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Background and Purpose: In animal models of sepsis, increased activation of the Nociceptin/Orphanin FQ (N/OFQ) receptor NOP is associated with mortality and NOP antagonists improved survival. We have explored the role of the N/OFQ‐NOP system in freshly isolated volunteer human B‐ and T‐cells incubated with lipopolysaccharide (LPS) and peptidoglycan G (PepG) as a model of in vitro sepsis. Experimental Approach: B‐ and T‐cell NOP expression was measured using the NOP fluorescent probe N/OFQATTO594, N/OFQ content was measured using immunofluorescence, N/OFQ release was tracked using a CHOhNOPGαiq5 biosensor assay and NOP function was measured using transwell migration and cytokine/chemokine release using a 25‐plex assay format. Cells were challenged with LPS/PepG. Key Results: CD19‐positive B‐cells bound N/OFQATTO594; they also contain N/OFQ. Stimulation with CXCL13/IL‐4 increased N/OFQ release. N/OFQ trended to reduced migration to CXCL13/IL‐4. Surface NOP expression was unaffected by LPS/PepG, but this treatment increased GM‐CSF release in an N/OFQ sensitive manner. CD3‐positive T‐cells did not bind N/OFQATTO594; they did contain N/OFQ. Stimulation with CXCL12/IL‐6 increased N/OFQ release. When incubated with LPS/PepG, NOP surface expression was induced leading to N/OFQATTO594 binding. In LPS/PepG‐treated cells, N/OFQ reduced migration to CXCL12/IL‐6. LPS/PepG increased GM‐CSF release in an N/OFQ sensitive manner. Conclusions and Implications: We suggest both a constitutive and sepsis‐inducible N/OFQ‐NOP receptor autocrine regulation of B‐ and T‐cell function, respectively. These NOP receptors variably inhibit migration and reduce GM‐CSF release. These data provide mechanistic insights to the detrimental role for increased N/OFQ signalling in sepsis and suggest a potential role for NOP antagonists as treatments. [ABSTRACT FROM AUTHOR]

Background: The peptide nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) are implicated in the modulation of emotional states. Previous human and rodent findings support NOP antagonists as antidepressants. However, the role played by the N/OFQ-NOP receptor system in resilience to stress is unclear.Aims: The present study investigated the effects of activation or blockade of NOP receptor signaling before exposure to acute stress.Methods: The behavioral effects of the administration before stress of the NOP agonists Ro 65-6570 (0.01-1 mg/kg) and MCOPPB (0.1-10 mg/kg), and the NOP antagonist SB-612111 (1-10 mg/kg) were assessed in mice exposed to inescapable electric footshock and forced swim as stressors. The behavioral phenotype of mice lacking the NOP receptor (NOP(-/-)) exposed to inescapable electric footshock was also investigated.Results: The activation of NOP receptor signaling with the agonists increased the percentage of mice developing helpless behavior and facilitated immobile posture. In contrast, the blockade of NOP receptor reduced the acquisition of depressive-like phenotypes, and similar resistance to develop helpless behaviors was observed in NOP(-/-) mice. Under the same stressful conditions, the antidepressant nortriptyline (20 mg/kg) did not change the acquisition of helpless behavior and immobile posture.Conclusions: These findings support the view that NOP activation during acute stress facilitates the development of depressive-related behaviors, whereas NOP blockade has a protective outcome. This study showed for first time that NOP antagonists are worthy of investigation as preemptive treatments in patients with severe risk factors for depression. [ABSTRACT FROM AUTHOR]

