FQ Peptide Research Articles

Safety, Tolerability, and Pharmacokinetics of Single- and Multiple-Ascending Doses of Sunobinop in Healthy Participants.

Sunobinop is an investigational, potent, selective partial agonist at the nociceptin/orphanin FQ peptide receptor in vitro. Three phase 1 studies were conducted to evaluate the safety, tolerability, and pharmacokinetics (PK) of escalating single- and multiple-dose administration of sunobinop in healthy participants. Study 1 was a randomized, double-blind, placebo-controlled, single-ascending dose study. Study 2 was a randomized, double-blind, placebo-controlled, multiple-ascending dose study. Study 3 was a randomized, open-label, single-dose, 4-way crossover study of oral and sublingual sunobinop comparing morning (AM) and bedtime (PM) administration. Seventy participants were included. Systemic exposure (peak plasma concentration [Cmax], area under the plasma concentration-time curve from time 0 to the time of last quantifiable concentration [AUC0-t], and area under the plasma concentration-time curve from time 0 extrapolated to infinity [AUCinf]) of sunobinop was characterized by dose proportionality from 0.6 to 2mg and increased less than proportionally from 3 to 30mg. The PKs of sunobinop were similar, regardless of AM or PM administration, for both the oral and sublingual formulations. The majority of absorbed sunobinop was excreted unchanged in the urine within 8 hours of dosing, thereby showing rapid elimination with no appreciable accumulation following 14 consecutive days of once-daily dosing and suggesting exclusive renal elimination. Most treatment-emergent adverse events (TEAEs) were mild in severity; 1 severe TEAE occurred and all TEAEs resolved by the end of the studies. Sunobinop was generally well-tolerated and safe across the range of doses evaluated and presents a clinical profile suitable for continued development.

Exploring the binding interactions of NOP receptor with designed natural phytochemical-neuropeptide conjugates: an in silico and SPR study

The Nociceptin/orphanin FQ peptide (NOP) receptor is considered a member of the opioid receptor subfamily of G-protein coupled receptors (GPCRs) which has been shown to be present in many parts of the central nervous system (CNS). It plays biologically diverse roles in pain modulation, immune response and in neurodegenerative diseases. In this work, phytochemical conjugates of two known neuropeptides, melanocyte inhibition factor (MiF-1) and mammalian amidated neuropeptide NPFF with pain modulating ability were developed. The binding interactions of those conjugates with NOP receptor was examined as an approach to develop novel natural compounds that can modulate NOP receptor activity. The selected phytochemicals are well-known for their antioxidant abilities and are derived either from natural alkaloids (betanin), polyphenols (gallic acid and sinapic acid) or terpenes (pomolic acid). Each of the phytochemicals selected are antioxidants which may play a role in mitigating diseases. Three conjugates of betanin were designed with each peptide by conjugating each of the three carboxylic acid groups of betanin with the peptides, while all others were mono-conjugates. Our results indicated that the betanin conjugates with both peptides showed strong binding interactions while the pomolate-peptide conjugates showed moderate binding. In general, NPFF and its conjugates showed stronger binding with the receptor. Docking and molecular dynamics studies revealed that binding interactions occurred at the binding pocket encompassing the transmembrane helices TM1, TM3 and TM7 in most cases, with the ligands binding deep within the hydrophobic core. The binding interactions were further confirmed experimentally through SPR analysis, which also showed higher binding with the betanin conjugates. MMGBSA studies indicated that the binding energies of MiF-1 conjugates were higher compared to neat MiF-1. However, in the case of NPFF, while the betanin conjugates showed enhancement, in some cases the binding energies were found to be slightly reduced compared to neat NPFF. Overall our studies reveal that such natural phytochemical derivatives that can bind to the NOP receptor when conjugated to the mammalian amidated neuropeptide NPFF and the short sequence of melanocyte inhibiting factor MiF-1 may be potentially developed for further laboratory studies for potential pharmaceutical applications.

