ABSTRACT
Neuroinflammation is associated with several neurological disorders including temporal lobe epilepsy. Seizures themselves can induce neuroinflammation. In an in vivo model of epilepsy, the supplementation of brain-derived neurotropic factor (BDNF) and fibroblast growth factor-2 (FGF-2) using a Herpes-based vector reduced epileptogenesis-associated neuroinflammation. The aim of this study was to test whether the attenuation of the neuroinflammation obtained in vivo with BDNF and FGF-2 was direct or secondary to other effects, for example, the reduction in the severity and frequency of spontaneous recurrent seizures. An in vitro model of neuroinflammation induced by lipopolysaccharide (LPS, 100 ng/mL) in a mouse primary mixed glial culture was used. The releases of cytokines and NO were analyzed via ELISA and Griess assay, respectively. The effects of LPS and neurotrophic factors on cell viability were determined by performing an MTT assay. BDNF and FGF-2 were tested alone and co-administered. LPS induced a significant increase in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and NO. BDNF, FGF-2, and their co-administration did not counteract these LPS effects. Our study suggests that the anti-inflammatory effect of BDNF and FGF-2 in vivo in the epilepsy model was indirect and likely due to a reduction in seizure frequency and severity.
Subject(s)
Brain-Derived Neurotrophic Factor , Cytokines , Fibroblast Growth Factor 2 , Lipopolysaccharides , Neuroinflammatory Diseases , Animals , Mice , Neuroinflammatory Diseases/metabolism , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factor 2/pharmacology , Brain-Derived Neurotrophic Factor/metabolism , Cytokines/metabolism , Cells, Cultured , Nerve Growth Factors/metabolism , Nerve Growth Factors/pharmacology , Neuroglia/metabolism , Neuroglia/drug effects , Cell Survival/drug effects , Disease Models, Animal , Mice, Inbred C57BLABSTRACT
Recently, mixed opioid/NOP agonists came to the spotlight for their favorable functional profiles and promising outcomes in clinical trials as novel analgesics. This study reports on two novel chimeric peptides incorporating the fragment Tyr-c[D-Lys-Phe-Phe]Asp-NH2 (RP-170), a cyclic peptide with high affinity for µ and κ opioid receptors (or MOP and KOP, respectively), conjugated with the peptide Ac-RYYRIK-NH2, a known ligand of the nociceptin/orphanin FQ receptor (NOP), yielding RP-170-RYYRIK-NH2 (KW-495) and RP-170-Gly3-RYYRIK-NH2 (KW-496). In vitro, the chimeric KW-496 gained affinity for KOP, hence becoming a dual KOP/MOP agonist, while KW-495 behaved as a mixed MOP/NOP agonist with low nM affinity. Hence, KW-495 was selected for further in vivo experiments. Intrathecal administration of this peptide in mice elicited antinociceptive effects in the hot-plate test; this action was sensitive to both the universal opioid receptor antagonist naloxone and the selective NOP antagonist SB-612111. The rotarod test revealed that KW-495 administration did not alter the mice motor coordination performance. Computational studies have been conducted on the two chimeras to investigate the structural determinants at the basis of the experimental activities, including any role of the Gly3 spacer.
Subject(s)
Analgesics, Opioid , Receptors, Opioid , Animals , Mice , Analgesics, Opioid/therapeutic use , Receptors, Opioid/agonists , Receptors, Opioid, kappa , Narcotic Antagonists/pharmacology , Receptors, Opioid, mu/agonists , Molecular Docking Simulation , Ligands , Dose-Response Relationship, Drug , Naloxone , Analgesics/pharmacology , Peptides/pharmacology , Chimera , Peptides, CyclicABSTRACT
The peptide nociceptin/orphanin FQ (N/OFQ) is the natural ligand of the N/OFQ receptor (NOP), which is widely expressed in the central and peripheral nervous system. Selective NOP antagonists are worthy of testing as innovative drugs to treat depression, Parkinson disease, and drug abuse. The aim of this study was to perform a detailed in vitro characterization of BTRX-246040 (also known as LY2940094, [2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4'-piperidine]-1'-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol), a novel NOP antagonist that has been already studied in humans. BTRX-246040 has been tested in vitro in the following assays: calcium mobilization in cells expressing NOP and classic opioid receptors and chimeric G proteins, bioluminescence resonance energy transfer assay measuring NOP interaction with G proteins and ß-arrestins, the label-free dynamic mass redistribution assay, and the electrically stimulated mouse vas deferens. BTRX-246040 was systematically compared with the standard NOP antagonist SB-612111. In all assays, BTRX-246040 behaves as a pure and selective antagonist at human recombinant and murine native NOP receptors displaying 3-10-fold higher potency than the standard antagonist SB-612111. BTRX-246040 is an essential pharmacological tool to further investigate the therapeutic potential of NOP antagonists in preclinical and clinical studies. SIGNIFICANCE STATEMENT: NOP antagonists may be innovative antidepressant drugs. In this research, the novel clinically viable NOP antagonist BTRX-246040 has been deeply characterized in vitro in a panel of assays. BTRX-246040 resulted a pure, potent, and selective NOP antagonist.
