Nalfurafine promotes myelination in vitro and facilitates recovery from cuprizone + rapamycin-induced demyelination in mice.

The kappa opioid receptor has been identified as a promising therapeutic target for promoting remyelination. In the current study, we evaluated the ability of nalfurafine to promote oligodendrocyte progenitor cell (OPC) differentiation and myelination in vitro, and its efficacy in an extended, cuprizone-induced demyelination model. Primary mouse (C57BL/6J) OPC-containing cultures were treated with nalfurafine (0.6-200 nM), clemastine (0.01-100 µM), T3 (30 ng/mL), or vehicle for 5 days. Using immunocytochemistry and confocal microscopy, we found that nalfurafine treatment increased OPC differentiation, oligodendrocyte (OL) morphological complexity, and myelination of nanofibers in vitro. Adult male mice (C57BL/6J) were given a diet containing 0.2% cuprizone and administered rapamycin (10 mg/kg) once daily for 12 weeks followed by 6 weeks of treatment with nalfurafine (0.01 or 0.1 mg/kg), clemastine (10 mg/kg), or vehicle. We quantified the number of OLs using immunofluorescence, gross myelination using black gold staining, and myelin thickness using electron microscopy. Cuprizone + rapamycin treatment produced extensive demyelination and was accompanied by a loss of mature OLs, which was partially reversed by therapeutic administration of nalfurafine. We also assessed these mice for functional behavioral changes in open-field, horizontal bar, and mouse motor skill sequence tests (complex wheel running). Cuprizone + rapamycin treatment resulted in hyperlocomotion, poorer horizontal bar scores, and less distance traveled on the running wheels. Partial recovery was observed on both the horizontal bar and complex running wheel tests over time, which was facilitated by nalfurafine treatment. Taken together, these data highlight the potential of nalfurafine as a remyelination-promoting therapeutic.

The Kappa Opioid Receptor Agonist 16-Bromo Salvinorin A Has Anti-Cocaine Effects without Significant Effects on Locomotion, Food Reward, Learning and Memory, or Anxiety and Depressive-like Behaviors.

Kappa opioid receptor (KOR) agonists have preclinical antipsychostimulant effects; however, adverse side effects have limited their therapeutic development. In this preclinical study, conducted in Sprague Dawley rats, B6-SJL mice, and non-human primates (NHPs), we evaluated the G-protein-biased analogue of salvinorin A (SalA), 16-bromo salvinorin A (16-BrSalA), for its anticocaine effects, side effects, and activation of cellular signaling pathways. 16-BrSalA dose-dependently decreased the cocaine-primed reinstatement of drug-seeking behavior in a KOR-dependent manner. It also decreased cocaine-induced hyperactivity, but had no effect on responding for cocaine on a progressive ratio schedule. Compared to SalA, 16-BrSalA had an improved side effect profile, with no significant effects in the elevated plus maze, light-dark test, forced swim test, sucrose self-administration, or novel object recognition; however, it did exhibit conditioned aversive effects. 16-BrSalA increased dopamine transporter (DAT) activity in HEK-293 cells coexpressing DAT and KOR, as well as in rat nucleus accumbens and dorsal striatal tissue. 16-BrSalA also increased the early phase activation of extracellular-signal-regulated kinases 1 and 2, as well as p38 in a KOR-dependent manner. In NHPs, 16-BrSalA caused dose-dependent increases in the neuroendocrine biomarker prolactin, similar to other KOR agonists, at doses without robust sedative effects. These findings highlight that G-protein-biased structural analogues of SalA can have improved pharmacokinetic profiles and fewer side effects while maintaining their anticocaine effects.

Strategies for Developing κ Opioid Receptor Agonists for the Treatment of Pain with Fewer Side Effects.