The aim of the study was to investigate the in vitro and in vivo pharmacological profile of cebranopadol, a novel agonist for opioid and nociceptin/orphanin FQ (N/OFQ) receptors (NOP). In vitro cebranopadol was assayed in calcium mobilization studies in cells coexpressing NOP or opioid receptors and chimeric G-proteins and in a bioluminescence resonance energy transfer (BRET) assay for studying receptor interaction with G-protein and b-arrestin 2. The mouse tail withdrawal and formalin tests were used for investigating cebranopadol antinociceptive properties. In calcium mobilization studies cebranopadol showed the following rank order of potency NOP = mu > kappa ≥ delta. In BRET studies, cebranopadol promoted NOP and mu receptors interaction with G-protein with similar high potency and efficacy. However, cebranopadol did not stimulated NOP–b-arrestin 2 interactions and displayed reduced potency at mu/b-arrestin 2. In vivo, cebranopadol exhibits highly potent and extremely long-lasting antinociceptive effects. The effects of cebranopadol in the tail withdrawal assay were sensitive to both SB-612111 and naloxone. Collectively the present results confirm and extend previous finding demonstrating that cebranopadol, by acting as mixed NOP/opioid receptor agonist, elicits robust analgesic effects in different pain models. [ABSTRACT FROM AUTHOR]

Abstract: Nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) were identified in the mid 90s as a novel peptidergic system structurally related to opioids. A growing body of preclinical evidence suggests that blockade of NOP receptors evokes antidepressant-like actions. These have been explored using a range of compounds (peptide and non peptide antagonists), across different species (rat and mouse) and assays (behavioral despair and chronic mild stress) suggesting a robust and consistent antidepressant-like effect. Moreover, rats and mice knockout for the NOP receptor gene display an antidepressant-like phenotype in behavioral despair assays. Electrophysiological, immunohistochemical and neurochemical studies point to an important role played by monoaminergic systems, particularly 5-HTergic, in mediating the antidepressant-like properties of NOP antagonists. However other putative mechanisms of action, including modulation of the CRF system, circadian rhythm and a possible neuroendocrine-immune control might be involved. A close relationship between the N/OFQ-NOP receptor system and stress responses is well described in the literature. Stressful situations also alter endocrine, behavioral and neurochemical parameters in rats and chronic administration of a NOP antagonist restored these alterations. Interestingly, clinical findings showed that plasma N/OFQ levels were significantly altered in major and post-partum depression, and bipolar disease patients. Collectively, data in the literature support the notion that blockade of NOP receptor signaling could be a novel and interesting strategy for the development of innovative antidepressants. [Copyright &y& Elsevier]

In this study, the Gαqi5 protein was used to force the human nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor to signal through the Ca2+ pathway in CHO cells. [Ca2+]i levels were monitored using the fluorometer FlexStation II and the Ca2+ dye Fluo 4 AM. Concentration response curves were generated with a panel of full and partial agonists, while NOP antagonists were assessed in inhibition-response curves. The following rank order of potency of antagonists was measured: $${\text{SB}} - {\text{612111}} >{\text{J}} - 113397 = {\text{Trap}} - 101 \geqslant {\text{UFP}} - 101 >\left[ {{\text{Nphe}}^{\text{1}} } \right]{{\text{N}} \mathord{\left/ {\vphantom {{\text{N}} {{\text{OFQ}}}}} \right. \kern-\nulldelimiterspace} {{\text{OFQ}}}}\left( {1 - 13} \right){\text{NH}}_{\text{2}} >>$$ naloxone, which is superimposable to literature findings. The rank order of potency of full and partial agonists is also similar to that obtained in previous studies with the exception of a panel of ligands (UFP-112, Ro 64-6198, ZP120, UFP-113) whose potency was relatively low in the Gαqi5–NOP receptor calcium assay. Interestingly, these NOP ligands are characterized by slow kinetic of interaction with the NOP receptor, as demonstrated by bioassay experiments. These results demonstrated that the FlexStation II–Gαqi5–NOP receptor calcium assay represents an adequate and useful screening for NOP receptor ligands, particularly for antagonists. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ peptide receptor (NOP). Binding studies have relied on [leucyl-3H]N/OFQ, but as this is an agonist G-protein coupling will affect affinity. In this paper, we describe a new [3H]labeled NOP antagonist, [Nphe1,4’-3H-Phe4,Arg14,Lys15]N/OFQ-NH2 ([3H]UFP-101). We have characterized [3H]UFP-101 at recombinant human NOP expressed in Chinese hamster ovary cells (CHOhNOP) and native rat NOP in cerebrocortex. Radioligand saturation and competition studies were performed on membranes, and [3H]UFP-101 (antagonist) was compared with [3H]N/OFQ (agonist). The effects of GTPγS (10 µM) and Na+ were investigated alone and in combination in competition experiments with both radioligands. In CHOhNOP, B max, and p K D, values were 561 and 580 fmol mg protein−1 and 9.97 and 10.19 for [3H]UFP-101 and [leucyl-3H]N/OFQ, respectively. In rat cerebrocortex B max and p K D, values were 65 and 88 fmol mg protein−1 and 10.12 and 10.34 for [3H]UFP-101 and [leucyl-3H]N/OFQ. The binding of both radioligands was displaced by a range of peptide and non-peptide NOP ligands at both isoforms with good correlation ( r 2 0.92 in Rat and 0.97 in CHOhNOP). Naloxone was inactive. The binding of both radioligands was Na+-dependent with [3H]UFP-101 being more sensitive (IC50 ~20 mM). Unlike the agonist [leucyl-3H]N/OFQ, the antagonist [3H]UFP-101 was unaffected by GTPγS. [3H]UFP-101 binds to human and rat NOP with high affinity and good agreement with standard [leucyl-3H]N/OFQ in competition experiments. In addition, the binding of [3H]UFP-101 is unaffected by GTPγS. This radioligand will be useful to further characterize NOP in a range of binding paradigms. [ABSTRACT FROM AUTHOR]

