Rare GPR37L1 variants reveal potential association between GPR37L1 and disorders of anxiety and migraine.

GPR37L1 is an orphan receptor that couples through heterotrimeric G proteins to regulate physiological functions. Since its role in humans is not fully defined, we used an unbiased computational approach to assess the clinical significance of rare GPR37L1 genetic variants found among 51,289 whole exome sequences from the DiscovEHR cohort. Briefly, rare GPR37L1 coding variants were binned according to predicted pathogenicity and analyzed by Sequence Kernel Association testing to reveal significant associations with disease diagnostic codes for epilepsy and migraine, among others. Since associations do not prove causality, rare GPR37L1 variants were then functionally analyzed in SK-N-MC cells to evaluate potential signaling differences and pathogenicity. Notably, receptor variants exhibited varying abilities to reduce cAMP levels, activate MAPK signaling, and/or upregulate receptor expression in response to the agonist prosaptide (TX14(A)), as compared to the wild-type receptor. In addition to signaling changes, knockout (KO) of GPR37L1 or expression of certain rare variants altered cellular cholesterol levels, which were also acutely regulated by administration of the agonist TX14(A) via activation of the MAPK pathway. Finally, to simulate the impact of rare nonsense variants found in the large patient cohort, a KO mouse line lacking Gpr37l1 was generated. Although KO animals did not recapitulate an acute migraine phenotype, loss of this receptor produced sex-specific changes in anxiety-related disorders often seen in chronic migraineurs. Collectively, these observations define the existence of rare GPR37L1 variants associated with neuropsychiatric conditions in the human population and identify the signaling changes contributing to pathological processes.Significance Statement G protein-coupled receptors (GPCRs) represent a diverse group of membrane receptors that contribute to a wide range of diseases and serve as effective drug targets. However, a number of these receptors have no identified ligands or functions, i.e., orphan receptors. Over the past decade, advances have been made, but there is a need for identifying new strategies to reveal their roles in health and disease. Our results highlight the utility of rare variant analyses of orphan receptors for identifying human disease associations, coupled with functional analyses in relevant cellular and animal systems, to ultimately reveal their roles as novel drug targets for treatment of neurological disorders that lack wide-spread efficacy.

Functional and anatomical analyses of active spinal circuits in a mouse model of chronic pain.

ABSTRACT: Decades of efforts in elucidating pain mechanisms, including pharmacological, neuroanatomical, and physiological studies have provided insights into how nociceptive information transmits from the periphery to the brain and the locations receiving nociceptive signals. However, little is known about which specific stimulus-dependent activated neurons, amongst heterogeneous neural environments, discriminatively evoke the cognate pain behavior. We here shed light on the population of neurons in the spinal cord activated by a painful stimulus to identify chronic pain-dependent activated neuronal subsets using Fos2A-iCreER (TRAP2) mice. We have found a large number of neurons activated by a normally nonpainful stimulus in the spinal cord of spinal nerve-ligated mice, compared with sham. Neuronal activation was observed in laminae I and II outer under heat hyperalgesia. A large number of neurons in laminae II inner were activated in both mechanical allodynia and heat hyperalgesia conditions, while mechanical allodynia tends to be the only stimulus that activates cells at lamina II inner dorsal region. Neuroanatomical analyses using spinal cell markers identified a large number of spinal inhibitory neurons that are recruited by both mechanical allodynia and heat hyperalgesia. Of interest, spinal neurons expressing calretinin, calbindin, and parvalbumin were activated differently with distinct pain modalities (ie, mechanical allodynia vs heat hyperalgesia). Chemogenetic inhibition of those activated neurons significantly and specifically reduced the response to the pain stimulus associated with the stimulus modality originally given to the animals. These findings support the idea that spinal neuronal ensembles underlying nociceptive transmission undergo dynamic changes to regulate selective pain responses.

Synthesis of α3ß4 Nicotinic Acetylcholine Receptor Modulators Derived from Aristoquinoline That Reduce Reinstatement of Cocaine-Seeking Behavior.

