Acute Thermal Nociception Research Articles

5-HT2A receptors are involved in the pharmaco-toxicological effects of the synthetic cannabinoids JWH-018 and 5F-PB22: In vivo studies in mice

Over the last years, Synthetic Cannabinoids (SCs) have been among the largest and most frequently seized groups of Novel Psychoactive Substances (NPS). These substances have been frequently detected in biological samples from patients involved in several intoxication and death cases. Their serious adverse effects have been related to their action as potent agonist of cannabinoid CB1 receptors. However, evidence concerning the potential interaction between SCs and serotoninergic mechanisms has emerged. Therefore, this study aims to evaluate the involvement of 5-HT2A receptors in the effects induced by acute systemic administration of 1-pentyl-3-(1-naphthoyl)indole (JWH-018; 1 mg/kg) and quinolin-8-yl 1-pentyfluoro-1H-indole-3-8-carboxylate (5F-PB22; 1 mg/kg). Sensorimotor (visual, acoustic, and tactile) responses, pain threshold (acute mechanical and thermal nociception), core temperature, breath rate and motor performance (stepping activity) have been assessed in CD-1 male mice. The present results pointed out that both substances deeply alter sensorimotor responses, nociceptive threshold, core temperature, breath rate and motor activity in mice. Noteworthy, pretreatment with the selective 5-HT2A receptors antagonist MDL100907 (0.1 mg/kg) at least partially prevented sensorimotor disruption, antinociception and hypothermic effects. Conversely, the respiratory and motor impairment was not prevented. Thus, it states the relevance of serotoninergic 5-HT2A mechanisms on pharmaco-toxicological effects induced by SCs.

Functional roles of neuromedin B and gastrin-releasing peptide in regulating itch and pain in the spinal cord of non-human primates

Despite accumulating evidence in rodents, the functional role of neuromedin B (NMB) in regulating somatosensory systems in primate spinal cord is unknown. We aimed to compare the expression patterns of NMB and its receptor (NMBR) and the behavioral effects of intrathecal (i.t.) NMB with gastrin-releasing peptide (GRP) on itch or pain in non-human primates (NHPs). We used six adult rhesus monkeys. The mRNA or protein expressions of NMB, GRP, and their receptors were evaluated by quantitative reverse transcription polymerase chain reaction, immunohistochemistry, or in situ hybridization. We determined the behavioral effects of NMB or GRP via acute thermal nociception, capsaicin-induced thermal allodynia, and itch scratching response assays. NMB expression levels were greater than those of GRP in the dorsal root ganglia and spinal dorsal horn. Conversely, NMBR expression was significantly lower than GRP receptor (GRPR). I.t. NMB elicited only mild scratching responses, whereas GRP caused robust scratching responses. GRP- and NMB-elicited scratching responses were attenuated by GRPR (RC-3095) and NMBR (PD168368) antagonists, respectively. Moreover, i.t. NMB and GRP did not induce thermal hypersensitivity and GRPR and NMBR antagonists did not affect peripherally elicited thermal allodynia. Consistently, NMBR expression was low in both itch- and pain-responsive neurons in the spinal dorsal horn. Spinal NMB-NMBR system plays a minimal functional role in the neurotransmission of itch and pain in primates. Unlike the functional significance of the GRP-GRPR system in itch, drugs targeting the spinal NMB-NMBR system may not effectively alleviate non-NMBR-mediated itch.

Sex-specific role for serotonin 5-HT2A receptor in modulation of opioid-induced antinociception and reward in mice

