Nociceptin/orphanin FQ Peptide Receptor Research Articles

Serine 363 is Required for NOPR Desensitization, Internalization, and Arrestin Signaling

Nociceptin/Orphanin FQ peptide receptor (NOPR) plays a key role in pain modulation, opiate tolerance, and responsivity to stress and anxiety. C‐terminal phosphorylation at serine (S), threonine (T), and tyrosine (Y) residues are required for μ and κ opioid receptor internalization, desensitization, arrestin recruitment, and mitogen‐activated protein kinase phosphorylation (pMAPK), so we generated mutations in the NOPR c‐terminus. We showed that NOPR‐YFP internalized shortly after nociceptin treatment while NOPR‐S363A blocked internalization. We determined time course and concentration‐dependence of NOPR‐YFP‐mediated pMAPK. NOPR‐YFP‐induced pERK peaked at 10min following treatment and pJNK peaked at 30min. NOPR‐S363A showed reduced pJNK levels. We determined that NOPR‐YFP internalization is blocked by arrestin3 and GRK3 shRNAs and that NOPR‐S363A internalization and pJNK can be rescued via a dominant positive arrestin3. These data implicate GRK/arrestin in NOPR MAPK signaling and highlight the potential for the development of functionally selective NOPR‐ligands. This work was partially supported by NIH grant R00DA034929 (to MRB), T32DA007261 (to SDC) and HHMI SURF Program (to NZ).

Inhibition of Toll-like receptor 4 (TLR4)-induced NF-kB activation by NOPr is lost in human glioblastoma cells chronically exposed to LPS

Glial cells can be activated at multiple sites along the pain pathway to produce pro-inflammatory mediators, thus contributing to the onset and maintenance of neuroinflammatory as well as chronic pain states. Toll-like receptor 4 (TLR4) is a glial key activator, responsible for the initial release of pro-inflammatory cytokines. The nociceptin/orphanin FQ peptide receptor (NOPr) is a GPCR which is expressed in neurons, lymphocytes, macrophages and in astrocytes and it is involved in a wide range of physiological responses within the nervous system and the immune system; the release of NOPr endogenous ligand, nociceptin (NC), is significantly increased in different models of neuroinflammation or chronic pain. These findings support a relevant role of NC/NOPr system in neuro-immune and neuron-glia interactions during glial adaptive response to pro-inflammatory stimuli. The aim of this research is to investigate NOPr expression and intracellular signaling in a model of human glial cells, either under basal condition or after exposition to the TLR4 activator, LPS. We found that NOPr activation by NC counteracts TLR4-mediated induction of NF-kB activation as well as the subsequent release of proinflammatory cytokines. A prolonged exposure (72 h) of glial cells to LPS, significantly down-regulated NOPr mRNA levels through transcriptional processes requiring the activation of p38MAPK. Under these conditions, NOPr-mediated inhibition of TLR4-dependent NF-kB activation was lost. These findings suggest an important role of NC/NOPr system in balancing glial activation by LPS.

Antihyperalgesic Effect of Buprenorphine Involves Nociceptin/Orphanin FQ Peptide–Receptor Activation in Rats With Spinal Nerve Injury–Induced Neuropathy

We evaluated the effect of buprenorphine, a mixed agonist for μ-opioid receptors and nociceptin/orphanin FQ peptide (NOP) receptors, in neuropathic rats, using the paw pressure test. Buprenorphine, administered i.p. at 50, but not 20, μg/kg, exhibited naloxone-reversible analgesic activity in naïve rats. In contrast, buprenorphine at 0.5 - 20 μg/kg produced a naloxonesensitive antihyperalgesic effect in the L5 spinal nerve-injured neuropathic rats. Intrathecal injection of [N-Phe(1)]nociceptin(1-13)NH2, a NOP-receptor antagonist, reversed the effect of buprenorphine in neuropathic rats, but not in naïve rats. Together, buprenorphine suppresses neuropathic hyperalgesia by activating NOP and opioid receptors, suggesting its therapeutic usefulness in treatment of neuropathic pain.

