We present the synthesis and pharmacological characterization of 3'-iodobenzoylamido epoxymorphinans, with a saturated ring C and devoid of 14-OH, based on a dihydronormorphine backbone with various N-17 alkyl substitutions. All synthesized compounds were characterized pharmacologically in radioligand binding assays, [35S]GTPγS functional assays, and for antinociception in mice. Among these analogues, MP1202, a pan-opioid receptor analogue, was characterized further in β-arrestin1/2 studies, G-protein recruitment and Gα-subtype profiling, as well as by evaluation of opioid-mediated effects in mice. MP1202 exhibits potent antinociceptive effects in mice without causing typical opioid side effects, such as respiratory depression and physical dependence. However, it displays conditioned place preference and aversion behaviors similar to those of classical mu and kappa agonists.

Schwann cells (SCs) are the primary glia of the peripheral nervous system. SCs are involved in many debilitating disorders, including diabetic peripheral neuropathy (DPN). Here, we present a strategy for deriving SCs from human pluripotent stem cells (hPSCs) that enables comprehensive studies of SC development, physiology, and disease. hPSC-derived SCs recapitulate the molecular features of primary SCs and are capable of in vitro and in vivo myelination. We established a model of DPN that revealed the selective vulnerability of SCs to high glucose. We performed a high-throughput screen and found that an antidepressant drug, bupropion, counteracts glucotoxicity in SCs. Treatment of hyperglycemic mice with bupropion prevents their sensory dysfunction, SC death, and myelin damage. Further, our retrospective analysis of health records revealed that bupropion treatment is associated with a lower incidence of neuropathy among diabetic patients. These results highlight the power of this approach for identifying therapeutic candidates for DPN.

Dry leaves of kratom (mitragyna speciosa) are anecdotally consumed as pain relievers and antidotes against opioid withdrawal and alcohol use disorders. There are at least 54 alkaloids in kratom; however, investigations to date have focused around mitragynine, 7-hydroxy mitragynine (7OH), and mitragynine pseudoindoxyl (MP). Herein, we probe a few minor indole and oxindole based alkaloids, reporting the receptor affinity, G-protein activity, and βarrestin-2 signaling of corynantheidine, corynoxine, corynoxine B, mitraciliatine, and isopaynantheine at mouse and human opioid receptors. We identify corynantheidine as a mu opioid receptor (MOR) partial agonist, whereas its oxindole derivative corynoxine was an MOR full agonist. Similarly, another alkaloid mitraciliatine was found to be an MOR partial agonist, while isopaynantheine was a KOR agonist which showed reduced βarrestin-2 recruitment. Corynantheidine, corynoxine, and mitraciliatine showed MOR dependent antinociception in mice, but mitraciliatine and corynoxine displayed attenuated respiratory depression and hyperlocomotion compared to the prototypic MOR agonist morphine in vivo when administered supraspinally. Isopaynantheine on the other hand was identified as the first kratom derived KOR agonist in vivo. While these minor alkaloids are unlikely to play the majority role in the biological actions of kratom, they represent excellent starting points for further diversification as well as distinct efficacy and signaling profiles with which to probe opioid actions in vivo.

Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here, we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex with MP1104, to rationally design novel mixed mu (MOR) and kappa (KOR) opioid receptor agonists with reduced arrestin signaling. Analysis of structure-activity relationships for new MP1104 analogs points to a region between transmembrane 5 (TM5) and extracellular loop (ECL2) as key for modulation of arrestin recruitment to both MOR and KOR. The lead compounds, MP1207 and MP1208, displayed MOR/KOR Gipartial agonism with diminished arrestin signaling, showed efficient analgesia with attenuated liabilities, including respiratory depression and conditioned place preference and aversion in mice. The findings validate a novel structure-inspired paradigm for achieving beneficial in vivo profiles for analgesia through different mechanisms that include bias, partial agonism, and dual MOR/KOR agonism.

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.