There is significant need to find effective, nonaddictive pain medications. κ Opioid receptor (KOPr) agonists have been studied for decades but have recently received increased attention because of their analgesic effects and lack of abuse potential. However, a range of side effects have limited the clinical development of these drugs. There are several strategies currently used to develop safer and more effective KOPr agonists. These strategies include identifying G-protein-biased agonists, developing peripherally restricted KOPr agonists without centrally mediated side effects, and developing mixed opioid agonists, which target multiple receptors at specific ratios to balance side-effect profiles and reduce tolerance. Here, we review the latest developments in research related to KOPr agonists for the treatment of pain. SIGNIFICANCE STATEMENT: This review discusses strategies for developing safer κ opioid receptor (KOPr) agonists with therapeutic potential for the treatment of pain. Although one strategy is to modify selective KOPr agonists to create peripherally restricted or G-protein-biased structures, another approach is to combine KOPr agonists with µ, δ, or nociceptin opioid receptor activation to obtain mixed opioid receptor agonists, therefore negating the adverse effects and retaining the therapeutic effect.

Kappa Opioid Receptor Agonist Mesyl Sal B Attenuates Behavioral Sensitization to Cocaine with Fewer Aversive Side-Effects than Salvinorin A in Rodents.

The acute activation of kappa opioid receptors (KOPr) produces antinociceptive and anti-cocaine effects, however, their side-effects have limited further clinical development. Mesyl Sal B is a potent and selective KOPr analogue of Salvinorin A (Sal A), a psychoactive natural product isolated from the plant Salvia divinorum. We assessed the antinociceptive, anti-cocaine, and side-effects of Mesyl Sal B. The anti-cocaine effects are evaluated in cocaine-induced hyperactivity and behavioral sensitization to cocaine in male Sprague Dawley rats. Mesyl Sal B was assessed for anhedonia (conditioned taste aversion), aversion (conditioned place aversion), pro-depressive effects (forced swim test), anxiety (elevated plus maze) and learning and memory deficits (novel object recognition). In male B6.SJL mice, the antinociceptive effects were evaluated in warm-water (50 °C) tail withdrawal and intraplantar formaldehyde (2%) assays and the sedative effects measured with the rotarod performance task. Mesyl Sal B (0.3 mg/kg) attenuated cocaine-induced hyperactivity and behavioral sensitization to cocaine without modulating sucrose self-administration and without producing aversion, sedation, anxiety, or learning and memory impairment in rats. However, increased immobility was observed in the forced swim test indicating pro-depressive effects. Mesyl Sal B was not as potent as Sal A at reducing pain in the antinociceptive assays. In conclusion, Mesyl Sal B possesses anti-cocaine effects, is longer acting in vivo and has fewer side-effects when compared to Sal A, however, the antinociceptive effects are limited.

Estimating cross-price elasticity of e-cigarettes using a simulated demand procedure.

INTRODUCTION: Our goal was to measure the cross-price elasticity of electronic cigarettes (e-cigarettes) and simulated demand for tobacco cigarettes both in the presence and absence of e-cigarette availability. METHOD: A sample of New Zealand smokers (N = 210) completed a Cigarette Purchase Task to indicate their demand for tobacco at a range of prices. They sampled an e-cigarette and rated it and their own-brand tobacco for favorability, and indicated how many e-cigarettes and regular cigarettes they would purchase at 0.5×, 1×, and 2× the current market price for regular cigarettes, assuming that the price of e-cigarettes remained constant. RESULTS: Cross-price elasticity for e-cigarettes was estimated as 0.16, and was significantly positive, indicating that e-cigarettes were partially substitutable for regular cigarettes. Simulated demand for regular cigarettes at current market prices decreased by 42.8% when e-cigarettes were available, and e-cigarettes were rated 81% as favorably as own-brand tobacco. However when cigarettes cost 2× the current market price, significantly more smokers said they would quit (50.2%) if e-cigarettes were not available than if they were available (30.0%). CONCLUSION: Results show that e-cigarettes are potentially substitutable for regular cigarettes and their availability will reduce tobacco consumption. However, e-cigarettes may discourage smokers from quitting entirely as cigarette price increases, so policy makers should consider maintaining a constant relative price differential between e-cigarettes and tobacco cigarettes.

Assessing the Temporal Stability of a Cigarette Purchase Task After an Excise Tax Increase for Factory-Made and Roll-Your-Own Smokers.