The Therapeutic Potential of Nociceptin/Orphanin FQ Receptor Agonists as Analgesics without Abuse Liability

Although mu opioid (MOP) receptor agonists are the most commonly used analgesics for the treatment of moderate to severe pain in the clinic, the side effects of MOP agonists such as abuse liability limit their value as a medication. Research to identify novel analgesics without adverse effects is pivotal to advance the health care of humans. The nociceptin/orphanin FQ peptide (NOP) receptor, the fourth opioid receptor subtype, mediates distinctive actions in nonhuman primates which suggests the possibility that activity at this receptor may result in strong analgesia in the absence of virtually all of the side effects associated with MOP agonists. The present review highlights the recent progress of pharmacological studies of NOP-related ligands in primates. Selective NOP agonists, either peptidic or nonpeptidic, produce full analgesia in various assays in primates, when delivered systemically or intrathecally. Yet small molecule NOP agonists do not serve as reinforcers, indicating a lack of abuse liability. Given that NOP agonists have low abuse liability and that coactivation of NOP and MOP receptors produces synergistic antinociception, it is worth developing bifunctional NOP/MOP ligands. The outcomes of these studies and recent developments provide new perspectives to establish a translational bridge for understanding the biobehavioral functions of NOP receptors in primates and for facilitating the development of NOP-related ligands as a new generation of analgesics without abuse liability in humans.

The nociceptin orphanin FQ peptide receptor agonist, Ro64-6198, impairs recognition memory formation through interaction with glutamatergic but not cholinergic receptor antagonists

We previously reported that the selective nociceptin orphanin peptide (NOP) receptor agonist, Ro64-6198, impairs mnemonic function through glutamatergic-dependent mechanisms. The aim of the current study was to determine whether the amnesic effects of Ro64-6198 involve a cholinergic component. The effects of systemic administration of Ro64-6198 (0.3 and 1mg/kg, i.p.), the cholinergic nicotinic receptor antagonist, mecamylamine (0.1 and 1mg/kg, s.c.), the cholinergic muscarinic receptor antagonist, scopolamine (0.1 and 0.3mg/kg, s.c.), and the glutamatergic NMDA receptor antagonist, MK-801 (0.03 and 0.1mg/kg, s.c.), were studied in the mouse object recognition task. All compounds tested were effective in disrupting formation of long-term (24-h delay) recognition memory. Drug interaction studies were then conducted to reveal the existence of functional interactions between NOP receptors and cholinergic and/or NMDA receptors. Co-administration of silent doses of Ro64-6198 (0.3mg/kg) and MK-801 (0.01mg/kg) produced clear-cut memory impairment. Similar synergistic effects were observed with the combination of mecamylamine (0.03mg/kg) and scopolamine (0.1mg/kg). In contrast, co-administration of Ro64-6198 (0.3mg/kg) with either mecamylamine (0.03 and 0.1mg/kg) or scopolamine (0.1mg/kg) was without any effect on recognition memory. These findings suggest that NOP receptor may modulate memory formation through a functional interaction with glutamatergic but not cholinergic receptors.

Roles of μ-Opioid Receptors and Nociceptin/Orphanin FQ Peptide Receptors in Buprenorphine-Induced Physiological Responses in Primates

