N/OFQ Receptor Research Articles

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.

Synergistic interaction between DAMGO-NH2 and NOP01 in peripherally acting antinociception in two mouse models of formalin pain

The most commonly used opioid analgesics are limited by their severe side-effects in the clinical treatment of pain. Preliminary reports indicate that the combination of classical opioids and N/OFQ receptor (NOP) ligands may be an effective strategy to reduce unwanted side-effects and improve antinociception. But the interaction of these two receptor ligands in pain regulation at the peripheral level remains unclear. In this study, the antinociception of a designed amide analogue of the mu opioid receptor (MOP) peptide agonist DAMGO, DAMGO-NH2, and its antinociceptive interaction with the peripherally limited NOP peptide agonist NOP01 was investigated in two mouse models of formalin pain. Our results showed that DAMGO-NH2 acted as a MOP agonist in in vitro functional assays. Moreover, local subcutaneous or intraplantar injection of DAMGO-NH2 exerted dose-related antinociception in both phases of the formalin orofacial and intraplantar pain, which could be mediated by the classical opioid receptor. Peripheral but not central pretreatment with the peripherally restricted opioid antagonist naloxone methiodide inhibited local DAMGO-NH2-induced antinociception, supporting the involvement of the peripheral opioid receptor in local DAMGO-NH2-induced antinociception. Furthermore, co-administration of the inactive doses of DAMGO-NH2 and NOP01 produced effective antinociception. More importantly, isobolographic analysis indicates that the combination of DAMGO-NH2 and NOP01 elicited supra-additive antinociception in these two models of formalin pain. In addition, the combination of DAMGO-NH2 and NOP01 did not change motor function of mice in rotarod test. In conclusion, these data suggest that peripheral DAMGO-NH2 and particularly its combination therapy with NOP01 may be effective for pain management.

Adverse maternal environment alters Oprl1 variant expression in mouse hippocampus.

An adverse maternal environment (AME) and Western diet (WD) in early life predispose offspring toward cognitive impairment in humans and mice. Cognitive impairment associates with hippocampal dysfunction. An important regulator of hippocampal function is the hippocampal Nociceptin/Orphanin FQ (N/OFQ) system. Previous studies find links between dysregulation of hippocampal N/OFQ receptor (NOP) expression and impaired cognitive function. NOP is encoded by the opioid receptor-like 1 (Oprl1) gene that contains multiple mRNA variants and isoforms. Regulation of Oprl1 expression includes histone modifications within the promoter. We tested the hypothesis that an AME and a postweaning WD increase the expression of hippocampal Oprl1 and select variants concurrent with altered histone code in the promoter. We created an AME-WD model combining maternal WD and prenatal environmental stress plus postweaning WD in the mouse. We analyzed the hippocampal expression of Oprl1, Oprl1 variants, and histone modifications in the Oprl1 promoter in offspring at postnatal day (P) 21 and P100. An AME and an AME-WD significantly increased the total hippocampal expression of Oprl1 and variant V4 concurrently with an increased accumulation of active histone marks in the promoter of male offspring. We concluded that an AME and an AME-WD alter hippocampal Oprl1 expression in offspring through an epigenetic mechanism in a variant-specific and sex-specific manner. Altered hippocampal Oprl1 expression may contribute to cognitive impairment seen in adult males in this model. Epigenetic regulation of Oprl1 is a potential mechanism by which an AME and a WD may contribute to neurocognitive impairment in male offspring.

Novel Mixed NOP/Opioid Receptor Peptide Agonists.

The nociceptin/orphanin FQ (N/OFQ)/N/OFQ receptor (NOP) system controls different biological functions including pain and cough reflex. Mixed NOP/opioid receptor agonists elicit similar effects to strong opioids but with reduced side effects. In this work, 31 peptides with the general sequence [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2 were synthesized and pharmacologically characterized for their action at human recombinant NOP/opioid receptors. The best results in terms of NOP versus mu opioid receptor potency were obtained by substituting both Tyr1 and Thr5 at the N-terminal portion of N/OFQ(1-13)-NH2 with the noncanonical amino acid Dmt. [Dmt1,5]N/OFQ(1-13)-NH2 has been identified as the most potent dual NOP/mu receptor peptide agonist so far described. Experimental data have been complemented by in silico studies to shed light on the molecular mechanisms by which the peptide binds the active form of the mu receptor. Finally, the compound exerted antitussive effects in an in vivo model of cough.

