Nociceptin Receptor Agonist Research Articles

Acute single non-sedative doses of NOP receptor agonists affect acquisition of object location memory but repeated high doses do not induce long-lasting deficits

The Nociceptin/Orphanin FQ (N/OFQ) system has been shown to modulate various aspects of long-term memory. It is therefore important to study the effects on memory impairment by nociceptin receptor (NOP) agonists under preclinical development. In the present study, we investigated the effect of systemic injection of two small molecule selective NOP agonists, AT-202 and AT-524, in the object location memory task in male and female mice. Since high doses of NOP agonists have been shown to induce sedation, we first determined the sedative doses for the two compounds and found them to be higher in female than in male mice. We then observed that sub-sedative doses of NOP agonists administered before learning, induced memory impairment during a test session performed 24 h later. Again, female mice were less sensitive to the amnesic effects than males. On the contrary, in male mice, NOP agonists did not produce amnesia when they were injected after learning, suggesting that they do not affect the consolidation of object location memory. Finally, repeated administration of high doses of NOP agonists over 7 days did not impair long-term spatial memory. Together, our data show for the first time that NOP receptor agonists impair the acquisition of object location memory with sex-dependent potency but do not affect memory consolidation, and that repeated stimulation of the receptor does not compromise long-term episodic-like spatial memory.

Differential In Vitro Pharmacological Profiles of Structurally Diverse Nociceptin Receptor Agonists in Activating G Protein and Beta-Arrestin Signaling at the Human Nociceptin Opioid Receptor.

Agonists at the nociceptin opioid peptide receptor (NOP) are under investigation as therapeutics for nonaddicting analgesia, opioid use disorder, Parkinson's disease, and other indications. NOP full and partial agonists have both been of interest, particularly since NOP partial agonists show a reduced propensity for behavioral disruption than NOP full agonists. Here, we investigated the in vitro pharmacological properties of chemically diverse NOP receptor agonists in assays measuring functional activation of the NOP receptor such as guanosine 5'-O-[gamma-thio]triphosphate (GTPγS) binding, cAMP inhibition, G protein-coupled inwardly rectifying potassium (GIRK) channel activation, phosphorylation, β-arrestin recruitment and receptor internalization. When normalized to the efficacy of the natural agonist nociceptin/orphanin FQ (N/OFQ), we found that different functional assays that measure intrinsic activity produce inconsistent levels of agonist efficacy, particularly for ligands that were partial agonists. Agonist efficacy obtained in the GTPγS assay tended to be lower than that in the cAMP and GIRK assays. These structurally diverse NOP agonists also showed differential receptor phosphorylation profiles at the phosphosites we examined and induced varying levels of receptor internalization. Interestingly, although the rank order for β-arrestin recruitment by these NOP agonists was consistent with their ability to induce receptor internalization, their phosphorylation signatures at the time point we investigated were not indicative of the levels of β-arrestin recruitment or internalization induced by these agonists. It is possible that other phosphorylation sites, yet to be identified, drive the recruitment of NOP receptor ensembles and subsequent receptor trafficking by some nonpeptide NOP agonists. These findings potentially help understand NOP agonist pharmacology in the context of ligand-activated receptor trafficking. SIGNIFICANCE STATEMENT: Chemically diverse agonist ligands at the nociceptin opioid receptor G protein-coupled receptor showed differential efficacy for activating downstream events after receptor binding, in a suite of functional assays measuring guanosine 5'-O-[gamma-thio]triphosphate binding, cAMP inhibition, G protein-coupled inwardly rectifying protein channel activation, β-arrestin recruitment, receptor internalization and receptor phosphorylation. These analyses provide a context for understanding nociceptin opioid peptide receptor (NOP) agonist pharmacology driven by ligand-induced differential NOP receptor signaling.

Cebranopadol: A First-in-Class Nociceptin Receptor Agonist for Managing Chronic Pain.

Cebranopadol: A First-in-Class Nociceptin Receptor Agonist for Managing Chronic Pain.