Subject(s)
Pyrans/pharmacology , Receptors, Opioid/physiology , Spiro Compounds/pharmacology , Animals , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Male , Mice , Nociceptin ReceptorABSTRACT
Opioids such as morphine-acting at the mu opioid receptor-are the mainstay for treatment of moderate to severe pain and have good efficacy in these indications. However, these drugs produce a plethora of unwanted adverse effects including respiratory depression, constipation, immune suppression and with prolonged treatment, tolerance, dependence and abuse liability. Studies in ß-arrestin 2 gene knockout (ßarr2(-/-)) animals indicate that morphine analgesia is potentiated while side effects are reduced, suggesting that drugs biased away from arrestin may manifest with a reduced-side-effect profile. However, there is controversy in this area with improvement of morphine-induced constipation and reduced respiratory effects in ßarr2(-/-) mice. Moreover, studies performed with mice genetically engineered with G-protein-biased mu receptors suggested increased sensitivity of these animals to both analgesic actions and side effects of opioid drugs. Several new molecules have been identified as mu receptor G-protein-biased agonists, including oliceridine (TRV130), PZM21 and SR-17018. These compounds have provided preclinical data with apparent support for bias toward G proteins and the genetic premise of effective and safer analgesics. There are clinical data for oliceridine that have been very recently approved for short term intravenous use in hospitals and other controlled settings. While these data are compelling and provide a potential new pathway-based target for drug discovery, a simpler explanation for the behavior of these biased agonists revolves around differences in intrinsic activity. A highly detailed study comparing oliceridine, PZM21 and SR-17018 (among others) in a range of assays showed that these molecules behave as partial agonists. Moreover, there was a correlation between their therapeutic indices and their efficacies, but not their bias factors. If there is amplification of G-protein, but not arrestin pathways, then agonists with reduced efficacy would show high levels of activity at G-protein and low or absent activity at arrestin; offering analgesia with reduced side effects or 'apparent bias'. Overall, the current data suggests-and we support-caution in ascribing biased agonism to reduced-side-effect profiles for mu-agonist analgesics.
Subject(s)
Analgesics, Opioid , Drug Approval , Pain/drug therapy , Analgesics, Opioid/chemistry , Analgesics, Opioid/therapeutic use , Animals , Drug Evaluation, Preclinical , Humans , Mice , Mice, Knockout , Pain/genetics , Pain/metabolism , Pain/pathology , beta-Arrestin 2/agonists , beta-Arrestin 2/genetics , beta-Arrestin 2/metabolismABSTRACT
The general aim of the work was the validation of a new synthetic methodology designed for obtaining bifunctional heterotetrabranched peptide ligands. Applying an easily accessible synthetic route, we provided a small series of heteromultimeric peptide conjugates targeting the nociceptin/orphanin FQ (N/OFQ) peptide receptors (NOP) and mu opioid receptors. Among these, H-PWT1-N/OFQ-[Dmt1]dermorphin demonstrated a similar and high agonist potency at the NOP and mu receptors. The achieved results confirmed the robustness of the approach that is extremely versatile and virtually applicable to different peptide sequences whose pharmacological activity can be combined for generating dual acting multimeric compounds. These innovative pharmacological tools will be extremely helpful for investigating the consequences of the simultaneous activation and/or blockage of different peptidergic receptors.