Buprenorphine is known as a μ-opioid peptide (MOP) receptor agonist, but its antinociception is compromised by the activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine-induced physiological responses in primates (rhesus monkeys). The effects of MOP antagonist (naltrexone), NOP antagonist [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397)], and NOP agonists [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4.5] decan-4-one (Ro 64-6198) and 3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octan-3-ol (SCH 221510)] on buprenorphine were studied in three functional assays for measuring analgesia, respiratory depression, and itch in primates. Over the dose range of 0.01 to 0.1 mg/kg, buprenorphine dose-dependently produced antinociception, respiratory depression, and itch/scratching responses, and there was a ceiling effect at higher doses (0.1-1 mg/kg). Naltrexone (0.03 mg/kg) produced similar degrees of rightward shifts of buprenorphine's dose-response curves for all three endpoints. Mean pK(B) values of naltrexone (8.1-8.3) confirmed that MOP receptors mediated mainly buprenorphine-induced antinociception, respiratory depression, and itch/scratching. In contrast, J-113397 (0.1 mg/kg) did not change buprenorphine-induced physiological responses, indicating that there were no functional NOP receptors in buprenorphine-induced effects. More importantly, both NOP agonists, Ro 64-6198 and SCH 221510, enhanced buprenorphine-induced antinociception without respiratory depression and itch/ scratching. The dose-addition analysis revealed that buprenorphine in combination with the NOP agonist synergistically produced antinociceptive effects. These findings provided functional evidence that the activation of NOP receptors did not attenuate buprenorphine-induced antinociception in primates; instead, the coactivation of MOP and NOP receptors produced synergistic antinociception without other side effects. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics.

Roles of MOP and NOP receptors in regulating buprenorphine‐induced physiological responses in monkeys

Buprenorphine is known as a mu opioid receptor (MOP) partial agonist, but its analgesia was attenuated by activation of nociceptin/orphanin FQ peptide (NOP) receptors in rodents. The aim of this study was to investigate the roles of MOP and NOP receptors in regulating buprenorphine‐induced physiological responses in monkeys. Systemic buprenorphine (0.01–0.1 mg/kg) dose‐dependently produced antinociception, respiratory depression, and itch/scratching responses. A MOP antagonist, naltrexone 0.03 mg/kg, produced similar degrees of rightward shifts of buprenorphine's dose‐response curves for all three endpoints and confirmed that they were MOP‐mediated effects. In contrast, a NOP antagonist, J‐113397 0.1 mg/kg, did not change buprenorphine‐induced effects, indicating that there were no functional NOP receptors in buprenorphine‐induced actions. More importantly, a NOP agonist Ro 64–6198 enhanced buprenorphine‐induced antinociception, and the dose‐addition analysis revealed that systemic combination of buprenorphine and Ro 64–6198 produced superadditive antinociceptive effects. These findings provided functional evidence that co‐activation of MOP and NOP receptors produced synergistic antinociception in primates. This study strongly supports the therapeutic potential of mixed MOP/NOP agonists as innovative analgesics (Supported by USPHS grant AR‐059193).

Pharmacological characterization of NOP receptor agonists as abuse‐free and constipation‐free analgesics in monkeys

The nociceptin/orphanin FQ peptide (NOP) receptor, the fourth opioid receptor subtype, mediates distinctive actions in monkeys that suggest a possibility that NOP agonists may result in strong analgesia in the absence of side effects that are found in mu opioid receptor (MOP) agonists in primates. The aim of this study was to investigate and compare the behavioral effects of NOP agonists, Ro 64‐6198 and SCH221510, in side‐by‐side comparisons with MOP agonists, morphine and alfentanil, in rhesus monkeys. Both NOP and MOP agonists dose‐dependently produced antinociceptive effects against a noxious stimulus. Antagonist studies demonstrated that NOP and MOP receptors independently mediate antinociceptive actions. However, at antinociceptive doses, MOP agonists, but not NOP agonists, produced respiratory depression. NOP agonists did not produce reinforcing effects comparable with those of MOP agonists under the self‐administration procedure. In addition, at equianalgesic doses, injections of 3 mg/kg of morphine, but not 0.01 mg/kg of SCH221510, significantly delayed the gastrointestinal transit rate of monkeys, indicating that NOP agonists lack of constipating effects of MOP agonists. These results suggest that activation of NOP receptors produces antinociception with reduced abuse liability and fewer side effects in primates (Supported by USPHS grant AR‐059193).