Growing evidence suggests that inhibition of the α3ß4 nicotinic acetylcholine receptor (nAChR) represents a promising therapeutic strategy to treat cocaine use disorder. Recently, aristoquinoline (1), an alkaloid from Aristotelia chilensis, was identified as an α3ß4-selective nAChR inhibitor. Here, we prepared 22 derivatives of 1 and evaluated their ability to inhibit the α3ß4 nAChR. These studies revealed structure-activity trends and several compounds with increased potency compared to 1 with few off-target liabilities. Additional mechanistic studies indicated that these compounds inhibit the α3ß4 nAChR noncompetitively, but do not act as channel blockers, suggesting they are negative allosteric modulators. Finally, using a cocaine-primed reinstatement paradigm, we demonstrated that 1 significantly attenuates drug-seeking behavior in an animal model of cocaine relapse. The results from these studies further support a role for the α3ß4 nAChR in the addictive properties of cocaine and highlight the possible utility of aristoquinoline derivatives in treating cocaine use disorder.

Rare GPR37L1 variants reveal potential roles in anxiety and migraine disorders.

GPR37L1 is an orphan receptor that couples through heterotrimeric G-proteins to regulate physiological functions. Since its role in humans is not fully defined, we used an unbiased computational approach to assess the clinical significance of rare GPR37L1 genetic variants found among 51,289 whole exome sequences from the DiscovEHR cohort. Briefly, rare GPR37L1 coding variants were binned according to predicted pathogenicity, and analyzed by Sequence Kernel Association testing to reveal significant associations with disease diagnostic codes for epilepsy and migraine, among others. Since associations do not prove causality, rare GPR37L1 variants were then functionally analyzed in SK-N-MC cells to evaluate potential signaling differences and pathogenicity. Notably, receptor variants exhibited varying abilities to reduce cAMP levels, activate MAPK signaling, and/or upregulate receptor expression in response to the agonist prosaptide (TX14(A)), as compared to the wild-type receptor. In addition to signaling changes, knockout of GPR37L1 or expression of certain rare variants altered cellular cholesterol levels, which were also acutely regulated by administration of the agonist TX14(A) via activation of the MAPK pathway. Finally, to simulate the impact of rare nonsense variants found in the large patient cohort, a knockout (KO) mouse line lacking Gpr37L1 was generated, revealing loss of this receptor produced sex-specific changes implicated in migraine-related disorders. Collectively, these observations define the existence of rare GPR37L1 variants in the human population that are associated with neuropsychiatric conditions and identify the underlying signaling changes that are implicated in the in vivo actions of this receptor in pathological processes leading to anxiety and migraine. SIGNIFICANCE STATEMENT: G-protein coupled receptors (GPCRs) represent a diverse group of membrane receptors that contribute to a wide range of diseases and serve as effective drug targets. However, a number of these receptors have no identified ligands or functions, i.e., orphan receptors. Over the past decade, advances have been made, but there is a need for identifying new strategies to reveal their roles in health and disease. Our results highlight the utility of rare variant analyses of orphan receptors for identifying human disease associations, coupled with functional analyses in relevant cellular and animal systems, to ultimately reveal their roles as novel drug targets for treatment of neurological disorders that lack wide-spread efficacy.

Preclinical effects of cannabidiol in an experimental model of migraine.

ABSTRACT: Migraine is a disabling disorder characterized by recurrent headaches, accompanied by abnormal sensory sensitivity and anxiety. Despite extensive historical use of cannabis in headache disorders, there is limited research on the nonpsychoactive cannabidiol (CBD) for migraine and there is no scientific evidence to prove that CBD is an effective treatment. The effects of CBD are examined here using a calcitonin gene-related peptide (CGRP)-induced migraine model that provides measures of cephalic allodynia, spontaneous pain, altered light sensitivity (photophobia), and anxiety-like behavior in C57BL/6J mice. A single administration of CGRP induced facial hypersensitivity in both female and male mice. Repeated CGRP treatment produced progressively decreased levels in basal thresholds of allodynia in females, but not in males. A single CBD administration protected both females and males from periorbital allodynia induced by a single CGRP injection. Repeated CBD administration prevented increased levels of basal allodynia induced by repeated CGRP treatment in female mice and did not lead to responses consistent with migraine headache as occurs with triptans. Cannabidiol, injected after CGRP, reversed CGRP-evoked allodynia. Cannabidiol also reduced spontaneous pain traits induced by CGRP administration in female mice. Finally, CBD blocked CGRP-induced anxiety in male mice, but failed in providing protection from CGRP-induced photophobia in females. These results demonstrate the efficacy of CBD in preventing episodic and chronic migraine-like states with reduced risk of causing medication overuse headache. Cannabidiol also shows potential as an abortive agent for treating migraine attacks and headache-related conditions such as spontaneous pain and anxiety.