Subject(s)
Peptides/chemistry , Peptides/pharmacology , Receptors, Opioid, mu/agonists , Receptors, Opioid/agonists , Animals , Benzaldehydes/chemistry , CHO Cells , Cricetulus , Humans , Maleimides/chemistry , Nociceptin ReceptorABSTRACT
Many studies point toward the nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP) as targets for the development of innovative drugs for treating anxiety- and mood-related disorders. Evidence supports the view that the activation of NOP receptors with agonists elicits anxiolytic-like effects, while its blockade with NOP antagonists promotes antidepressant-like actions in rodents. Genetic studies showed that NOP receptor knockout mice display an antidepressant-like phenotype, and NOP antagonists are inactive in these animals. In contrast, the genetic blockade of NOP receptor signaling generally displays an increase of anxiety states in the elevated plus-maze test. In this chapter we summarized the most relevant findings of NOP receptor ligands in the modulation of anxiety and mood disorders, and the putative mechanisms of action are discussed.
Subject(s)
Antidepressive Agents/pharmacology , Mood Disorders , Opioid Peptides/pharmacology , Receptors, Opioid/metabolism , Animals , Antidepressive Agents/therapeutic use , Anxiety , Ligands , Mice , Opioid Peptides/metabolism , Receptors, Opioid/chemistry , Receptors, Opioid/drug effects , Nociceptin ReceptorABSTRACT
Morphine, which acts through opioid receptors, is one of the most efficient analgesics for the alleviation of severe pain. However, its usefulness is limited by serious side effects, including analgesic tolerance, constipation, and dependence liability. The growing awareness that multifunctional ligands which simultaneously activate two or more targets may produce a more desirable drug profile than selectively targeted compounds has created an opportunity for a new approach to developing more effective medications. Here, in order to better understand the role of the neurokinin system in opioid-induced antinociception, we report the synthesis, structure-activity relationship, and pharmacological characterization of a series of hybrids combining opioid pharmacophores with either substance P (SP) fragments or neurokinin receptor (NK1) antagonist fragments. On the bases of the in vitro biological activities of the hybrids, two analogs, opioid agonist/NK1 antagonist Tyr-[d-Lys-Phe-Phe-Asp]-Asn-d-Trp-Phe-d-Trp-Leu-Nle-NH2 (2) and opioid agonist/NK1 agonist Tyr-[d-Lys-Phe-Phe-Asp]-Gln-Phe-Phe-Gly-Leu-Met-NH2 (4), were selected for in vivo tests. In the writhing test, both hybrids showed significant an antinociceptive effect in mice, while neither of them triggered the development of tolerance, nor did they produce constipation. No statistically significant differences in in vivo activity profiles were observed between opioid/NK1 agonist and opioid/NK1 antagonist hybrids.
Subject(s)
Analgesics , Narcotic Antagonists , Neurokinin-1 Receptor Antagonists , Nociception/drug effects , Oligopeptides , Receptors, Neurokinin-1 , Receptors, Opioid , Analgesics/pharmacology , Animals , Cell Line , Drug Tolerance , Male , Mice , Mice, Inbred BALB C , Narcotic Antagonists/chemistry , Narcotic Antagonists/pharmacology , Neurokinin-1 Receptor Antagonists/chemistry , Neurokinin-1 Receptor Antagonists/pharmacology , Oligopeptides/chemistry , Oligopeptides/pharmacology , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/metabolism , Receptors, Opioid/agonists , Receptors, Opioid/metabolismABSTRACT
Ghrelin modulates several biological functions via selective activation of the growth hormone secretagogue receptor (GHSR). GHSR agonists may be useful for the treatment of anorexia and cachexia, while antagonists and inverse agonists may represent new drugs for the treatment of metabolic and substance use disorders. Thus, the identification and pharmacodynamic characterization of new GHSR ligands is of high interest. In the present work the label-free dynamic mass redistribution (DMR) assay has been used to evaluate the pharmacological activity of a panel of GHSR ligands. This includes the endogenous peptides ghrelin, desacyl-ghrelin and LEAP2(1-14). Among synthetic compounds, the agonists anamorelin and HM01, the antagonists HM04 and YIL-781, and the inverse agonist PF-05190457 have been tested, together with HM03, R011, and H1498 from patent literature. The DMR results have been compared to those obtained in parallel experiments with the calcium mobilization assay. Ghrelin, anamorelin, HM01, and HM03 behaved as potent full GHSR agonists. YIL-781 behaved as a partial GHSR agonist and R011 as antagonist in both the assays. LEAP2(1-14) resulted a GHSR inverse agonist in DMR but not in calcium mobilization assay. PF-05190457, HM04, and H1498 behaved as GHSR inverse agonists in DMR experiments, while they acted as antagonists in calcium mobilization studies. In conclusion, this study provided a systematic pharmacodynamic characterization of several GHSR ligands in two different pharmacological assays. It demonstrated that the DMR assay can be successfully used particularly to discriminate between antagonists and inverse agonists. This study may be useful for the selection of the most appropriate compounds to be used in future studies.
