Peptide-derived ligands for the discovery of safer opioid analgesics.

Drugs targeting the µ-opioid receptor (MOR) remain the most efficacious analgesics for the treatment of pain, but activation of MOR with current opioid analgesics also produces harmful side effects, notably physical dependence, addiction, and respiratory depression. Opioid peptides have been accepted as promising candidates for the development of safer and more efficacious analgesics. To develop peptide-based opioid analgesics, strategies such as modification of endogenous opioid peptides, development of multifunctional opioid peptides, G protein-biased opioid peptides, and peripherally restricted opioid peptides have been reported. This review seeks to provide an overview of the opioid peptides that produce potent antinociception with much reduced side effects in animal models and highlight the potential advantages of peptides as safer opioid analgesics.

NCP, a dual kappa and mu opioid receptor agonist, is a potent analgesic against inflammatory pain without reinforcing or aversive properties.

While agonists of mu (MOR) and kappa (KOR) opioid receptors have analgesic effects, they produce euphoria and dysphoria, respectively. Other side effects include respiratory depression and addiction for MOR agonists and sedation for KOR agonists. We reported that 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-{[4'-(2'-cyanopyridyl)]carboxamido}cmorphinan (NCP) displayed potent KOR full agonist and MOR partial agonist activities (58%) with 6.5x KOR-over-MOR selectivity in vitro Herein, we characterized pharmacological effects of NCP in rodents. In mice, NCP exerted analgesic effects against inflammatory pain in both the formalin test and the acetic acid writhing test, with A50 values of 47.6 and 14.4 microg/kg (s.c.), respectively. The analgesic effects in the acetic acid writhing test were mediated by the KOR. NCP at doses much higher than those effective in reducing inflammatory pain did not produce antinociception in the hot plate and tail flick tests, inhibit compound 48/80-induced scratching, cause conditioned place aversion (CPA) or preference, impair rotarod performance, inhibit locomotor activity, cause respiratory depression, or precipitate morphine withdrawal. However, NCP (10~100 microg/kg) inhibited gastrointestinal transit with a maximum of ~40% inhibition. In MOR knockout mice, NCP caused CPA, demonstrating that its lack of CPA is due to combined actions on the MOR and KOR. Following s.c. injection, NCP penetrated into the mouse brain. In rats trained to self-administer heroin, NCP (1~320 microg/kg/infusion) did not function as a reinforcer. Thus, NCP produces potent analgesic effects via KOR without side effects except constipation. Therefore, dual full KOR/partial MOR agonists with moderate KOR-over-MOR selectivity may be promising as non-addictive analgesics for inflammatory pain. Significance Statement Developing non-addictive analgesics is crucial for reducing opioid overdose deaths, minimizing drug misuse, and promoting safer pain management practices. Herein, pharmacology of a potential non-addictive analgesic, NCP, is reported. NCP has full KOR agonist / partial MOR agonist activities with a 6.5 x selectivity for KOR over MOR. Unlike MOR agonists, analgesic doses of NCP do not lead to self-administration or respiratory depression. Furthermore, NCP does not produce aversion, hypolocomotion, or motor incoordination, side effects typically associated with KOR activation.

Paternal morphine exposure enhances morphine self-administration and induces region-specific neural adaptations in reward-related brain regions of male offspring.

Background: A growing body of preclinical studies report that preconceptional experiences can have a profound and long-lasting impact on adult offspring behavior and physiology. However, less is known about paternal drug exposure and its effects on reward sensitivity in the next generation. Methods: Adult male rats self-administered morphine for 65 days; controls received saline. Sires were bred to drug-naïve dams to produce first-generation (F1) offspring. Morphine, cocaine, and nicotine self-administration were measured in adult F1 progeny. Molecular correlates of addiction-like behaviors were measured in reward-related brain regions of drug naïve F1 offspring. Results: Male, but not female offspring produced by morphine-exposed sires exhibited dose-dependent increased morphine self-administration and increased motivation to earn morphine infusions under a progressive ratio schedule of reinforcement. This phenotype was drug-specific as self-administration of cocaine, nicotine, and sucrose were not altered by paternal morphine history. The male offspring of morphine-exposed sires also had increased expression of mu-opioid receptors in the ventral tegmental area but not in the nucleus accumbens. Conclusions: Paternal morphine exposure increased morphine addiction-like behavioral vulnerability in male but not female progeny. This phenotype is likely driven by long-lasting neural adaptations within the reward neural brain pathways.