Activation of the nociceptin/orphanin-FQ receptor promotes NREM sleep and EEG slow wave activity.

Sleep/wake control involves several neurotransmitter and neuromodulatory systems yet the coordination of the behavioral and physiological processes underlying sleep is incompletely understood. Previous studies have suggested that activation of the Nociceptin/orphanin FQ (N/OFQ) receptor (NOPR) reduces locomotor activity and produces a sedation-like effect in rodents. In the present study, we systematically evaluated the efficacy of two NOPR agonists, Ro64-6198 and SR16835, on sleep/wake in rats, mice, and Cynomolgus macaques. We found a profound, dose-related increase in non-Rapid Eye Movement (NREM) sleep and electroencephalogram (EEG) slow wave activity (SWA) and suppression of Rapid Eye Movement sleep (REM) sleep in all three species. At the highest dose tested in rats, the increase in NREM sleep and EEG SWA was accompanied by a prolonged inhibition of REM sleep, hypothermia, and reduced locomotor activity. However, even at the highest dose tested, rats were immediately arousable upon sensory stimulation, suggesting sleep rather than an anesthetic state. NOPR agonism also resulted in increased expression of c-Fos in the anterodorsal preoptic and parastrial nuclei, two GABAergic nuclei that are highly interconnected with brain regions involved in physiological regulation. These results suggest that the N/OFQ-NOPR system may have a previously unrecognized role in sleep/wake control and potential promise as a therapeutic target for the treatment of insomnia.

A novel G protein-biased agonist at the µ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase.

BACKGROUND AND PURPOSE: G protein-biased µ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased µ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. EXPERIMENTAL APPROACH: The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced µ receptor desensitisation was probed using kinase inhibitors. KEY RESULTS: Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased µ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular µ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid µ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. CONCLUSION AND IMPLICATIONS: Compound 1 is a novel G protein-biased µ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

PPL-103: A mixed opioid partial agonist with desirable anti-cocaine properties.

Cocaine use disorder (CUD) is a persistent public health problem for which no effective medications are available. PPL-103 is an opioid receptor ligand with partial agonist activity at mu, kappa and delta opioid receptors, with a greater efficacy for kappa and low efficacy at mu receptors. Because chronic cocaine use induces changes in the kappa opioid receptor/dynorphin system, we hypothesized that a kappa partial agonist, such as PPL-103, would attenuate the aversive properties of the upregulated kappa system, resulting in effective treatment approach for CUD. We tested the effects of PPL-103 on cocaine self-administration models that recapitulate core aspects of CUD in humans. We found that PPL-103 reduced both long and short access cocaine self-administration, motivation to respond for cocaine, and binge-like cocaine taking, in rats. Operant responding for food, fentanyl and locomotor behavior were not altered at doses that decreased cocaine infusions. Repeated PPL-103 treatment did not lead to tolerance development. PPL-103 also reduced both priming- and cue-induced reinstatement of cocaine seeking, being more effective in the former. Surprisingly, PPL-103 reduced self-administration parameters and reinstatement in rats previously treated with the long-acting kappa receptor antagonist JDTic more potently than in non-JDTic treated animals, whereas naltrexone injected to rats subsequent to JDTic administration increased self-administration, suggesting that the partial mu agonist activity, rather than kappa agonism is important for reduction in cocaine taking and seeking. However, partial kappa activation seems to increase safety by limiting dysphoria, tolerance and addiction development. PPL-103 displays a desirable profile as a possible CUD pharmacotherapy.