INTRODUCTION: Cigarette purchase tasks (CPTs) are used increasingly to measure simulated demand curves for tobacco. However, there is currently limited information about the temporal stability of demand curves obtained from these tasks. METHOD: We interviewed a sample (N = 210) of smokers in New Zealand both before and after a 10% increase in the tobacco excise tax that took effect on January 1, 2013. Participants were interviewed in November-December 2012 (wave 1) and February-March 2013 (wave 2). At each interview, participants completed a high-resolution CPT with 64 prices ranging from NZ $0.00 to NZ $5.00/cigarette, and questionnaires regarding their smoking habit. RESULTS: Roll-your-own smokers had higher levels of nicotine dependence and tobacco demand based on CPT responses than factory-made smokers. Although demand curves for waves 1 and 2 were similar, intentions to purchase cigarettes were significantly less at wave 2 for three prices (NZ $0.85, NZ $0.90, and NZ $0.95) that were just higher than the actual price after the tax increase, for both roll-your-own and factory-made smokers. Measures of elasticity (α) derived from Hursh and Silberberg's model were significantly greater at wave 2 than wave 1, and there was a significant reduction in smoking habit as measured by cigarettes/day and the Fagerström Test for Nicotine Dependence at wave 2. CONCLUSIONS: Purchase tasks can discriminate between smokers based on their tobacco preference, and although results are relatively stable over time, they depend on contextual factors such as the current real price for tobacco.

Predicting decreases in smoking with a cigarette purchase task: evidence from an excise tax rise in New Zealand.

BACKGROUND: Tobacco excise taxes are known to be effective in reducing smoking at the population level, but less research has examined how individual smokers respond to changes in tax policy. We ask whether price elasticities for individual smokers, derived from simulated demand curves obtained with a cigarette purchase task (CPT), can predict changes in smoking after a tax increase. METHOD: Smokers (N=357) were recruited from four New Zealand cities and interviewed before and after a 10% tobacco excise tax increase. RESULTS: Simulated demand curves from the CPT were curvilinear and well described by an exponential model. Smokers reported significant reductions in cigarettes/day and addiction scores at Wave 2 (n=226). Local elasticities derived from the demand curves significantly predicted decreases in cigarettes/day after controlling for covariates. CONCLUSIONS: Elasticities from simulated demand curves can predict decreases in consumption for individual smokers after an excise tax increase. Understanding individual differences in tobacco demand curves may help to predict how different groups of smokers will respond to price increases.

The effects of nicotine and tobacco particulate matter on dopamine uptake in the rat brain.

Cigarette smoking is the leading cause of preventable death worldwide. Recently, tobacco extracts have been shown to have a different pharmacological profile to nicotine alone and there is increasing evidence of a role for non-nicotinic components of cigarette smoke in smoking addiction. Nicotine is known to affect the uptake of dopamine in the brain of laboratory animals, but studies in the literature are often contradictory and little is known of the effects on non-nicotinic tobacco components on dopamine uptake. This study has examined the acute and chronic effects of nicotine and a tobacco extract (TPM) on dopamine uptake by the dopamine and norepinephrine transporters (DAT and NET) ex vivo using rotating disk electrode voltammetry, and quantified DAT and NET protein and mRNA expression in key brain regions. Nicotine (0.35 mg/kg) significantly decreased DAT function in the nucleus accumbens (NAc) at 30 min with no change in protein expression. This effect was sensitive to mecamylamine and DHßE but not MLA, indicating that it is dependent on α4 subunit containing nicotinic receptors. Furthermore, TPM, but not nicotine, increased DAT function in the dorsal striatum at 1 h in a nicotinic receptor independent manner with no change in DAT protein expression. At 1 h DAT mRNA in the ventral tegmental area was decreased by both acute and chronic TPM treatments.

Enhanced and complementary benefits of a nalfurafine and fingolimod combination to treat immune-driven demyelination.

