal that dexamethasone mitigateds neuropathic pain through upregulation of dynorphin A in spinal microglia, likely involving the membrane glucocorticoid receptor/cAMP/PKA/p38 MAPK/CREB signaling pathway.

LY2444296, a κ-opioid receptor antagonist, selectively reduces alcohol drinking in male and female Wistar rats with a history of alcohol dependence

Alcohol use disorder (AUD) remains a major public health concern. The dynorphin (DYN)/κ-opioid receptor (KOP) system is involved in actions of alcohol, particularly its withdrawal-associated negative affective states. This study tested the ability of LY2444296, a selective, short-acting, KOP antagonist, to decrease alcohol self-administration in dependent male and female Wistar rats at 8 h abstinence. Animals were trained to orally self-administer 10% alcohol (30 min/day for 21 sessions) and were made dependent via chronic intermittent alcohol vapor exposure for 6 weeks or exposed to air (nondependent). After 6 weeks, the effect of LY2444296 (0, 3, and 10 mg/kg, p.o.) was tested on alcohol self-administration at 8 h of abstinence. A separate cohort of rats was prepared in parallel, and their somatic withdrawal signs and alcohol self-administration were measured after LY2444296 administration at 8 h, 2 weeks, and 4 weeks abstinence. LY2444296 at 3 and 10 mg/kg significantly reduced physical signs of withdrawal in dependent rats at 8 h abstinence, only. Furthermore, 3 and 10 mg/kg selectively decreased alcohol self-administration in dependent rats at only 8 h abstinence. These results highlight the DYN/KOP system in actions of alcohol during acute abstinence, suggesting KOP antagonism could be beneficial for mitigating acute withdrawal signs and, in turn, significantly reduce excessive alcohol consumption associated with AUD.

The Role of δ2-Opioid Receptors in the Regulation of Tolerance of Isolated Cardiomyocytes to Hypoxia and Reoxygenation.

Hypoxia (20 min) and reoxygenation (30 min) were simulated on isolated rat cardiomyocytes to evaluate the cytoprotective effect of selective δ2-opioid receptor agonist deltorphin II, opioid receptor antagonist naloxone methiodide, μ-opioid receptor antagonist CTAP, κ-opioid receptor antagonist nor-binaltorphimine, ε1-opioid receptor antagonist BNTX, and δ2-opioid receptors naltriben. Deltorphin II was administered 5 min before reoxygenation, antagonists were administered 10 min before reoxygenation. The cytoprotective effect of deltorphin II was assessed by the number of cardiomyocytes survived after hypoxia/reoxygenation, as well as by the lactate dehydrogenase content in the incubation medium. It has been established that the cytoprotective effect of deltorphin II occurs at a concentration of 64 nmol/liter and is associated with activation of δ2-opioid receptors.

Comorbid alcohol use disorder and posttraumatic stress disorder: A proof-of-concept randomized placebo-controlled trial of buprenorphine and naltrexone combination treatment.

Effective pharmacologic treatments for comorbid alcohol use disorder (AUD) and posttraumatic stress disorder (PTSD) are lacking. Kappa (κ) opioid receptor antagonists may address this unmet need. Buprenorphine is a κ-opioid antagonist and a partial agonist of mu (μ) opioid receptors. Whereas naltrexone blocks all μ-mediated effects combining it with buprenorphine yields a pharmacologic net effect of opioid receptor antagonism. Because no κ-opioid receptor antagonist it available for clinical use, we tested this combination in a proof-of-concept study. Consenting participants were enrolled in a Phase II, multisite, double-blind, randomized, placebo-controlled trial evaluating the effectiveness of sublingual (SL) buprenorphine combined with extended-release (XR) injectable naltrexone for the treatment of comorbid AUD and PTSD. Eligible participants (n = 75) were randomized (1:1:1) to receive either buprenorphine 2 mg/day plus naltrexone-XR (n = 35), buprenorphine 8 mg/day plus naltrexone-XR (n = 6) or SL plus injectable placebo (n = 34) for 12 weeks. The buprenorphine 8 mg/day plus naltrexone-XR arm was dropped early in the trial due to the negative impact of COVID-19 on enrollment. A binary primary outcome of response at week 8 was defined as a decrease from baseline of ≥10 points on the past week Clinician-Administered PTSD Scale (CAPS-5) and a reduction of ≥1 of past month alcohol risk level, as defined by the World Health Organization (WHO) and measured by the Timeline Follow-Back. Based on the results of a futility analysis, enrollment was stopped prior to reaching the initial goal of 90 participants. At the week eight primary timepoint, there were no statistically significant differences between buprenorphine plus naltrexone-XR and placebo group for the primary composite outcome (OR = 0.63; p-value = 0.52), or the subcomponents of the PTSD outcome (OR = 0.76; p-value = 0.69) and AUD outcome (OR = 0.17; p-value = 0.08). The placebo arm had a significantly higher proportion of participants with ≥1 WHO risk level reduction than the buprenorphine plus naltrexone-XR arm (OR = 0.18, p value = 0.02). This is the first study to evaluate the potential of κ-opioid receptor antagonism for the treatment of comorbid AUD and PTSD. The combination of buprenorphine and naltrexone-XR showed no significant improvement over placebo for the composite, PTSD, or alcohol measures.