Expression heterogeneity, tumor immune characteristics and the prognosis effects of OPRL1 in patients with tumors: a pan-cancer study combined with bioinformation analyses and in vitro validation

PurposeOpioids are currently the most frequently prescribed analgesics in clinical practice. However, their effect on cancer progression remains a topic of debate. Opioid receptors (ORs) are present in various types of tumor cells and their expression levels vary depending on the type of tumor. This study aims to explore and preliminarily characterize the association between four different ORs (μ, δ, κ, and nociception/orphanin FQ peptide receptor) and the prognosis of different types of tumors for comparison, with a focus on nociception/ orphanin FQ peptide receptor.MethodsThe expression levels of four ORs in normal tissues and immune cells were obtained from Human Protein Atlas (HPA) RNA-seq dataset, Monaco dataset, and Consensus dataset. Pan-cancer analysis was performed using the The Cancer Genome Atlas (TCGA) dataset, which included the expression of four ORs in different cancer types, significant copy-number alterations (sCNA), gene mutations of the four ORs, survival analysis, co-expression genes analysis, functional enrichment analyses, and correlations between ORs and immune cell infiltration levels. Based on the results of bioinformatic analysis, we selected 10 cancer cell lines for validation in vitro using specific agonists for the four ORs.ResultsOPRL1 (opioid related nociceptin receptor 1 gene) exhibited the highest abundance across different types of cancers, while OPRM1 (opioid receptor mu 1 gene) and OPRD1 (opioid receptor delta 1 gene) were barely detectable in multiple cancer types. Pan-cancer survival analysis revealed the overall worse/better prognosis of the four ORs in certain cancer types. Elevated levels of OPRM1 appear to be associated with poorer outcomes in breast invasive carcinoma and kidney renal clear cell carcinoma. Elevated OPRD1 levels are connected to worsen outcomes in kidney renal clear cell carcinoma and liver hepatocellular carcinoma, but better prognosis in bladder urothelial carcinoma. Increased OPRK1 (opioid receptor kappa 1 gene) expression is linked to a poorer prognosis in kidney renal papillary cell carcinoma. Furthermore, high OPRL1 expression relates to worse outcomes in bladder urothelial carcinoma and liver hepatocellular carcinoma, but better outcomes in breast invasive carcinoma and pancreatic adenocarcinoma. Functional enrichment analyses found that immune-related pathways were enriched in OPRK1 and OPRL1, with OPRL1 exhibiting the highest correlation with immune cell infiltration. Different effects on cell growth, migration, and invasion were observed in different cancer types upon the administration of agonists for the four ORs.ConclusionOPRL1 may play a vital role in monocytes and regulating the immune response and tumor-infiltrating macrophages. Due to its high abundance in different types of tumors, it may hold greater clinical significance for oncology patients. OPRK1 also participates in immune-related pathways. OPRL1 could potentially serve as therapeutic targets for different types of cancers.Graphical

The Nociceptin/Orphanin FQ peptide receptor antagonist, SB-612111, improves cerebral blood flow in a rat model of traumatic brain injury.

Traumatic brain injury (TBI) affects more than 2.5 million people in the U.S. each year and is the leading cause of death and disability in children and adults ages 1 to 44. Approximately 90% of TBI cases are classified as mild but may still lead to acute detrimental effects such as impaired cerebral blood flow (CBF) that result in prolonged impacts on brain function and quality of life in up to 15% of patients. We previously reported that nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor antagonism reversed mild blast TBI-induced vestibulomotor deficits and prevented hypoxia. To explore mechanisms by which the NOP receptor-N/OFQ pathway modulates hypoxia and other TBI sequelae, the ability of the NOP antagonist, SB-612111 (SB), to reverse TBI-induced CBF and associated injury marker changes were tested in this study. Male Wistar rats randomly received sham craniotomy or craniotomy + TBI via controlled cortical impact. Injury severity was assessed after 1h (modified neurological severity score (mNSS). Changes in CBF were assessed 2h post-injury above the exposed cortex using laser speckle contrast imaging in response to the direct application of increasing concentrations of vehicle or SB (1, 10, and 100µM) to the brain surface. TBI increased mNSS scores compared to baseline and confirmed mild TBI (mTBI) severity. CBF was significantly impaired on the ipsilateral side of the brain following mTBI, compared to contralateral side and to sham rats. SB dose-dependently improved CBF on the ipsilateral side after mTBI compared to SB effects on the respective ipsilateral side of sham rats but had no effect on contralateral CBF or in uninjured rats. N/OFQ levels increased in the cerebral spinal fluid (CSF) following mTBI, which correlated with the percent decrease in ipsilateral CBF. TBI also activated ERK and cofilin within 3h post-TBI; ERK activation correlated with increased CSF N/OFQ. In conclusion, this study reveals a significant contribution of the N/OFQ-NOP receptor system to TBI-induced dysregulation of cerebral vasculature and suggests that the NOP receptor should be considered as a potential therapeutic target for TBI.

Recovery from Traumatic Brain Injury Is Nociceptin/Orphanin FQ Peptide Receptor Genotype-, Sex-, and Injury Severity-Dependent.