1 Nocistatin (NST) antagonizes several actions of nociceptin orphanin FQ (N/OFQ), but acts on distinct receptors. As N/OFQ exerts anxiolytic-like actions in various tests, its behavioural actions in the elevated plus-maze (EPM) test were compared with those of bovine NST. 2 Five minutes after i.c.v. treatment, mice were placed on the EPM for 5 min and entries into and lime spent on open and closed arms were recorded alongside other parameters. 3 NST (0.1 -3 pmol) reduced percentages of entries into (control 39.6±3.1%, peak effect at 1 pmol NST 8.5±2.9%) and time spent on open arms (control 30.8±2.3%. NST 2.7 ± 1.5%), The C-terminal hexapeptide of NST (NST-C6; 0.01 -10 pmol) closely mimicked these actions of NST, with peak effects at 0.1 pmol. 4 N/OFQ (1-100 pmol) increased percentages of entries into (control 38.5±3.4%: peak effect at 10 pmol N/OFQ 67.9 ± 4.9%) and time spent on open arms (control 32.0±3.8%; N/OFQ 74.9± 5.8%). Closed arm entries, an index of locomotor activity, were unchanged by all peptides. 5 Effects of NST or NST-C6, but not N/OFQ, were still detectable 15 min after injection. Behaviour of animals co-injected with NST (1 pmol) or NST-C6 (0.1 pmol) plus N/OFQ (10 pmol) was indistinguishable from that of controls. 6 These results reveal potent anxiogenic-like actions of NST and NST-C6, and confirm the anxiolytic-like properties of N/OFQ. As NST and N/OFQ both derive from preproN/OF, anxiety may be modulated in opposing directions depending on how this precursor is processed. [ABSTRACT FROM AUTHOR]