Objectives: Multiple sclerosis (MS) is a neurodegenerative disease characterised by inflammation and damage to myelin sheaths. While all current disease-modifying treatments (DMTs) are very effective at reducing relapses, they do not slow the progression of the disease, and there is little evidence that these treatments are able to repair or remyelinate damaged axons. Recent evidence suggests that activating kappa opioid receptors (KORs) has a beneficial effect on the progression of MS, and this study investigates the effects of KOR agonists treatment in combination with two current DMTs. Methods: Using the well-established murine model for immune-driven demyelination of MS, experimental autoimmune encephalomyelitis, the effect of KOR agonists in combination with DMTs fingolimod or dimethyl fumarate on disease progression, immune cell infiltration and activation as well as myelination were analysed. Results: Fingolimod in combination with the KOR agonist, nalfurafine, significantly increased each individual beneficial effect as measured by increased recovery of mice and reduced relapses. These beneficial effects correlated with a reduction in immune cell infiltration into the CNS as well as peripheral immune cell alterations including a reduction in autoreactive CD4+ T-cell cytokine production as well as increased myelination in the spinal cords of co-treated animals. In contrast, while the use of dimethyl fumarate in combination with nalfurafine did not adversely affect the benefits of nalfurafine, the combination did not significantly enhance those benefits. Conclusion: This study indicates that KOR agonists can be used in combination with fingolimod and dimethyl fumarate with the nalfurafine-fingolimod combination providing enhanced benefits.

The Kappa Opioid Receptor: A Promising Therapeutic Target for Multiple Pathologies.

Kappa-opioid receptors (KOR) are widely expressed throughout the central nervous system, where they modulate a range of physiological processes depending on their location, including stress, mood, reward, pain, inflammation, and remyelination. However, clinical use of KOR agonists is limited by adverse effects such as dysphoria, aversion, and sedation. Within the drug-development field KOR agonists have been extensively investigated for the treatment of many centrally mediated nociceptive disorders including pruritis and pain. KOR agonists are potential alternatives to mu-opioid receptor (MOR) agonists for the treatment of pain due to their anti-nociceptive effects, lack of abuse potential, and reduced respiratory depressive effects, however, dysphoric side-effects have limited their widespread clinical use. Other diseases for which KOR agonists hold promising therapeutic potential include pruritis, multiple sclerosis, Alzheimer's disease, inflammatory diseases, gastrointestinal diseases, cancer, and ischemia. This review highlights recent drug-development efforts targeting KOR, including the development of G-protein-biased ligands, mixed opioid agonists, and peripherally restricted ligands to reduce side-effects. We also highlight the current KOR agonists that are in preclinical development or undergoing clinical trials.

Sex Differences in the Behavioural Aspects of the Cuprizone-Induced Demyelination Model in Mice.

Multiple sclerosis is an autoimmune disease characterised by demyelination in the central nervous system. The cuprizone-induced demyelination model is often used in mice to test novel treatments for multiple sclerosis. However, despite significant demyelination, behavioural deficits may be subtle or have mixed results depending on the paradigm used. Furthermore, the sex differences within the model are not well understood. In the current study, we have sought to understand the behavioural deficits associated with the cuprizone-induced demyelination model in both male and female C57BL/6J mice. Using Black gold II stain, we found that cuprizone administration over 6 weeks caused significant demyelination in the corpus callosum that was consistent across both sexes. Cuprizone administration caused increased mechanical sensitivity when measured using an electronic von Frey aesthesiometer, with no sex differences observed. However, cuprizone administration decreased motor coordination, with more severe deficits seen in males in the horizontal bar and passive wire hang tests. In contrast, female mice showed more severe deficits in the motor skill sequence test. Cuprizone administration caused more anxiety-like behaviours in males compared to females in the elevated zero maze. Therefore, this study provides a better understanding of the sex differences involved in the behavioural aspects of cuprizone-induced demyelination, which could allow for a better translation of results from the laboratory to the clinic.

Sex Differences in Kappa Opioid Receptor Agonist Mediated Attenuation of Chemotherapy-Induced Neuropathic Pain in Mice.

Chemotherapy-induced neuropathic pain is a common side effect for cancer patients which has limited effective treatment options. Kappa opioid receptor (KOR) agonists are a promising alternative to currently available opioid drugs due to their low abuse potential. In the current study, we have investigated the effects of Salvinorin A (SalA) analogues, 16-Ethynyl SalA, 16-Bromo SalA and ethyoxymethyl ether (EOM) SalB, and in a preclinical model of paclitaxel-induced neuropathic pain in male and female C57BL/6J mice. Using an acute dose-response procedure, we showed that compared to morphine, 16-Ethynyl SalA was more potent at reducing mechanical allodynia; and SalA, 16-Ethynyl SalA, and EOM SalB were more potent at reducing cold allodynia. In the mechanical allodynia testing, U50,488 was more potent in males and SalA was more potent in females. There were no sex differences in the acute cold allodynia testing. In the chronic administration model, treatment with U50,488 (10 mg/kg) reduced the mechanical and cold allodynia responses to healthy levels over 23 days of treatment. Overall, we have shown that KOR agonists are effective in a model of chemotherapy-induced neuropathic pain, indicating that KOR agonists could be further developed to treat this debilitating condition.