Opioid analgesics, most of which act through mu opioid receptors, have long represented valuable therapeutic agents to treat severe pain. Concerted drug development efforts for over a 100 years have resulted in a large variety of opioid analgesics used in the clinic, but all of them continue to exhibit the side effects, especially respiratory depression, that have long plagued the use of morphine. The recent explosion in fatalities resulting from overdose of prescription and synthetic opioids has dramatically increased the need for safer analgesics, but recent developments in mu receptor research have provided new strategies to develop such drugs. This chapter reviews recent advances in developing novel opioid analgesics from an understanding of mu receptor structure and function. This includes a summary of the mechanism of agonist binding deduced from the crystal structure of mu receptors. It will also highlight the development of novel agonist mechanisms, including biased agonists, bivalent ligands, and allosteric modulators of mu receptor function, and describe how receptor phosphorylation modulates these pathways. Finally, it will summarize research on the alternative pre-mRNA splicing mechanisms that produces a multiplicity of mu receptor isoforms. Many of these isoforms exhibit different pharmacological specificities and brain circuitry localization, thus providing an opportunity to develop novel drugs with increased therapeutic windows.

The original version of this article unfortunately contained a typo in co-author name.

Mitragyna speciosa, also known as kratom, is a plant containing opioidergic alkaloids and is currently being used to self‐medicate chronic pain, opioid dependence, and is also used as a heroin substitute. Kratom, though unscheduled, may be less dangerous when compared to prescription opioids due to the preferential interaction of kratom alkaloids with G‐proteins instead of beta‐arrestin proteins. We hypothesized that kratom alkaloids and synthetic opioids with similar biased pharmacology can be used to treat alcohol use disorder. We completed in vitro pharmacological characterization for two kratom strain extracts, four kratom alkaloids (mitragynine, 7‐hydroxymitragynine, paynantheine, and speciogynine), and a series of synthetic carefentanil‐amide opioids by determining their ability to interact selectively with G‐proteins instead of beta‐arrestin at the mu, delta and kappa opioid receptors. In addition, in C57BL/6 mice we assessed if kratom (alkaloids) and opioids were capable of reducing alcohol use and determined if the alkaloids had rewarding properties. We found that kratom alkaloids are strongly biased towards the G‐protein pathway at all three opioid receptors. More specifically, we found the delta opioid receptor plays an important role in the mechanism of action as interpreted from reduced efficacy in knockout mice. MP102, one of the synthesized carfentanilamide opioids, displayed a pharmacological profile similar to the kratom alkaloids but with preference for delta opioid receptors, and also reduced alcohol intake and likewise produced less significant place preference than morphine. Our findings demonstrate that kratom use can decrease alcohol use, but its use still carries substantial risk. The development of further delta‐opioid‐selective synthetic opioids may provide an avenue for alcohol use treatment that does not produce strong rewards.Support or Funding InformationRMvR support: NIH grants AA025368, AA026949, AA026675, DA045897, a NARSAD young investigator award from The Brain and Behavior Research Foundation, the Purdue Institute for Drug Discovery and the Dept. of Med.Chem. and Molecular Pharmacology. SM support: NIH grants DA045884, DA046487, AA026949 and W81XWH‐17‐1‐0256 & start‐up funds from the Center for Clinical Pharmacology in St. Louis. Funded in part through NIH/NCI Cancer Center Support Grant P30. CA008748.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sigma-1 receptors (S1Rs) are overexpressed in almost all human cancers, especially in breast cancers. 1-(4-Iodophenyl)-3-(2-adamantyl)guanidine (IPAG) is a validated high-affinity S1R antagonist. The objective of the current study is to evaluate the potential of iodine-124-labeled IPAG ([124I]IPAG) to image S1R-overexpressing tumors. [124I]IPAG was synthesized from a tributyltin precursor dissolved in ethanol using chloramine-T as oxidant. Purity was analyzed using HPLC. In vitro and in vivo studies were performed using the breast cancer cell line MCF-7. Competitive inhibition studies were performed using haloperidol and cold IPAG. Tumors were established in athymic nude mice by injecting 107 cells subcutaneously. Mice were imaged on micro-positron emission tomography (PET) at 4, 24, 48, 72, and 144 h post i.v. injection. Biodistribution studies were performed at same time points. In vivo tracer dilution studies were performed using excess of IPAG and haloperidol. The efficacy of [124I]IPAG to image tumors was evaluated in LNCaP tumor–bearing mice as well. [124I]IPAG was synthesized in quantitative yield and in vitro studies indicated that [124I]IPAG binding was specific to S1R. PET imaging studies in MCF7 tumor–bearing mice reveal that [124I]IPAG accumulates in tumor and is preferentially retained while clearing from non-target organs. The tumor to background increases with time, and tumors could be clearly visualized starting from 24 h post administration. Similar results were obtained in mice bearing LNCaP tumors. In vivo tracer dilution studies showed that the uptake of [124I]IPAG could be competitively inhibited by excess of IPAG and haloperidol. [124I]IPAG was synthesized successfully in high yields, and in vitro and in vivo studies demonstrate specificity of [124I]IPAG. [124I]IPAG shows specific accumulation in tumors with increasing tumor to background ratio at later time points and therefore has high potential for imaging S1R-overexpressing cancers.