Brain and Whole-Body Imaging of Nociceptin/Orphanin FQ Peptide Receptor in Humans Using the PET Ligand 11C-NOP-1A

Nociceptin/orphanin FQ peptide (NOP) receptor is a new class of opioid receptor that may play a pathophysiologic role in anxiety and drug abuse and is a potential therapeutic target in these disorders. We previously developed a high-affinity PET ligand, (11)C-NOP-1A, which yielded promising results in monkey brain. Here, we assessed the ability of (11)C-NOP-1A to quantify NOP receptors in human brain and estimated its radiation safety profile. After intravenous injection of (11)C-NOP-1A, 7 healthy subjects underwent brain PET for 2 h and serial sampling of radial arterial blood to measure parent radioligand concentrations. Distribution volume (V(T); a measure of receptor density) was determined by compartmental (1- and 2-tissue) and noncompartmental (Logan analysis and Ichise's bilinear analysis [MA1]) methods. A separate group of 9 healthy subjects underwent whole-body PET to estimate whole-body radiation exposure (effective dose). After (11)C-NOP-1A injection, the peak concentration of radioactivity in brain was high (∼5-7 standardized uptake values), occurred early (∼10 min), and then washed out quickly. The unconstrained 2-tissue-compartment model gave excellent V(T) identifiability (∼1.1% SE) and fitted the data better than a 1-tissue-compartment model. Regional V(T) values (mL·cm(-3)) ranged from 10.1 in temporal cortex to 5.6 in cerebellum. V(T) was well identified in the initial 70 min of imaging and remained stable for the remaining 50 min, suggesting that brain radioactivity was most likely parent radioligand, as supported by the fact that all plasma radiometabolites of (11)C-NOP-1A were less lipophilic than the parent radioligand. Voxel-based MA1 V(T) values correlated well with results from the 2-tissue-compartment model, showing that parametric methods can be used to compare populations. Whole-body scans showed radioactivity in brain and in peripheral organs expressing NOP receptors, such as heart, pancreas, and spleen. (11)C-NOP-1A was significantly metabolized and excreted via the hepatobiliary route. Gallbladder had the highest radiation exposure (21 μSv/MBq), and the effective dose was 4.3 μSv/MBq. (11)C-NOP-1A is a promising radioligand that reliably quantifies NOP receptors in human brain. The effective dose in humans is low and similar to that of other (11)C-labeled radioligands, allowing multiple scans in 1 subject.

Structural Determinants of Opioid and NOP Receptor Activity in Derivatives of Buprenorphine

The unique pharmacological profile of buprenorphine has led to its considerable success as an analgesic and as a treatment agent for drug abuse. Activation of nociceptin/orphanin FQ peptide (NOP) receptors has been postulated to account for certain aspects of buprenorphine's behavioral profile. In order to investigate the role of NOP activation further, a series of buprenorphine analogues has been synthesized with the aim of increasing affinity for the NOP receptor. Binding and functional assay data on these new compounds indicate that the area around C20 in the orvinols is key to NOP receptor activity, with several compounds displaying higher affinity than buprenorphine. One compound, 1b, was found to be a mu opioid receptor partial agonist of comparable efficacy to buprenorphine but with higher efficacy at NOP receptors.

Brain and Whole-Body Imaging in Rhesus Monkeys of 11C-NOP-1A, a Promising PET Radioligand for Nociceptin/Orphanin FQ Peptide Receptors