The imidazodiazepine, KRM-II-81: An example of a newly emerging generation of GABAkines for neurological and psychiatric disorders.

GABAkines, or positive allosteric modulators of γ-aminobutyric acid-A (GABAA) receptors, are used for the treatment of anxiety, epilepsy, sleep, and other disorders. The search for improved GABAkines, with reduced safety liabilities (e.g., dependence) or side-effect profiles (e.g., sedation) constituted multiple discovery and development campaigns that involved a multitude of strategies over the past century. Due to the general lack of success in the development of new GABAkines, there had been a decades-long draught in bringing new GABAkines to market. Recently, however, there has been a resurgence of efforts to bring GABAkines to patients, the FDA approval of the neuroactive steroid brexanolone for post-partum depression in 2019 being the first. Other neuroactive steroids are in various stages of clinical development (ganaxolone, zuranolone, LYT-300, Sage-324, PRAX 114, and ETX-155). These GABAkines and non-steroid compounds (GRX-917, a TSPO binding site ligand), darigabat (CVL-865), an α2/3/5-preferring GABAkine, SAN711, an α3-preferring GABAkine, and the α2/3-preferring GABAkine, KRM-II-81, bring new therapeutic promise to this highly utilized medicinal target in neurology and psychiatry. Herein, we also discuss possible conditions that have enabled the transition to a new age of GABAkines. We highlight the pharmacology of KRM-II-81 that has the most preclinical data reported. KRM-II-81 is the lead compound in a new series of orally bioavailable imidazodiazepines entering IND-enabling safety studies. KRM-II-81 has a preclinical profile predicting efficacy against pharmacoresistant epilepsies, traumatic brain injury, and neuropathic pain. KRM-II-81 also produces anxiolytic- and antidepressant-like effects in rodent models. Other key features of the pharmacology of this compound are its low sedation rate, lack of tolerance development, and the ability to prevent the development of seizure sensitization.

The effects of nicotine and cigarette smoke on the monoamine transporters.

Cigarette smoking is the leading cause of preventable illness worldwide; however, smoking addiction remains poorly understood and cessation therapies based on nicotine replacement have limited success. The monoamine transporters are the primary mechanism for regulating the levels of dopamine, serotonin and norepinephrine in the synapse, and have been implicated in addiction and associated behaviors. Furthermore, the non-nicotinic smoking cessation therapy bupropion acts at least in part by blocking the dopamine and norepinephrine transporters. Despite this, little work has been conducted into the effects of nicotine and cigarette smoke on the monoamine transporters. This review will outline research that has been conducted to date on cigarette smoke, nicotine and the monoamine transporters. This will include monoamine transporter regulation by nicotine and cigarette smoke, genetic associations of the transporters with smoking behavior, and the potential for monoamine transporters to be targets in the development of smoking cessation pharmacotherapies.

Nalfurafine reduces neuroinflammation and drives remyelination in models of CNS demyelinating disease.

OBJECTIVES: Multiple sclerosis (MS) is a neurodegenerative disease characterised by inflammation and damage to the myelin sheath, resulting in physical and cognitive disability. There is currently no cure for MS, and finding effective treatments to prevent disease progression has been challenging. Recent evidence suggests that activating kappa opioid receptors (KOR) has a beneficial effect on the progression of MS. Although many KOR agonists like U50,488 are not suitable for clinical use because of a poor side-effect profile, nalfurafine is a potent, clinically used KOR agonist with a favorable side-effect profile. METHODS: Using the experimental autoimmune encephalomyelitis (EAE) model, the effect of therapeutically administered nalfurafine or U50,488 on remyelination, CNS infiltration and peripheral immune responses were compared. Additionally, the cuprizone model was used to compare the effects on non-immune demyelination. RESULTS: Nalfurafine enabled recovery and remyelination during EAE. Additionally, it was more effective than U50,488 and promoted disease reduction when administered after chronic demyelination. Blocking KOR with the antagonist, nor-BNI, impaired full recovery by nalfurafine, indicating that nalfurafine mediates recovery from EAE in a KOR-dependent fashion. Furthermore, nalfurafine treatment reduced CNS infiltration (especially CD4+ and CD8+ T cells) and promoted a more immunoregulatory environment by decreasing Th17 responses. Finally, nalfurafine was able to promote remyelination in the cuprizone demyelination model, supporting the direct effect on remyelination in the absence of peripheral immune cell invasion. CONCLUSIONS: Overall, our findings support the potential of nalfurafine to promote recovery and remyelination and highlight its promise for clinical use in MS.