Traumatic brain injury (TBI) is a leading cause of death and disability in the United States, and survivors often experience mental and physical health consequences that reduce quality of life. We previously reported that blockade of the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor reduced tissue damage markers produced by blast TBI. The goal of this study was to determine the extent to which N/OFQ and NOP receptor levels change following mild (mTBI) and moderate TBI (modTBI) and whether the absence of the NOP receptor attenuates TBI-induced sequelae. Male and female NOP receptor knockout (KO) or wild-type (WT) rats received craniotomy-only (sham) or craniotomy plus mTBI, or modTBI impact to the left cerebral hemisphere. Neurologic and vestibulomotor deficits and nociceptive hyperalgesia and allodynia found in WT male and female rats following mTBI and modTBI were greatly reduced or absent in NOP receptor KO rats. NOP receptor levels increased in brain tissue from injured males but remained unchanged in females. Neurofilament light chain (NF-L) and glial fibrillary acidic protein (GFAP) expression were reduced in NOP receptor KO rats compared with WT following TBI. Levels of N/OFQ in injured brain tissue correlated with neurobehavioral outcomes and GFAP in WT males, but not with KO male or WT and KO female rats. This study reveals a significant contribution of the N/OFQ-NOP receptor system to TBI-induced deficits and suggests that the NOP receptor should be regarded as a potential therapeutic target for TBI. SIGNIFICANCE STATEMENT: This study revealed that nociceptin/orphanin FQ peptide (NOP) receptor knockout animals experienced fewer traumatic brain injury (TBI)-induced deficits than their wild-type counterparts in a sex- and injury severity-dependent manner, suggesting that NOP receptor antagonists may be a potential therapy for TBI.

BTRX-246040 Acts Through the Ventrolateral Periaqueductal Gray to Exert Antidepressant-Relevant Actions in Mice.

BTRX-246040, a nociceptin/orphanin FQ peptide receptor antagonist, is being developed for the treatment of depressive patients. However, the underlying mechanism of this potential antidepressant is still largely unclear. Here, we studied the antidepressant-related actions of BTRX-246040 in the ventrolateral periaqueductal gray (vlPAG). The tail suspension test, forced swim test, female urine sniffing test, sucrose preference test, and learned helplessness (LH) combined with pharmacological approaches were employed to examine the antidepressant-like effects and drug effects on LH-induced depressive-like behavior in C57BL/6J mice. Electrophysiological recordings in vlPAG neurons were used to study synaptic activity. Intraperitoneal administration of BTRX-246040 produced antidepressant-like behavioral effects in a dose-dependent manner. Systemic BTRX-246040 (10 mg/kg) resulted in an increased frequency and amplitude of miniature excitatory postsynaptic currents (EPSCs) in the vlPAG. Moreover, slice perfusion of BTRX-246040 directly elevated the frequency and amplitude of miniature EPSCs and enhanced the evoked EPSCs in the vlPAG, which were blocked by pretreatment with the nociceptin/orphanin FQ peptide receptor agonist Ro 64-6198. In addition, intra-vlPAG application of BTRX-246040 produced antidepressant-like behavioral effects in a dose-dependent manner. Moreover, intra-vlPAG pretreatment with 6-cyano-7-nitroquinoxaline-2,3-dione reversed both systemic and local BTRX-246040-mediated antidepressant-like behavioral effects. Furthermore, both systemic and local BTRX-246040 decreased the LH phenotype and reduced LH-induced depressive-like behavior. The results suggested that BTRX-246040 may act through the vlPAG to exert antidepressant-relevant actions. The present study provides new insight into a vlPAG-dependent mechanism underlying the antidepressant-like actions of BTRX-246040.

Nociceptin Receptor-Related Agonists as Safe and Non-addictive Analgesics.

As clinical use of currently available opioid analgesics is often impeded by dose-limiting adverse effects, such as abuse liability and respiratory depression, new approaches have been pursued to develop safe, effective, and non-addictive pain medications. After the identification of the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor more than 25 years ago, NOP receptor-related agonists have emerged as a promising target for developing novel and effective opioids that modulate the analgesic and addictive properties of mu-opioid peptide (MOP) receptor agonists. In this review, we highlight the effects of the NOP receptor-related agonists compared with those of MOP receptor agonists in experimental rodent and more translational non-human primate (NHP) models and the development status of key NOP receptor-related agonists as potential safe and non-addictive analgesics. Several lines of evidence demonstrated that peptidic and non-peptidic NOP receptor agonists produce potent analgesic effects by intrathecal delivery in NHPs. Moreover, mixed NOP/MOP receptor partial agonists (e.g., BU08028, BU10038, and AT-121) display potent analgesic effects when administered intrathecally or systemically, without eliciting adverse effects, such as respiratory depression, itch behavior, and signs of abuse liability. More importantly, cebranopadol, a mixed NOP/opioid receptor agonist with full efficacy at NOP and MOP receptors, produces robust analgesic efficacy with reduced adverse effects, conferring promising outcomes in clinical studies. A balanced coactivation of NOP and MOP receptors is a strategy that warrants further exploration and refinement for the development of novel analgesics with a safer and effective profile.