Nociceptin/orphanin FQ receptor system blockade as an innovative strategy for increasing resilience to stress

The ability to successfully cope with stress is known as 'resilience', and resilient individuals are less prone to develop psychopathologies. Understanding the neurobiological mechanisms of resilience may be instrumental to improve current therapies and benefit high-risk subjects. This review summarizes the complex interplay that exists between physiological and pathological responses to stressful events and the nociceptin/orphanin FQ (N/OFQ) – N/OFQ receptor (NOP) system, including: the effects of stress in regulating N/OFQ release and NOP expression; the ability of the N/OFQ-NOP system to modulate the hypothalamic-pituitary-adrenal axis; behavioral studies; and evidence in humans correlating this peptidergic system with psychopathologies. Available findings support the view that N/OFQ signaling stimulates the hypothalamic-pituitary-adrenal axis, thus increasing stress circulating hormones and corticotropin-releasing factor signaling. Additionally, activation of the NOP receptor inhibits monoamine transmission, including 5-HT, and this may contribute to maladaptive outcomes of stress. Ultimately, the N/OFQ system seems to have an important role in stress vulnerability, and blockade of NOP signaling may provide an innovative strategy for the treatment of stress related psychopathologies.

Botulinum toxin A alleviates orofacial nociception induced by orthodontic tooth movement through nociceptin/orphanin-FQ pathway in rats

ObjectivesTo investigate the effect and mechanism of botulinum neurotoxin type A (BoNT/A) in the modulation of orofacial nociception induced by orthodontic tooth movement in rats. MethodsAn orofacial nociception model was established in male Sprague-Dawley rats by ligating closed-coil springs between incisors and ipsilateral molars. There were two group sets of animals. For the first group set, 120 rats were randomly divided into four groups: no-force group (n = 30), force + saline group (n = 30), force + low dose BoNT/A group (1U/6 μL, n = 30), and force + high dose BoNT/A group (1U/6 μL, n = 30). BoNT/A and saline were injected into periodontal ligament to explore the nociceptive effect of BoNT/A. Ipsilateral trigeminal ganglia (TG) were harvested for detecting the expression levels of nociceptin/orphanin-FQ (N/OFQ). For the second group set, 36 rats were randomly divided into three force groups: BoNT/A + saline group (n = 12), BoNT/A + UFP-101 group (n = 12), and saline + UFP-101 group (n = 12). A potent N/OFQ receptor (NOP) antagonist (UFP-101) was used to examine the role of N/OFQ in BoNT/A-induced antinociception. Tooth-movement nociception level of all groups was evaluated by bite force and rat grimace scale (RGS) at baseline, day 1, day 3, day 5, day 7, day 14. ResultsThe behavioral assessments showed the orofacial nociception level in the force + low dose BoNT/A group and force + high dose BoNT/A group were lower than that in the force + saline group. No significant difference was observed in orofacial nociception among no-force group, force + low dose and force + high dose group. The expression levels of N/OFQ in TG were elevated from day 1 and maintained a high level, presenting in descending order among the force + high dose, force + low dose, force + saline and no-force group, respectively. The nociception level of the BoNT/A + UFP-101 group was higher than that of the BoNT/A + saline group. No significant difference was observed between the BoNT/A + UFP-101 group and the saline + UFP-101 group. ConclusionsBoNT/A can exert an antinociceptive effect on orofacial nociception induced by tooth movement by stimulating the expression of N/OFQ in TG.

Detailed In Vitro Pharmacological Characterization of the Clinically Viable Nociceptin/Orphanin FQ Peptide Receptor Antagonist BTRX-246040.