A novel Oprm1-Cre mouse maintains endogenous expression, function and enables detailed molecular characterization of µ-opioid receptor cells.

Key targets of both the therapeutic and abused properties of opioids are µ-opioid receptors (MORs). Despite years of research investigating the biochemistry and signal transduction pathways associated with MOR activation, we do not fully understand the cellular mechanisms underlying opioid addiction. Given that addictive opioids such as morphine, oxycodone, heroin, and fentanyl all activate MORs, and current therapies such as naloxone and buprenorphine block this activation, the availability of tools to mechanistically investigate opioid-mediated cellular and behavioral phenotypes are necessary. Therefore, we derived, validated, and applied a novel MOR-specific Cre mouse line, inserting a T2A cleavable peptide sequence and the Cre coding sequence into the MOR 3'UTR. Importantly, this line shows specificity and fidelity of MOR expression throughout the brain and with respect to function, there were no differences in behavioral responses to morphine when compared to wild type mice, nor are there any alterations in Oprm1 gene expression or receptor density. To assess Cre recombinase activity, MOR-Cre mice were crossed with the floxed GFP-reporters, RosaLSLSun1-sfGFP or RosaLSL-GFP-L10a. The latter allowed for cell type specific RNA sequencing via TRAP (Translating Ribosome Affinity Purification) of striatal MOR+ neurons following opioid withdrawal. The breadth of utility of this new tool will greatly facilitate the study of opioid biology under varying conditions.

Signaling underlying kappa opioid receptor-mediated behaviors in rodents.

Kappa opioid receptor (KOR) agonists are potentially useful as analgesic and anti-pruritic agents, for prevention and treatment of substance use disorders, and for treatment of demyelinating diseases. However, side effects of KOR agonists, including psychotomimesis, dysphoria, and sedation, have caused early termination of clinical trials. Understanding the signaling mechanisms underlying the beneficial therapeutic effects and the adverse side effects may help in the development of KOR agonist compounds. In this review, we summarize the current knowledge in this regard in five sections. First, studies conducted on mutant mouse lines (GRK3-/-, p38alpha MAPK-/-, ß-arrestin2-/-, phosphorylation-deficient KOR) are summarized. In addition, the abilities of four distinct KOR agonists, which have analgesic and anti-pruritic effects with different side effect profiles, to cause KOR phosphorylation are discussed. Second, investigations on the KOR agonist nalfurafine, both in vitro and in vivo are reviewed. Nalfurafine was the first KOR full agonist approved for clinical use and in the therapeutic dose range it did not produce significant side effects associated with typical KOR agonists. Third, large-scale high-throughput phosphoproteomic studies without a priori hypotheses are described. These studies have revealed that KOR-mediated side effects are associated with many signaling pathways. Fourth, several novel G protein-biased KOR agonists that have been characterized for in vitro biochemical properties and agonist biases and in vivo behavior effects are described. Lastly, possible mechanisms underlying KOR-mediated CPA, hypolocomotion and motor incoordination are discussed. Overall, it is agreed upon that the analgesic and anti-pruritic effects of KOR agonists are mediated via G protein signaling. However, there is no consensus on the mechanisms underlying their side effects. GRK3, p38 MAPK, ß-arrestin2, mTOR pathway, CB1 cannabinoid receptor and protein kinase C have been implicated in one side effect or another. For drug discovery, after initial in vitro characterization, in vivo pharmacological characterizations in various behavior tests are still the most crucial steps and dose separation between beneficial therapeutic effects and adverse side effects are the critical determinant for the compounds to be moved forward for clinical development.

Reactivation of cocaine contextual memory engages mechanistic target of rapamycin/S6 kinase 1 signaling.