Opioids are among the most effective analgesics and the mainstay of pain management. However, concerns about safety and abuse liability have challenged their widespread use by the medical community. Opioid-sparing therapies include drugs that in combination with opioids have the ability to enhance analgesia while decreasing opioid requirement as well as their side effects. Sex differences in antinociceptive responses to opioids have received increasing attention in recent years. However, the molecular mechanisms underlying sex differences related to opioid-sparing adjuncts remain largely unexplored. Using warm water tail-withdrawal as a mouse model of acute thermal nociception, our data suggest that adjunctive administration of the serotonin 5-HT2A receptor (5-HT2AR) antagonist volinanserin dose-dependently enhanced potency of the opioid analgesic oxycodone in male, but not female, mice. This antinociceptive-like response induced by oxycodone was also augmented in 5-HT2AR knockout (5-HT2AR−/−) male, but not female mice; an effect that was reversed by Cre-loxP-mediated selective expression of 5-HT2AR in dorsal root ganglion (DRG) neurons of 5-HT2AR−/− littermates. Pharmacological inhibition with volinanserin or genetic deletion in 5-HT2AR−/− animals potentiated the ability of oxycodone to reduce DRG excitability in male mice. Adjunctive volinanserin did not affect oxycodone-induced conditioned place preference (CPP), whereas it reduced oxycodone-induced locomotor sensitization in male and female mice. Together, these results suggest that adjunctive volinanserin augments opioid-induced antinociception, but not abuse-related behavior, through a sex-specific signaling crosstalk mechanism that requires 5-HT2AR expression in mouse DRG neurons. Ultimately, our results may pave the way for the clinical evaluation of volinanserin as a potential sex-specific opioid adjuvant.

This Month in Anesthesiology

This Month in Anesthesiology

Antinociceptive, reinforcing, and pruritic effects of the G-protein signalling-biased mu opioid receptor agonist PZM21 in non-human primates

BackgroundA novel G-protein signalling-biased mu opioid peptide (MOP) receptor agonist, PZM21, was recently developed with a distinct chemical structure. It is a potent Gi/o activator with minimal β-arrestin-2 recruitment. Despite intriguing activity in rodent models, PZM21 function in non-human primates is unknown. The aim of this study was to investigate PZM21 actions after systemic or intrathecal administration in primates. MethodsAntinociceptive, reinforcing, and pruritic effects of PZM21 were compared with those of the clinically used MOP receptor agonists oxycodone and morphine in assays of acute thermal nociception, capsaicin-induced thermal allodynia, itch scratching responses, and drug self-administration in gonadally intact, adult rhesus macaques (10 males, six females). ResultsAfter subcutaneous administration, PZM21 (1.0–6.0 mg kg−1) and oxycodone (0.1–0.6 mg kg−1) induced dose-dependent thermal antinociceptive effects (P<0.05); PZM21 was 10 times less potent than oxycodone. PZM21 exerted oxycodone-like reinforcing effects and strength as determined by two operant schedules of reinforcement in the intravenous drug self-administration assay. After intrathecal administration, PZM21 (0.03–0.3 mg) dose-dependently attenuated capsaicin-induced thermal allodynia (P<0.05). Although intrathecal PZM21 and morphine induced MOP receptor-mediated antiallodynic effects, both compounds induced robust, long-lasting itch scratching. ConclusionsPZM21 induced antinociceptive, reinforcing, and pruritic effects similar to clinically used MOP receptor agonists in primates. Although structure-based discovery of PZM21 identified a novel avenue for studying G-protein signalling-biased ligands, biasing an agonist towards G-protein signalling pathways did not determine or alter reinforcing (i.e. abuse potential) or pruritic effects of MOP receptor agonists in a translationally relevant non-human primate model.

Efficacy assessment of salicylidene salicylhydrazide in chemotherapy associated peripheral neuropathy

Chemotherapy-induced peripheral neuropathy (CIPN) is an increasingly important problem for cancer survivors and is the foremost cause of drug-induced morbidity. In this study, the antinociceptive efficacy of salicylidene salicylhydrazide (SSH) in CIPN was investigated. SSH was evaluated for acute toxicity, antinociceptive effectiveness against tonic and phasic pain modalities, anti-inflammatory propensity, and effect on motoric balance. SSH was tested in the mouse models of oxaliplatin, paclitaxel, and vincristine associated established neuropathic nociceptive paradigms. The tested doses of SSH (10–75 mg/kg) strongly suppressed the expression of acetic acid-induced tonic visceral nociception, formalin-induced biphasic nociception, and acute phasic thermal nociception. SSH selectively antagonized the capsaicin-elicited nociceptive behavior. SSH produced a significant reduction in the phlogistic agents-induced temporal inflammatory escalation involving prostaglandins, serotonin, and histamine. SSH was devoid of any adverse-effects that impair the neurological processes involved in the arousal and coordination of movements. The neuropathic nociception inflicted by chemotherapeutic agents were expressed as reduced sensitivity to non-noxious mechanical stimuli (mechanical allodynia), increased nociceptive response to cold (cold allodynia), and decreased nociceptive latency to heat (heat hyperalgesia). SSH (50 and 75 mg/kg) significantly suppressed the expression of CIPN-induced established neuropathic allodynia and hyperalgesia and the anti-neuropathic effects were equipotent to gabapentin. These findings concluded that SSH is a novel analgesic that can be useful for treating peripheral neuropathic pain conditions linked with chemotherapy with the advantage of being free of neurological adverse-effects encountered with gabapentinoids.