The peptide nociceptin/orphanin FQ (N/OFQ) is the natural ligand of the N/OFQ receptor (NOP), which is widely expressed in the central and peripheral nervous system. Selective NOP antagonists are worthy of testing as innovative drugs to treat depression, Parkinson disease, and drug abuse. The aim of this study was to perform a detailed in vitro characterization of BTRX-246040 (also known as LY2940094, [2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4'-piperidine]-1'-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol), a novel NOP antagonist that has been already studied in humans. BTRX-246040 has been tested in vitro in the following assays: calcium mobilization in cells expressing NOP and classic opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer assay measuring NOP interaction with G proteins and β-arrestins, the label-free dynamic mass redistribution assay, and the electrically stimulated mouse vas deferens. BTRX-246040 was systematically compared with the standard NOP antagonist SB-612111. In all assays, BTRX-246040 behaves as a pure and selective antagonist at human recombinant and murine native NOP receptors displaying 3-10-fold higher potency than the standard antagonist SB-612111. BTRX-246040 is an essential pharmacological tool to further investigate the therapeutic potential of NOP antagonists in preclinical and clinical studies. SIGNIFICANCE STATEMENT: NOP antagonists may be innovative antidepressant drugs. In this research, the novel clinically viable NOP antagonist BTRX-246040 has been deeply characterized in vitro in a panel of assays. BTRX-246040 resulted a pure, potent, and selective NOP antagonist.

Targeting opioid dysregulation in depression for the development of novel therapeutics.

Since the serendipitous discovery of the first class of modern antidepressants in the 1950's, all pharmacotherapies approved by the Food and Drug Administration for major depressive disorder (MDD) have shared a common mechanism of action, increased monoaminergic neurotransmission. Despite the widespread availability of antidepressants, as many as 50% of depressed patients are resistant to these conventional therapies. The significant length of time required to produce meaningful symptom relief with these medications, 4-6 weeks, indicates that other mechanisms are likely involved in the pathophysiology of depression which may yield more viable targets for drug development. For decades, no viable candidate target with a different mechanism of action to that of conventional therapies proved successful in clinical studies. Now several exciting avenues for drug development are under intense investigation. One of these emerging targets is modulation of endogenous opioid tone. This review will evaluate preclinical and clinical evidence pertaining to opioid dysregulation in depression, focusing on the role of the endogenous ligands endorphin, enkephalin, dynorphin, and nociceptin/orphanin FQ (N/OFQ) and their respective receptors, mu (MOR), delta (DOR), kappa (KOR), and the N/OFQ receptor (NOP) in mediating behaviors relevant to depression and anxiety. Finally, putative opioid based antidepressants that are under investigation in clinical trials, ALKS5461, JNJ-67953964 (formerly LY2456302 and CERC-501) and BTRX-246040 (formerly LY-2940094) will be discussed. This review will illustrate the potential therapeutic value of targeting opioid dysregulation in developing novel therapies for MDD.

NOP Receptor Agonist Ro 64-6198 Decreases Escalation of Cocaine Self-Administration in Rats Genetically Selected for Alcohol Preference.

Cocaine dependence is a psychiatric condition for which effective medications are still lacking. Published data indicate that an increase in nociceptin/orphanin FQ (N/OFQ) transmission by NOP receptor activation attenuates cocaine-induced place conditioning and the locomotor sensitization effects of cocaine. This suggests that the activation of the N/OFQ receptor (NOP) may attenuate the motivation for psychostimulants. To further explore this possibility, we investigated the effect of the potent and selective NOP receptor agonist Ro 64-6198 on cocaine intake under 1 h short access (ShA) and 6 h long access (LgA) operant self-administration conditions in rats. We used Marchigian Sardinian alcohol-preferring (msP) rats and Wistar control rats. msP rats were used because we recently found that this rat line, originally selected for excessive alcohol drinking and preference, exhibits a greater propensity to escalate cocaine self-administration following LgA training. msP rats are also characterized by innate overexpression of the N/OFQ-NOP system compared with Wistar rats. Wistar and msP rats both exhibited an increase in cocaine self-administration under LgA conditions, with a higher trend toward escalation in msP rats. In Wistar rats, the intraperitoneal administration of Ro 64-6198 (0. 1 and 3 mg/kg) significantly decreased ShA cocaine self-administration. In Wistar rats that underwent LgA cocaine self-administration training, Ro 64-6198 induced no significant effect either during the first hour of self-administration or after the entire 6 h session. In msP rats, Ro 64-6198 significantly reduced cocaine self-administration both under ShA conditions and in the first hour of the LgA session. At the end of the 6 h session, the effect of Ro 64-6198 was no longer observed in msP rats. The highest dose of Ro 64-6198 (3 mg/kg) did not affect saccharin self-administration in msP rats but reduced saccharin self-administration in Wistar rats. Altogether, these data suggest that NOP receptor activation attenuates cocaine self-administration, and this effect tends to be more pronounced in a rat line with innately higher NOP receptor expression and that more robustly escalates cocaine intake.