Patent Highlights December 2018–January 2019

Patent Highlights December 2018–January 2019

Buprenorphine C7‐Esters with Improved Nociceptin Receptor Agonist Potency as Analgesics

There is an urgent need for analgesics with reduced abuse liability, withdrawal, respiratory depression and constipation to treat chronic pain patients. While clinical opiates produce analgesia and associated side effects through Mu Opioid Receptor (MOR) agonist activity, preclinical evidence indicates activity at the Kappa‐ (KOR), Delta‐ (DOR), and Nociception Receptors (NOP) subtypes can minimize side effects. Compounds displaying NOP agonist and weak MOR partial agonist activity produce antinociception with minimal abuse liability in primates and therefore are desirable therapeutic target. In the present study we evaluated buprenorphine analogues with C7 ester substituents for opioid receptor affinity by competition binding assay and for function using the [35S]‐GTPgS assay at MOR, NOP, DOR and KOR. Compounds showed a nearly 1000‐fold range in binding and [35S]‐GTPgS activity at NOP, ranging from low nanomolar to micromolar. One compound, containing a phenyl ester at C7, and lacking a keto group at C6, showed the highest potency NOP activity (EC50= 0.97 +/− 0.22 nM, EMAX= 47% +/− 11) with weak partial agonist potency at MOR (EC50= 3.9 +/− 1.3 nM, EMAX= 23% +/− 7). Since this compound has less MOR agonist efficacy than buprenorphine, it would be predicted to have minimal abuse liability. The series also showed a synergistic relationship in NOP activity between the C7 functional group and the C6 oxidation state. For example, the presence of a keto group at C6 decreased NOP potency significantly (EC50= 164 +/− 51 nM, EMAX= 47% +/− 9). This pattern was observed with numerous C7 functional groups. All compounds in the series were putative antagonists at DOR and KOR as they bound with high affinity but did stimulate significant activity in the [35S]‐GTPgS assay. This series of compounds provides different degrees of partial agonism at MOR and NOP and should help to determine the minimal MOR activity necessary for effective antinociception while minimizing potential abuse liability.Support or Funding InformationSupported by R01 DA07315 and 035316.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Nociceptin Receptor (NOP) Agonist AT-312 Blocks Acquisition of Morphine- and Cocaine-Induced Conditioned Place Preference in Mice.

Treatment of drug addiction remains an unmet medical need due to the dearth of approved pharmacotherapies. There are no approved treatments for cocaine addiction, whereas the current opioid crisis has revealed the stark reality of the limited options to treat prescription and illicit opioid abuse. Preclinical studies in rodents and nonhuman primates have shown that orphanin FQ/nociceptin (N/OFQ), the endogenous ligand for the nociceptin opioid receptor (NOP) reduces the rewarding effects of several abused substances, including opioids, psychostimulants and alcohol. A few nonpeptide small-molecule NOP agonists have also shown efficacy in attenuating the rewarding effects of various abused drugs. We previously demonstrated that a high affinity small-molecule NOP agonist AT-312 selectively reduced the rewarding effects of ethanol in the conditioned place preference paradigm in mice. In the present study, we examined if AT-312 (3 mg/kg, i.p. or s.c. respectively), would alter the rewarding action of morphine (7.5 mg/kg, s.c.) or cocaine (15 mg/kg, i.p.). The effect of AT-312 on morphine- and cocaine-induced motor stimulation was also assessed on the conditioning days. The role of the NOP receptor in the effects of AT-312 was further confirmed by conducting the place conditioning experiments in NOP knockout mice and compared to their wild-type controls. Our results showed that AT-312 significantly reduced the acquisition of morphine and cocaine CPP in wild-type mice but not in mice lacking NOP receptors. AT-312 also suppressed morphine-induced and completely abolished cocaine-induced motor stimulation in NOP wild-type mice, but not in NOP knockout mice. These results show that small-molecule NOP receptor agonists have promising efficacy for attenuating the rewarding effects of morphine and cocaine, and may have potential as pharmacotherapy for opioid and psychostimulant addiction or for treating polydrug addiction.