Bipolar disorder is a psychiatric pathology characterized by biphasic mood episodes of mania or hypomania and depression. The pharmacotherapy of bipolar disorder has significant adverse effects impairing treatment adherence and patient quality of life. The N/OFQ-NOP receptor system has been widely implicated with mood disorders. Clinical and preclinical findings suggest antidepressants actions for NOP antagonists. More recently, the administration of NOP agonists has shown to promote depressant states. The present study aimed to investigate the effects of non-peptide NOP ligands in methylphenidate-induced manic-like behavior in mice. The NOP agonist Ro 65–6570 (0.01–1 mg/kg, ip), at the higher dose, did not affect spontaneous locomotion per se, but prevented the methylphenidate (10 mg/kg, sc)-induced hyperlocomotion. The NOP partial agonist AT-090 (0.001–0.03 mg/kg, ip) and the NOP antagonist SB-612111 (1–10 mg/kg, ip) did not significantly affect the psychostimulant-induced hyperactivity. Experiments performed with mice lacking the NOP receptor (NOP(−/−)) demonstrated that the treatment with methylphenidate induced similar hyperlocomotion in NOP(−/−) and NOP(+/+) mice. In conclusion, these findings suggest a potential role for NOP agonists in the prevention of manic states, especially by counteracting the hyperactivity symptom of bipolar patients. However, more studies are necessary in order to evaluate these compounds in other features of bipolar disorder. • The antimanic drug valproate prevented the methylphenidate-induced hypelocomotion. • The NOP full agonist Ro 65–6570 at higher doses attenuated methylphenidateinduced hyperlocomotion. • The NOP partial agonist AT-090 and the NOP antagonist SB-612111 did not affect manic-like behaviors. [ABSTRACT FROM AUTHOR]

• A complex interplay exists between responses to stress and the N/OFQ-NOP system. • Stress modulates N/OFQ release and NOP expression. • The N/OFQ-NOP system modulates the HPA axis. • NOP activation contributes to maladaptive outcomes of stress. • NOP blockade may be useful for the treatment of stress related disorders. 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. [ABSTRACT FROM AUTHOR]

Highlights • NOP agonists increased male mouse aggressiveness. • The NOP antagonist SB-612111 did not change mouse aggressiveness. • NOP(-/-) mice did not display any behavioral phenotype in the resident-intruder test. • Lithium, valproate and carbamazepine attenuated mouse aggressiveness. • An inhibitor of 5-HT synthesis, PCPA, increased mouse aggressive behavior. Abstract Aggressive behaviors can be considered symptoms of bipolar disorder, schizophrenia, post-traumatic stress, intermittent explosive, and personality disorders. Nociceptin/orphanin FQ (N/OFQ) is a peptide acting as endogenous ligand of the NOP receptor. Preclinical and clinical findings suggest the NOP receptor as an innovative target for the treatment of psychopathologies, such as anxiety, depression, and drug abuse. This study investigated the effects of NOP ligands and the behavioral phenotype of mice lacking the NOP receptor in an animal model of aggressiveness, the resident-intruder test. Mood stabilizers, such as valproate, lithium, and carbamazepine reduced aggressive behaviors of resident mice, while diazepam was inactive. In contrast, para-chlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis, increased aggressiveness in mice. Similar to PCPA, the treatment with the NOP agonists Ro 65-6570 and AT-090 also increased aggressive behaviors. The systemic administration of the NOP antagonist SB-612111 did not modify the behavior of resident mice, but it prevented the aggressive behavior of Ro 65-6570. NOP receptor knockout mice did not display any behavioral difference compared to wild-type animals in the resident-intruder test. None of the treatments affected non-agonistic behaviors and spontaneous locomotion. In conclusion, NOP receptor agonists increased aggressiveness, while the pharmacological and genetic blockade of NOP receptor signaling did not modify agonistic behaviors. Ultimately, the aggressive profile of action of NOP agonists should be taken into account in the development of innovative psychiatric drugs targeting the NOP receptor. [ABSTRACT FROM AUTHOR]