Nociceptin/orphanin FQ receptor modulates painful and fatigue symptoms in a mouse model of fibromyalgia.

Generalized pain and fatigue are both hallmarks of fibromyalgia, a syndrome with an indefinite etiology. The treatment options for fibromyalgia are currently limited, probably because of its intricate pathophysiology. Thus, further basic and clinical research on this condition is currently needed. This study investigated the effects of nociceptin/orphanin FQ (N/OFQ) receptor (NOPr) ligands and the modulation of the NOP system in the preclinical mouse model of reserpine-induced fibromyalgia. The effects of administration of the natural agonist N/OFQ and the selective NOPr antagonists (UFP-101 and SB-612111) were evaluated in fibromyalgia-related symptoms in reserpine-treated mice. The expression of prepronociceptin/orphanin FQ and NOPr was assessed in central and peripheral sites at different time points after reserpine administration. Nociceptin/orphanin FQ displayed dual effects in the behavioral changes in the reserpine-elicited fibromyalgia model. The peptide NOPr antagonist UFP-101 produced analgesic and antifatigue effects, by preventing alterations in brain activity and skeletal muscle metabolism, secondary to fibromyalgia induction. The nonpeptide NOPr antagonist SB-612111 mirrored the favorable effects of UFP-101 in painful and fatigue alterations induced by reserpine. A time-related up- or downregulation of prepronociceptin/orphanin FQ and NOPr was observed in supraspinal, spinal, and peripheral sites of reserpine-treated mice. Our data shed new lights on the mechanisms underlying the fibromyalgia pathogenesis, supporting a role for N/OFQ-NOP receptor system in this syndrome.

NOP-Related Mechanisms in Substance Use Disorders.

Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that was deorphanized in 1995 and has been widely studied since. The role of the N/OFQ system in drug abuse has attracted researchers' attention since its initial discovery. The first two scientific papers describing the effect of intracranial injection of N/OFQ appeared 20years ago and reported efficacy of the peptide in attenuating alcohol intake, whereas heroin self-administration was insensitive. Since then more than 100 scientific articles investigating the role of the N/OFQ and N/OFQ receptor (NOP) system in drug abuse have been published. The present article provides an historical overview of the advances in the field with focus on three major elements. First, the most robust data supportive of the efficacy of NOP agonists in treating drug abuse come from studies in the field of alcohol research, followed by psychostimulant and opioid research. In contrast, activation of NOP appears to facilitate nicotine consumption. Second, emerging data challenge the assumption that activation of NOP is the most appropriate strategy to attenuate consumption of substances of abuse. This assumption is based first on the observation that animals carrying an overexpression of NOP system components are more prone to consume substances of abuse, whereas NOP knockout rats are less motivated to self-administer heroin, alcohol, and cocaine. Third, administration of NOP antagonists also reduces alcohol consumption. In addition, NOP blockade reduces nicotine self-administration. Hypothetical mechanisms explaining this apparent paradox are discussed. Finally, we focus on the possibility that co-activation of NOP and mu opioid (MOP) receptors is an alternative strategy, readily testable in the clinic, to reduce the consumption of psychostimulants, opiates, and, possibly, alcohol.

Electrophysiological Actions of N/OFQ.

Whilst the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) has similar intracellular coupling mechanisms to opioid receptors, it has distinct modulatory effects on physiological functions such as pain. These actions range from agonistic to antagonistic interactions with classical opioids within the spinal cord and brain, respectively. Understanding the electrophysiological actions of N/OFQ has been crucial in ascertaining the mechanisms by which these agonistic and antagonistic interactions occur. These similarities and differences between N/OFQ and opioids are due to the relative location of NOP versus opioid receptors on specific neuronal elements within these CNS regions. These mechanisms result in varied cellular actions including postsynaptic modulation of ion channels and presynaptic regulation of neurotransmitter release.