Nociceptin and the NOP receptor in aversive learning in mice

The endogenous neuropeptide nociceptin (N/OFQ), which mediates its actions via the nociceptin receptor (NOP), is implicated in multiple behavioural and physiological functions. This study examined the effects of the NOP agonists N/OFQ and the synthetic agonist Ro 64–6198, the antagonists NNN and NalBzoH, as well as deletion of the Pronociceptin gene on emotional memory in mice. The animals were tested in the passive avoidance (PA) task, dependent on hippocampal and amygdala functions. N/OFQ injected intraventricularly (i.c.v.) prior to training produced a biphasic effect on PA retention; facilitation at a low dose and impairment at higher doses. Ro 64–6198 also displayed a biphasic effect with memory facilitation at lower doses and impairment at a high dose. None of the agonists influenced PA training latencies. NNN did not significantly modulate retention in the PA task but antagonized the inhibitory effects of N/OFQ. NalBzoH facilitated memory retention in a dose-dependent manner and blocked the impairing effects of N/OFQ. However, neither NNN nor NalBzoH blocked the memory-impairing effects of Ro 64–6198. Finally, the Pnoc knockout mice exhibited enhanced PA retention latencies compared to the wild type mice. The biphasic effect of the natural ligand and Ro 64–6198 and the failure of the antagonists to block the action of Ro 64–6198 indicate complexity in ligand-receptor interaction. These results indicate that brain nociceptin and its NOP has a subtle role in regulation of mechanisms of relevance for treatment of disorders with processing disturbances of aversive events e.g. Alzheimer's disease, anxiety, depression and PTSD.

Bifunctional opioid/nociceptin hybrid KGNOP1 effectively attenuates pain-related behaviour in a rat model of neuropathy

A bifunctional peptide containing an opioid and nociceptin receptor-binding pharmacophore, H-Dmt-D-Arg-Aba-β-Ala-Arg-Tyr-Tyr-Arg-Ile-Lys-NH2 (KGNOP1), was tested for its analgesic properties when administered intrathecally in naïve and chronic constriction injury (CCI)-exposed rats with neuropathy-like symptoms. KGNOP1 significantly increased the acute pain threshold, as measured by the tail-flick test, and also increased the threshold of a painful reaction to mechanical and thermal stimuli in CCI-exposed rats. Both of the effects could be blocked by pre-administration of [Nphe1]-Nociceptin (1-13)-NH2 (NPhe) or naloxone, antagonists for nociceptin and opioid receptors, respectively. This led us to conclude that KGNOP1 acts as a dual opioid and nociceptin receptor agonist in vivo. The analgesic effect of KGNOP1 proved to be more powerful than clinical drugs such as morphine and buprenorphine. Repeated daily intrathecal injections of KGNOP1 led to the development of analgesic tolerance, with the antiallodynic action being completely abolished on day 6. Nevertheless, the development of tolerance to the antihyperalgesic effect was delayed in comparison to morphine, which lost its efficacy as measured by the cold plate test after 3days of daily intrathecal administration, whereas KGNOP1 was efficient up to day 6. A single intrathecal injection of morphine to KGNOP1-tolerant rats did not raise the pain threshold in any of the behavioural tests; in contrast, a single intrathecal dose of KGNOP1 significantly suppressed allodynia and hyperalgesia in morphine-tolerant rats.

Nociceptin receptor activation does not alter acquisition, expression, extinction and reinstatement of conditioned cocaine preference in mice

Growing evidence indicates that targeting nociceptin receptor (NOP) signaling may have therapeutic efficacy in treating alcohol and opioid addiction. However, little is known about the therapeutic value of selective NOP agonists for the treatment of cocaine dependence. Recently, we identified a highly selective, brain-penetrant NOP small molecule agonist (SR-8993), and using this compound, we previously showed that nociceptin receptor activation attenuated consolidation of fear-related memories. Here, we sought to determine whether SR-8993 also affects the rewarding properties of cocaine. Using a conditioned place preference (CPP) procedure, we show that SR-8993 (3 or 10mg/kg) failed to disrupt acquisition or expression of cocaine CPP (7.5 or 15mg/kg) in C57BL/6 mice. Additionally, SR-8993 did not affect rate of extinction or reinstatement (yohimbine- and cocaine-induced) of cocaine CPP. These studies indicate that selective activation of NOP may not be sufficient in reducing behavioral responses to cocaine.