ABSTRACT
RATIONALE: Recently, we demonstrated that the activation of the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) signaling facilitates depressive-like behaviors. Additionally, literature findings support the ability of the N/OFQ-NOP system to modulate the hypothalamic-pituitary-adrenal (HPA) axis. OBJECTIVES: Considering that dysfunctional HPA axis is strictly related to stress-induced psychopathologies, we aimed to study the role of the HPA axis in the pro-depressant effects of NOP agonists. METHODS: Mice were treated prior to stress with the NOP agonist Ro 65-6570, and immobility time in the forced swimming task and corticosterone levels were measured. Additionally, the role of endogenous glucocorticoids and CRF was investigated using the glucocorticoid receptor antagonist mifepristone and the CRF1 antagonist antalarmin in the mediation of the effects of Ro 65-6570. RESULTS: The NOP agonist in a dose-dependent manner further increased the immobility of mice in the second swimming session compared to vehicle. By contrast, under the same conditions, the administration of the NOP antagonist SB-612111 before stress reduced immobility, while the antidepressant nortriptyline was inactive. Concerning in-serum corticosterone in mice treated with vehicle, nortriptyline, or SB-612111, a significant decrease was observed after re-exposition to stress, but no differences were detected in Ro 65-6570-treated mice. Administration of mifepristone or antalarmin blocked the Ro 65-6570-induced increase in the immobility time in the second swimming session. CONCLUSIONS: Present findings suggest that NOP agonists increase vulnerability to depression by hyperactivating the HPA axis and then increasing stress circulating hormones and CRF1 receptor signaling.
Subject(s)
Cycloheptanes , Imidazoles , Opioid Peptides , Piperidines , Receptors, Opioid , Spiro Compounds , Mice , Animals , Receptors, Opioid/physiology , Opioid Peptides/metabolism , Glucocorticoids/pharmacology , Nortriptyline/pharmacology , Nociceptin Receptor , Corticosterone/pharmacology , Hypothalamo-Hypophyseal System/metabolism , Mifepristone/pharmacology , Pituitary-Adrenal System/metabolismABSTRACT
ABSTRACT: Migraine is a disabling disorder characterized by recurrent headaches, accompanied by abnormal sensory sensitivity and anxiety. Despite extensive historical use of cannabis in headache disorders, there is limited research on the nonpsychoactive cannabidiol (CBD) for migraine and there is no scientific evidence to prove that CBD is an effective treatment. The effects of CBD are examined here using a calcitonin gene-related peptide (CGRP)-induced migraine model that provides measures of cephalic allodynia, spontaneous pain, altered light sensitivity (photophobia), and anxiety-like behavior in C57BL/6J mice. A single administration of CGRP induced facial hypersensitivity in both female and male mice. Repeated CGRP treatment produced progressively decreased levels in basal thresholds of allodynia in females, but not in males. A single CBD administration protected both females and males from periorbital allodynia induced by a single CGRP injection. Repeated CBD administration prevented increased levels of basal allodynia induced by repeated CGRP treatment in female mice and did not lead to responses consistent with migraine headache as occurs with triptans. Cannabidiol, injected after CGRP, reversed CGRP-evoked allodynia. Cannabidiol also reduced spontaneous pain traits induced by CGRP administration in female mice. Finally, CBD blocked CGRP-induced anxiety in male mice, but failed in providing protection from CGRP-induced photophobia in females. These results demonstrate the efficacy of CBD in preventing episodic and chronic migraine-like states with reduced risk of causing medication overuse headache. Cannabidiol also shows potential as an abortive agent for treating migraine attacks and headache-related conditions such as spontaneous pain and anxiety.