Nociceptin/Orphanin FQ Opioid Peptide-Receptor Expression in the Endometriosis-Associated Nerve Fibers-Possible Treatment Option?

Endometriosis (EM) is a chronic inflammatory disease affecting millions of women worldwide. Chronic pelvic pain is one of the main problems of this condition, leading to quality-of-life impairment. Currently, available treatment options are not able to treat these women accurately. A better understanding of the pain mechanisms would be beneficial to integrate additional therapeutic management strategies, especially specific analgesic options. To understand pain in more detail, nociceptin/orphanin FQ peptide (NOP) receptor expression was analyzed in EM-associated nerve fibers (NFs) for the first time. Laparoscopically excised peritoneal samples from 94 symptomatic women (73 with EM and 21 controls) were immunohistochemically stained for NOP, protein gene product 9.5 (PGP9.5), substance P (SP), calcitonin gene-related peptide (CGRP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP). Peritoneal NFs of EM patients and healthy controls were positive for NOP and often colocalized with SP-, CGRP-, TH-, and VIP-positive nerve fibers, suggesting that NOP is expressed in sensory and autonomic nerve fibers. In addition, NOP expression was increased in EM associate NF. Our findings highlight the potential of NOP agonists, particularly in chronic EM-associated pain syndromes and deserve further study, as the efficacy of NOP-selective agonists in clinical trials.

Nociceptin/Orphanin FQ receptor expression in primary human umbilical vein endothelial cells is not regulated by exposure to breast cancer cell media or angiogenic stimuli.

Opioid receptors are naloxone-sensitive (MOP [mu: μ], DOP [delta: δ], and KOP [kappa: κ]) and naloxone-insensitive Nociceptin/Orphanin FQ (N/OFQ) peptide receptor (NOP). Clinically, most opioid analgesics target MOP. Angiogenesis is the formation of new blood vessels and involves endothelial cell activation, proliferation, and migration. The effect of opioids on this process is controversial with no data for NOP receptor ligands. We used patient-derived human umbilical vein endothelial cells (HUVECs) treated with media from the Michigan Cancer Foundation-7 (MCF-7) breast cancer cells or vascular endothelial growth factor (VEGF; 10 ng ml-1) and fibroblast growth factor (FGF; 10 ng ml-1) as angiogenic stimuli. We have measured (i) NOP/MOP messenger RNA, (ii) receptor protein using N/OFQATTO594 and DermorphinATTO488 as fluorescent probes for NOP and MOP, and (iii) NOP/MOP function in a wound healing assay (crude measure of migration that occurs during angiogenesis). HUVEC lines from 32 patients were used. Using all 32 lines, mRNA for NOP but not MOP was detected. This was unaffected by media from MCF-7 cells or VEGF/FGF. There was no binding of either N/OFQATTO594(NOP) or DermorphinATTO488(MOP) in the absence or presence of angiogenic stimuli (six lines tested). In the absence of MOP mRNA, this was expected. Whilst MCF-7 conditioned medium (not VEGF/FGF) reduced wound healing per se (14 lines tested), there was no effect of N/OFQ (NOP ligand) or morphine (MOP ligand). Media from MCF-7 breast cancer cells or VEGF/FGF as angiogenic stimuli did not influence NOP translation into receptor protein. MOP was absent. In the absence of constitutive or inducible MOP/NOP, there was no effect on wound healing as a measure of angiogenesis.

Buprenorphine reduces methamphetamine intake and drug seeking behavior via activating nociceptin/orphanin FQ peptide receptor in rats.