Structure-Based Virtual Screening of the Nociceptin Receptor: Hybrid Docking and Shape-Based Approaches for Improved Hit Identification

The antagonist-bound crystal structure of the nociceptin receptor (NOP), from the opioid receptor family, was recently reported along with those of the other opioid receptors bound to opioid antagonists. We recently reported the first homology model of the ‘active-state’ of the NOP receptor, which when docked with ‘agonist’ ligands showed differences in the TM helices and residues, consistent with GPCR activation after agonist binding. In this study, we explored the use of the active-state NOP homology model for structure-based virtual screening to discover NOP ligands containing new chemical scaffolds. Several NOP agonist and antagonist ligands previously reported are based on a common piperidine scaffold. Given the structure–activity relationships for known NOP ligands, we developed a hybrid method that combines a structure-based and ligand-based approach, utilizing the active-state NOP receptor as well as the pharmacophoric features of known NOP ligands, to identify novel NOP binding scaffolds by virtual screening. Multiple conformations of the NOP active site including the flexible second extracellular loop (EL2) loop were generated by simulated annealing and ranked using enrichment factor (EF) analysis and a ligand–decoy dataset containing known NOP agonist ligands. The enrichment factors were further improved by combining shape-based screening of this ligand–decoy dataset and calculation of consensus scores. This combined structure-based and ligand-based EF analysis yielded higher enrichment factors than the individual methods, suggesting the effectiveness of the hybrid approach. Virtual screening of the CNS Permeable subset of the ZINC database was carried out using the above-mentioned hybrid approach in a tiered fashion utilizing a ligand pharmacophore-based filtering step, followed by structure-based virtual screening using the refined NOP active-state models from the enrichment analysis. Determination of the NOP receptor binding affinity of a selected set of top-scoring hits resulted in identification of several compounds with measurable binding affinity at the NOP receptor, one of which had a new chemotype for NOP receptor binding. The hybrid ligand-based and structure-based methodology demonstrates an effective approach for virtual screening that leverages existing SAR and receptor structure information for identifying novel hits for NOP receptor binding. The refined active-state NOP homology models obtained from the enrichment studies can be further used for structure-based optimization of these new chemotypes to obtain potent and selective NOP receptor ligands for therapeutic development.

Activation of the endogenous nociceptin system by selective nociceptin receptor agonist SCH 221510 produces antitransit and antinociceptive effect: a novel strategy for treatment of diarrhea-predominant IBS.

Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional gastrointestinal (GI) disorder, defined by the presence of loose stools and abdominal pain. In search for a novel anti-IBS-D therapy, here we investigated the nociceptin receptor (NOP)-dependent effects in the GI tract. A novel potent and selective NOP agonist SCH 221510 was used in the study. The effect of NOP activation on mouse intestinal motility was characterized in vitro and in vivo, in physiological conditions and in animal models of hypermotility and diarrhea. Well-established mouse models of visceral pain were used to characterize the antinociceptive effect of the NOP activation. To provide additional evidence that the endogenous nociceptin system is a relevant target for IBS, NOP expression and nociceptin levels were quantified in serum and colonic biopsies from IBS-D patients. SCH 221510 produced a potent NOP-mediated inhibitory effect on mouse intestinal motility in vitro and in vivo in physiological conditions. The NOP agonist displayed an antidiarrheal and analgesic action after oral administration in animal models mimicking the symptoms of IBS-D. Studies on human samples revealed a strong decrease in endogenous nociceptin system expression in IBS-D patients compared with healthy controls. Collectively, mouse and human data suggest that the endogenous nociceptin system is involved in IBS-D and may become a target for anti-IBS-D treatments using potent and selective synthetic NOP agonists.