Development of a Selective Peptide κ-Opioid Receptor Antagonist by Late-Stage Functionalization with Cysteine Staples.

The κ-opioid receptor (KOR) is an attractive target for the development of novel drugs. KOR agonists are potentially safer pain medications, whereas KOR antagonists are promising drug candidates for the treatment of neuropsychiatric disorders. Hitherto, the vast majority of selective drug leads that have been developed for KOR are small molecules. In this study, novel peptide probes were designed by using an endogenous dynorphin A1-13 sequence as a template for peptide stapling via late-stage cysteine functionalization. Leveraging this strategy, we developed a stable and potent KOR antagonist, CSD-CH2(1,8)-NH2, with approximately 1000-fold improved selectivity for KOR over μ- and δ-opioid receptors. Its potent competitive KOR antagonism was verified in KOR-expressing cells, peripheral dorsal root ganglion neurons, and using the tail-flick and rotarod tests in mice. This work highlights the value of cysteine stapling to develop selective peptide probes to modulate central KOR function, as innovative peptide drug candidates for the treatment of KOR-related illnesses.

The Pharmacokinetics of Subcutaneous Methylnaltrexone Bromide in Rhesus Macaques (Macaca mulatta).

Opioids are an integral component of pain management for nonhuman primates. These potent analgesics also adverse gastrointestinal (GI) effects that include constipation, bloating, and delayed gastric emptying. Methylnaltrexone bromide (MNTX) is a selective, peripherally acting μ- and κ-opioid receptor antagonist that can be used to mitigate the GI effects associated with opioid administration. Unlike naltrexone, a similar drug in this class, MNTX possesses an N-methyl-quaternary amine group that prevents it from crossing the blood brain barrier. This blockage allows inhibition of peripheral GI opioid receptors without affecting opioid-mediated analgesia in the central nervous system. We conducted a pharmacokinetic analysis of MNTX in serum and CSF of 6 healthy juvenile male rhesus macaques after subcutaneous administration of a 0.15-mg/kg dose. We hypothesized that the macaques would demonstrate a Tmax of 0.5 h, similar to that of humans, and that no MNTX would be detected in the CSF. This treatment resulted in a peak serum concentration of 114 ± 44 ng/mL at 0.25 ± 0.00 h; peak CSF at concentrations were 0.34 ± 0.07 ng/mL at the Tmax. These data show that subcutaneous administration of MNTX to rhesus macaques may block peripheral adverse effects of opioids without interfering with their central analgesic effects.

Strategies for the Production of [11C]LY2795050 for Clinical Use.

This report describes a comparison of four different routes for the clinical-scale radiosynthesis of the κ-opioid receptor antagonist [11C]LY2795050. Palladium-mediated radiocyanation and radiocarbonylation of an aryl iodide precursor as well as copper-mediated radiocyanation of an aryl iodide and an aryl boronate ester have been investigated. Full automation of all four methods is reported, each of which provides [11C]LY2795050 in sufficient radiochemical yield, molar activity, and radiochemical purity for clinical use. The advantages and disadvantages of each radiosynthesis method are compared and contrasted.

AI-assisted de novo design of selective κ-opioid receptor antagonists for the treatment of opioid addiction.