Buprenorphine, which has been approved for the treatment of opioid dependence, reduces cocaine consumption by co-activating μ-opioid receptors and nociceptin/orphanin FQ peptide (NOP) receptors. However, the role of buprenorphine in methamphetamine (METH) reinforcement and drug-seeking behavior remains unclear. This study investigated the effects of buprenorphine on METH self-administration and reinstatement of METH-seeking behavior in rats. We found that buprenorphine pretreatment had an inhibitory effect on METH self-administration behavior, and that buprenorphine at a dose of 0.3 mg/kg could inhibit motivation to respond for METH. Pretreatment with the NOP receptor antagonist thienorphine (0.5 mg/kg) or SB-612111 (1 mg/kg) could reverse the inhibitory effect of buprenorphine (0.1 mg/kg) on the METH self-administration. Moreover, treatment with buprenorphine (0.1 mg/kg and 0.3 mg/kg) significantly reduced the drug-seeking behavior induced by context or by METH priming but failed to reduce the drug-seeking behavior induced by conditional cues. Additionally, the NOP receptor antagonist SB-612111 reversed the inhibitory action of buprenorphine on the drug-seeking behavior induced by METH priming. The results demonstrated that buprenorphine reduced either METH intake or the drug-seeking behavior by activating NOP receptors, providing empirical evidence for the clinical use of buprenorphine in the treatment of METH relapse and addiction.

OREX-1038: a potential new treatment for pain with low abuse liability and limited adverse effects.

Drugs targeting mu opioid receptors are the mainstay of clinical practice for treating moderate-to-severe pain. While they can offer excellent analgesia, their use can be limited by adverse effects, including constipation, respiratory depression, tolerance, and abuse liability. Multifunctional ligands acting at mu opioid and nociceptin/orphanin FQ peptide receptors might provide antinociception with substantially improved adverse-effect profiles. This study explored one of these ligands, OREX-1038 (BU10038), in several assays in rodents and nonhuman primates. Binding and functional studies confirmed OREX-1038 to be a low-efficacy agonist at mu opioid and nociceptin/orphanin FQ peptide receptors and an antagonist at delta and kappa opioid receptors with selectivity for opioid receptors over other proteins. OREX-1038 had long-acting antinociceptive effects in postsurgical and complete Freund's adjuvant (CFA)-induced thermal hyperalgesia assays in rats and a warm water tail-withdrawal assay in monkeys. OREX-1038 was active for at least 24 h in each antinociception assay, and its effects in monkeys did not diminish over 22 days of daily administration. This activity was coupled with limited effects on physiological signs (arterial pressure, heart rate, and body temperature) and no evidence of withdrawal after administration of naltrexone or discontinuation of treatment in monkeys receiving OREX-1038 daily. Over a range of doses, OREX-1038 was only transiently self-administered, which diminished rapidly to nonsignificant levels; overall, both OREX-1038 and buprenorphine maintained less responding than remifentanil. These results support the concept of dual mu and nociceptin/orphanin FQ peptide receptor partial agonists having improved pharmacological profiles compared with opioids currently used to treat pain.

Attenuated G protein signaling and minimal receptor phosphorylation as a biochemical signature of low side-effect opioid analgesics

Multi-receptor targeting has been proposed as a promising strategy for the development of opioid analgesics with fewer side effects. Cebranopadol and AT-121 are prototypical bifunctional ligands targeting the nociceptin/orphanin FQ peptide receptor (NOP) and µ-opioid receptor (MOP) that elicit potent analgesia in humans and nonhuman primates, respectively. Cebranopadol was reported to produce typical MOP-related side effects such as respiratory depression and reward, whereas AT-121 appeared to be devoid of these liabilities. However, the molecular basis underlying different side effect profiles in opioid analgesics remains unknown. Here, we examine agonist-induced receptor phosphorylation and G protein signaling profiles of a series of chemically diverse mixed MOP/NOP agonists, including cebranopadol and AT-121. We found that these compounds produce strikingly different MOP phosphorylation profiles. Cebranopadol, AT-034 and AT-324 stimulated extensive MOP phosphorylation, whereas AT-201 induced selective phosphorylation at S375 only. AT-121, on the other hand, did not promote any detectable MOP phosphorylation. Conversely, none of these compounds was able to elicit strong NOP phosphorylation and low NOP receptor phosphorylation correlated with partial agonism in a GIRK-channel assay. Our results suggest a close correlation between MOP receptor phosphorylation and side effect profile. Thus, bifunctional MOP/NOP opioid ligands combining low efficacy G protein signaling at both NOP and MOP with no detectable receptor phosphorylation appear to be devoid of side-effects such as respiratory depression, abuse liability or tolerance development, as with AT-121.

Mechanistic Characterization of the Pharmacological Profile of HS-731, a Peripherally Acting Opioid Analgesic, at the µ-, δ-, κ-Opioid and Nociceptin Receptors.