Intrathecal nociceptin displaces a PET ligand for its receptor in the brain at analgesic doses in rhesus monkeys (658.1)

The nociceptin receptor (NOP) has been investigated as a potential therapeutic target for pain control. Nociceptin, the endogenous ligand for NOP, produces analgesia in rodents and primates when given intrathecally (i.t.) (Ko et al. 2006, Xu et al. 1996). Recently NOP radiotracers have been developed for use with Positron Emission Tomography (PET) (Kimura et al. 2011, Hostetler et al. 2013). Although behavioral studies have shown that NOP agonists may be therapeutically useful, it is unknown whether nociceptin given i.t. binds to receptors supraspinally, and if so, which areas in the brain display the greatest ligand‐target interaction. The purpose of this study was to measure the amount of radiotracer displacement in the brain at NOP following an analgesic dose of nociceptin. Two female rhesus monkeys were anesthetized, and underwent PET scans with the NOP radiotracer 11C NOP‐1A. Eight minutes following tracer delivery, the monkey was injected i.t. with either saline or 100 nM nociceptin. Preliminary data show that 100 nM nociceptin delivered into the spinal canal displaces the radiotracer 11C NOP‐1A in a variety of brain nuclei. Prominent regions of displacement include the cortex, striatum, and thalamus. These data suggest that analgesia with NOP agonists can be produced with relatively low receptor occupancy and further supports NOP as a novel drug target for analgesia.Grant Funding Source: Supported by: The Michigan Memorial Pheonix Project and NIDA Traning Grant T32‐DA007281

Anti-Inflammatory and Antinociceptive Action of an Orally Available Nociceptin Receptor Agonist SCH 221510 in a Mouse Model of Inflammatory Bowel Diseases

The nociceptin receptors (NOPs) are expressed in the gastrointestinal (GI) tract on muscle cell membranes and neurons, as well as the immune cells that infiltrate the mucosa. The involvement of NOPs in the pathophysiology of GI inflammation has been suggested, but due to the lack of selective NOP agonists, it never fully elucidated. Our aim was to characterize the anti-inflammatory and antinociceptive effect of the NOP agonist, SCH 221510 [3-endo-8-[bis(2-methylphenyl)methyl]-3-phenyl-8-azabicyclo [3.2.1]octan-3-ol], as a potential therapeutic strategy in the treatment of inflammatory bowel diseases (IBD). The anti-inflammatory action of SCH 221510 was determined after intraperitoneal, oral, and intracolonic administration of SCH 221510 (0.1-3.0 mg/kg once or twice daily) in mice treated with 2,4,6-trinitrobenzenesulfonic acid (TNBS). Antinociceptive action of SCH 221510 was evaluated in the mouse model of mustard oil (MO)-induced abdominal pain. Relative NOP mRNA expression was assessed in patients with IBD using real-time reverse transcriptase-polymerase chain reaction. We found that the expression of NOP mRNA was significantly decreased in patients with IBD. The administration (0.1 and 1.0 mg/kg i.p. twice daily and 3 mg/kg p.o. twice daily) of SCH 221510 attenuated TNBS colitis in mice. This effect was blocked by a selective NOP antagonist [J-113397 [(±)-1-[(3R*,4R*)-1-(cyclooctylmethyl)-3-(hydroxymethyl)-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one]]. The intracolonic injections of SCH 221510 did not improve colitis in mice. The antinociceptive effect of SCH 221510 was observed after oral administration of SCH 221510 in MO-induced pain tests in mice with acute colitis. In conclusion, our results show a potent anti-inflammatory and antinociceptive effect upon selective activation of NOP receptors and suggest that the NOP agonist SCH 221510 is a promising drug candidate for future treatment of IBD.

Effects of the γ-secretase inhibitor semagacestat on hippocampal neuronal network oscillation

Neurological and psychiatric disorders are frequently associated with disruption of various cognitive functions, but development of effective drug treatments for these conditions has proven challenging. One of the main obstacles is the poor predictive validity of our preclinical animal models. In the present study the effects of the γ-secretase inhibitor semagacestat was evaluated in preclinical in vivo electrophysiological models. Recently disclosed Phase III findings on semagacestat indicated that Alzheimer’s disease (AD) patients on this drug showed significantly worsened cognitive function compared to those treated with placebo. Since previous studies have shown that drugs impairing cognitive function (including scopolamine, NMDA (N-methyl-D-aspartate) receptor antagonists, and nociceptin receptor agonists) disrupt or decrease power of elicited theta oscillation in the hippocampus, we tested the effects of acute and sub-chronic administration of semagacestat in this assay. Field potentials were recorded across the hippocampal formation with NeuroNexus multi-site silicon probes in urethane anesthetized male C57BL/6 mice; hippocampal CA1 theta oscillation was elicited by electrical stimulation of the brainstem nucleus pontis oralis. Sub-chronic administration of semagacestat twice daily over 12 days at a dose known to reduce beta-amyloid peptide (Aβ) level [100 mg/kg, p.o. (per oral)] diminished power of elicited hippocampal theta oscillation. Acute, subcutaneous administration of semagacestat (100 mg/kg) produced a similar effect on hippocampal activity. We propose that the disruptive effect of semagacestat on hippocampal function could be one of the contributing mechanisms to its worsening of cognition in patients with AD. As it has been expected, both acute and sub-chronic administrations of semagacestat significantly decreased Aβ40 and Aβ42 levels but the current findings do not reveal the mode of action of semagacestat in disrupting hippocampal oscillignificantly reduced braination.