Several neuropeptidergic systems act as modulators of cognitive performances. Among them, nociceptin, an opioid-like peptide also known as orphanin FQ (N/OFQ), has recently gained attention. Stimulation of its receptor, the N/OFQ opioid receptor (NOP), which is expressed in brain regions involved in emotion, memory and stress response, has inhibitory effects on the acquisition and/or consolidation of spatial and emotional memory in rodents. Recently, N/OFQ was also proposed to be linked to the pathogenesis of Post-Traumatic Stress Disorder in humans. However, until now the effect of the activation of the N/OFQ-NOP system on already consolidated memory, such as during retrieval and reconsolidation phases, has never been explored. In the present study, we investigated the consequences of systemic injection of NOP agonists or i.c.v. injection of the N/OFQ peptide on the retrieval and the reconsolidation of contextual fear memory in mice. We demonstrate that the activation of the N/OFQ system impairs the reconsolidation of context-dependent but not cue-dependent aversive memories. We also show that this amnestic effect is associated with decreased c-Fos expression in the hippocampus and amygdala. Our data thus provide the first evidence that the NOP receptor could be targeted during the reconsolidation process to weaken maladaptive memories. The N/OFQ-NOP system might constitute in the future an interesting pharmacological target for interfering with so-called “pathological memories”, in particular those involving maladaptive contextual memories. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ is the natural ligand of a Gi-protein coupled receptor named NOP. This peptidergic system is involved in the regulation of mood states and inflammatory responses. The present study aimed to investigate the consequences of blocking NOP signaling in lipopolysaccharide (LPS)-induced sickness and depressive-like behaviors in mice. LPS 0.8 mg/kg, ip, significantly induced sickness signs such as weight loss, decrease of water and food intake and depressive-like behavior in the tail suspension test. Nortriptyline (ip, 60 min prior the test) reversed the LPS-induced depressive states. The NOP receptor antagonist SB-612111, 30 min prior LPS, did not modify LPS-induced sickness signs and depressive-like behavior. However, when injected 24 h after LPS, NOP antagonists (UFP-101, icv, and SB-612111, ip) significantly reversed the mood effects of LPS. LPS evoked similar sickness signs and significantly increased tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) plasma levels 6 h post-injection in wild-type ((NOP(+/+)) and NOP knockout ((NOP(−/−)) mice. However, LPS treatment elicited depressive-like effects in NOP(+/+) but not in NOP(−/−) mice. In conclusion, the pharmacological and genetic blockade of NOP signaling does not affect LPS evoked sickness signs while reversing depressive-like behavior. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ (N/OFQ) is a heptadecapeptide acting as the endogenous ligand of the N/OFQ peptide receptor (NOP). N/OFQ(1–13)-NH 2 is the shortest N/OFQ sequence maintaining the same potency and efficacy as the natural peptide. Thus N/OFQ(1–13)-NH 2 was used as chemical template for investigating the structure activity relationship of threonine in position 5. 28 [X 5 ]N/OFQ(1–13)-NH 2 derivatives, in which Thr was substituted with natural and unnatural residues, were synthesized and characterized pharmacologically for their effects at the human NOP receptor. Two different functional assays were used: agonist stimulated [ 35 S]GTPγS binding in cell membranes and calcium mobilization in whole cells co-expressing chimeric G proteins. All [X 5 ]N/OFQ(1–13)-NH 2 derivatives behaved as full NOP agonists showing large differences in their potency. There was an excellent correlation between the results obtained in the two assays. The results of this study suggest that: position 5 does not play a pivotal role in receptor activation; the secondary alcoholic function of Thr is not important for receptor binding; side chain size, lipo/hydrophilic balance as well as hydrogen bond capability are also not crucial for receptor binding; an aliphatic amino function positively charged with at least 3 carbon atom distance from the peptide backbone has a huge disrupting effect on receptor binding. In conclusion this study demonstrates that a simple ethyl side chain as in compound 23 is sufficient in N/OFQ position 5 for maintaining bioactivity. [ABSTRACT FROM AUTHOR]