Antinociceptive Effect of the Citrus Flavonoid Eriocitrinon Postoperative Pain Conditions.

BackgroundPostoperative pain remains a major clinical problem as there are limited analgesic strategies that have been proven to be effective in preventing and relieving this type of pain. Natural products, including flavonoids, have distinct pharmacological properties and play an important role in the discovery of analgesic drugs.Materials and MethodsIn this study, the flavonoid eriocitrin (eriodictyol 7-O-rutinoside), which is the main flavonoid in lemon fruit (Citrus limon), was mechanistically investigated for its prospective antinociceptive effect in a mouse model of postoperative pain. The antinociceptive property was evaluated by utilizing both tonic (acetic acid-induced writhing behavior) and phasic (hot-plate) nociception modalities. The hindpaw incisional surgery was performed and hyperalgesia was assessed using von Frey filaments.ResultsThe tested doses of eriocitrin significantly attenuated (P<0.01, P<0.001) the chemically-induced tonic visceral nociception (5, 10, 15, and 30 mg/kg) and acute phasic thermal nociception (10, 15, and 30 mg/kg). A significant dose-dependent reduction in the incisional nociceptive hyperalgesia was exhibited by eriocitrin, with a marked antinociception observed at doses of 15 mg/kg (P<0.05 during 30–60 minutes) and 30 mg/kg (P<0.05, P<0.01 during 30–120 minutes).ConclusionThe antinociceptive effect of eriocitrin (30 mg/kg) was strongly blocked by the antagonists of the opioid receptor, naltrexone, and GABAA receptor, bicuculline, thereby suggesting the involvement of opioidergic and GABAergic mechanisms in the nociception, reducing proclivity of eriocitrin during transmission of incisional nociception. These results concluded that eriocitrin has a potent antinociceptive effect in postoperative pain conditions, probably mediated through opioid and GABAA receptors.

Adjunctive effect of the serotonin 5-HT2C receptor agonist lorcaserin on opioid-induced antinociception in mice

Opioid-sparing adjuncts are treatments that aim to reduce the overall dose of opioids needed to achieve analgesia, hence decreasing the burden of side effects through alternative mechanisms of action. Lorcaserin is a serotonin 5-HT2C receptor (5-HT2CR) agonist that has recently been reported to reduce abuse-related effects of the opioid analgesic oxycodone. The goal of our studies was to evaluate the effects of adjunctive lorcaserin on opioid-induced analgesic-like behavior using the tail-flick reflex (TFR) test as a mouse model of acute thermal nociception. We show that whereas subcutaneous (s.c.) administration of lorcaserin alone was inactive on the TFR test, adjunctive lorcaserin (s.c.) significantly increased the potency of oxycodone as an antinociceptive drug. This effect was prevented by the 5-HT2CR antagonist SB242084. A similar lorcaserin (s.c.)-induced adjunctive phenotype was observed upon administration of the opioid analgesics morphine and fentanyl. Remarkably, we also show that, opposite to the effects observed via s.c. administration, intrathecal (i.t.) administration of lorcaserin alone induced antinociceptive TFR behavior, an effect that was not prevented by the opioid receptor antagonist naloxone. This route of administration (i.t.) also led to a significant augmentation of oxycodone-induced antinociception. Lorcaserin (s.c.) did not alter the brain or blood concentrations of oxycodone, which suggests that its adjunctive effects on opioid-induced antinociception do not depend upon changes in opioid metabolism. Together, these data indicate that lorcaserin-mediated activation of the 5-HT2CR may represent a new pharmacological approach to augment opioid-induced antinociception.This article is part of the special issue entitled ‘Serotonin Research: Crossing Scales and Boundaries’.

Behavioral pharmacology of the mixed-action delta-selective opioid receptor agonist BBI-11008: studies on acute, inflammatory and neuropathic pain, respiration, and drug self-administration.