Our laboratory developed (S)-3-(2'-fluoro-6',7'-dihydrospiro[piperidine-4,4'-thieno[3,2-c]pyran]-1-yl)-2-(2-fluorobenzyl)-N-methylpropanamide ((11)C-NOP-1A), a new radioligand for the nociceptin/orphanin FQ peptide (NOP) receptor, with high affinity (K(i), 0.15 nM) and appropriate lipophilicity (measured logD, 3.4) for PET brain imaging. Here, we assessed the utility of (11)C-NOP-1A for quantifying NOP receptors in the monkey brain and estimated the radiation safety profile of this radioligand based on its biodistribution in monkeys. Baseline and blocking PET scans were acquired from head to thigh for 3 rhesus monkeys for approximately 120 min after (11)C-NOP-1A injection. These 6 PET scans were used to quantify NOP receptors in the brain and to estimate radiation exposure to organs of the body. In the blocked scans, a selective nonradioactive NOP receptor antagonist (SB-612111; 1 mg/kg intravenously) was administered before (11)C-NOP-1A. In all scans, arterial blood was sampled to measure the parent radioligand (11)C-NOP-1A. Distribution volume (V(T); a measure of receptor density) was calculated with a compartment model using brain and arterial plasma data. Radiation-absorbed doses were calculated using the MIRD Committee scheme. After (11)C-NOP-1A injection, peak uptake of radioactivity in the brain had a high concentration (∼5 standardized uptake value), occurred early (∼12 min), and thereafter washed out quickly. V(T) (mL · cm(-3)) was highest in the neocortex (∼20) and lowest in hypothalamus and cerebellum (∼13). SB-612111 blocked approximately 50%-70% of uptake and reduced V(T) in all brain regions to approximately 7 mL · cm(-3). Distribution was well identified within 60 min of injection and stable for the remaining 60 min, consistent with only parent radioligand and not radiometabolites entering the brain. Whole-body scans confirmed that the brain had specific (i.e., displaceable) binding but could not detect specific binding in peripheral organs. The effective dose for humans estimated from the baseline scans in monkeys was 5.0 μSv/MBq. (11)C-NOP-1A is a useful radioligand for quantifying NOP receptors in the monkey brain, and its radiation dose is similar to that of other (11)C-labeled ligands for neuroreceptors. (11)C-NOP-1A appears to be a promising candidate for measuring NOP receptors in the human brain.

Orphanin FQ/Nociceptin Activates Nuclear Factor Kappa B

Endogenous neuropeptide orphanin FQ/nociceptin (OFQ/N) and its receptor, nociceptin orphanin FQ peptide receptor (NOPr), play a modulatory role throughout the body including nociceptive sensitivity, motor function, spatial learning, and the immune system. NOPr is an inhibitory G protein coupled receptor (GPCR) that modulates expression and release of inflammatory mediators from immune cells and in the CNS. Inhibitory GPCRs have been shown to activate the immune and central nervous system regulator, nuclear factor kappa B (NFκB), whose family consists of several subunits. When activated, NFκB translocates to the nucleus and can modify transcription. To determine if OFQ/N modulates NFκB activity, SH-SY5Y human neuroblastoma cells were treated with OFQ/N and assessed for changes in nuclear accumulation, DNA binding, and transcriptional activation. For the first time, we show that OFQ/N increases the nuclear accumulation (1.9-2.8-fold) and the DNA binding of NFκB (2.9-fold) by 2h as determined by immunoblotting and electromobility shift assay, respectively. OFQ/N induction of NFκB binding to DNA is protein kinase C-dependent and NOPr-specific. OFQ/N stimulated binding of both NFκB p50 and p65 subunits to their consensus binding site on DNA. OFQ/N also induces transcriptional activation of an NFκB reporter gene 2.2-fold by 2h with an EC(50) of 6.3nM. This activation of NFκB by OFQ/N suggests a likely mechanism for its modulation of the central nervous and immune systems.

Synthesis and evaluation of radioligands for imaging brain nociceptin/orphanin FQ peptide (NOP) receptors with positron emission tomography.