Opioid medications are widely prescribed to alleviate pain and suffering for millions of patients, but the utility of these drugs is limited by serious adverse effects including abuse liability, dependence, and overdose. At present, the non-medical (i.e., recreational) abuse of opioids is a worldwide public health threat. In the United States alone, more than 47,000 opioid-related overdose deaths occurred during 2017 (Scholl et al., 2018), and most of these fatalities were associated with synthetic opioids, especially fentanyl and its various analogs. Fentanyl is a mu-opioid receptor (MOP) agonist that is 50-100 times more potent than morphine as an analgesic agent. The origins of the current opioid crisis are complex, and effective solutions will require multidisciplinary cooperation among law enforcement personnel, first responders, treatment providers, policymakers, and scientists (Madras, 2018). To this end, basic research in pharmacology can provide critical information for addressing the opioid crisis (Baumann et al., 2018). In this Special Issue of Neuropharmacology, entitled “New Vistas in Opioid Pharmacology”, we bring together an international panel of experts who report research findings related to three topics: 1] neuropharmacology of heroin, fentanyl and its analogs; 2] development of safer pain medications; and 3] novel pharmacotherapies for substance use disorders.

Mitragynina speciosa, more commonly known as kratom, is a plant native to Southeast Asia, the leaves of which have been used traditionally as a stimulant, analgesic, and treatment for opioid addiction. Recently, growing use of the plant in the United States and concerns that kratom represents an uncontrolled drug with potential abuse liability, have highlighted the need for more careful study of its pharmacological activity. The major active alkaloid found in kratom, mitragynine, has been reported to have opioid agonist and analgesic activity in vitro and in animal models, consistent with the purported effects of kratom leaf in humans. However, preliminary research has provided some evidence that mitragynine and related compounds may act as atypical opioid agonists, inducing therapeutic effects such as analgesia, while limiting the negative side effects typical of classical opioids. Here we report evidence that an active metabolite plays an important role in mediating the analgesic effects of mitragynine. We find that mitragynine is converted in vitro in both mouse and human liver preparations to the much more potent mu-opioid receptor agonist 7-hydroxymitragynine, and that this conversion is mediated by cytochrome P450 3A isoforms. Further, we show that 7-hydroxymitragynine is formed from mitragynine in mice and that brain concentrations of this metabolite are sufficient to explain most or all of the opioid-receptor-mediated analgesic activity of mitragynine. At the same time, mitragynine is found in the brains of mice at very high concentrations relative to its opioid receptor binding affinity, suggesting that it does not directly activate opioid receptors. The results presented here provide a metabolism-dependent mechanism for the analgesic effects of mitragynine and clarify the importance of route of administration for determining the activity of this compound. Further, they raise important questions about the interpretation of existing data on mitragynine and highlight critical areas for further research in animals and humans.