Nociceptin/Orphanin FQ (N/OFQ) conjugated to ATTO594: a novel fluorescent probe for the N/OFQ (NOP) receptor.

Background and PurposeThe nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is a member of the opioid receptor family and is involved in a number of physiological responses, pain and immune regulation as examples. In this study, we conjugated a red fluorophore‐ATTO594 to the peptide ligand N/OFQ (N/OFQATTO594) for the NOP receptor and explored NOP receptor function at high (in recombinant systems) and low (on immune cells) expression.Experimental ApproachWe assessed N/OFQATTO594 receptor binding, selectivity and functional activity in recombinant (CHO) cell lines. Live cell N/OFQATTO594 binding was measured in (i) HEK cells expressing NOP and NOPGFP receptors, (ii) CHO cells expressing the hNOPGαqi5 chimera (to force coupling to measurable Ca2+ responses) and (iii) freshly isolated human polymorphonuclear cells (PMN).Key ResultsN/OFQATTO594 bound to NOP receptor with nM affinity and high selectivity. N/OFQATTO594 activated NOP receptor by reducing cAMP formation and increasing Ca2+ levels in CHOhNOPGαqi5 cells. N/OFQATTO594 was also able to visualize NOP receptors at low expression levels on PMN cells. In NOP‐GFP‐tagged receptors, N/OFQATTO594 was used in a FRET protocol where GFP emission activated ATTO, visualizing ligand–receptor interaction. When the NOPGFP receptor is activated by N/OFQATTO594, movement of ligand and receptor from the cell surface to the cytosol can be measured.Conclusions and ImplicationsIn the absence of validated NOP receptor antibodies and issues surrounding the use of radiolabels (especially in low expression systems), these data indicate the utility of N/OFQATTO594 to study a wide range of N/OFQ‐driven cellular responses.

In vitro pharmacological characterization of a novel unbiased NOP receptor-selective nonpeptide agonist AT-403.

Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP), a member of the opioid receptor family. We recently identified a new high affinity and highly selective NOP agonist AT‐403. In this study, we characterized the functional profile of AT‐403 and compared it to other known nonpeptide NOP agonists Ro 65‐6570, Ro 2q, SCH‐221510, MCOPPB, AT‐202 and SCH‐486757, using the following assays: GTP γ[35S] stimulated binding, calcium mobilization assay in cells‐expressing human NOP or classical opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, and the electrically stimulated mouse vas deferens bioassay. All compounds behaved as NOP full agonists consistently showing the following rank order of potency MCOPPB > AT‐403 > Ro 65‐6570 = Ro 2q > SCH‐221510 > AT‐202 > SCH‐486757. AT‐403 and MCOPPB displayed the highest NOP selectivity both at human and murine receptors. Interestingly, while all the other nonpeptide NOP agonists displayed bias toward G protein‐mediated signaling in the BRET assay, AT‐403, similar to the natural ligand N/OFQ, behaved as an unbiased agonist, activating G‐protein‐mediated function as well as arrestin recruitment. AT‐403 may be a useful nonpeptide tool compound to study the pharmacology of NOP activation in disease states.

Structure- and conformation-activity studies of nociceptin/orphanin FQ receptor dimeric ligands

The peptide nociceptin/orphanin FQ (N/OFQ) and the N/OFQ receptor (NOP) constitute a neuropeptidergic system that modulates various biological functions and is currently targeted for the generation of innovative drugs. In the present study dimeric NOP receptor ligands with spacers of different lengths were generated using both peptide and non-peptide pharmacophores. The novel compounds (12 peptide and 7 nonpeptide ligands) were pharmacologically investigated in a calcium mobilization assay and in the mouse vas deferens bioassay. Both structure- and conformation-activity studies were performed. Results demonstrated that dimerization did not modify the pharmacological activity of both peptide and non-peptide pharmacophores. Moreover, when dimeric compounds were obtained with low potency peptide pharmacophores, dimerization recovered ligand potency. This effect depends on the doubling of the C-terminal address sequence rather than the presence of an additional N-terminal message sequence or modifications of peptide conformation.