Positron emission tomography (PET) coupled to an effective radioligand could provide an important tool for understanding possible links between neuropsychiatric disorders and brain NOP (nociceptin/orphanin FQ peptide) receptors. We sought to develop such a PET radioligand. High-affinity NOP ligands were synthesized based on a 3-(2'-fluoro-4',5'-dihydrospiro[piperidine-4,7'-thieno[2,3-c]pyran]-1-yl)-2(2-halobenzyl)-N-alkylpropanamide scaffold and from experimental screens in rats, with ex vivo LC-MS/MS measures, three ligands were identified for labeling with carbon-11 and evaluation with PET in monkey. Each ligand was labeled by (11)C-methylation of an N-desmethyl precursor and studied in monkey under baseline and NOP receptor-preblock conditions. The three radioligands, [(11)C](S)-10a-c, gave similar results. Baseline scans showed high entry of radioactivity into the brain to give a distribution reflecting that expected for NOP receptors. Preblock experiments showed high early peak levels of brain radioactivity, which rapidly declined to a much lower level than seen in baseline scans, thereby indicating a high level of receptor-specific binding in baseline experiments. Overall, [(11)C](S)-10c showed the most favorable receptor-specific signal and kinetics and is now selected for evaluation in human subjects.

The First Universal Opioid Ligand, (2S)-2-[(5R,6R,7R,14S)-N-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxymorphinan-7-yl]-3,3-dimethylpentan-2-ol (BU08028): Characterization of the In Vitro Profile and In Vivo Behavioral Effects in Mouse Models of Acute Pain and Cocaine-Induced Reward

Certain behavioral features of buprenorphine, including a bell-shaped curve for antinociception and attenuation of alcohol consumption, are thought to be mediated by activation of nociceptin/orphanin FQ peptide (NOP) receptors, despite moderate affinity and low efficacy at NOP receptors. We hypothesized that ligands with buprenorphine's physical properties, but possessing increased NOP receptor affinity and efficacy, would improve the profile as a drug abuse medication and reduce addiction liability. Using this strategy, we designed several compounds with universally high affinity, i.e., less than 10 nM at μ, δ, κ, and NOP receptors. Among these, (2S)-2-[(5R,6R,7R,14S)-N-cyclopropylmethyl-4,5-epoxy-6,14-ethano-3-hydroxy-6-methoxymorphinan-7-yl]-3,3-dimethylpentan-2-ol (BU08028) has high affinity at all opioid receptors and increased NOP receptor efficacy in vitro in the [³⁵S]GTPγS binding assay, however, while still being a partial agonist. In vivo, BU08028 was evaluated in an acute thermal antinociception assay, for its ability to induce conditioned place preference (CPP), and for its effect on cocaine-induced CPP. BU08028 is a very potent long-lasting analgesic. It produces an increase in locomotor activity and a significant CPP. As a pretreatment to cocaine, BU08028 does not alter cocaine CPP but causes a further increase in cocaine-induced locomotor activity. The analgesic, rewarding, and stimulant effects are probably caused by μ receptor stimulation. It is likely that with BU08028, a partial agonist at both NOP and μ receptors, μ-mediated activity overpowers NOP-mediated effects. Thus, it is possible that a different buprenorphine analog that is a universal high-affinity opioid ligand but with "full agonist" activity at NOP may counteract traditional opioid-mediated effects such as antinociception and reward.

The effects of nociceptin/orphanin FQ receptor agonist Ro 64-6198 and diazepam on antinociception and remifentanil self-administration in rhesus monkeys