Mechanistic target of rapamycin (mTOR) C1 and its downstream effectors have been implicated in synaptic plasticity and memory. Our prior work demonstrated that reactivation of cocaine memory engages a signaling pathway consisting of Akt, glycogen synthase kinase-3ß (GSK3ß), and mTORC1. The present study sought to identify other components of mTORC1 signaling involved in the reconsolidation of cocaine contextual memory, including eukaryotic translation initiation factor 4E (eIF4E)-eIF4G interactions, p70 S6 kinase polypeptide 1 (p70S6K, S6K1) activity, and activity-regulated cytoskeleton (Arc) expression. Cocaine contextual memory was established in adult CD-1 mice using conditioned place preference. After cocaine place preference was established, mice were briefly re-exposed to the cocaine-paired context to reactivate the cocaine memory and brains examined. Western blot analysis showed that phosphorylation of the mTORC1 target, p70S6K, in nucleus accumbens and hippocampus was enhanced 60 min following reactivation of cocaine memories. Inhibition of mTORC1 with systemic administration of rapamycin or inhibition of p70S6K with systemic PF-4708671 after reactivation of cocaine contextual memory abolished the established cocaine place preference. Immunoprecipitation assays showed that reactivation of cocaine memory did not affect eIF4E-eIF4G interactions in nucleus accumbens or hippocampus. Levels of Arc mRNA were significantly elevated 60 and 120 min after cocaine memory reactivation and returned to baseline 24 h later. These findings demonstrate that mTORC1 and p70S6K are required for reconsolidation of cocaine contextual memory.

Agonist-Promoted Phosphorylation and Internalization of the Kappa Opioid Receptor in Mouse Brains: Lack of Connection With Conditioned Place Aversion.

Selective kappa opioid receptor (KOR) agonists are promising antipruritic agents and analgesics. However, clinical development of KOR agonists has been limited by side effects, including psychotomimetic effects, dysphoria, and sedation, except for nalfurafine, and recently. CR845 (difelikefalin). Activation of KOR elicits G protein- and ß-arrestin-mediated signaling. KOR-induced analgesic and antipruritic effects are mediated by G protein signaling. However, different results have been reported as to whether conditioned place aversion (CPA) induced by KOR agonists is mediated by ß-arrestin signaling. In this study, we examined in male mice if there was a connection between agonist-promoted CPA and KOR phosphorylation and internalization, proxies for ß-arrestin recruitment in vivo using four KOR agonists. Herein, we demonstrated that at doses producing maximal effective analgesic and antiscratch effects, U50,488H, MOM-SalB, and 42B, but not nalfurafine, promoted KOR phosphorylation at T363 and S369 in mouse brains, as detected by immunoblotting with phospho-KOR-specific antibodies. In addition, at doses producing maximal effective analgesic and antiscratch effects, U50,488H, MOM-SalB, and 42B, but not nalfurafine, caused KOR internalization in the ventral tegmental area of a mutant mouse line expressing a fusion protein of KOR conjugated at the C-terminus with tdTomato (KtdT). We have reported previously that the KOR agonists U50,488H and methoxymethyl salvinorin B (MOM-SalB) cause CPA, whereas nalfurafine and 42B do not, at doses effective for analgesic and antiscratch effects. Taken together, these data reveal a lack of connection between agonist-promoted KOR-mediated CPA with agonist-induced KOR phosphorylation and internalization in male mice.

Modulation of Morphine Analgesia, Antinociceptive Tolerance, and Mu-Opioid Receptor Binding by the Cannabinoid CB2 Receptor Agonist O-1966.