In vitro functional characterization of novel nociceptin/orphanin FQ receptor agonists in recombinant and native preparations

Nociceptin/Orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ receptor (NOP). In this study novel nonpeptide NOP ligands were characterized in vitro in receptor binding and [35S]GTPγS stimulated binding in membranes of cells expressing human NOP and classical opioid receptors, calcium mobilization assay in cells coexpressing the receptors and chimeric G proteins, bioluminescence resonance energy transfer (BRET) based assay for studying NOP receptor interaction with G protein and arrestin, the electrically stimulated mouse vas deferens and the mouse colon bioassays. The action of the AT compounds were compared with standard NOP agonists (N/OFQ and Ro 65–6570) and the NOP selective antagonist SB-612111. AT compounds displayed high NOP affinity and behaved as NOP agonists in all the functional assays consistently showing the following rank order of potency AT-127≥AT-090≥AT-035>AT-004= AT-001. AT compounds behaved as NOP full agonists in the calcium mobilization and mouse colon assays and as partial agonists in the [35S]GTPγS and BRET assays. Interestingly AT-090 and AT-127, contrary to standard nonpeptide agonists that display G protein biased agonism, behaved as an unbiased agonists. AT-090 and AT-127 displayed higher NOP selectivity than Ro 65-6570 at native mouse receptors. AT-090 and AT-127 might be useful pharmacological tools for investigating the therapeutic potential of NOP partial agonists.

Changes in nociceptin/orphanin FQ levels in rat brain regions after acute and chronic cannabinoid treatment in conjunction with the development of antinociceptive tolerance.

It has been indicated that acute and chronic morphine administrations enhance nociceptin/orphanin FQ (N/OFQ) levels in the brain, which might play role in the development of tolerance to the antinociceptive effect of morphine. Accordingly, N/OFQ receptor (NOP) antagonists have been shown to prevent the development of antinociceptive tolerance to morphine. Our aim is to observe whether cannabinoids, similarly to opioids, enhance N/OFQ levels in pain-related brain regions and whether antagonism of NOP receptors attenuates the development of tolerance to the antinociceptive effect of cannabinoids. Hot plate and Tail flick tests are used to assess the antinociceptive response in Sprague-Dawley rats. N/OFQ levels are measured in cortex, amygdala, hypothalamus, periaqueductal gray, nucleus raphe magnus and locus coeruleus of rat brains using Western blotting and immunohistochemistry. Within 9 days, animals became completely tolerant to the antinociceptive effect of the cannabinoid agonist WIN 55,212-2 (2, 4, 6 mg/kg, i.p.). Chronic administration of JTC-801, a NOP receptor antagonist, at a dose that exerted no effect on its own (1 mg/kg, i.p.), attenuated development of tolerance to the antinociceptive effect of WIN 55,212-2 (4 mg/kg, i.p.). Western blotting and immunohistochemistry results showed that N/OFQ levels significantly increased in amygdala, periaqueductal gray, nucleus raphe magnus and locus coeruleus of rat brains when WIN 55,212-2 was combined with JTC-801. We hypothesize that, similar to opioids, chronic cannabinoid + NOP antagonist administration may enhance N/OFQ levels and NOP receptor antagonism prevents development of tolerance to cannabinoid antinociception.

Evidence for nociceptin/orphanin FQ (NOP) but not µ (MOP), δ (DOP) or κ (KOP) opioid receptor mRNA in whole human blood

BackgroundWhile it is well known that opioids depress the immune system, the site(s) of action for this depression is highly controversial. Immune modulation could occur directly at the immune cell or centrally via the hypothalamic-pituitary-adrenal axis. In a number of studies using individual enriched immune cell populations we have failed to detect classical µ (MOP), δ (DOP) and κ (KOP) receptors. The non-classical nociceptin/orphanin FQ (N/OFQ) receptor (NOP) is expressed on all cells examined thus far. Our hypothesis was that immune cells do not express classical opioid receptors and that using whole blood would definitively answer this question. MethodsWhole blood (containing all immune cell types) was incubated with opioids (morphine and fentanyl) commonly encountered in anaesthesia and with agents mimicking sepsis [lipopolysaccharide (LPS) and peptidoglycan G (PepG)]. Opioid receptor mRNA expression was assessed by endpoint polymerase chain reaction (PCR) with gel visualisation and quantitative PCR. ResultsClassical MOP, DOP, and KOP receptors were not detected in any of the samples tested either at rest or when challenged with opioids, LPS or PepG. Commercial primers for DOP did not perform well in quantitative PCR, so the absence of expression was confirmed using a traditional gel-based approach. NOP receptors were detected in all samples; expression was unaffected by opioids and reduced by LPS/PepG combinations. ConclusionsClassical opioid receptors are not expressed on circulating immune cells.