Daunting estimates of overdose fatalities, costs of healthcare, lost productivity, and criminal justice involvement for the misuse of prescription opioids have converted opioid use disorders into a major national crisis in the United States. Among likely efficacious pharmacological interventions for the treatment of opioid dependence are those that can attenuate brain reward deficits experienced during periods of abstinence. Pharmacological blockade of κ-opioid receptors (KOR) has been shown to abolish brain reward deficits detected in rodents during withdrawal from opioids, as well as to reduce the escalation of opioid use in rats with extended access to opioids. Although KOR antagonists represent promising candidates for the treatment of opioid addiction, very few potent selective KOR antagonists are known to date and most of them exhibit significant safety concerns. Here, we used a deep learning framework for the de novo design of ligands with predefined molecular properties to predict new chemotypes with KOR antagonistic activity. Specifically, we pre-trained a deep generative tensorial model to learn a mapping of the chemical space from a ZINC subset of purchasable drug-like molecules, as well as sets of effective (IC50 < 1 µM) or less effective (IC50 ≥ 1 µM) known KOR inhibitors from the ChEMBL database, while simultaneously encoding the relationship between the molecules and their properties. Subsequently, we biased the resulting generative model towards the development of putative KOR antagonists with a reinforcement learning algorithm that rewarded similarity to the antagonist binding mode revealed by the JDTic-bound KOR crystal structure, as well as novelty, synthetic accessibility, absence of unstable and reactive moieties, drug-like solubility, and blood-brain barrier permeability. The generated molecules were prioritized for chemical synthesis and functional evaluation based on their predicted optimal interactions with the receptor.

Ginsenoside Rb1, a principal effective ingredient of Panax notoginseng, produces pain antihypersensitivity by spinal microglial dynorphin A expression

Panax notoginseng (Chinese ginseng, Sanqi), one of the major ginseng species, has been traditionally used to alleviate different types of chronic pain. The raw P. notoginseng powder is commonly available in China as a non-prescription drug to treat various aliments including arthritic pain. However, strong scientific evidence is needed to illustrate its pain antihypersensitive effects, effective ingredients and mechanism of action. The oral P. notoginseng powder dose-dependently alleviated formalin-induced tonic hyperalgesia, and its total ginsenosides remarkably inhibited neuropathic pain hypersensitivity. Ginsenoside Rb1, the most abundant ginsenoside of P. notoginseng, dose-dependently produced neuropathic pain antihypersensitivity. Conversely, ginsenosides Rg1, Re and notoginseng R1, the other major saponins from P. notoginseng, failed to inhibit formalin-induced tonic pain or mechanical allodynia in neuropathic pain. Ginsenoside Rb1 metabolites ginsenosides Rg3, Compound-K and protopanaxadiol also had similar antineuropathic pain efficacy to ginsenoside Rb1. Additionally, intrathecal ginsenoside Rb1 specifically stimulated dynorphin A expression which was colocalized with microglia but not neurons or astrocytes in the spinal dorsal horn and primary cultured cells. Pretreatment with microglial metabolic inhibitor minocycline, dynorphin A antiserum and specific κ-opioid receptor antagonist GNTI completely blocked Rb1-induced mechanical antiallodynia in neuropathic pain. Furthermore, the specific glucocorticoid receptor (GR) antagonist Dex-21-mesylate (but not GPR30 estrogen receptor antagonist G15) also entirely attenuated ginsenoside Rb1-related antineuropathic pain effects. All these results, for the first time, show that P. notoginseng alleviates neuropathic pain and ginsenoside Rb1 is its principal effective ingredient. Furthermore, ginsenoside Rb1 inhibits neuropathic pain by stimulation of spinal microglial dynorphin A expression following GR activation.

A novel scaffold opioid ligand as a κ-opioid receptor antagonist: a pharmacological and computational study

A novel scaffold opioid ligand as a κ-opioid receptor antagonist: a pharmacological and computational study

Intracerebroventricular Administration of Dermorphin-Dynorphin Analogs Producing Antidepressant-Like Effects through Activation of μ1- and κ-Opioid Receptors in Mice.