ABSTRACT
Neuropeptide S (NPS) regulates various biological functions by selectively activating the NPS receptor (NPSR). Recently, epidemiological studies revealed an association between NPSR single nucleotide polymorphisms and susceptibility to panic disorders. Here we investigated the effects of NPS in mice subjected to the elevated T maze (ETM), an assay which has been proposed to model anxiety and panic. Diazepam [1 mg/kg, intraperitoneally (i.p.)] elicited clear anxiolytic effects reducing the latency to emerge from the closed to the open (CO) arm without modifying the latencies from the open to the closed (OC) arm. By contrast, chronic fluoxetine (10 mg/kg i.p., once a day for 21 days) selectively increased OC latency, suggesting a panicolytic-like effect. NPS given intracerebroventricularly at 0.001-1 nmol elicited both anxiolytic- and panicolytic-like effects. However, although the NPS anxiolytic dose-response curve displayed the classical sigmoidal shape, the dose-response curve of the putative panicolytic-like effect was bell shaped with peak effect at 0.01 nmol. The behaviour of wild-type [NPSR(+/+)] and receptor knock out [NPSR(-/-)] mice in the ETM task was superimposable. NPS at 0.01 nmol elicited anxiolytic- and panicolytic-like effects in NPSR(+/+) but not in NPSR(-/-) mice. In conclusion, this study demonstrated that NPS, via selective activation of the NPSR, promotes both anxiolytic- and panicolytic-like actions in the mouse ETM.
Subject(s)
Anxiety/chemically induced , Maze Learning/drug effects , Neuropeptides/toxicity , Panic Disorder/chemically induced , Animals , Anti-Anxiety Agents/therapeutic use , Antidepressive Agents, Second-Generation/therapeutic use , Anxiety/drug therapy , Diazepam/therapeutic use , Dose-Response Relationship, Drug , Fluoxetine/therapeutic use , Male , Mice , Mice, Knockout , Panic Disorder/drug therapy , Reaction Time , Receptors, Neuropeptide/geneticsABSTRACT
The present study investigated the in vitro pharmacology of the human kappa opioid receptor using multiple assays, including calcium mobilization in cells expressing chimeric G proteins, the dynamic mass redistribution (DMR) label-free assay, and a bioluminescence resonance energy transfer (BRET) assay that allows measurement of receptor interaction with G protein and ß-arrestin 2. In all assays, dynorphin A, U-69,593, and [D-Pro10]dyn(1-11)-NH2 behaved as full agonists with the following rank order of potency [D-Pro10]dyn(1-11)-NH2 > dynorphin A ≥ U-69,593. [Dmt1,Tic2]dyn(1-11)-NH2 behaved as a moderate potency pure antagonist in the kappa-ß-arrestin 2 interaction assay and as low efficacy partial agonist in the other assays. Norbinaltorphimine acted as a highly potent and pure antagonist in all assays except kappa-G protein interaction, where it displayed efficacy as an inverse agonist. The pharmacological actions of novel kappa ligands, namely the dynorphin A tetrameric derivative PWT2-Dyn A and the palmitoylated derivative Dyn A-palmitic, were also investigated. PWT2-Dyn A and Dyn A-palmitic mimicked dynorphin A effects in all assays showing similar maximal effects but 3-10 fold lower potency. In conclusion, in the present study, multiple in vitro assays for the kappa receptor have been set up and pharmacologically validated. In addition, PWT2-Dyn A and Dyn A-palmitic were characterized as potent full agonists; these compounds are worthy of further investigation in vivo for those conditions in which the activation of the kappa opioid receptor elicits beneficial effects e.g. pain and pruritus.
ABSTRACT
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.