Targeting Nociceptin Receptor to Treat Pain in Sickle Cell Disease

AbstractAbstract 373Morphine and its congeners remain a sub-optimal approach to treat pain in sickle cell disease (SCD) due to side effects, risk of addiction, and development of tolerance. To avoid these liabilities associated with use of morphine and mu opioid receptor (MOR) agonist analgesics, we examined the ability of nociceptin receptor (NOPR) ligands to treat pain in SCD. The NOPR and its endogenous ligand nociceptin/orphanin FQ (N/OFQ) belonging to the opioid receptor (OR) and opioid family respectively, are involved in nociceptive signaling. Small molecule NOPR agonists are non-addicting, while providing analgesia in acute and chronic pain models. We used AT-200 ([1-(1-cyclooctylpiperidin-4-yl)-indolin-2-one), which has high binding affinity and agonist efficacy at NOPR and low efficacy, partial agonist activity at MOR, to treat chronic and hypoxia/reoxygenation (H/R)-evoked pain, in sickle HbSS-BERK and control HbAA-BERK mice, expressing sickle and normal human hemoglobin, respectively. AT-200 was injected subcutaneously at a dose of 10 mg/Kg. Equal number of mice were treated with vehicle in parallel. Pain behaviors were analyzed over a period of time in the following paradigms [i] under normoxia after a single dose of AT-200, [ii] after injecting AT-200 following the incitement of H/R, and [iii] AT-200 given everyday for 7 days. For comparison, we used 20 mg/Kg morphine treatment following H/R. Pain measures that were characterized included deep tissue pain by grip force measurement, cutaneous hyperalgesia by mechanical sensitivity to von Frey filaments and thermal sensitivity to heat and cold as described by us for sickle mice (Kohli et al., Blood 2010). Blood flow and dorsal skin temperature were measured using laser doppler velocimetry and infrared thermography, respectively. The measure of mechanical threshold and suprathreshold to a 1.0 g von Frey fiber showed a significant reduction in mechanical sensitivity even 24h after AT-200 treatment as compared to vehicle under normoxia (p<0.05 for threshold and p<0.005 for suprathreshold) in sickle mice. H/R evoked a persistent increase in mechanical hyperalgesia in sickle mice up to 24h and in control mice up to 4h, and both were ameliorated by AT-200. Morphine reduced mechanical hyperalgesia following H/R in sickle mice up to 4h but not 24h and its effect was significantly less than that of AT-200. H/R-incitement increased deep pain for 24h in sickle mice and for 30 min in control mice and AT-200 ameliorated both. Interestingly, morphine significantly blocked deep pain at 30 min following H/R (p<0.005), but remained ineffective thereafter in sickle mice. Since deep pain appears is a characteristic feature of vasoocclusive pain in sickle mice as compared to cutaneous pain, it is likely that AT-200 acts as a superior analgesic with a long-lasting effect to treat sickle-specific vasoocclusive pain, compared to morphine. Heat and cold sensitivity were persistently elevated in sickle mice up to 24h post-H/R but returned to baseline (before H/R) in control mice. Both, AT-200 and morphine inhibited the H/R-induced thermal sensitivity up to 24h, in sickle mice, but the effect of AT-200 was significantly more remarkable than morphine. Blood flow and skin temperature increased significantly following H/R in sickle mice (p<0.05 for both vs baseline before H/R) and were significantly reduced by AT-200 and morphine equally, suggesting that both drugs have a similar acute effect on vascular physiology and inflammation in sickle mice. The enhanced analgesic effectiveness of AT-200 compared to morphine, in sickle mice could be due to a ∼2-fold increase in NOPR expression in the dorsal root ganglion of sickle mice compared to control, observed by qPCR in this study. Chronic pain conditions, including pain in sickle mice, are characterized by reduced MOR expression peripherally and in the spinal cord. However, spinal NOPR expression by qPCR did not show a significant difference between sickle and control mice. AT-200 was effective in treating chronic cutaneous, deep tissue and thermal hyperalgesia even after 7 days of continuous treatment without causing tolerance. Thus, AT-200 provided an analgesic effect in chronic pain and that evoked by H/R, without the development of analgesic tolerance in sickle mice. Targeting NOPR appears to be a promising strategy warranting further development of novel NOPR agonists to treat pain in SCD. Disclosures:No relevant conflicts of interest to declare.