PPL-138 (BU10038): A bifunctional NOP/mu partial agonist that reduces cocaine self-administration in rats.

The search for new and effective treatments for cocaine use disorder (CUD) is a priority. We determined whether PPL-138 (BU10038), a compound with partial agonist activity at both nociceptin opioid peptide (NOP) and mu-opioid receptors, reduces cocaine consumption, reinstatement, and whether the compound itself produces reinforcing effects in rats. Using an intermittent access (IntA) cocaine self-administration procedure, we found that PPL-138 (0.1 and 0.3 mg/kg) effectively decreased the total number of cocaine infusions and burst-like cocaine intake in both male and female rats. Responses for food in an IntA model of food self-administration were not altered for either sex, although locomotor activity was increased in female but not male rats. Blockade of NOP receptors with the selective antagonist J-113397 (5 mg/kg) did not prevent the PPL-138-induced suppression of cocaine self-administration, whereas blockade of mu-opioid receptors by naltrexone (1 mg/kg) reversed such effect. Consistently, treatment with morphine (1, 3, and 10 mg/kg) dose-dependently reduced IntA cocaine self-administration measures. PPL-138 also reduced reinstatement of cocaine seeking at all doses examined. Although an initial treatment with PPL-138 (2.5, 10, and 40 µg/kg/infusion) appeared rewarding, the compound did not maintain self-administration behavior. Animals treated with PPL-138 showed initial suppression of cocaine self-administration, which was eliminated following repeated daily dosing. However, suppression of cocaine self-administration was retained when subsequent PPL-138 treatments were administered 48 h apart. These findings demonstrate that the approach of combining partial NOP/mu-opioid activation successfully reduces cocaine use, but properties of PPL-138 seem to depend on the timing of drug administration.

Contributions of the International Narcotics Research Conference to Opioid Research Over the Past 50 years.

The International Narcotics Research Conference (INRC), founded in 1969, has been a successful forum for research into the actions of opiates, with an annual conference since 1971. Every year, scientists from around the world have congregated to present the latest data on novel opiates, opiate receptors and endogenous ligands, mechanisms of analgesic activity and unwanted side effects, etc. All the important discoveries in the opiate field were discussed, often first, at the annual INRC meeting. With an apology to important events and participants not discussed, this review presents a short history of INRC with a discussion of groundbreaking discoveries in the opiate field and the researchers who presented from the first meeting up to the present.

The NOP Receptor System in Neurological and Psychiatric Disorders: Discrepancies, Peculiarities and Clinical Progress in Developing Targeted Therapies.

The nociceptin opioid peptide (NOP) receptor and its endogenous ligand nociceptin/orphanin FQ (N/OFQ) are the fourth members of the opioid receptor and opioid peptide families. Although they have considerable sequence homology to the other family members, they are not considered opioid per se because they do not have pharmacological profiles similar to the other family members. The number of NOP receptors in the brain is higher than the other family members, and NOP receptors can be found throughout the brain. Because of the widespread distribution of NOP receptors, N/OFQ and other peptide and small molecule agonists and antagonists have extensive CNS activities. Originally thought to be anti-opioid, NOP receptor agonists block some opioid activities, potentiate others, and modulate other activities not affected by traditional opiates. Because the effect of receptor activation can be dependent upon site of administration, state of the animal, and other variables, the study of NOP receptors has been fraught with contradictions and inconsistencies. In this article, the actions and controversies pertaining to NOP receptor activation and inhibition are discussed with respect to CNS disorders including pain (acute, chronic, and migraine), drug abuse, anxiety and depression. In addition, progress towards clinical use of NOP receptor-directed compounds is discussed.

NOP receptor agonist attenuates nitroglycerin-induced migraine-like symptoms in mice.