Acutely, non-selective cannabinoid (CB) agonists have been shown to increase morphine antinociceptive effects, and we and others have also demonstrated that non-selective CB agonists attenuate morphine antinociceptive tolerance. Activation of cannabinoid CB2 receptors reverses allodynia and hyperalgesia in models of chronic pain, and co-administration of morphine with CB2 receptor selective agonists has been shown to be synergistic. CB2 receptor activation has also been shown to reduce morphine-induced hyperalgesia in rodents, an effect attributed to CB2 receptor modulation of inflammation. In the present set of experiments, we tested both the acute and chronic interactions between morphine and the CB2 receptor selective agonist O-1966 treatments on antinociception and antinociceptive tolerance in C57Bl6 mice. Co-administration of morphine and O-1966 was tested under three dosing regimens: simultaneous administration, morphine pre-treated with O-1966, and O-1966 pre-treated with morphine. The effects of O-1966 on mu-opioid receptor binding were determined using [3H]DAMGO and [35S]GTPγS binding assays, and these interactions were further examined by FRET analysis linked to flow cytometry. Results yielded surprising evidence of interactions between the CB2 receptor selective agonist O-1966 and morphine that were dependent upon the order of administration. When O-1966 was administered prior to or simultaneous with morphine, morphine antinociception was attenuated and antinociceptive tolerance was exacerbated. When O-1966 was administered following morphine, morphine antinociception was not affected and antinociceptive tolerance was attenuated. The [35S]GTPγS results suggest that O-1966 interrupts functional activity of morphine at the mu-opioid receptor, leading to decreased potency of morphine to produce acute thermal antinociceptive effects and potentiation of morphine antinociceptive tolerance. However, O-1966 administered after morphine blocked morphine hyperalgesia and led to an attenuation of morphine tolerance, perhaps due to well-documented anti-inflammatory effects of CB2 receptor agonism.

Does GEC1 Enhance Expression and Forward Trafficking of the Kappa Opioid Receptor (KOR) via Its Ability to Interact with NSF Directly?

We reported previously that GEC1 (glandular epithelial cell 1), a member of microtubule-associated proteins (MAPs), interacted directly with the C-tail of KOR (KCT) and tubulin and enhanced cell surface expression of KOR in CHO cells by facilitating its trafficking along the export pathway. Two GEC1 analogs (GABARAP and GATE16) were also shown to increase KOR expression. In addition, to understand the underlying mechanism, we demonstrated that N-ethylmaleimide-sensitive factor (NSF), an essential component for membrane fusion, co-immunoprecipitated with GEC1 from brain extracts. In this study, using pull-down techniques, we have found that (1) GEC1 interacts with NSF directly and prefers the ADP-bound NSF to the ATP-bound NSF; (2) D1 and/or D2 domain(s) of NSF interact with GEC1, but the N domain of NSF does not; (3) NSF does not interact with KCT directly, but forms a protein complex with KCT via GEC1; (4) NSF and/or α-SNAP do not affect KCT-GEC1 interaction. Thus, GEC1 (vs the α-SNAP/SNAREs complex) binds to NSF in distinctive ways in terms of the ADP- or ATP-bound form and domains of NSF involved. In conclusion, GEC1 may, via its direct interactions with KOR, NSF, and tubulin, enhance trafficking and fusion of KOR-containing vesicles selectively along the export pathway, which leads to increase in surface expression of KOR. GABARAP and GATE16 may enhance KOR expression in a similar way.

Agonist-promoted kappa opioid receptor (KOR) phosphorylation has behavioral endpoint-dependent and sex-specific effects.

We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (mKOR) in vitro at four residues in the C-terminal domain. In this study, we generated a mutant mouse line in which all the four residues were mutated to Ala (K4A) to examine the in vivo functional significance of agonist-induced KOR phosphorylation. U50,488H promoted KOR phosphorylation in brains of the wildtype (WT), but not K4A, male and female mice. Autoradiography of [3H] 69,593 binding to KOR in brain sections showed that WT and K4A mice had similar KOR distribution and expression levels in brain regions without sex differences. In K4A mice, U50,488H inhibited compound 48/80-induced scratching and attenuated novelty-induced hyperlocomotion to similar extents as in WT mice without sex differences. Interestingly, repeated pretreatment with U50,488H (80 mg/kg, s.c.) resulted in profound tolerance to the anti-scratch effects of U50,488H (5 mg/kg, s.c.) in WT mice of both sexes and female K4A mice, while in male K4A mice tolerance was attenuated. Moreover, U50,488H (2 mg/kg) induced conditioned place aversion (CPA) in WT mice of both sexes and male K4A mice, but not in female K4A mice. In contrast, U50,488H (5 mg/kg) caused CPA in male, but not female, mice, regardless of genotype. Thus, agonist-promoted KOR phosphorylation plays important roles in U50,488H-induced tolerance and CPA in a sex-dependent manner, without affecting acute U50,488H-induced anti-pruritic and hypo-locomotor effects. These results are the first to demonstrate sex differences in the effects of GPCR phosphorylation on the GPCR-mediated behaviors.