Homology modeling and molecular dynamics simulations of the active state of the nociceptin receptor reveal new insights intoagonist binding and activation

The opioid receptor-like receptor, also known as the nociceptin receptor (NOP), is a class A G protein-coupled receptor (GPCR) in the opioid receptor family. Although NOP shares a significant homology with the other opioid receptors, it does not bind known opioid ligands and has been shown to have a distinct mechanism of activation compared to the closely related opioid receptors mu, delta, and kappa. Previously reported homology models of the NOP receptor, based on the inactive-state GPCR crystal structures, give limited information on the activation and selectivity features of this fourth member of the opioid receptor family. We report here the first active-state homology model of the NOP receptor based on the opsin GPCR crystal structure. An inactive-state homology model of NOP was also built using a multiple template approach. Molecular dynamics simulation of the active-state NOP model and comparison to the inactive-state model suggest that NOP activation involves movements of transmembrane (TM)3 and TM6 and several activation microswitches, consistent with GPCR activation. Docking of the selective nonpeptidic NOP agonist ligand Ro 64-6198 into the active-state model reveals active-site residues in NOP that play a role in the high selectivity of this ligand for NOP over the other opioid receptors. Docking the shortest active fragment of endogenous agonist nociceptin/orphaninFQ (residues 1-13) shows that the NOP extracellular loop 2 (EL2) loop interacts with the positively charged residues (8-13) of N/OFQ. Both agonists show extensive polar interactions with residues at the extracellular end of the TM domain and EL2 loop, suggesting agonist-induced reorganization of polar networks, during receptor activation.

The anxiolytic-like profile of the nociceptin receptor agonist, endo-8-[bis(2-chlorophenyl)methyl]-3-phenyl-8-azabicyclo[3.2.1]octane-3-carboxamide (SCH 655842): Comparison of efficacy and side effects across rodent species

The endogenous opioid-like peptide, nociceptin, produces anxiolytic-like effects that are mediated via the nociceptin (NOP) receptor. Similarly, synthetic, non-peptide NOP agonists produce robust anxiolytic-like effects although these effects are limited by marked side effects. In the present studies, the effects of a novel NOP receptor agonist, SCH 655842, were examined in rodent models sensitive to anxiolytic drugs and tests measuring potential adverse affects. Oral administration of SCH 655842 produced robust, anxiolytic-like effects in three species, i.e., rat, guinea pig, and mouse. Specifically, SCH 655842 was effective in rat conditioned lick suppression (3–10 mg/kg) and fear-potentiated startle (3–10 mg/kg) tests, a guinea pig pup vocalization test (1–3 mg/kg), as well as in mouse Geller–Seifter (30 mg/kg) and marble burying (30 mg/kg) tests. The anxiolytic-like effect of SCH 655842 in the conditioned lick suppression test was attenuated by the NOP antagonist, J-113397. In mice, SCH 655842 reduced locomotor activity and body temperature at doses similar to the anxiolytic-like dose and these effects were absent in NOP receptor knockout mice. In rats, SCH 655842 did not produce adverse behavioral effects up to doses of 70–100 mg/kg. Pharmacokinetic studies in the rat confirmed dose-related increases in plasma and brain levels of SCH 655842 across a wide oral dose range. Taken together, SCH 655842 may represent a NOP receptor agonist with improved tolerability compared to other members of this class although further studies are necessary to establish whether this extends to higher species.