Migraine is an extraordinarily prevalent and disabling headache disorder that affects one billion people worldwide. Throbbing pain is one of several migraine symptoms including sensitivity to light (photophobia), sometimes to sounds, smell and touch. The basic mechanisms underlying migraine remain inadequately understood, and current treatments (with triptans being the primary standard of care) are not well tolerated by some patients. NOP (Nociceptin OPioid) receptors, the fourth member of the opioid receptor family, are expressed in the brain and periphery with particularly high expression known to be in trigeminal ganglia (TG). The aim of our study was to further explore the involvement of the NOP receptor system in migraine. To this end, we used immunohistochemistry to examine NOP receptor distribution in TG and trigeminal nucleus caudalus (TNC) in mice, including colocalization with specific cellular markers, and used nitroglycerin (NTG) models of migraine to assess the influence of the selective NOP receptor agonist, Ro 64-6198, on NTG-induced pain (sensitivity of paw and head using von Frey filaments) and photophobia in mice. Our immunohistochemical studies with NOP-eGFP knock-in mice indicate that NOP receptors are on the majority of neurons in the TG and are also very highly expressed in the TNC. In addition, Ro 64-6198 can dose dependently block NTG-induced paw and head allodynia, an effect that is blocked by the NOP antagonist, SB-612111. Moreover, Ro 64-6198, can decrease NTG-induced light sensitivity in mice. These results suggest that NOP receptor agonists should be futher explored as treatment for migraine symptoms. This article is part of the special issue on Neuropeptides.

Potent and selective NOP receptor activation reduces cocaine self-administration in rats by lowering hedonic set point.

Developing new medications for the treatment of cocaine dependence continues to be a research priority. Compelling evidence indicates that mixed opioid receptor agonists, particularly bifunctional compounds that target nociceptin/orphanin FQ peptide (NOP) and mu opioid receptors, may be useful for the treatment of cocaine addiction. Here, we verify that potent and selective pharmacological activation of NOP receptors is sufficient to reduce relevant facets of cocaine addiction in animal models. Accordingly, we determined whether systemic injections of the small molecule AT-312 (0, 1, 3 mg/kg) could reduce operant cocaine self-administration, motivation for cocaine, and vulnerability to cocaine relapse in rats. Results indicate that a potent and selective NOP receptor agonist was equally efficacious in reducing the number of cocaine infusions in short (1-hour), as well as long (6-hour) access sessions. When tested on an economic-demand reinforcement schedule, AT-312 reduced Q0 , the parameter that describes the amount of drug consumed at zero price, while leaving the parameter α, a measure of motivation for drug consumption, unaltered. Furthermore, AT-312 successfully reduced conditioned reinstatement of cocaine seeking. In contrast, the NOP receptor agonist did not modify food self-administration. Blockade of the NOP receptor with the antagonist SB-612111 prevented the effect of AT-312 in decreasing cocaine-reinforced responding under a 2-hour fixed ratio 1 schedule, suggesting a NOP receptor-mediated mechanism. This work demonstrates that potent and selective activation of NOP receptors is sufficient to decrease cocaine taking and seeking behaviors in rats.

NOP Receptor Antagonists Decrease Alcohol Drinking in the Dark in C57BL/6J Mice.

BACKGROUND: The nociceptin/orphanin FQ opioid peptide (NOP) receptor and its endogenous ligand N/OFQ have been implicated in the regulation of drug and alcohol use disorders (AUD). In particular, evidence demonstrated that NOP receptor activation blocks reinforcing and motivating effects of alcohol across a range of behavioral measures, including alcohol intake, conditioned place preference, and vulnerability to relapse. METHODS: Here, we show the effects of pharmacological activation and inhibition of NOP receptors on binge-like alcohol consumption, as measured by the "drinking in the dark" (DID) model in C57BL/6J mice. RESULTS: We found that 2 potent and selective NOP agonists AT-202 (0, 0.3, 1, 3 mg/kg) and AT-312 (0, 0.3, 1 mg/kg) did not affect binge alcohol drinking at doses that do not affect locomotor activity. AT-202 also failed to alter DID behavior when administered to mice previously exposed to chronic alcohol treatment with an alcohol-containing liquid diet. Conversely, treatment with either the high affinity NOP receptor antagonist SB-612111 (0, 3, 10, 30 mg/kg) or the selective antagonist LY2817412 (0, 3, 10, 30 mg/kg) decreased binge drinking. SB-612111 was effective at all doses examined, and LY2817412 was effective at 30 mg/kg. Consistently, NOP receptor knockout mice consumed less alcohol compared to wild type. SB-612111 reduced DID and increased sucrose consumption at doses that do not appear to affect locomotor activity. However, the high dose of SB-612111 (30 mg/kg) reduced alcohol intake but failed to inhibit preference in a 2-bottle choice DID model that can assess moderate alcohol intake. CONCLUSIONS: The present results suggest that NOP receptor inhibition rather than activation may represent a valuable approach for treatment of AUD characterized by excessive alcohol consumption such as binge drinking.