The present study characterized the behavioral pharmacology of a novel, mixed-action delta-selective (78:1) opioid receptor agonist, BBI-11008. This glycopeptide drug candidate was tested in assays assessing antinociception (acute, inflammatory, and neuropathic pain-like conditions) and side-effect endpoints (respiratory depression and drug self-administration). BBI-11008 had a 78-fold greater affinity for the delta opioid receptor than the mu receptor, and there was no binding to the kappa opioid receptor. BBI-11008 (3.2-100; 10-32mgkg-1, i.v.) and morphine (1-10; 1-3.2mgkg-1, i.v.) produced antinociceptive and anti-allodynic effects in assays of acute thermal nociception and complete Freund's adjuvant (CFA)-induced inflammatory pain, with BBI-11008 being less potent than morphine in both assays. BBI-11008 (1-18mgkg-1, i.v.) had similar efficacy to gabapentin (10-56mgkg-1, i.v.) in a spinal nerve ligation (SNL) model of neuropathic pain. In the respiration assay, with increasing %CO2 exposure, BBI-11008 produced an initial increase (32mgkg-1, s.c.) and then decrease (56mgkg-1, s.c.) in minute volume (MV) whereas morphine (3.2-32mgkg-1, s.c.) produced dose-dependent decreases in MV. In the drug self-administration procedure, BBI-11008 did not maintain self-administration at any dose tested. These results suggest that the glycopeptide drug candidate possesses broad-spectrum antinociceptive and anti-allodynic activity across a range of pain-like conditions. Relative to morphine or fentanyl, the profile for BBI-11008 in the respiration and drug self-administration assays suggests that BBI-11008 may have less pronounced deleterious side effects. Continued assessment of this compound is warranted.

Supraspinal and Peripheral, but Not Intrathecal, σ1R Blockade by S1RA Enhances Morphine Antinociception.

Sigma-1 receptor (σ1R) antagonism increases the effects of morphine on acute nociceptive pain. S1RA (E-52862) is a selective σ1R antagonist widely used to study the role of σ1Rs. S1RA alone exerted antinociceptive effect in the formalin test in rats and increased noradrenaline levels in the spinal cord, thus accounting for its antinociceptive effect. Conversely, while systemic S1RA failed to elicit antinociceptive effect by itself in the tail-flick test in mice, it did potentiate the antinociceptive effect of opioids in this acute pain model. The present study aimed to investigate the site of action and the involvement of spinal noradrenaline on the potentiation of opioid antinociception by S1RA on acute thermal nociception using the tail-flick test in rats. Local administration was performed after intrathecal catheterization or intracerebroventricular and rostroventral medullar (RVM) cannulae implantation. Noradrenaline levels in the spinal cord were evaluated using the concentric microdialysis technique in awake, freely-moving rats. Systemic or supraspinal administration of S1RA alone, while having no effect on antinociception, enhanced the effect of morphine in rats. However, spinal S1RA administration did not potentiate the antinociceptive effect of morphine. Additionally, the peripherally restricted opioid agonist loperamide was devoid of antinociceptive effect but produced antinociception when combined with S1RA. Neurochemical studies revealed that noradrenaline levels in the dorsal horn of the spinal cord were not increased at doses exerting potentiation of the antinociceptive effect of the opioid. In conclusion, the site of action of σ1R for opioid modulation on acute thermal nociception is located at the peripheral and supraspinal levels, and the opioid-potentiating effect is independent of the spinal noradrenaline increase produced by S1RA.

Chalcone derivatives: synthesis, in vitro and in vivo evaluation of their anti-anxiety, anti-depression and analgesic effects

Anxiety disorders, depression and pain are highly prevalent pathologies. Their pharmacotherapy is associated with unwanted side effects; hence there is a clinical need to develop more effective drugs with fewer adverse reactions.Chalcones are one of the major classes of naturally occurring compounds. Chalcones and their derivatives have a huge importance in medicinal chemistry, displaying a wide range of pharmacological activities including anti-inflammatory, antimicrobial, antioxidant, cytotoxic and antitumor actions.The aim of this work was to evaluate chalcone effects on different targets involved in these pathologies. We have synthesized a series of simple chalcone derivatives taking common structural requirements described in literature related to their anxiolytic-like, antidepressant-like and/or antinociceptive properties into account.Furthermore, their potential in vitro effects towards different targets involved in these pathologies were evaluated. We have obtained twenty chalcones with moderate to high yields and assessed their ability to bind distinctive receptors, from rat brain homogenates, by displacement of labelled specific ligands: [3H] FNZ (binding site of benzodiazepines/GABAA), [3H] 8-OH-DPAT (serotonin 5-HT1A) and [3H] DAMGO (μ-opioid). Those compounds that showed the better in vitro activities were evaluated in mice using different behavioural tasks. In vivo results showed that 5′-methyl-2′-hydroxychalcone (9) exerted anxiolytic-like effects in mice in the plus maze test. While chalcone nuclei (1) revealed antidepressant-like activities in the tail suspension test. In addition, the novel 5′-methyl-2′-hydroxy-3′-nitrochalcone (12) exhibited antinociceptive activity in acute chemical and thermal nociception tests (writhing and hot plate tests). In conclusion, chalcones are thus promising compounds for the development of novel drugs with central nervous system (CNS) actions.