The opioid system in the central nervous system regulates depressive-like behavior in animals. Opioid receptors and their endogenous ligands have been focused on as novel therapeutic targets for depression. We synthesized dermorphin (DRM)-dynorphin (DYN) analogs (DRM-DYN001-004) using the message-address concept concerning interactions with opioid receptors. It has previously been reported that DRM-DYN001, 003, and 004 have shown high affinities for μ- and κ-opioid receptors, whereas all analogs had a lower affinity for the δ-opioid receptor than for other receptors using a receptor binding assay. However, it remains unknown whether these analogs show antidepressant-like effects in mice. We examined the effects of DRM-DYN analogs on the duration of immobile behavior in a tail suspension test. Intracerebroventricular administration of DRM-DYN001 in mice shortened the duration of immobile behavior, but did not affect locomotion. The DRM-DYN001-induced antidepressant-like effect was inhibited by co-administration of naloxone (non-selective opioid receptor antagonist), naloxonazine (selective μ1-opioid receptor antagonist), or nor-BNI (κ-opioid receptor antagonist), but not naltrindole (δ-opioid receptor antagonist). These data suggest that DRM-DYN001 exerts an antidepressant-like effect via activation of the central μ1- and κ-opioid receptors in mice and may represent a new lead peptide for further investigation for the development of novel therapeutic approaches for depression.

Gestational buprenorphine exposure disrupts dopamine neuron activity and related behaviors in adulthood.

Opioid misuse among pregnant women is rapidly increasing in the United States. The number of maternal opioid-related diagnoses increased by 131% in the last 10 years, resulting in an increased number of infants exposed to opioids in utero and a subsequent increase in infants developing neonatal abstinence syndrome (NAS). The most prescribed treatment to combat maternal opioid use disorder is buprenorphine, a partial μ-opioid receptor agonist and κ-opioid receptor antagonist. Buprenorphine treatment effectively reduces NAS but has been associated with disrupted cortical development and neurodevelopmental consequences in childhood. Less is known about the long-term neurodevelopmental consequences following buprenorphine exposure in utero. Previous research has shown that gestational buprenorphine exposure can induce anxiety-like and depressive-like phenotypes in adult rats, suggesting that exposure to buprenorphine in utero may render individuals more susceptible to psychiatric illness in adulthood. A common pathology observed across multiple psychiatric illnesses is dopamine system dysfunction. Here, we administered the highly-abused opioid, oxycodone (10 mg/kg, i.p.) or a therapeutic used to treat opioid use disorder, buprenorphine (1 mg/kg, i.p) to pregnant Sprague Dawley rats from gestational day 11 through 21, then examined neurophysiological alterations in the mesolimbic dopamine system and dopamine-dependent behaviors in adult offspring. We found that gestational exposure to buprenorphine or oxycodone increases dopamine neuron activity in adulthood. Moreover, prenatal buprenorphine exposure disrupts the afferent regulation of dopamine neuron activity in the ventral tegmental area (VTA). Taken together, we posit that gestational buprenorphine or oxycodone exposure can have profound effects on the mesolimbic dopamine system in adulthood.

Dynorphin A (1–8) inhibits oxidative stress and apoptosis in MCAO rats, affording neuroprotection through NMDA receptor and κ-opioid receptor channels

The contents of Dynorphin A(1–8) decreased gradually in ischemic cortices in rats and an intracerebroventricular administration of synthetic Dynorphin A(1–8) reduced the volume of cerebral infarction in our previous research. However, the specific protective mechanism is unclear and Dynorphin A(1–8) is unlikely to cross the blood-brain barrier (BBB) by noninvasive oral or intravenous administration as a macromolecule neuropeptide. In this study, intranasal administration was used to middle cerebral artery occlusion(MCAO) rats to assessed the therapeutic effects of Dynorphin A(1–8) by evaluating behavior, volume of cerebral infarct, cerebral edema ratio, histological observation. Then apoptosis neuron rate was detected by TUNEL staining. Immunohistochemical staining was carried out to explore the alteration of Bcl-2, Bax and Caspase-3. Finally, κ-opioid receptor antagonist and N-methyl-d-aspartate(NMDA) receptor antagonist were used to explore its possible mechanism. We found that MCAO rats under intranasal administration of Dynorphin A(1–8) showed better behavioral improvement, higher extent of Bcl-2, activity of SOD along with much lower level of infarction volume, brain water content, number of cell apoptosis, extent of Bax and Caspase-3, and concentration of MDA compared with those in MCAO model group and intravenous Dynorphin A(1–8) group. Administration of nor-BNI or MK-801 reversed these neuroprotective effects of intranasal Dynorphin A(1–8). In summary, Dynorphin A(1–8), with advantages of intranasal administration, could be effectively delivered to central nervous system(CNS). Dynorphin A(1–8) inhibited oxidative stress and apoptosis against cerebral ischemia/reperfusion injury, affording neuroprotection through NMDA receptor and κ-opioid receptor channels.