NOP-Related Mechanisms in Pain and Analgesia.

Since the discovery of the NOP receptor and N/OFQ as the endogenous ligand, evidence has appeared demonstrating the involvement of this receptor system in pain. This was not surprising for members of the opioid receptor and peptide families, particularly since both the receptor and N/OFQ are highly expressed in brain regions involved in pain, spinal cord, and dorsal root ganglia. What has been surprising is the complicated picture that has emerged from 25 years of research. The original finding that N/OFQ decreased tail flick and hotplate latency, when administered i.c.v., led to the hypothesis that NOP receptor antagonists could have analgesic activity without abuse liability. However, as data accumulated, it became clear that not only the potency but the activity per se was different when N/OFQ or small molecule NOP agonists were administered in the brain versus the spinal cord and it also depended upon the pain assay used. When administered systemically, NOP receptor agonists are generally ineffective in attenuating heat pain but are antinociceptive in an acute inflammatory pain model. Most antagonists administered systemically have no antinociceptive activity of their own, even though selective peptide NOP antagonists have potent antinociceptive activity when administered i.c.v. Chronic pain models provide different results as well, as small molecule NOP receptor agonists have potent anti-allodynic and anti-hyperalgesic activity after systemic administration. A considerable number of electrophysiological and anatomical experiments, in particular with NOP-eGFP mice, have been conducted in an attempt to explain the complicated profile resulting from NOP receptor modulation, to examine receptor plasticity, and to elucidate mechanisms by which selective NOP agonists, bifunctional NOP/mu agonists, or NOP receptor antagonists modulate acute and chronic pain.

BU10038 as a safe opioid analgesic with fewer side-effects after systemic and intrathecal administration in primates.

BACKGROUND: The marked increase in mis-use of prescription opioids has greatly affected our society. One potential solution is to develop improved analgesics which have agonist action at both mu opioid peptide (MOP) and nociceptin/orphanin FQ peptide (NOP) receptors. BU10038 is a recently identified bifunctional MOP/NOP partial agonist. The aim of this study was to determine the functional profile of systemic or spinal delivery of BU10038 in primates after acute and chronic administration. METHODS: A series of behavioural and physiological assays have been established specifically to reflect the therapeutic (analgesia) and side-effects (abuse potential, respiratory depression, itch, physical dependence, and tolerance) of opioid analgesics in rhesus monkeys. RESULTS: After systemic administration, BU10038 (0.001-0.01 mg kg-1) dose-dependently produced long-lasting antinociceptive and antihypersensitive effects. Unlike the MOP agonist oxycodone, BU10038 lacked reinforcing effects (i.e. little or no abuse liability), and BU10038 did not compromise the physiological functions of primates including respiration, cardiovascular activities, and body temperature at antinociceptive doses and a 10-30-fold higher dose (0.01-0.1 mg kg-1). After intrathecal administration, BU10038 (3 µg) exerted morphine-comparable antinociception and antihypersensitivity without itch scratching responses. Unlike morphine, BU10038 did not cause the development of physical dependence and tolerance after repeated and chronic administration. CONCLUSIONS: These in vivo findings demonstrate the translational potential of bifunctional MOP/NOP receptor agonists such as BU10038 as a safe, non-addictive analgesic with fewer side-effects in primates. This study strongly supports that bifunctional MOP/NOP agonists may provide improved analgesics and an alternative solution for the ongoing prescription opioid crisis.

Agonist-selective NOP receptor phosphorylation correlates in vitro and in vivo and reveals differential post-activation signaling by chemically diverse agonists.