μ-Opioid receptors in primary sensory neurons are essential for opioid analgesic effect on acute and inflammatory pain and opioid-induced hyperalgesia.

μ-Opioid receptors (MORs) are expressed peripherally and centrally, but the loci of MORs responsible for clinically relevant opioid analgesia are uncertain. Crossing Oprm1flox/flox and AdvillinCre/+ mice completely ablates MORs in dorsal root ganglion neurons and reduces the MOR expression level in the spinal cord. Presynaptic MORs expressed at primary afferent central terminals are essential for synaptic inhibition and potentiation of sensory input by opioids. MOR ablation in primary sensory neurons diminishes analgesic effects produced by systemic and intrathecal opioid agonists and abolishes chronic opioid treatment-induced hyperalgesia. These findings demonstrate a critical role of MORs expressed in primary sensory neurons in opioid analgesia and suggest new strategies to increase the efficacy and reduce adverse effects of opioids. The pain and analgesic systems are complex, and the actions of systemically administered opioids may be mediated by simultaneous activation of μ-opioid receptors (MORs, encoded by the Oprm1 gene) at multiple, interacting sites. The loci of MORs and circuits responsible for systemic opioid-induced analgesia and hyperalgesia remain unclear. Previous studies using mice in which MORs are removed from Nav1.8- or TRPV1-expressing neurons provided only an incomplete and erroneous view about the role of peripheral MORs in opioid actions in vivo. In the present study, we determined the specific role of MORs expressed in primary sensory neurons in the analgesic and hyperalgesic effects produced by systemic opioid administration. We generated Oprm1 conditional knockout (Oprm1-cKO) mice in which MOR expression is completely deleted from dorsal root ganglion neurons and substantially reduced in the spinal cord, which was confirmed by immunoblotting and immunocytochemical labelling. Both opioid-induced inhibition and potentiation of primary sensory input were abrogated in Oprm1-cKO mice. Remarkably, systemically administered morphine potently inhibited acute thermal and mechanical nociception and persistent inflammatory pain in control mice but had little effect in Oprm1-cKO mice. The analgesic effect of intrathecally administered morphine was also profoundly reduced in Oprm1-cKO mice. Additionally, chronic morphine treatment-induced hyperalgesia was absent in Oprm1-cKO mice. Our findings directly challenge the notion that clinically relevant opioid analgesia is mediated mostly by centrally expressed MORs. MORs in primary sensory neurons, particularly those expressed presynaptically at the first sensory synapse in the spinal cord, are crucial for both opioid analgesia and opioid-induced hyperalgesia.

C57BL/6 substrain differences in inflammatory and neuropathic nociception and genetic mapping of a major quantitative trait locus underlying acute thermal nociception.