The Salvinorin Analogue, Ethoxymethyl Ether Salvinorin B, Promotes Remyelination in Preclinical Models of Multiple Sclerosis.

Multiple sclerosis is a neurodegenerative disease associated with demyelination and neuroinflammation in the central nervous system. There is an urgent need to develop remyelinating therapies to better treat multiple sclerosis and other demyelinating diseases. The kappa opioid receptor (KOR) has been identified as a potential target for the development of remyelinating therapies; however, prototypical KOR agonists, such as U50,488 have side effects, which limit clinical use. In the current study, we investigated a Salvinorin A analog, ethoxymethyl ether Salvinorin B (EOM SalB) in two preclinical models of demyelination in C57BL/6J mice. We showed that in cellular assays EOM SalB was G-protein biased, an effect often correlated with fewer KOR-mediated side effects. In the experimental autoimmune encephalomyelitis model, we found that EOM SalB (0.1-0.3 mg/kg) effectively decreased disease severity in a KOR-dependent manner and led to a greater number of animals in recovery compared to U50,488 treatment. Furthermore, EOM SalB treatment decreased immune cell infiltration and increased myelin levels in the central nervous system. In the cuprizone-induced demyelination model, we showed that EOM SalB (0.3 mg/kg) administration led to an increase in the number of mature oligodendrocytes, the number of myelinated axons and the myelin thickness in the corpus callosum. Overall, EOM SalB was effective in two preclinical models of multiple sclerosis and demyelination, adding further evidence to show KOR agonists are a promising target for remyelinating therapies.

Oxidative Metabolism as a Modulator of Kratom's Biological Actions.

The leaves of Mitragyna speciosa (kratom), a plant native to Southeast Asia, are increasingly used as a pain reliever and for attenuation of opioid withdrawal symptoms. Using the tools of natural products chemistry, chemical synthesis, and pharmacology, we provide a detailed in vitro and in vivo pharmacological characterization of the alkaloids in kratom. We report that metabolism of kratom's major alkaloid, mitragynine, in mice leads to formation of (a) a potent mu opioid receptor agonist antinociceptive agent, 7-hydroxymitragynine, through a CYP3A-mediated pathway, which exhibits reinforcing properties, inhibition of gastrointestinal (GI) transit and reduced hyperlocomotion, (b) a multifunctional mu agonist/delta-kappa antagonist, mitragynine pseudoindoxyl, through a CYP3A-mediated skeletal rearrangement, displaying reduced hyperlocomotion, inhibition of GI transit and reinforcing properties, and (c) a potentially toxic metabolite, 3-dehydromitragynine, through a non-CYP oxidation pathway. Our results indicate that the oxidative metabolism of the mitragynine template beyond 7-hydroxymitragynine may have implications in its overall pharmacology in vivo.

N-docosahexaenoyl ethanolamine (synaptamide) has antinociceptive effects in male mice.

