Ross van de Wetering

The development of safer mu opioid receptor (MOR) agonists with reduced side effects is a key focus of pain research. Some studies have suggested that MOR agonists with reduced β-arrestin 2 (βArr2) signaling (i.e. G-protein biased agonists) may have greater therapeutic windows. However, there have been a several conflicting reports, and it is not clear what role, if any, βArr2 signaling plays in MOR-mediated analgesia, tolerance, or side effects. Therefore, we used βArr2 knockout mice to systematically investigate the causal role of βArr2 signaling in antinociception, antinociceptive tolerance, respiratory depression, constipation, and reward induced by morphine and the two novel MOR agonists, kurkinorin and kurkinol. Kurkinorin and kurkinol exhibited potent antinociceptive effects that were reversed by MOR knockout. Unlike morphine or kurkinorin, our most G-protein biased agonist, kurkinol, showed no significant tolerance after seven days of ~2×ED₅₀ dosing. However, in a chemotherapy-induced neuropathic pain model, all three compounds were ineffective after 20 days of ~ED₅₀ dosing, indicative of tolerance. All compounds exhibited significant MOR-dependent side effects, though kurkinorin had reduced gastrointestinal and respiratory depressive effects compared to morphine despite exhibiting less G-protein bias. Knockout of βArr2 significantly increased antinociceptive potency for morphine and kurkinorin but not kurkinol, and otherwise had no significant impact on tolerance or any side effect tested. These results largely suggest that βArr2 signaling does not drive MOR-mediated antinociceptive tolerance, respiratory depression, constipation, or reward and do not support the development of G-protein biased compounds as a broadly effective strategy to reduce side effects.

Kappa opioid receptor (KOR) agonists have well-established antinociceptive effects. However, many KOR agonists have negative side effects, which limit their therapeutic potential. Some researchers have suggested that the development of biased agonists that preferentially stimulate KOR G-protein pathways over β-arrestin pathways may yield drugs with fewer adverse side effects. This was investigated in the current study. We describe the synthesis and characterization of three U50,488 analogues, 1, 2, and 3. We evaluated the acute and chronic antinociceptive effects of these compounds in mice using the warm-water tail flick assay and in a paclitaxel-induced neuropathic pain model. Side effects were investigated using open-field, passive wire hang, rotarod, elevated zero maze, conditioned place aversion, and whole-body plethysmography, with some tests being conducted in KOR or β-arrestin2 knock out mice. All compounds were highly potent, full agonists of the KOR, with varying signaling biases in vitro. In the warm-water tail withdrawal assay, these agonists were ~10 times more potent than U50,488, but not more efficacious. All KOR agonists reversed paclitaxel-induced neuropathic pain, without tolerance. Compound 3 showed no significant side effects on any test. Signaling bias did not correlate with the antinociceptive or side effects of any compounds and knockout of β-arrestin2 had no effect on U50,488-induced sedation or motor incoordination. These findings highlight the therapeutic potential of 3, with its lack of side effects typically associated with KOR agonists, and also suggest that G-protein signaling bias is a poor predictor of KOR agonist-induced side effects.

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.

Introduction: Metabolomics produces vast quantities of data but determining which metabolites are the most relevant to the disease or disorder of interest can be challenging. Objectives: This study sought to demonstrate how behavioral models of psychiatric disorders can be combined with metabolomics research to overcome this limitation. Methods: We designed a preclinical, untargeted metabolomics procedure, that focuses on the determination of central metabolites relevant to substance use disorders that are (a) associated with changes in behavior produced by acute drug exposure and (b) impacted by repeated drug exposure. Untargeted metabolomics analysis was carried out on liquid chromatography-mass spectrometry data obtained from 336 microdialysis samples. Samples were collected from the medial striatum of male Sprague-Dawley (N = 21) rats whilst behavioral data were simultaneously collected as part of a (±)-3,4-methylenedioxymethamphetamine (MDMA)-induced behavioral sensitization experiment. Analysis was conducted by orthogonal partial least squares, where the Y variable was the behavioral data, and the X variables were the relative concentrations of the 737 detected features. Results: MDMA and its derivatives, serotonin, and several dopamine/norepinephrine metabolites were the greatest predictors of acute MDMA-produced behavior. Subsequent univariate analyses showed that repeated MDMA exposure produced significant changes in MDMA metabolism, which may contribute to the increased abuse liability of the drug as a function of repeated exposure. Conclusion: These findings highlight how the inclusion of behavioral data can guide metabolomics data analysis and increase the relevance of the results to the phenotype of interest.

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.

Repeated exposure to drugs produces a plethora of persistent brain changes, some of which underlie the development of drug addiction. An important objective of addiction research is to identify the brain changes that might mediate the transition from drug use to drug misuse. The persistent accumulation of the transcription factor, ∆FosB, following repeated drug exposure provides a means of achieving this objective. Experiments were conducted on sexually mature male Sprague‐Dawley rats. The effects of extensive 3,4‐methylenedioxymethamphetamine (MDMA) self‐administration on immunohistochemical measurements of ∆FosB accumulation in 12 brain regions was compared with a matched, drug‐naive, control group. Other groups were pretreated with MDMA (0.0 or 10.0 mg/kg, ip, once daily for 5 days), and the locomotor‐activating effect of MDMA (200 μg/side) microinjected bilaterally into brain regions selected on the basis of the ∆FosB results was subsequently determined. MDMA self‐administration significantly increased ∆FosB expression in the nucleus accumbens core, ventromedial and dorsomedial caudate‐putamen, anterior cingulate, prelimbic, infralimbic, and orbitofrontal cortex, and both the central and basolateral amygdala, but not in the ventrolateral or dorsolateral caudate‐putamen. Increases in the nucleus accumbens shell were substantial but were not significant following statistical correction for multiple comparisons. MDMA pretreatment enhanced MDMA‐produced hyperactivity only when administered into the nucleus accumbens or the medial, but not the lateral, caudate‐putamen, mirroring the ∆FosB results. These data compare favorably to results following repeated exposure to other drugs of abuse and support the idea of common neuroplastic changes following repeated drug exposure.

Repeated exposure to ±3, 4-methylenedioxymethamphetamine (MDMA) produces sensitization to MDMA-produced hyperactivity, but the mechanisms underlying the development of this sensitized response or the relationship to the reinforcing effects of MDMA is unknown.