Beta-arrestin 2 rather than G protein efficacy determines the anxiolytic-versus antidepressant-like effects of nociceptin/orphanin FQ receptor ligands

Background and purposeNociceptin/orphanin FQ (N/OFQ) receptor (NOP) agonists produce anxiolytic-like effects in rodents while antagonists promote antidepressant-like effects. The aim of this study was to investigate the effect on anxiety and depression of NOP receptor partial agonists such as the peptides [F/G]N/OFQ(1-13)NH2 and UFP-113 and the non-peptide AT-090. Experimental approachIn vitro AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 were tested for their ability to promote NOP/G-protein and NOP/β-arrestin 2 interaction, using a bioluminescence resonance energy transfer assay. In vivo, they were tested in mice in the elevated plus maze (EPM) and in the forced swim (FST) tests. NOP partial agonists effects were systematically compared to those of full agonists (N/OFQ and Ro 65-6570) and antagonists (UFP-101 and SB-612111). Key resultsIn vitro, AT-090, UFP-113, and [F/G]N/OFQ(1-13)NH2 promoted NOP/G protein interaction, with maximal effects lower than those evoked by N/OFQ and Ro 65-6570. AT-090 behaved as a NOP partial agonist also in inducing β-arrestin 2 recruitment, while UFP-113 and [F/G]N/OFQ(1-13)NH2 were inactive in this assay. In vivo, AT-090 induced anxiolytic-like effects in the EPM but was inactive in the FST. Opposite results were obtained with UFP-113 and [F/G]N/OFQ(1-13)NH2. Conclusions and implicationsNOP ligands producing similar effects on NOP/G protein interaction (partial agonism) but showing different effects on β-arrestin 2 recruitment (partial agonism vs antagonism) elicited different actions on anxiety and mood. These results suggest that the action of a NOP ligand on emotional states is better predicted based on its β-arrestin 2 rather than G-protein efficacy.

Spinal antinociceptive effects of the novel NOP receptor agonist PWT2-nociceptin/orphanin FQ in mice and monkeys.

Using an innovative chemical approach, peptide welding technology (PWT), a tetrabranched derivative of nociceptin/orphanin FQ (N/OFQ) has been generated and pharmacologically characterized. Both in vitro and in vivo PWT2-N/OFQ displayed the same pharmacological profile to the natural ligand. It was more potent and produced longer-lasting effects. The aim of the present study was to investigate the spinal effects of PWT2-N/OFQ in nociceptive and neuropathic pain models in mice and non-human primates. Tail withdrawal assay in mice and monkeys was used as a nociceptive pain model and mechanical threshold in mice subjected to chronic constriction injury was used as a neuropathic pain model. The antinociceptive effects of spinally administered N/OFQ and PWT2-N/OFQ were assessed in these models. PWT2-N/OFQ mimicked the spinal antinociceptive effects of N/OFQ both in nociceptive and neuropathic pain models in mice as well as in non-human primates displaying 40-fold higher potency and a markedly prolonged duration of action. The effects of N/OFQ and PWT2-N/OFQ were sensitive to the N/OFQ receptor (NOP) antagonist SB-612111, but not to opioid receptor antagonists. The present study has demonstrated that PWT2-N/OFQ mimicked the antinociceptive effects of the natural peptide in rodents and non-human primates acting as a potent and longer-lasting NOP-selective agonist. More generally, PWT derivatives of biologically active peptides can be viewed as innovative pharmacological tools for investigating those conditions and states in which selective and prolonged receptor stimulation promotes beneficial effects.