Circular RNAs (circRNAs) are a distinct category of single-stranded, covalently closed RNAs formed by backsplicing. The functions of circRNAs are incompletely known and are under active investigation. Here, we report that in addition to traditional linear mRNAs (linRNA), mouse, rat, and human opioid receptor genes generate exonic circRNA isoforms. Using standard molecular biologic methods, Oprm1 circRNAs (circOprm1) were detected in RNAs of rodent and human brains and spinal cords, as well as human neuroblastoma cells, suggesting evolutionary conservation. Sequencing confirmed backsplicing using canonical splice sites. Oprm1 circRNAs were sense-stranded circRNAs resistant to RNase R digestion. The relative abundance of Oprm1 circRNA to linRNA determined by quantitative reverse transcription polymerase chain reaction varied among mouse brain regions, with circRNA isoforms predominating in rostral structures and less abundant in brain stem. Chronic morphine exposure in mice increased brain circOprm1.e2.3 and circOprm1.e2.e3.e4(302) levels by 1.5- to 1.6-fold relative to linRNA. Sequence analysis predicted numerous microRNA binding sites within Oprm1 circRNA sequences, suggesting a potential role in microRNA sequestration through sponging. In addition, we observed that other opioid receptor genes including δ, κ, and nociceptin receptor genes produced similar circRNAs. In conclusion, all members of the opioid receptor gene family express circRNAs, with Oprm1 circRNA levels exceeding those of linear forms in some regions. Significance Statement The modulation of Oprm1 circular RNA (circRNA) expression by morphine, coupled with the high abundance and existence of potential miRNA binding sites with circRNA sequences suggests the potential role of Oprm1 circRNAs in chronic opioid effects such as tolerance.

Kappa opioid receptor (KOPr) agonists have preclinical anti-cocaine and antinociceptive effects. However, adverse effects including dysphoria, aversion, sedation, anxiety and depression limit their clinical development. MP1104, an analogue of 3-iodobenzoyl naltrexamine, is a potent dual agonist at KOPr and delta opioid receptor (DOPr), with full agonist efficacy at both these receptors. In this study, we evaluate the ability of MP1104 to modulate cocaine-induced behaviors and side-effects preclinically. In male Sprague-Dawley rats trained to self-administer cocaine, MP1104 (0.3 and 1 mg/kg) reduced cocaine-primed reinstatement of drug-seeking behavior and caused significant downward shift of the dose-response curve in cocaine self-administration tests (0.3 and 0.6 mg/kg). The anti-cocaine effects exerted by MP1104 are in part due to increased dopamine (DA) uptake by the dopamine transporter (DAT) in the dorsal striatum (dStr) and nucleus accumbens (NAc). MP1104 (0.3 and 0.6 mg/kg) showed no significant anxiogenic effects in the elevated plus maze, pro-depressive effects in the forced swim test, or conditioned place aversion. Furthermore, pretreatment with a DOPr antagonist, led to MP1104 producing aversive effects. This data suggests that the DOPr agonist actions of MP1104 attenuate the KOPr-mediated aversive effects of MP1104. The overall results from this study show that MP1104, modulates DA uptake in the dStr and NAc, and exerts potent anti-cocaine properties in self-administration tests with reduced side-effects compared to pure KOPr agonists. This data supports the therapeutic development of dual KOPr/DOPr agonists to reduce the side-effects of selective KOPr agonists. This article is part of the Special Issue entitled 'Opioid Neuropharmacology: Advances in treating pain and opioid addiction'.