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of an inhibitory G protein coupled receptor named N/OFQ peptide receptor (NOP). Clinical and preclinical findings suggest that the blockade of the NOP signaling induces antidepressant-like effects. Additionally, the blockade of the NOP receptor during inescapable stress exposure prevented the acquisition of the helplessness phenotype, suggesting that NOP antagonists are able to increase stress resilience. BTRX-246040 (aka LY2940094) is a NOP receptor antagonist with high affinity, potency and selectivity for the NOP over classical opioid receptors. BTRX-246040 is under development for the treatment of depression, eating disorders and alcohol abuse and it already entered clinical trials. In the present study, the antidepressant effects of BTRX-246040 were evaluated in mice subjected to the forced swimming test and to the learned helplessness model of depression. Additionally, the ability of BTRX-246040 to prevent the development of the helpless behavior and to modulate adult hippocampal neurogenesis has been investigated. BTRX-246040 (30 mg/kg, i.p.) produced antidepressant-like effects in the forced swimming test and in the learned helplessness model. More interestingly, when given before the stress induction sessions it was able to prevent the development of the helplessness behavior. Under these experimental conditions, BTRX-246040 did not modulate adult hippocampal neurogenesis, neither in naive nor in stressed mice. This study, performed with a clinically viable ligand, further corroborates growing evidence indicating that the blockade of the NOP signaling may provide an innovative strategy for the treatment of stress related psychopathologies. • BTRX-246040 is a NOP receptor antagonist. • BTRX-246040 produced antidepressant-like effects in mice. • BTRX-246040 prevented the development of the helpless behavior in stressed mice. • BTRX-246040 did not altered mice neurogenesis under these experimental conditions. [ABSTRACT FROM AUTHOR]

Abstract: Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ peptide (NOP) receptor. Recently knockout rats for the NOP receptor gene (NOP(−/−)) have been generated; these animals were used in the present study to investigate their emotional (open field, elevated plus maze, and forced swimming test), locomotor (drag and rotarod test), and nociceptive (plantar and formalin test) phenotypes in comparison with their NOP(+/+) littermates. In addition, N/OFQ sensitivity has been assessed in electrically stimulated vas deferens tissues taken from NOP(+/+) and NOP(−/−) rats. In the elevated plus maze and forced swimming tests NOP(−/−) rats showed anxiety- and anti-depressant-like phenotype, respectively. No differences were found in the open field test. NOP(−/−) rats outperformed their NOP(+/+) littermates in two motor behaviour assays. Genetic ablation of the NOP receptor gene produced a statistically significant increase in nociceptive behaviour of the mutant rats in the formalin test. Finally, in the electrically stimulated rat vas deferens taken from NOP(+/+) tissues, N/OFQ inhibited in a concentration-dependent manner the electrically induced twitches while the peptide was inactive in tissues taken from NOP(−/−) animals. These results, in line with previous findings obtained with selective NOP receptor antagonists in mice and rats and with mouse knockout studies, clearly indicate that endogenous N/OFQ–NOP receptor signalling plays an important role in controlling anxiety- and mood-related behaviours, exercise-driven locomotor activity and nociception. These observations are relevant for defining the therapeutic indications (and contraindications) of NOP receptor antagonists. [Copyright &y& Elsevier]

Abstract: Compound 24, 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4′-piperidin-1-yl]propyl} pyrrolidine-2-carboxamide was recently identified as a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In this study, the in vitro and in vivo pharmacological profiles of Compound 24 were investigated. In vitro studies were performed measuring receptor and [35S]GTPγS binding and calcium mobilization in cells expressing the recombinant NOP receptor as well as using N/OFQ sensitive tissues. In vivo studies were conducted using the tail withdrawal assay in mice. Compound 24 produced a concentration-dependent displacement of [3H]N/OFQ binding to CHOhNOP cell membranes showing high affinity (pK i 9.62) and selectivity (1000 fold) over classical opioid receptors. Compound 24 antagonized with high potency the following in vitro effects of N/OFQ: stimulation of [35S]GTPγS binding in CHOhNOP cell membranes (pA2 9.98), calcium mobilization in CHOhNOP cells expressing the Gαqi5 chimeric protein (pK B 8.73), inhibition of electrically evoked twitches in the mouse (pA2 8.44) and rat (pK B 8.28) vas deferens, and in the guinea pig ileum (pK B 9.12). In electrically stimulated tissues, Compound 24 up to 1 µM did not modify the effects of classical opioid receptor agonists. Finally in vivo, in the mouse tail withdrawal assay, Compound 24 at 10 mg/kg antagonized the pronociceptive and antinociceptive effects of 1 nmol N/OFQ given supraspinally and spinally, respectively. Under the same experimental conditions Compound 24 did not affect the antinociceptive action of 3 nmol endomorphin-1 injected intrathecally. The present study demonstrated that Compound 24 is a pure, competitive, and highly potent non-peptide NOP receptor selective antagonist. [Copyright &y& Elsevier]