Considerations on Using Antibodies for Studying the Dynorphins/Kappa Opioid Receptor System.

Antibodies are important tools for protein and peptide research, including for the kappa opioid receptor (KOR) and dynorphins (Dyns). Well-characterized antibodies are essential for rigorous and reproducible research. However, lack of validation of antibody specificity has been thought to contribute significantly to the reproducibility crisis in biomedical research. Since 2003, many scientific journals have required documentation of validation of antibody specificity and use of knockout mouse tissues as a negative control is strongly recommended. Lack of specificity of antibodies against many G protein-coupled receptors (GPCRs) after extensive testing has been well-documented, but antibodies generated against partial sequences of the KOR have not been similarly investigated. For the dynorphins, differential processing has been described in distinct brain areas, resulting in controversial findings in immunohistochemistry (IHC) when different antibodies were used. In this chapter, we summarized accepted approaches for validation of antibody specificity. We discussed two KOR antibodies most commonly used in IHC and described generation and characterization of KOR antibodies and phospho-KOR specific antibodies in western blotting or immunoblotting (IB). In addition, applying antibodies targeting prodynorphin or mature dynorphin A illustrates the diversity of results obtained regarding the distribution of dynorphins in distinct brain areas.

Preclinical Studies on Nalfurafine (TRK-820), a Clinically Used KOR Agonist.

Nalfurafine has been used clinically in Japan for treatment of itch in kidney dialysis patients and in patients with chronic liver diseases. A one-year post-marketing study showed nalfurafine to be safe and efficacious without producing side effects of typical KOR agonists such as anhedonia and psychotomimesis. In this chapter, we summarize in vitro characterization and in vivo preclinical studies on nalfurafine. In vitro, nalfurafine is a highly potent and moderately selective KOR full agonist; however, whether it is a biased KOR agonist is a matter of debate. In animals, nalfurafine produced anti-pruritic effects in a dose range lower than that caused side effects, including conditioned place aversion (CPA), hypolocomotion, motor incoordination, consistent with the human data. In addition, nalfurafine showed antinociceptive effects in several pain models at doses that did not cause the side effects mentioned above. It appears to be effective against inflammatory pain and mechanical pain, but less so against thermal pain, particularly high-intensity thermal pain. U50,488H and nalfurafine differentially modulated several signaling pathways in a brain region-specific manners. Notably, U50,488H, but not nalfurafine, activated the mTOR pathway, which contributed to U50,488H-induced CPA. Because of its lack of side effects associated with typical KOR agonists, nalfurafine has been investigated as a combination therapy with an MOR ligand for pain treatment and for its effects on opioid use disorder and alcohol use disorder, and results indicate potential usefulness for these indications. Thus, although in vitro data regarding uniqueness of nalfurafine in terms of signaling at the KOR are somewhat equivocal, in vivo results support the assertion that nalfurafine is an atypical KOR agonist with a significantly improved side-effect profile relative to typical KOR agonists.

Fundamentals of the Dynorphins/Kappa Opioid Receptor System: From Distribution to Signaling and Function.

This chapter provides a general introduction to the dynorphins (DYNs)/kappa opioid receptor (KOR) system, including DYN peptides, neuroanatomy of the DYNs/KOR system, cellular signaling, and in vivo behavioral effects of KOR activation and inhibition. It is intended to serve as a primer for the book and to provide a basic background for the chapters in the book.

Verifying the role of 3-hydroxy of 17-cyclopropylmethyl-4,5α-epoxy-3,14ß-dihydroxy-6ß-[(4'-pyridyl) carboxamido]morphinan derivatives via their binding affinity and selectivity profiles on opioid receptors.