The synthetic nonpeptide NOP (nociceptin/orphanin FQ peptide) receptor agonist Ro 64-6198 produces antinociception in rhesus monkeys. In rodents, it has much more variable effects on pain responses, but has response rate-increasing effects on punished operant behavior and decreases drug reward. The aim of this study was to compare Ro 64-6198 with the benzodiazepine diazepam in tests of analgesia, drug self-administration, and response-increasing effects in rhesus monkeys. Ro 64-6198 (0.001-0.01 mg/kg, i.v.) produced antinociception against an acute noxious stimulus (50°C water) in the absence of sedation, whereas diazepam (0.32-3.2 mg/kg, i.v.) did not have analgesic effects without sedation. Diazepam (1.0-5.6 mg/kg, i.v.) and the largest dose of Ro 64-6198 (0.32 mg/kg, i.v.) decreased lever pressing maintained by intravenous self-administration of the mu-opioid agonist, remifentanil, but neither effect could be distinguished from sedative effects. Although neither drug consistently increased responding during nonreinforcement, such effects were observed more frequently following diazepam administration. The effects of Ro 64-6198 on lever pressing were blocked by the NOP-receptor antagonist, J-113397, but not by the benzodiazepine antagonist, flumazenil. These findings suggest that the effects of Ro 64-6198 on operant lever pressing are mediated by NOP receptors and that larger doses are required to impact operant behavior when compared directly with those that produce antinociception. Therefore, the present findings support previous literature suggesting NOP receptors are a viable target for pain management.

The effects of Ro 64‐6198 and diazepam on antinociception and remifentanil self‐administration in rhesus monkeys

The aim of this study was to compare the behavioral effects of a synthetic nonpeptidic NOP (nociceptin/orphanin FQ Peptide) receptor agonist, Ro 64‐6198, with a benzodiazepine, diazepam, in rhesus monkeys. Ro 64‐6198 (0.001–0.01 mg/kg, i.v.) produced antinociception against an acute noxious stimulus (50 degrees C water), but diazepam (0.32–3.2 mg/kg, i.v.) did not produce such effects at any dose tested. Higher doses of Ro 64‐6198 (0.32 mg/kg, i.v.) and diazepam (1.0–5.6 mg/kg, i.v.) decreased lever pressing maintained by intravenous self‐administration of the mu‐opioid agonist, remifentanil. Although neither drug increased nonreinforced responding with complete consistency, such effects were more frequent with diazepam. These effects of Ro 64‐6198 on lever pressing were blocked by an NOP‐receptor antagonist, J‐113397, but not by the benzodiazepine antagonist, flumazenil. Conversely, the effects of diazepam on lever pressing were blocked by flumazenil but not J‐113397. These findings suggest the effects of Ro 64‐6198 on operant lever pressing are mediated by NOP receptors and that higher doses are required compared to those producing antinociception. Moreover, Ro 64‐6198 was less likely to disinhibit nonreinforced operant responding than diazepam, suggesting diazepam might produce greater anxiolytic‐like effects than Ro 64‐6198 in rhesus monkeys.

Chronic treatment with the selective NOP receptor antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) reverses the behavioural and biochemical effects of unpredictable chronic mild stress in rats

The present study was designed to assess the antidepressant effects of UFP-101, a selective nociceptin/orphanin FQ peptide (NOP) receptor antagonist, in a validated animal model of depression: the chronic mild stress (CMS). UFP-101 (5, 10 and 20 nmol/rat; i.c.v., once a day for 21 days) dose- and time-dependently reinstated sucrose consumption in stressed animals without affecting the same parameter in non-stressed ones. In the forced swimming test, UFP-101 reduced immobility of stressed rats from day 8 of treatment. After a 3-week treatment, rats were killed for biochemical evaluations. UFP-101 abolished increase in serum corticosterone induced by CMS and reverted changes in central 5-HT/5-HIAA ratio. The behavioural and biochemical effects of UFP-101 mimicked those of imipramine, the reference antidepressant drug, administered at the dose of 15 mg/kg (i.p.). Co-administration of nociceptin/orphanin FQ (5 nmol/rat, from day 12 to 21) prevented the effects of UFP-101. Brain-derived neurotrophic factor mRNA and protein in hippocampus were not reduced by CMS nor did UFP-101 modify these parameters. This study demonstrated that chronic treatment with UFP-101 produces antidepressant-like effects in rats subjected to CMS supporting the proposal that NOP receptors represent a candidate target for the development of innovative antidepressant drugs.