Accumulated preclinical and clinical data show that peripheral restricted opioids provide pain relief with reduced side effects. The peripherally acting opioid analgesic HS-731 is a potent dual μ-/δ-opioid receptor (MOR/DOR) full agonist, and a weak, partial agonist at the κ-opioid receptor (KOR). However, its binding mode at the opioid receptors remains elusive. Here, we present a comprehensive in silico evaluation of HS-731 binding at all opioid receptors. We provide insights into dynamic interaction patterns explaining the different binding and activity of HS-731 on the opioid receptors. For this purpose, we conducted docking, performed molecular dynamics (MD) simulations and generated dynamic pharmacophores (dynophores). Our results highlight two residues important for HS-731 recognition at the classical opioid receptors (MOR, DOR and KOR), particular the conserved residue 5.39 (K) and the non-conserved residue 6.58 (MOR: K, DOR: W and KOR: E). Furthermore, we assume a salt bridge between the transmembrane helices (TM) 5 and 6 via K2275.39 and E2976.58 to be responsible for the partial agonism of HS-731 at the KOR. Additionally, we experimentally demonstrated the absence of affinity of HS-731 to the nociceptin/orphanin FQ peptide (NOP) receptor. We consider the morphinan phenol Y1303.33 responsible for this affinity lack. Y1303.33 points deep into the NOP receptor binding pocket preventing HS-731 binding to the orthosteric binding pocket. These findings provide significant structural insights into HS-731 interaction pattern with the opioid receptors that are important for understanding the pharmacology of this peripheral opioid analgesic.

Loss of Nociceptin/Orphanin FQ peptide receptor protects against traumatic brain injury (TBI)‐induced deficits in male and female rats

Introduction : Traumatic brain injury (TBI) is a leading cause of death and disability in the United States and often chronically affects mental and physical health and overall quality of life. We previously reported that acute treatment of blast TBI with a Nociceptin/OrphaninFQ (N/OFQ) peptide receptor (NOP) antagonist improved vestibulomotor deficits in male rats, corresponding to TBI-induced increases in NOP and its endogenous neuropeptide N/OFQ. The goal of this study is to determine whether the absence of NOP protects against neurological and behavioral sequelae of TBI in male and female KO rats compared to WT. Methods Male and female Wistar NOP receptor knockout (NOP KO) and wildtype (WT) rats received either a sham craniotomy, mild TBI (mTBI) or moderate TBI (ModTBI) controlled cortical impact (CCI) to the left cerebral hemisphere. Injury severity and recovery were assessed using modified neurological severity score (mNSS) on days 1 and 8 post-TBI. Vestibulomotor function was determined using the rotarod. Nociceptive sensitivity to mechanical and thermal stimuli were determined by measuring hind paw withdrawal threshold (PWT) to plastic filaments and by measuring tail flick latency (TFL) from radiant heat, respectively, prior to injury (baseline) and on days 2, 4, and 7 post-TBI. The presence of anxiety-like symptoms was assessed using the elevated plus maze (EPM) on day 7 post-TBI. Data were analyzed by two-way ANOVA with or without repeated measures (as appropriate). Results Rats of both genotypes and sexes showed complete neurological recovery (mNSS<1) following mTBI, and partial recovery following ModTBI by day 8. Mild and ModTBI produced significant vestibular deficits in rat rotarod performance on days 1-8 post-TBI in WT male (n=7-8). In WT females (n=8), ModTBI produced vestibular deficits on days 1-7, whereas mTBI only produced vestibular deficits on days 1-4. Interestingly, neither male (n=5-6) nor female (n=3) mTBI NOP KO rats exhibited vestibulomotor deficits. ModTBI caused significant vestibular deficits only on day 1 post-TBI in male and female NOP KO rats. Rotarod performance in WT females was significantly worse than KO females on days 2-4 post TBI following moderate injury (*p<0.05), and on days 4-7 following mild injury (*p<0.05). WT males (n=7-8) and females (n=8) exhibited thermal hyperalgesia and mechanical allodynia throughout the study, with little difference between mild and moderate TBI groups. KO males (n=3) developed no thermal hyperalgesia after either mild or ModTBI. Only ModTBI rats developed mechanical allodynia, and it lasted throughout the study. KO females (n=3) showed both thermal and mechanical hypersensitivity on day 2 and only mechanical hypersensitivity on day 4. No differences in anxiety-like behaviors were found between any of the groups. Conclusion The N/OFQ-NOP system contributes to vestibular deficits and hyperalgesia associated with TBI; the absence of NOP protects against TBI-induced vestibulomotor deficits and nociceptive hypersensitivity.

Evaluation of [Cys(ATTO 488)8]Dermorphin-NH2 as a novel tool for the study of μ-opioid peptide receptors.