Subject(s)
Opioid Peptides , Receptors, Opioid , Animals , Antidepressive Agents/pharmacology , Hippocampus/metabolism , Ligands , Mice , Neurogenesis , Opioid Peptides/metabolism , Receptors, Opioid/metabolismABSTRACT
The Neuropeptide S (NPS) system is a rather 'young' transmitter system that was discovered and functionally described less than 20 years ago. This review highlights the progress that has been made in elucidating its pharmacology, anatomical distribution, and functional involvement in a variety of physiological effects, including behavior and immune functions. Early on, genetic variations of the human NPS receptor (NPSR1) have attracted attention and we summarize current hypotheses of genetic linkage with disease and human behaviors. Finally, we review the therapeutic potential of future drugs modulating NPS signaling. This review serves as an introduction to the broad collection of original research papers and reviews from experts in the field that are presented in this Special Issue.
ABSTRACT
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.
Subject(s)
Opioid Peptides/physiology , Receptors, Opioid/physiology , Stress, Psychological/etiology , Animals , Humans , Hypothalamo-Hypophyseal System/physiology , Narcotic Antagonists/pharmacology , Pituitary-Adrenal System/physiology , Stress, Psychological/drug therapy , Nociceptin Receptor , NociceptinABSTRACT
Opioid-induced constipation is the most prevalent adverse effect of opioid drugs. Peripherally acting mu opioid receptor antagonists (PAMORAs), including naloxegol, are indicated for the treatment of opioid-induced constipation. The aim of this study was the in vitro and in vivo pharmacological characterization of naloxegol in comparison with naloxone. In vitro experiments were performed to measure calcium mobilization in cells coexpressing opioid receptors and chimeric G proteins and mu receptor interaction with G protein and ß-arrestin 2 using bioluminescence resonance energy transfer. In vivo experiments were performed in mice to measure pain threshold using the tail withdrawal assay and colonic transit using the bead expulsion assay. In vitro, naloxegol behaved as a selective and competitive mu receptor antagonist similarly to naloxone, being 3-10-fold less potent. In vivo, naloxone was effective in blocking fentanyl actions when given subcutaneously (sc), but not per os (po). In contrast, naloxegol elicited very similar effects with sc or po administration counteracting in a dose dependent manner the constipating effects of fentanyl without interfering with the fentanyl mediated analgesia. Thus, a useful PAMORA action could be obtained with naloxegol both after po and sc administration.
Subject(s)
Constipation/drug therapy , Morphinans/pharmacology , Narcotic Antagonists/pharmacology , Polyethylene Glycols/pharmacology , Administration, Oral , Analgesics, Opioid/administration & dosage , Analgesics, Opioid/adverse effects , Analgesics, Opioid/pharmacology , Animals , Behavior, Animal/drug effects , CHO Cells , Calcium/metabolism , Constipation/chemically induced , Cricetulus , Fentanyl/administration & dosage , Fentanyl/adverse effects , Fentanyl/pharmacology , Injections, Subcutaneous , Male , Mice , Morphinans/administration & dosage , Morphine/pharmacology , Naloxone/administration & dosage , Naloxone/pharmacology , Narcotic Antagonists/administration & dosage , Pain/drug therapy , Polyethylene Glycols/administration & dosage , Receptors, Opioid, mu/antagonists & inhibitors , Receptors, Opioid, mu/drug effectsABSTRACT
Nociceptin/orphanin FQ controls several functions, including pain transmission, via stimulation of the N/OFQ peptide (NOP) receptor. Here we tested the hypothesis that NOP biased agonism may be instrumental for identifying innovative analgesics. In vitro experiments were performed with the dynamic mass redistribution label free assay and the NOP non-peptide agonists Ro 65-6570, AT-403 and MCOPPB. In vivo studies were performed in wild type and ß-arrestin 2 knockout mice using the formalin, rotarod and locomotor activity tests. In vitro all compounds mimicked the effects of N/OFQ behaving as potent NOP full agonists. In vivo Ro 65-6570 demonstrated a slightly higher therapeutic index (antinociceptive vs. motor impairment effects) in knockout mice. However, all NOP agonists displayed very similar therapeutic index in normal mice despite significant differences in G protein biased agonism. In conclusion the different ability of inducing G protein vs. ß-arrestin 2 recruitment of a NOP agonist cannot be applied to predict its antinociceptive vs. motor impairment properties.