Behavioral Effects of a Synthetic Agonist Selective for Nociceptin/Orphanin FQ Peptide Receptors in Monkeys

Behavioral effects of a nonpeptidic NOP (nociceptin/orphanin FQ Peptide) receptor agonist, Ro 64-6198, have not been studied in primate species. The aim of the study was to verify the receptor mechanism underlying the behavioral effects of Ro 64-6198 and to systematically compare behavioral effects of Ro 64-6198 with those of a mu-opioid receptor agonist, alfentanil, in monkeys. Both Ro 64-6198 (0.001-0.06 mg/kg, s.c.) and alfentanil (0.001-0.06 mg/kg, s.c.) produced antinociception against an acute noxious stimulus (50 degrees C water) and capsaicin-induced allodynia. An NOP receptor antagonist, J-113397 (0.01-0.1 mg/kg, s.c.), dose-dependently produced rightward shifts of the dose-response curve of Ro 64-6198-induced antinociception. The apparent pA(2) value of J-113397 was 8.0. Antagonist studies using J-113397 and naltrexone revealed that Ro 64-6198 produced NOP receptor-mediated antinociception independent of mu-opioid receptors. In addition, alfentanil dose-dependently produced respiratory depression and itch/scratching responses, but antinociceptive doses of Ro 64-6198 did not produce such effects. More important, Ro 64-6198 did not produce reinforcing effects comparable with those of alfentanil, cocaine, or methohexital under self-administration procedures in monkeys. These results provide the first functional evidence that the activation of NOP receptors produces antinociception without reinforcing effects in primates. Non-peptidic NOP receptor agonists may have therapeutic value as novel analgesics without abuse liability in humans.

Opioid, cannabinoid CB 1 and NOP receptors do not mediate APAP-induced hypothermia in rats

Acetaminophen (APAP) produces antinociception and hypothermia. Because the antinociceptive effect in rats is partially dependent on opioid and cannabinoid CB 1 receptor activation, we determined if activation of these receptors also contributes to the hypothermic effect of APAP. Rats injected with APAP (100, 250, 375 or 500 mg/kg, i.p.) displayed dose-related hypothermia. For combined administration, the hypothermic effect of APAP (400 mg/kg, i.p.) was not altered by pretreatment with: naltrexone (10 mg/kg, s.c.), a non-selective opioid antagonist; naltrindole (1 mg/kg, s.c.), a delta opioid antagonist; nor-binaltorphimine (10 mg/kg, i.p.), a kappa opioid antagonist; SR 141716A (3 mg/kg, i.m.), a cannabinoid CB 1 receptor antagonist; or JTC-801(1 mg/kg, i.p.), a nociceptin/orphanin FQ peptide (NOP) receptor antagonist. The demonstration that APAP produces hypothermia independent of opioid, cannabinoid CB 1 or NOP receptor activation is contrary to its antinociceptive effect, which requires opioid and cannabinoid CB 1 receptor activation.

Cloning and characterization of the rhesus monkey nociceptin/orphanin FQ receptor

We succeeded in cloning the rhesus monkey nociceptin/orphanin FQ peptide (NOP) receptor. The nucleotide sequence and amino acid sequence of the rhesus monkey NOP receptor were 95.9% and 97.8%, respectively, identical to the human NOP receptor. There was no significant difference between the rhesus monkey NOP receptor and the human NOP receptor in the binding affinity of [(125)I] [Thy(14)]nociceptin and the binding of [(35)S]guanosine 5'-O-(gamma thio)triphospate ([(35)S]GTPgammaS) stimulated by nociceptin/orphanin FQ (N/OFQ). A selective NOP receptor antagonist, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one ((+)-J-113397) inhibited the [(35)S]GTPgammaS binding activated by N/OFQ using the membrane of the rhesus monkey NOP receptor. The antagonistic activity of (+)-J-113397 to the rhesus monkey NOP receptor was comparable to that to the human NOP receptor. Thus, N/OFQ acts via activation of the NOP receptor in both human and rhesus monkeys without significant species differences.