Comparison of the Antinociceptive and Antirewarding Profiles of Novel Bifunctional Nociceptin Receptor/μ-Opioid Receptor Ligands: Implications for Therapeutic Applications

The nociceptin receptor (NOPr), a member of the opioid receptor family, is a target for the treatment of pain and drug abuse. Nociceptin/orphanin FQ (N/OFQ), the endogenous peptide for NOPr, not only modulates opioid antinociception, but also blocks the rewarding effects of several abused drugs, such as morphine, cocaine, and amphetamine. We hypothesized that NOPr agonists, with bifunctional activity at the mu-opioid receptor (MOPr), may function as nonaddicting analgesics or as drug abuse medications. Bifunctional small-molecule NOPr agonists possessing different selectivities and efficacies at MOPr were evaluated in an acute thermal antinociception assay, and for their ability to induce conditioned place preference (CPP) and their effect on morphine-induced CPP. 1-(1-Cyclooctylpiperidin-4-yl)-indolin-2-one) (SR14150), a high-affinity NOPr partial agonist, with low MOPr affinity and efficacy, produced analgesia that was naloxone-reversible. SR14150 did not induce CPP alone, nor did it attenuate morphine-induced CPP. 3-Ethyl-1-(1-(4-isopropylcyclohexyl)piperidin-4-yl)-indolin-2-one (SR16507), which has high affinity for both NOPr and MOPr, full agonist activity at NOPr, and partial agonist activity at MOPr, was also a potent analgesic and produced CPP alone, but also modestly attenuated morphine CPP. 1-(1-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)piperidinl-4-yl)-indolin-2-one (SR16835), a NOPr full agonist and low-affinity MOPr partial agonist, was not antinociceptive, did not produce CPP alone, but attenuated morphine CPP. Our results suggest that NOPr full-agonist activity is required to modulate opioid-induced reward, whereas a bifunctional NOPr/MOPr partial agonist profile may be suitable as a nonaddicting analgesic. The opioid-modulating effects of the NOPr ligands may be used effectively to produce better medications for treatment of drug abuse and pain.

P.l.c.052 Paclitaxel induced pain and its character

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the opioid receptor-like receptor or nociceptin receptor (NOP). We have compared a novel non–peptide NOP agonist Ro64-6198 with N/OFQ in a series of GTPγ35S binding and inhibition of forskolin stimulated cAMP formation assays. GTPγ35S binding assays were performed in membranes prepared from Chinese hamster ovary cells expressing the recombinant human NOP (CHOhNOP). cAMP inhibition studies were performed in whole CHOhNOP cells. Both Ro64-6198 and N/OFQ stimulated GTPγ35S binding with pEC50 values(95%CL) of 7.61(0.18) and 8.58(0.21) respectively. Both Ro64-6198 and N/OFQ inhibited cAMP formation with pEC50 values of 8.45(0.09) and 9.28(0.28) respectively. In each assay Ro64-6198 and N/OFQ were full agonists. Ro64-6198 stimulation of GTPγ35S binding and inhibition of cAMP formation was competitively antagonised by the NOP antagonists [Nphe1]NC(1–13)NH2 (10μM), J-113397 (100nM) and III-BTD (1μM) with pKB values of 7.04(0.34) and 6.29(0.10), 8.65(0.34) and 7.90(0.30) and 7.59(0.22) and 7.60(0.22) respectively. Despite the slightly reduced potency of Ro64-6198 compared with N/OFQ, by virtue of high selectivity and relative metabolic stability this molecule will be of considerable use in studies of the actions of the NOP.

Discovery of Orally Active 3-Pyridinyl-tropane As a Potent Nociceptin Receptor Agonist for the Management of Cough

A series of 3-pyridinyl-tropane analogues based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. From the SAR study and our lead optimization efforts, compound 10 was found to possess potent oral antitussive activity in the capsaicin-induced guinea pig model. The rationale for compound selection and the biological profile of the optimized lead (10) are disclosed.