The μ-opioid peptide (MOP) receptor is a member of the opioid receptor family and an important clinical target for analgesia. Measuring MOP receptor location and tracking its turnover traditionally used radiolabels or antibodies with attendant problems of utility of radiolabels in whole cells and poor antibody selectivity. To address these issues we have synthesized and characterised a novel ATTO488 based fluorescent Dermorphin analogue; [Cys(ATTO 488)8]Dermorphin-NH2 (DermATTO488). We initially assessed the binding profile of DermATTO488 in HEK cells expressing human MOP and CHO cells expressing human MOP, δ-opioid peptide (DOP), κ-opioid peptide (KOP) and Nociceptin/Orphanin FQ peptide (NOP) receptors using radioligand binding. Functional activity of the conjugated peptide was assessed by measuring (i) the ability of the ligand to engage G-protein by measuring the ability to stimulate GTPγ[35S] binding and (ii) the ability to stimulate phosphorylation of ERK1/2. Receptor location was visualised using confocal scanning laser microscopy. Dermorphin and DermATTO488 bound to HEKMOP (pKi: 8.29 and 7.00; p<0.05), CHOMOP (pKi: 9.26 and 8.12; p<0.05) and CHODOP (pKi: 7.03 and 7.16; p>0.05). Both ligands were inactive at KOP and NOP. Dermorphin and DermATTO488 stimulated the binding of GTPγ[35S] with similar pEC50 (7.84 and 7.62; p>0.05) and Emax (1.52 and 1.34fold p>0.05) values. Moreover, Dermorphin and DermATTO488 produced a monophasic stimulation of ERK1/2 phosphorylation peaking at 5mins (6.98 and 7.64-fold; p>0.05). Finally, in confocal microscopy DermATTO488 bound to recombinant MOP receptors on CHO and HEK cells in a concentration dependent manner that could be blocked by pre-incubation with unlabelled Dermorphin or Naloxone. Collectively, addition to ATTO488 to Dermorphin produced a ligand not dissimilar to Dermorphin; with ~10fold selectivity over DOP. This new ligand DermATTO488 retained functional activity and could be used to visualise MOP receptor location.

Cebranopadol, a novel first-in-class drug candidate: Method validation and first exploratory pharmacokinetic study in rabbits.

Cebranopadol is a novel, centrally acting, potent, first-in-class analgesic drug candidate with a unique mode of action that combines nociceptin/orphanin FQ peptide receptor and opioid peptide receptor agonism. The present study aimed to develop and validate a novel UHPLC-MS/MS method to quantify cebranopadol in rabbit plasma and to assess its pharmacokinetics in rabbits after subcutaneous (s.c.) administration. Twelve adult females were administered with 200µg/kg s.c. injection. Blood samples were withdrawn at 15, 30 and 45min and 1, 1.5, 2, 4, 6, 8, 10 and 24hr after administration. The plasma samples were extracted with a liquid/liquid extraction. The new analytical method complied with the EMA requirements for the bioanalytical method validation. The method was selective, repeatable, accurate, precise and robust with a lower limit of quantification of 0.1ng/ml. In all the rabbits, cebranopadol was quantifiable from 0.25 to 10hr. Mean Cmax and Tmax were 871ng/ml and 0.25hr, respectively. Further studies including the i.v. administration are necessary to fully evaluate the pharmacokinetic features of this novel active compound.

Therapeutic potentials of safe opioid analgesics targeting nociceptin/orphanin FQ peptide receptor

After the identification of nociceptin/orphanin FQ (N/OFQ) peptide (NOP) and its cognate receptor, the unique functional profiles of the N/OFQ-NOP receptor system have been uncovered. NOP receptors are distributed in the key regions that regulate pain and reward processing in the central nervous system. In non-human primates (NHPs), activation of the NOP receptor causes antinociception and anti-hypersensitivity via spinal and supraspinal effects. Moreover, activation of the NOP receptor attenuates dopaminergic transmission and potentiates mu-opioid peptide (MOP) receptor-mediated analgesia. Here, we highlight the functional profiles of bifunctional NOP and MOP receptor agonists based on their promising effects for the treatment of pain and drug abuse. Bifunctional NOP/MOP receptor "partial" agonists, such as AT-121, BU08028, and BU10038, exert potent analgesic effects without MOP receptor-related side effects such as abuse liability, respiratory depression, physical dependence, and itching in NHPs. These novel NOP/MOP receptor agonists reduce rewarding and the reinforcing effects of abused drugs. Furthermore, a mixed NOP/opioid receptor "full" agonist, cebranopadol, is undergoing several clinical trials, and the therapeutic advantage of the coactivation of NOP and MOP receptors has also been confirmed in humans. Therefore, this class of drugs that coactivate NOP and MOP receptors proposes a wide therapeutic range with fewer side effects, indicating a greater potential for the development of novel safer opioid analgesics.