Agonists of the nociceptin/orphanin FQ opioid peptide (NOP) receptor, a member of the opioid receptor family, are under active investigation as novel analgesics, but their modes of signaling are less well characterized than those of other members of the opioid receptor family. Therefore, we investigated whether different NOP receptor ligands showed differential signaling or functional selectivity at the NOP receptor. Using newly developed phosphosite-specific antibodies to the NOP receptor, we found that agonist-induced NOP receptor phosphorylation occurred primarily at four carboxyl-terminal serine (Ser) and threonine (Thr) residues, namely, Ser346, Ser351, Thr362, and Ser363, and proceeded with a temporal hierarchy, with Ser346 as the first site of phosphorylation. G protein-coupled receptor kinases 2 and 3 (GRK2/3) cooperated during agonist-induced phosphorylation, which, in turn, facilitated NOP receptor desensitization and internalization. A comparison of structurally distinct NOP receptor agonists revealed dissociation in functional efficacies between G protein-dependent signaling and receptor phosphorylation. Furthermore, in NOP-eGFP and NOP-eYFP mice, NOP receptor agonists induced multisite phosphorylation and internalization in a dose-dependent and agonist-selective manner that could be blocked by specific antagonists. Our study provides new tools to study ligand-activated NOP receptor signaling in vitro and in vivo. Differential agonist-selective NOP receptor phosphorylation by chemically diverse NOP receptor agonists suggests that differential signaling by NOP receptor agonists may play a role in NOP receptor ligand pharmacology.

Synthesis, Biological Evaluation, and SAR Studies of 14ß-phenylacetyl Substituted 17-cyclopropylmethyl-7, 8-dihydronoroxymorphinones Derivatives: Ligands With Mixed NOP and Opioid Receptor Profile.

A series of 14ß-acyl substituted 17-cyclopropylmethyl-7,8-dihydronoroxymorphinone compounds has been synthesized and evaluated for affinity and efficacy for mu (MOP), kappa (KOP), and delta (DOP) opioid receptors and nociceptin/orphanin FQ peptide (NOP) receptors. The majority of the new ligands displayed high binding affinities for the three opioid receptors, and moderate affinity for NOP receptors. The affinities for NOP receptors are of particular interest as most classical opioid ligands do not bind to NOP receptors. The predominant activity in the [35S]GTPγS assay was partial agonism at each receptor. The results are consistent with our prediction that an appropriate 14ß side chain would access a binding site within the NOP receptor and result in substantially higher affinity than displayed by the parent compound naltrexone. Molecular modeling studies, utilizing the recently reported structure of the NOP receptor, are also consistent with this interpretation.

Influence of neuropathic pain on nicotinic acetylcholine receptor plasticity and behavioral responses to nicotine in rats.

Tobacco smoking is particularly evident in individuals experiencing chronic pain. This complex relationship is poorly understood at both molecular and behavioral levels. Here, we describe experiments aimed at understanding whether a chronic pain state induces neuroadaptations into the brain or peripheral nerves that involve nicotinic acetylcholine receptors (nAChRs) and whether these neuroadaptations directly lead to increased vulnerability to nicotine addiction or to the development of coping strategies to relieve pain symptoms. We found that ligation of the rat L5 spinal nerve led to a dramatic downregulation in the mRNA expression levels of all nAChR subunits examined in dorsal root ganglia and a time-dependent downregulation of discrete subunits, particularly in the cingulate cortex and the amygdala. Spinal nerve ligation and sham-operated rats showed minor or no changes in patterns of acquisition and motivation for nicotine taking. Spinal nerve ligation rats also showed similar vulnerability to nicotine seeking as sham animals when reinstatement was induced by nicotine-associated cues, but failed to reinstate lever pressing when relapse was induced by nicotine priming. Spinal nerve ligation and sham rats were equally sensitive to nicotine-induced anxiety-like behavior and antinociception; however, nicotine produced a potent and long-lasting antiallodynic effect in spinal nerve ligation rats. These results demonstrate that chronic pain leads to plasticity of nAChRs that do not directly facilitate nicotine addictive behaviors. Instead, nicotine potently decreases allodynia, an effect that could lead to increased nicotine consumption in chronic pain subjects.