Butorphanol Promotes Macrophage Phenotypic Transition to Inhibit Inflammatory Lung Injury via κ Receptors.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is characterized by diffuse inflammation of the lung parenchyma and refractory hypoxemia. Butorphanol is commonly used clinically for perioperative pain relief, but whether butorphanol can regulate LPS-induced alveolar macrophage polarization is unclear. In this study, we observed that butorphanol markedly attenuated sepsis-induced lung tissue injury and mortality in mice. Moreover, butorphanol also decreased the expression of M1 phenotype markers (TNF-α, IL-6, IL-1β and iNOS) and enhanced the expression of M2 marker (CD206) in alveolar macrophages in the bronchoalveolar lavage fluid (BALF) of LPS-stimulated mice. Butorphanol administration reduced LPS-induced numbers of proinflammatory (M1) macrophages and increased numbers of anti-inflammatory (M2) macrophages in the lungs of mice. Furthermore, we found that butorphanol-mediated suppression of the LPS-induced increases in M1 phenotype marker expression (TNF-α, IL-6, IL-1β and iNOS) in bone marrow-derived macrophages (BMDMs), and this effect was reversed by κ-opioid receptor (KOR) antagonists. Moreover, butorphanol inhibited the interaction of TLR4 with MyD88 and further suppressed NF-κB and MAPKs activation. In addition, butorphanol prevented the Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF)-mediated IFN signaling pathway. These effects were ameliorated by KOR antagonists. Thus, butorphanol may promote macrophage polarization from a proinflammatory to an anti-inflammatory phenotype secondary to the inhibition of NF-κB, MAPKs, and the TRIF-mediated IFN signaling pathway through κ receptors.

Protopanaxadiol alleviates neuropathic pain by spinal microglial dynorphin A expression following glucocorticoid receptor activation.

New remedies are required for the treatment of neuropathic pain due to insufficient efficacy of available therapies. This study provides a novel approach to develop painkillers for chronic pain treatment. The rat formalin pain test and spinal nerve ligation model of neuropathic pain were used to evaluate antinociception of protopanaxadiol. Primary cell cultures, immunofluorescence staining, and gene and protein expression were also performed for mechanism studies. Gavage protopanaxadiol remarkably produces pain antihypersensitive effects in neuropathic pain, bone cancer pain and inflammatory pain, with efficacy comparable with gabapentin. Long-term PPD administration does not induce antihypersensitive tolerance, but prevents and reverses the development and expression of morphine analgesic tolerance. Oral protopanaxadiol specifically stimulates spinal expression of dynorphin A in microglia but not in astrocytes or neurons. Protopanaxadiol gavage-related pain antihypersensitivity is abolished by the intrathecal pretreatment with the microglial metabolic inhibitor minocycline, dynorphin antiserum or specific κ-opioid receptor antagonist GNTI. Intrathecal pretreatment with glucocorticoid receptor)antagonists RU486 and dexamethasone-21-mesylate, but not GPR-30 antagonist G15 or mineralocorticoid receptor antagonist eplerenone, completely attenuates protopanaxadiol-induced spinal dynorphin A expression and pain antihypersensitivity in neuropathic pain. Treatment with protopanaxadiol, the glucocorticoid receptor agonist dexamethasone and membrane-impermeable glucocorticoid receptor agonist dexamethasone-BSA in cultured microglia induces remarkable dynorphin A expression, which is totally blocked by pretreatment with dexamthasone-21-mesylate. All the results, for the first time, indicate that protopanaxadiol produces pain antihypersensitivity in neuropathic pain probably through spinal microglial dynorphin A expression after glucocorticoid receptor activation and hypothesize that microglial membrane glucocorticoid receptor/dynorphin A pathway is a potential target to discover and develop novel painkillers in chronic pain.