Abstract: ZP120 is a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In previous studies, the effects of ZP120 were found to be sensitive to J-113397 in mouse tissues while resistant to UFP-101 in rat tissues. The aim of this study was to further investigate the ZP120 pharmacological profile using mouse and rat preparations, J-113397 and UFP-101, as well as NOP receptor knockout (NOP−/−) mice. Electrically stimulated mouse and rat vas deferens were used to characterize the pharmacology of ZP120 in vitro. For in vivo studies the tail-withdrawal assay was performed in wild type (NOP+/+) and NOP knockout (NOP−/−) mice. In the mouse and rat vas deferens ZP120 mimicked the effects of N/OFQ showing higher potency but lower maximal effects. In both preparations, J-113397 antagonized N/OFQ and ZP120 effects showing similar pK B values (≈7.8). UFP-101 antagonized the actions of N/OFQ (pK B values ≈7.3) but did not modify the effects of ZP120. The inhibitory effects of N/OFQ and ZP120 were no longer evident in vas deferens tissues taken from NOP−/− mice. In NOP+/+ mice subjected to the tail-withdrawal assay, ZP120 (1nmol) mimicked the pronociceptive action of N/OFQ (10nmol), producing longer lasting effects. The effects of both peptides were absent in NOP−/− animals. The NOP receptor ligand ZP120 is a high potency NOP selective partial agonist able to evoke long-lasting effects; its diverse antagonist sensitivity in comparison with N/OFQ may derive from different modality of binding to the NOP receptor. [Copyright &y& Elsevier]

Abstract: Nociceptin/orphanin FQ (N/OFQ) and nocistatin are two neuropeptides originated from the same precursor prepronociceptin/orphanin FQ (ppN/OFQ). N/OFQ is the endogenous ligand of the NOP receptor, while the target of action of nocistatin is still unknown. N/OFQ modulates various biological functions, including anxiety. Conversely, nocistatin either behaves as a functional N/OFQ antagonist or evokes per se effects opposite to those of N/OFQ. Here we investigated the interaction between the anxiolytic-like effects of N/OFQ and the anxiogenic-like action of nocistatin with those evoked by GABAA receptor ligands in the mouse elevated plus maze. The anxiogenic-like effects of the GABAA receptor antagonist pentylenetetrazol (20mg/kg; intraperitoneal, i.p.) were abolished by the co-treatment with N/OFQ (10pmol; intracerebroventricular, i.c.v.) while potentiated by the administration of nocistatin (0.01pmol; i.c.v.). The anxiolytic-like effects of the benzodiazepine receptor agonist diazepam (0.75mg/kg, i.p.) were reversed by nocistatin (0.1pmol; i.c.v.), whereas signs of sedation were observed when mice were co-treated with diazepam and N/OFQ (3pmol). Interesting enough, the i.p. treatment with flumazenil (1mg/kg) blocked the anxiolytic-like effects of N/OFQ (10pmol; i.c.v.), but not the anxiogenic effect elicited by nocistatin. Collectively, our findings suggest that the effects on anxiety elicited by pentylenetetrazol and diazepam can be counteracted or potentiated in the presence of N/OFQ and nocistatin. In addition, the effects on anxiety of N/OFQ, but not nocistatin, appear to be dependent on the benzodiazepine site of the GABAA receptor. [Copyright &y& Elsevier]

Abstract: [(pF)Phe4Aib7Arg14Lys15]N/OFQ-NH2 (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC50 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP−/− mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1–100pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP−/− mice. Equi-effective high doses of UFP-112 (0.1nmol) and N/OFQ (10nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16h. N/OFQ produced a clear inhibitory effect which lasted for 60min while UFP-112 elicited longer lasting effects (>6h). In conscious rats, UFP-112 (0.1 and 10nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors. [Copyright &y& Elsevier]