BACKGROUND: Levorphanol is a potent analgesic that has been used for decades. Most commonly used for acute and cancer pain, it also is effective against neuropathic pain. The recent appreciation of the importance of functional bias and the uncovering of multiple µ opioid receptor splice variants may help explain the variability of patient responses to different opioid drugs. METHODS: Here, we evaluate levorphanol in a variety of traditional in vitro receptor binding and functional assays. In vivo analgesia studies using the radiant heat tail flick assay explored the receptor selectivity of the responses through the use of knockout (KO) mice, selective antagonists, and viral rescue approaches. RESULTS: Receptor binding studies revealed high levorphanol affinity for all the μ, δ, and κ opioid receptors. In 35 S-GTPγS binding assays, it was a full agonist at most µ receptor subtypes, with the exception of MOR-1 O , but displayed little activity in β-arrestin2 recruitment assays, indicating a preference for G-protein transduction mechanisms. A KO mouse and selective antagonists confirmed that levorphanol analgesia was mediated through classical µ receptors, but there was a contribution from 6 transmembrane targets, as illustrated by a lower response in an exon 11 KO mouse and its rescue with a virally transfected 6 transmembrane receptor splice variant. Compared to morphine, levorphanol had less respiratory depression at equianalgesic doses. CONCLUSIONS: While levorphanol shares many of the same properties as the classic opioid morphine, it displays subtle differences that may prove helpful in its clinical use. Its G-protein signaling bias is consistent with its diminished respiratory depression, while its incomplete cross tolerance with morphine suggests it may prove valuable clinically with opioid rotation.

The differential modulation of agonist and antagonist binding to opioid receptors (ORs) by sodium (Na+) has been known for decades. To shed light on the molecular determinants, thermodynamics, and kinetics of Na+ translocation through the μ-OR (MOR), we used a multi-ensemble Markov model framework combining equilibrium and non-equilibrium atomistic molecular dynamics simulations of Na+ binding to MOR active or inactive crystal structures embedded in an explicit lipid bilayer. We identify an energetically favorable, continuous ion pathway through the MOR active conformation only, and provide, for the first time: i) estimates of the energy differences and required timescales of Na+ translocation in inactive and active MORs, ii) estimates of Na+-induced changes to agonist binding validated by radioligand measurements, and iii) testable hypotheses of molecular determinants and correlated motions involved in this translocation, which are likely to play a key role in MOR signaling.

Background A single‐copy mu opioid receptor ( OPRM1 ) gene generates a vast array of mu opioid receptor splice variants through extensive alternative splicing that is conserved from rodent to human. These variants can be divided into two sets: exon 11 (E11) and exon 1 (E1) associated variants. The functional differences between the E1 and E11 sets of variants have been demonstrated using gene targeted mouse models. Rationale/Significance While mouse models are valuable, rats have many advantages both in behavioral modeling and in vivo manipulation. Hypothesis E11‐ and E1‐associated splice variants play important and distinct roles In opioid pharmacology. Results We have generated two conditional Oprm1 knockout (KO) models, E11‐KO and E1‐KO, in Sprague Dawley rats using a novel Easi‐CRISPR approach. Cre‐mediated E11 and E1 deletions was confirmed through breeding a CAG‐Cre rat. Initial behavioral studies showed that in both E11 and E1 KO rats, buprenorphine analgesia was lost. Buprenorphine CPP was significantly reduced in E11 KO rats. Morphine analgesia was completely lost in the E1‐KO rat, but intact in the E11‐KO rat. Discussion Our results suggest that buprenorphine analgesia is dependent on both E11 and E1‐associated variants, a similar scenario seen in the E11‐KO and E1‐KO mouse models. E11‐associated variants also involved in buprenorphine reward in rat. These conditional Oprm1 KO rat models will provide valuable resources for the research community and would expand our ability explore mu opioid pharmacology with approaches and techniques not feasible in mice. Support or Funding Information Supported by grants from NIH (DA40858, DA06241, DA042888 and CA08748) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Computational chemists typically assay drug candidates by virtually screening compounds against crystal structures of a protein despite the fact that some targets, like the $\mu$ Opioid Receptor and other members of the GPCR family, traverse many non-crystallographic states. We discover new conformational states of $\mu OR$ with molecular dynamics simulation and then machine learn ligand-structure relationships to predict opioid ligand function. These artificial intelligence models identified a novel $\mu$ opioid chemotype.

Background: A single-copy mu opioid receptor (OPRM1) gene generates a vast array of mu opioid receptor splice variants through extensive alternative splicing that is conserved from rodent to human. These variants can be divided into two sets: exon 11 (E11) and exon 1 (E1) associated variants. The functional differences between the E1 and E11 sets of variants have been demonstrated using gene targeted mouse models.