Potential of Nociceptin/Orphanin FQ Peptide Analogs for Drug Development.

Nociceptin receptor (NOP) belongs to the family of opioid receptors but was discovered and characterized much later than the so called classical opioid receptors, μ, δ and κ (or MOP, DOP and KOP, resp.). Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of this receptor and it controls numerous important functions in the central nervous system and in the periphery, so its analogs may be developed as innovative drugs for the treatment of a variety of conditions and pathological states. Availability of potent and selective ligands with high affinity to NOP receptor is essential to fully understand the role of NOP-N/OFQ system in the body, which in turn may lead to designing novel therapeutics. Here, we have focused on reviewing the structure of potent peptide-based agonists, antagonists, biased analogs and bivalent ligands that target NOP receptor.

Nociceptin/orphanin FQ peptide receptor mediates inhibition of N-type calcium currents in vestibular afferent neurons of the rat.

The vestibular system is modulated by various neuromodulators including opioid peptides. The current study was conducted to determine whether activation of nociceptin/orphanin FQ peptide (NOP) receptors modulates voltage-gated calcium currents and action potential discharge of rat vestibular afferent neurons. We performed whole cell patch-clamp recordings on cultured vestibular afferent neurons from P7-P10 Long-Evans rats. Application of nociceptin/orphanin FQ (N/OFQ), a 17-amino acid neuropeptide that is the endogenous ligand for NOP receptor, inhibits the high-voltage activated (HVA) component of the calcium current in a concentration-dependent manner with a half inhibitory concentration of 26 nM. Said inhibitory action on the calcium current is voltage-dependent, which was made clear by the fact that it was reverted in 80% by a depolarizing prepulse. Furthermore, the effect of N/OFQ was blocked by application of the specific NOP-antagonist UFP101, by preincubation with G-protein blocker pertussis toxin, and by coapplication of the specific N-type calcium-current blocker ω-conotoxin-MVIIA. N/OFQ application causes an increase in the duration and maximum rate of repolarization of action potentials. It also decreases repetitive discharge and discharge elicited by sinusoidal stimulation. These results show that in vestibular afferents, NOP receptor activation inhibits N-type calcium current by activating G proteins, mostly through the Gβγ subunit. This suggests that NOP activation produces a presynaptic modulation of primary vestibular afferent neurons' output into the vestibular nuclei, thus taking part in the integration and gain setting of vestibular information in second-order vestibular nucleus neurons.NEW & NOTEWORTHY Our results show that in primary vestibular afferent neurons, activation of the nociceptin/orphanin FQ peptide receptor inhibits the N-type calcium current by a mechanism mediated by G proteins. We propose that calcium current inhibition modulates neurotransmitter release from vestibular afferents, producing a presynaptic modulation of vestibular input to vestibular nuclei, thus contributing to gain control in the vestibular afferent input.

Nociceptin/Orphanin FQ Peptide Receptor-Related Ligands as Novel Analgesics.

Despite similar distribution patterns and intracellular events observed in the nociceptin/ orphanin FQ peptide (NOP) receptor and other opioid receptors, NOP receptor activation displays unique pharmacological profiles. Several researchers have identified a variety of peptide and nonpeptide ligands to determine the functional roles of NOP receptor activation and observed that NOP receptor- related ligands exhibit pain modality-dependent pain processing. Importantly, NOP receptor activation results in anti-nociception and anti-hypersensitivity at the spinal and supraspinal levels regardless of the experimental settings in non-human primates (NHPs). Given that the NOP receptor agonists synergistically enhance mu-opioid peptide (MOP) receptor agonist-induced anti-nociception, it has been hypothesized that dual NOP and MOP receptor agonists may display promising functional properties as analgesics. Accumulating evidence indicates that the mixed NOP/opioid receptor agonists demonstrate favorable functional profiles. In NHP studies, bifunctional NOP/MOP partial agonists (e.g., AT-121, BU08028, and BU10038) exerted potent anti-nociception via NOP and MOP receptor activation; however, dose-limiting adverse effects associated with the MOP receptor activation, including respiratory depression, itch sensation, physical dependence, and abuse liability, were not observed. Moreover, a mixed NOP/opioid receptor agonist, cebranopadol, presented promising outcomes in clinical trials as a novel analgesic. Collectively, the dual agonistic actions on NOP and MOP receptors, with appropriate binding affinities and efficacies, may be a viable strategy to develop innovative and safe analgesics.