The administration of the opioid buprenorphine decreases motivational error signals

While opioid addiction has reached pandemic proportions, we still lack a good understanding of how the administration of opioids interacts with cognitive functions. Error processing – the ability to detect erroneous actions and correct one’s behaviour afterwards - is one such cognitive function that might be susceptible to opioidergic influences. Errors are hypothesised to induce aversive negative arousal, while opioids have been suggested to reduce aversive arousal induced by unpleasant and stressful stimuli. Thus, this study investigated whether the acute administration of an opioid would affect error processing. In a double-blind between-subject study, 42 male volunteers were recruited and received either 0.2 mg buprenorphine (a partial µ-opioid receptor agonist and κ-opioid receptor antagonist) or a placebo pill before they performed a stimulus-response task provoking errors. Electroencephalograms (EEG) were recorded while participants performed the task. We observed no group differences in terms of reaction times, error rates, and affective state ratings during the task between buprenorphine and control participants. Additional measures of adaptive control, however, showed interfering effects of buprenorphine administration. On the neural level, decreased Pe (Error Positivity) amplitudes were found in buprenorphine compared to control participants following error commission. Further, frontal delta oscillations were decreased in the buprenorphine group after all responses. Our neural results jointly demonstrate a general reduction in error processing in those participants who received an opioid before task completion, thereby suggesting that opioids might have indeed the potential to dampen motivational error signals. Importantly, the effects of the opioid were evident in more elaborate error processing stages, thereby impacting on processes of conscious error appraisal and evidence accumulation.

Transmucosal Buprenorphine in the Treatment of Dyspnea: Case Series and Review of the Literature.

Full μ-opioid agonists are commonly employed in advanced disease to relieve dyspnea of various etiologies. Although there are ongoing debates and studies regarding the relative efficacy of different agents, a factor limiting more general use is a concern about side effects, in particular respiratory depression. Buprenorphine is a partial μ receptor agonist and a κ-opioid receptor antagonist, with a better safety profile than full μ receptor agonists. We conducted a literature search, which did not reveal any studies looking at the use of buprenorphine to treat dyspnea. We also report a case series of three patients with dyspnea of various etiologies treated with buprenorphine, with an apparent excellent response of dyspnea to treatment, without any significant side effects. Given those results, as well as the potential impact of κ-opioid receptor antagonism on dyspnea-associated anxiety, we conclude that there is a need for controlled studies of buprenorphine against full μ-opioid agonists for the symptomatic treatment of dyspnea in palliative care.

Low-Threshold Mechanosensitive VGLUT3-Lineage Sensory Neurons Mediate Spinal Inhibition of Itch by Touch.

Innocuous mechanical stimuli, such as rubbing or stroking the skin, relieve itch through the activation of low-threshold mechanoreceptors. However, the mechanisms behind this inhibition remain unknown. We presently investigated whether stroking the skin reduces the responses of superficial dorsal horn neurons to pruritogens in male C57BL/6J mice. Single-unit recordings revealed that neuronal responses to chloroquine were enhanced during skin stroking, and this was followed by suppression of firing below baseline levels after the termination of stroking. Most of these neurons additionally responded to capsaicin. Stroking did not suppress neuronal responses to capsaicin, indicating state-dependent inhibition. Vesicular glutamate transporter 3 (VGLUT3)-lineage sensory nerves compose a subset of low-threshold mechanoreceptors. Stroking-related inhibition of neuronal responses to chloroquine was diminished by optogenetic inhibition of VGLUT3-lineage sensory nerves in male and female Vglut3-cre/NpHR-EYFP mice. Conversely, in male and female Vglut3-cre/ChR2-EYFP mice, optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited firing responses of spinal neurons to pruritogens after the termination of stimulation. This inhibition was nearly abolished by spinal delivery of the κ-opioid receptor antagonist nor-binaltorphimine dihydrochloride, but not the neuropeptide Y receptor Y1 antagonist BMS193885. Optogenetic stimulation of VGLUT3-lineage sensory nerves inhibited pruritogen-evoked scratching without affecting mechanical and thermal pain behaviors. Therefore, VGLUT3-lineage sensory nerves appear to mediate inhibition of itch by tactile stimuli.SIGNIFICANCE STATEMENT Rubbing or stroking the skin is known to relieve itch. We investigated the mechanisms behind touch-evoked inhibition of itch in mice. Stroking the skin reduced the activity of itch-responsive spinal neurons. Optogenetic inhibition of VGLUT3-lineage sensory nerves diminished stroking-evoked inhibition, and optogenetic stimulation of VGLUT3-lineage nerves inhibited pruritogen-evoked firing. Together, our results provide a mechanistic understanding of touch-evoked inhibition of itch.