In the present study, the role of 3-hydroxy group of a series of epoxymorphinan derivatives in their binding affinity and selectivity profiles toward the opioid receptors (ORs) has been investigated. It was found that the 3-hydroxy group was crucial for the binding affinity of these derivatives for all three ORs due to the fact that all the analogues 1a-e exhibited significantly higher binding affinities compared to their counterpart 3-dehydroxy ones 6a-e. Meanwhile most compounds carrying the 3-hydroxy group possessed similar selectivity profiles for the kappa opioid receptor over the mu opioid receptor as their corresponding 3-dehydroxy derivatives. [35S]-GTPγS functional assay results indicated that the 3-hydroxy group of these epoxymorphinan derivatives was important for maintaining their potency on the ORs with various effects. Further molecular modeling studies helped comprehend the remarkably different binding affinity and functional profiles between compound 1c (NCP) and its 3-dehydroxy analogue 6c.

Exploring the putative mechanism of allosteric modulations by mixed-action kappa/mu opioid receptor bitopic modulators.

The modulation and selectivity mechanisms of seven mixed-action kappa opioid receptor (KOR)/mu opioid receptor (MOR) bitopic modulators were explored. Molecular modeling results indicated that the 'message' moiety of seven bitopic modulators shared the same binding mode with the orthosteric site of the KOR and MOR, whereas the 'address' moiety bound with different subdomains of the allosteric site of the KOR and MOR. The 'address' moiety of seven bitopic modulators bound to different subdomains of the allosteric site of the KOR and MOR may exhibit distinguishable allosteric modulations to the binding affinity and/or efficacy of the 'message' moiety. Moreover, the 3-hydroxy group on the phenolic moiety of the seven bitopic modulators induced selectivity to the KOR over the MOR.

Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

Kappa opioid receptor (KOR) agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as non-addictive anti-pruritic and analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the beneficial antinociceptive and antipruritic effects of KOR agonists, but attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated signaling in the mouse brain, we observed PKC-dependent modulation of G protein-coupled receptor kinases and Wnt pathways at 5 min; stress signaling, cytoskeleton, mTOR signaling and receptor phosphorylation, including cannabinoid receptor CB1 at 30 min. We further demonstrated that inhibition of CB1 attenuated KOR-mediated CPA. Our results demonstrated the feasibility of in vivo biochemical dissection of signaling pathways that lead to side effects.

Comparison of Pharmacological Properties between the Kappa Opioid Receptor Agonist Nalfurafine and 42B, Its 3-Dehydroxy Analogue: Disconnect between in Vitro Agonist Bias and in Vivo Pharmacological Effects.

Nalfurafine, a moderately selective kappa opioid receptor (KOR) agonist, is used in Japan for treatment of itch without causing dysphoria or psychotomimesis. Here we characterized the pharmacology of compound 42B, a 3-dehydroxy analogue of nalfurafine and compared with that of nalfurafine. Nalfurafine and 42B acted as full KOR agonists and partial µ opioid receptor (MOR) agonists, but 42B showed much lower potency for both receptors and lower KOR/MOR selectivity, different from previous reports. Molecular modeling revealed that water-mediated hydrogen-bond formation between 3-OH of nalfurafine and KOR accounted for its higher KOR potency than 42B. The higher potency of both at KOR over MOR may be due to hydrogen-bond formation between nonconserved Y7.35 of KOR and their carbonyl groups. Both showed modest G protein signaling biases. In mice, like nalfurafine, 42B produced antinociceptive and antiscratch effects and did not cause conditioned place aversion (CPA) in the effective dose ranges. Unlike nalfurafine, 42B caused motor incoordination and hypolocomotion. As both agonists showed G protein biases, yet produced different effects on locomotor activity and motor incoordination, the findings and those in the literature suggest caution in correlating in vitro biochemical data with in vivo behavior effects. The factors contributing to the disconnect, including pharmacodynamic and pharmacokinetic issues, are discussed. In addition, our results suggest that among the KOR-induced adverse behaviors, CPA can be separated from motor incoordination and hypolocomotion.

The kappa opioid receptor agonist U50,488H did not affect brain-stimulation reward while it elicited conditioned place aversion in mice.