Abstract: Studies showed that nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) agonists produce anxiolytic-like actions, while little is known about the effects of blockade of NOP receptor signaling in anxiety. To this aim, we investigated the behavioral phenotype of NOP receptor gene knockout mice (NOP−/−) in different assays. In the elevated plus-maze and light-dark box, NOP−/− mice displayed increased anxiety-related behavior. In the novelty-suppressed feeding behavior and elevated T-maze, NOP−/− mice showed anxiolytic-like phenotype, while no differences were found in the open-field, hole-board, marble-burying, and stress-induced hyperthermia. Altogether, these findings suggest that the N/OFQ-NOP receptor system modulates anxiety-related behavior in a complex manner. [Copyright &y& Elsevier]

Abstract: To date, J-113397 represents the most potent and selective non peptide NOP receptor antagonist widely used in pharmacological studies. However, the synthesis, purification, and enantiomer separation of this molecule, which contains two chiral centers, is rather difficult and low-yielding. Here, we synthesized and tested a series of simplified J-113397 analogues to investigate the importance of the stereochemistry and the influence of the substituents at position 3 of the piperidine nucleus and on the nitrogen atom of the benzimidazolidinone nucleus. The compound coded as Trap-101, an achiral analogue of J-113397, combines a pharmacological profile similar to that of the parent compound with a practical, high-yielding preparation. [Copyright &y& Elsevier]

Abstract: Nociceptin/orphanin FQ (N/OFQ) has been reported to inhibit neurogenic contractions in various tissues, including guinea pig airways. In the present study, we investigated the ability of N/OFQ to affect cholinergic contractions of human bronchi elicited by electrical field stimulation (EFS). Tissues were obtained from 23 patients undergoing surgery for lung cancer. EFS (20Hz, 320mA, 1.5ms, 10s) was applied five times every 20min. Contractions induced by EFS were abolished by either TTX (1μM) or atropine (1μM) and concentration-dependently (10nM–1μM) inhibited by N/OFQ (E max, 11.5±1.8% inhibition). The inhibitory effects of N/OFQ were mimicked by the N/OFQ receptor (NOP) ligand [Arg14, Lys15]N/OFQ which displayed however, higher significant maximal effects (17.7±2.9% inhibition, P <0.05). The actions of N/OFQ and [Arg14, Lys15]N/OFQ were not affected by naloxone (1μM) while prevented by the selective NOP receptor antagonist UFP-101 (10μM). Moreover, the inhibitory effects of NOP agonists were no longer evident in tissues treated with tertiapin (10μM), an inhibitor of inward-rectifier potassium channels. In conclusion, the present data demonstrate that N/OFQ inhibited acetylcholine (ACh) release in the human bronchi via NOP receptor activation. This effect may involve stimulation of potassium currents. [Copyright &y& Elsevier]

Abstract: Nociceptin/orphanin FQ (N/OFQ) and N/OFQ peptide (NOP) receptors are implicated in many physiological functions including pain regulation. This study quantitatively investigated the interaction of a novel NOP receptor antagonist, UFP-101 ([Nphe1,Arg14,Lys15]N/OFQ-NH2), with N/OFQ in the ventrolateral periaqueductal gray, a crucial midbrain area for pain regulation. N/OFQ concentration-dependently activated G-protein coupled inwardly rectifying K+ (GIRK) channels in ventrolateral neurons of periaqueductal gray slices. UFP-101 antagonized N/OFQ-induced GIRK channel activation in a concentration-dependent manner and produced a parallel shift of the concentration–response curve of N/OFQ. The pA 2 value estimated from Schild plot is 6.92±0.06. At concentrations up to 1 μM, UFP-101 had no effect on membrane current per se and did not affect the GIRK current activated by [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin, a μ-opioid receptor agonist. It is concluded that UFP-101 is a potent and competitive peptide antagonist of NOP receptors that mediate GIRK channel activation in ventrolateral periaqueductal gray neurons. [Copyright &y& Elsevier]