Zerumbone-Induced Analgesia Modulated via Potassium Channels and Opioid Receptors in Chronic Constriction Injury-Induced Neuropathic Pain

Zerumbone, a monocyclic sesquiterpene from the wild ginger plant Zingiber zerumbet (L.) Smith, attenuates allodynia and hyperalgesia. Currently, its mechanisms of action in neuropathic pain conditions remain unclear. This study examines the involvement of potassium channels and opioid receptors in zerumbone-induced analgesia in a chronic constriction injury (CCI) neuropathic pain mice model. Male Institute of Cancer Research (ICR) mice were subjected to CCI and behavioral responses were tested on day 14. Responses toward mechanical allodynia and thermal hyperalgesia were tested with von Frey’s filament and Hargreaves’ tests, respectively. Symptoms of neuropathic pain were significantly alleviated following treatment with zerumbone (10 mg/kg; intraperitoneal, i.p.). However, when the voltage-dependent K+ channel blocker tetraethylammonium (TEA, 4 mg/kg; i.p.), ATP-sensitive K+ channel blocker, glibenclamide (GLIB, 10 mg/kg; i.p.); small-conductance Ca2+-activated K+ channel inhibitor apamin (APA, 0.04 mg/kg; i.p.), or large-conductance Ca2+-activated K+ channel inhibitor charybdotoxin (CHAR, 0.02 mg/kg; i.p.) was administered prior to zerumbone (10 mg/kg; i.p.), the antiallodynic and antihyperalgesic effects of zerumbone were significantly reversed. Additionally, non-specific opioid receptors antagonist, naloxone (NAL, 10 mg/kg; i.p.), selective µ-, δ- and κ-opioid receptor antagonists; β-funaltrexamine (β-FN, 40 mg/kg; i.p.), naltrindole (20 mg/kg; s.c.), nor-binaltorphamine (10 mg/kg; s.c.) respectively attenuated the antiallodynic and antihyperalgesic effects of zerumbone. This outcome clearly demonstrates the participation of potassium channels and opioid receptors in the antineuropathic properties of zerumbone. As various clinically used neuropathic pain drugs also share this similar mechanism, this compound is, therefore, a highly potential substitute to these therapeutic options.

Multifunctional opioid receptor agonism and antagonism by a novel macrocyclic tetrapeptide prevents reinstatement of morphine-seeking behaviour.

The macrocyclic tetrapeptide natural product CJ-15,208 (cyclo[Phe-d-Pro-Phe-Trp]) is a multifunctional μ-opioid receptor and κ-opioid receptor agonist and κ-opioid receptor antagonist that produces antinociception and prevents stress-induced reinstatement of extinguished cocaine-conditioned place preference (CPP). We hypothesized that an analogue of CJ-15,208, cyclo[Pro-Sar-Phe-d-Phe], would demonstrate multifunctional μ-opioid receptor and κ-opioid receptor ligand activity, producing potent antinociception with fewer liabilities than selective μ-opioid receptor agonists, while preventing both drug- and stress-induced reinstatement of morphine-induced CPP. The opioid receptor agonist and antagonist activity of cyclo[Pro-Sar-Phe-d-Phe] was characterized after i.c.v. and i.p. administration to C57BL/6J or transgenic opioid receptor "knockout" mice using the 55°C warm-water tail-withdrawal assay. Liabilities of locomotor coordination, respiration and spontaneous ambulation, and direct rewarding or aversive properties were assessed. Finally, the ability of cyclo[Pro-Sar-Phe-d-Phe] to block morphine- and stress-induced reinstatement of extinguished CPP was determined. cyclo[Pro-Sar-Phe-d-Phe] demonstrated dose-dependent, short-lasting antinociception, with an ED50 (and 95% confidence interval) of 0.15 (0.05-0.21) nmol i.c.v. and 1.91 (0.40-3.54) mg·kg-1 i.p., mediated by μ- and κ-opioid receptors. The macrocyclic tetrapeptide also demonstrated potent dose-dependent κ-opioid receptor antagonist-like activity at 2.5, but not at 4.5, h after administration. cyclo[Pro-Sar-Phe-d-Phe] displayed reduced liabiities compared with morphine, attributed to its additional activity at κ-receptors. Pretreatment with cyclo[Pro-Sar-Phe-d-Phe] prevented stress- and drug-induced reinstatement of extinguished morphine-place preference responses in a time-dependent manner. These data suggest that cyclo[Pro-Sar-Phe-d-Phe] is a promising lead compound for both the treatment of pain with reduced sideeffects and preventing both drug- and stress-induced relapse in morphine-abstinent subjects.