ABSTRACT
Neuropeptide S modulates important neurobiological functions including locomotion, anxiety, and drug abuse through interaction with its G protein-coupled receptor known as neuropeptide S receptor (NPSR). NPSR antagonists are potentially useful for the treatment of substance abuse disorders against which there is an urgent need for new effective therapeutic approaches. Potent NPSR antagonists in vitro have been discovered which, however, require further optimization of their in vivo pharmacological profile. This work describes a new series of NPSR antagonists of the oxazolo[3,4-a]pyrazine class. The guanidine derivative 16 exhibited nanomolar activity in vitro and 5-fold improved potency in vivo compared to SHA-68, a reference pharmacological tool in this field. Compound 16 can be considered a new tool for research studies on the translational potential of the NPSergic system. An in-depth molecular modeling investigation was also performed to gain new insights into the observed structure-activity relationships and provide an updated model of ligand/NPSR interactions.
Subject(s)
Oxazoles/pharmacology , Pyrazines/pharmacology , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, Neuropeptide/antagonists & inhibitors , Animals , HEK293 Cells , Humans , Locomotion/drug effects , Mice, Knockout , Molecular Docking Simulation , Molecular Structure , Oxazoles/chemical synthesis , Oxazoles/metabolism , Protein Binding , Pyrazines/chemical synthesis , Pyrazines/metabolism , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/metabolism , Structure-Activity RelationshipABSTRACT
The nociceptin/orphanin FQ (N/OFQ)/N/OFQ receptor (NOP) system controls different biological functions including pain and cough reflex. Mixed NOP/opioid receptor agonists elicit similar effects to strong opioids but with reduced side effects. In this work, 31 peptides with the general sequence [Tyr/Dmt1,Xaa5]N/OFQ(1-13)-NH2 were synthesized and pharmacologically characterized for their action at human recombinant NOP/opioid receptors. The best results in terms of NOP versus mu opioid receptor potency were obtained by substituting both Tyr1 and Thr5 at the N-terminal portion of N/OFQ(1-13)-NH2 with the noncanonical amino acid Dmt. [Dmt1,5]N/OFQ(1-13)-NH2 has been identified as the most potent dual NOP/mu receptor peptide agonist so far described. Experimental data have been complemented by in silico studies to shed light on the molecular mechanisms by which the peptide binds the active form of the mu receptor. Finally, the compound exerted antitussive effects in an in vivo model of cough.
Subject(s)
Peptides/chemistry , Receptors, Opioid, mu/agonists , Receptors, Opioid/agonists , Animals , Binding Sites , Cough/chemically induced , Cough/drug therapy , Disease Models, Animal , Guinea Pigs , Humans , Hydrophobic and Hydrophilic Interactions , Male , Molecular Dynamics Simulation , Peptides/metabolism , Peptides/therapeutic use , Receptors, Opioid/metabolism , Receptors, Opioid, mu/genetics , Receptors, Opioid, mu/metabolism , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Structure-Activity Relationship , Nociceptin ReceptorABSTRACT
The role of stress in the etiology of depression has been largely reported. In this line, exogenous glucocorticoids are employed to mimic the influence of stress on the development of depression. The N/OFQ-NOP receptor system has been implicated in the modulation of stress and emotional behaviors. In fact, the blockade of NOP receptors induces antidepressant effects and increases resilience to acute stress. This study investigated the effects of the NOP receptor blockade on dexamethasone-treated mice exposed to acute and prolonged swimming stress. Swiss and NOP(+/+) and NOP(-/-) mice were treated with dexamethasone, and the protective effects of the NOP antagonist SB-612111 (10 mg/kg, ip) or imipramine (20 mg/kg, ip) were investigated in three swimming sessions. The re-exposure to swim stress increased immobility time in Swiss and NOP(+/+), but not in NOP(-/-) mice. Acute and repeated dexamethasone administration induced a further increase in the immobility time, and facilitated body weight loss in Swiss mice. Single administration of SB-612111, but not imipramine, prevented swimming stress- and dexamethasone-induced increase in the immobility time. Repeated administrations of SB-612111 prevented the deleterious effects of 5 days of dexamethasone treatment. Imipramine also partially prevented the effects of repeated glucocorticoid administration on the immobility time, but did not affect the body weight loss. NOP(-/-) mice were more resistant than NOP(+/+) mice to inescapable swimming stress, but not dexamethasone-induced increase in the immobility time and body weight loss. In conclusion, the blockade of the NOP receptor facilitates an active stress copying response and attenuates body weight loss due to repeated stress.