BACKGROUND: N-docosahexaenoyl ethanolamine (DHEA; also known as synaptamide) binds to both the cannabinoid-1 and 2 (CB1 and CB2) cannabinoid receptors and has anti-inflammatory properties in vitro. However, the in vivo effects of DHEA remain unknown. Therefore, this study was designed to understand the effects of DHEA in models of pain and inflammation in mice. METHODS: The intraplantar formaldehyde assay, hot water tail withdrawal assay and hotplate model were used to assess the antinociceptive properties of DHEA in mice. The mechanism of action was studied by antagonising the cannabinoid receptors, transient receptor potential vanilloid 1 (TRPV1) ion channel, peroxisome proliferator-activated receptors (PPARs) and G-protein receptor 55 (GPR55). RESULTS: N-docosahexaenoyl ethanolamine (2-10 mg/kg) reduced the levels of nociceptive and inflammatory pain-related behaviour over 60 min in the intraplantar formaldehyde assay via both intraperitoneal and local intraplantar administration. The area under the curve analysis showed the overall antinociceptive effects of DHEA (10 mg/kg) were not modulated by pre-treatment with antagonists for the cannabinoid receptors, TRPV1ion channel, PPARα, PPARγ or GPR55. However, the time-course analysis showed that within the early inflammatory phase, antagonism of the CB2 receptor, PPARα and PPARγ led to a partial reversal of the antinociceptive effects of DHEA. In the hot water tail withdrawal and hotplate models of thermal nociception, DHEA (2-10 mg/kg) did not have any antinociceptive effects. CONCLUSIONS: N-docosahexaenoyl ethanolamine reduced the level of formaldehyde-induced nociceptive and inflammatory pain-related behaviour; however, was not active in thermal nociceptive models. This study highlights the potential of DHEA for the treatment of acute inflammatory pain. SIGNIFICANCE: This study shows that both intraperitoneal and intraplantar administration of DHEA reduces the level of formaldehyde-induced nociceptive and inflammatory pain.

Evaluation of Biased and Balanced Salvinorin A Analogs in Preclinical Models of Pain.

In the search for safer, non-addictive analgesics, kappa opioid receptor (KOPr) agonists are a potential target, as unlike mu-opioid analgesics, they do not have abuse potential. Salvinorin A (SalA) is a potent and selective KOPr agonist, however, clinical utility is limited by the short duration of action and aversive side effects. Biasing KOPr signaling toward G-protein activation has been highlighted as a key cellular mechanism to reduce the side effects of KOPr agonists. The present study investigated KOPr signaling bias and the acute antinociceptive effects and side effects of two novel analogs of SalA, 16-Bromo SalA and 16-Ethynyl SalA. 16-Bromo SalA showed G-protein signaling bias, whereas 16-Ethynyl SalA displayed balanced signaling properties. In the dose-response tail-withdrawal assay, SalA, 16-Ethynyl SalA and 16-Bromo SalA were more potent than the traditional KOPr agonist U50,488, and 16-Ethynyl SalA was more efficacious. 16-Ethynyl SalA and 16-Bromo SalA both had a longer duration of action in the warm water tail-withdrawal assay, and 16-Ethynyl had greater antinociceptive effect in the hot-plate assay, compared to SalA. In the intraplantar 2% formaldehyde test, 16-Ethynyl SalA and 16-Bromo SalA significantly reduced both nociceptive and inflammatory pain-related behaviors. Moreover, 16-Ethynyl SalA and 16-Bromo SalA had no anxiogenic effects in the marble burying task, and 16-Bromo SalA did not alter behavior in the elevated zero maze. Overall, 16-Ethynyl SalA significantly attenuated acute pain-related behaviors in multiple preclinical models, while the biased KOPr agonist, 16-Bromo SalA, displayed modest antinociceptive effects, and lacked anxiogenic effects.

Synthetic Studies of Neoclerodane Diterpenes from Salvia divinorum: Design, Synthesis, and Evaluation of Analogues with Improved Potency and G-protein Activation Bias at the µ-Opioid Receptor.

Previous structure-activity relationship (SAR) studies identified the first centrally acting, non-nitrogenous µ-opioid receptor (MOR) agonist, kurkinorin (1), derived from salvinorin A. In an effort to further probe the physiological effects induced upon activation of MORs with this nonmorphine scaffold, a variety of analogues were synthesized and evaluated in vitro for their ability to activate G-proteins and recruit ß-arrestin-2 upon MOR activation. Through these studies, compounds that are potent agonists at MORs and either biased toward ß-arrestin-2 recruitment or biased toward G-protein activation have been identified. One such compound, 25, has potent activity and selectivity at the MOR over KOR with bias for G-protein activation. Impressively, 25 is over 100× more potent than morphine and over 5× more potent than fentanyl in vitro and elicits antinociception with limited tolerance development in vivo. This is especially significant given that 25 lacks a basic nitrogen and other ionizable groups present in other opioid ligand classes.