OBJECTIVE: Selective kappa opioid receptor (KOR) agonists were shown to produce a dose-dependent depression of brain-stimulation reward (BSR) in the rat intracranial self-stimulation (ICSS) tests. However, limited studies using mice produced less conclusive results. Here the effects of U50,488H were re-examined on BSR in mice with a larger cohort of animals. RESULTS: Forty C57BL/6J male mice were implanted with the electrodes in medial forebrain bundle. About a week after surgery, mice were subject to ICSS training. Only eighteen passed the two-phase procedures, at which point they readily spun the wheels to obtain reinforcing effect of BSR, and were used for the ICSS tests. Compared with saline (s.c.), U50,488H (2 mg/kg, s.c.) did not have effects on the BSR thresholds within 1 h post-treatment, while it decreased the maximum wheel-spinning rates in a time-dependent manner. In contrast, cocaine (5 mg/kg, s.c.) decreased the BSR thresholds time-dependently without affecting the maximum wheel-spinning rates in the same cohort of mice, demonstrating the validity of our mouse ICSS models. For comparison, U50,488H (2 mg/kg, s.c.) induced significant conditioned place aversion (CPA) in a different cohort of mice without surgeries. Thus, ICSS may not be an appropriate test for KOR agonist-induced aversion in mice.

Characterization of a Knock-In Mouse Line Expressing a Fusion Protein of κ Opioid Receptor Conjugated with tdTomato: 3-Dimensional Brain Imaging via CLARITY.

Activation of κ opioid receptor (KOR) produces analgesia, antipruritic effect, sedation and dysphoria. To characterize neuroanatomy of KOR at high resolutions and circumvent issues of specificity of KOR antibodies, we generated a knock-in mouse line expressing KOR fused at the C terminus with the fluorescent protein tdTomato (KtdT). The selective KOR agonist U50,488H caused anti-scratch effect and hypolocomotion, indicating intact KOR neuronal circuitries. Clearing of brains with CLARITY revealed three-dimensional (3-D) images of distribution of KOR, and any G-protein-coupled receptors, for the first time. 3-D brain images of KtdT and immunohistochemistry (IHC) on brain sections with antibodies against tdTomato show similar distribution to that of autoradiography of [3H]U69,593 binding to KOR in wild-type mice. KtdT was observed in regions involved in reward and aversion, pain modulation, and neuroendocrine regulation. KOR is present in several areas with unknown roles, including the claustrum (CLA), dorsal endopiriform nucleus, paraventricular nucleus of the thalamus (PVT), lateral habenula (LHb), and substantia nigra pars reticulata (SNr), which are discussed. Prominent KtdT-containing fibers were observed to project from caudate putamen (CP) and nucleus accumbens (ACB) to substantia innominata (SI) and SNr. Double IHC revealed co-localization of KtdT with tyrosine hydroxylase (TH) in brain regions, including CP, ACB, and ventral tegmental area (VTA). KOR was visualized at the cellular level, such as co-localization with TH and agonist-induced KOR translocation into intracellular space in some VTA neurons. These mice thus represent a powerful and heretofore unparalleled tool for neuroanatomy of KOR at both the 3-D and cellular levels.

Pharmacological characterization of 17-cyclopropylmethyl-3,14-dihydroxy-4,5-epoxy-6-[(3'-fluoro-4'-pyridyl)acetamido]morphinan (NFP) as a dual selective MOR/KOR ligand with potential applications in treating opioid use disorder.

For thousands of years opioids have been the first-line treatment option for pain management. However, the tolerance and addiction potential of opioids limit their applications in clinic. NFP, a MOR/KOR dual-selective opioid antagonist, was identified as a ligand that significantly antagonized the antinociceptive effects of morphine with lesser withdrawal effects than naloxone at similar doses. To validate the potential application of NFP in opioid addiction treatment, a series of in vitro and in vivo assays were conducted to further characterize its pharmacological profile. In calcium mobilization assays and MOR internalization studies, NFP showed the apparent capacity to antagonize DAMGO-induced calcium flux and etorphine-induced MOR internalization. In contrast to the opioid agonists DAMGO and morphine, cells pretreated with NFP did not show apparent desensitization and down regulation of the MOR. Though in vitro bidirectional transport studies showed that NFP might be a P-gp substrate, in warm-water tail-withdrawal assays it was able to antagonize the antinociceptive effects of morphine indicating its potential central nervous system activity. Overall these results suggest that NFP is a promising dual selective opioid antagonist that may have the potential to be used therapeutically in opioid use disorder treatment.