Sensitivity to different pain modalities has a genetic basis that remains largely unknown. Employing closely related inbred mouse substrains can facilitate gene mapping of nociceptive behaviors in preclinical pain models. We previously reported enhanced sensitivity to acute thermal nociception in C57BL/6J (B6J) versus C57BL/6N (B6N) substrains. Here, we expanded on nociceptive phenotypes and observed an increase in formalin-induced inflammatory nociceptive behaviors and paw diameter in B6J versus B6N mice (Charles River Laboratories). No strain differences were observed in mechanical or thermal hypersensitivity or in edema following the Complete Freund’s Adjuvant model of inflammatory pain, indicating specificity in the inflammatory nociceptive stimulus. In the chronic constrictive nerve injury, a model of neuropathic pain, no strain differences were observed in baseline mechanical threshold or in mechanical hypersensitivity up to one month post-chronic constrictive nerve injury. We replicated the enhanced thermal nociception in the 52.5°C hot plate test in B6J versus B6N mice from The Jackson Laboratory. Using a B6J × B6N-F2 cross (N = 164), we mapped a major quantitative trait locus underlying hot plate sensitivity to chromosome 7 that peaked at 26 Mb (log of the odds [LOD] = 3.81, p < 0.01; 8.74 Mb-36.50 Mb) that was more pronounced in males. Genes containing expression quantitative trait loci associated with the peak nociceptive marker that are implicated in pain and inflammation include Ryr1, Cyp2a5, Pou2f2, Clip3, Sirt2, Actn4, and Ltbp4 (false discovery rate < 0.05). Future studies involving positional cloning and gene editing will determine the quantitative trait gene(s) and potential pleiotropy of this locus across pain modalities.

The analgesic efficacy of morphine varies with rat strain and experimental pain model: implications for target validation efforts in pain drug discovery

BackgroundTranslating efficacy of analgesic drugs from animal models to humans remains challenging. Reasons are multifaceted, but lack of sufficiently rigorous preclinical study design criteria and phenotypically relevant models may be partly responsible. To begin to address this fundamental issue, we assessed the analgesic efficacy of morphine in three inbred rat strains (selected based on stress reactivity and affective/pain phenotypes), and outbred Sprague Dawley (SD) rats supplied from two vendors.MethodsSensitivity to morphine (0.3–6.0 mg/kg, s.c.) was evaluated in the hot plate test of acute thermal nociception, the Complete Freund's Adjuvant (CFA) model of inflammatory‐induced mechanical hyperalgesia, and in a locomotor motility assay in male rats from the following strains; Lewis (LEW), Fischer (F344), Wistar Kyoto (WKY), and SD's from Envigo and Charles River.ResultsF344 and SD rats were similarly sensitive to morphine in hot plate and CFA‐induced inflammatory hyperalgesia (Minimum Effective Dose (MED) = 3.0 mg/kg). WKY rats developed a less robust mechanical hypersensitivity after CFA injection, and were less sensitive to morphine in both pain tests (MED = 6.0 mg/kg). LEW rats were completely insensitive to morphine in the hot plate test, in contrast to the reversal of CFA‐induced hyperalgesia (MED = 3.0 mg/kg). All strains exhibited a dose‐dependent reduction in locomotor activity at 3.0–6.0 mg/kg.ConclusionSensory phenotyping in response to acute thermal and inflammatory‐induced pain, and sensitivity to morphine in various inbred and outbred rat strains indicates that different pathophysiological mechanisms are engaged after injury. This could have profound implications for translating preclinical drug discovery efforts into pain patients.SignificanceThe choice of rat strain used in preclinical pain research can profoundly affect the outcome of experiments in relation to (a) nociceptive threshold responses, and (b) efficacy to analgesic treatment, in assays of acute and tonic inflammatory nociceptive pain.

In vitro and in vivo Pharmacological Activities of 14-O-Phenylpropyloxymorphone, a Potent Mixed Mu/Delta/Kappa-Opioid Receptor Agonist With Reduced Constipation in Mice.

Pain, particularly chronic pain, is still an unsolved medical condition. Central goals in pain control are to provide analgesia of adequate efficacy and to reduce complications associated with the currently available drugs. Opioids are the mainstay for the treatment of moderate to severe pain. However, opioid pain medications also cause detrimental side effects, thus highlighting the need of innovative and safer analgesics. Opioids mediate their actions via the activation of opioid receptors, with the mu-opioid receptor as the primary target for analgesia, but also for side effects. One long-standing focus of drug discovery is the pursuit for new opioids exhibiting a favorable dissociation between analgesia and adverse effects. In this study, we describe the in vitro and in vivo pharmacological profiles of the 14-O-phenylpropyl substituted analog of the mu-opioid agonist 14-O-methyloxymorphone (14-OMO). The consequence of the substitution of the 14-O-methyl in 14-OMO with a 14-O-phenylpropyl group on in vitro binding and functional activity, and in vivo behavioral properties (nociception and gastrointestinal motility) was investigated. In binding studies, 14-O-phenylpropyloxymorphone (POMO) displayed very high affinity at mu-, delta-, and kappa-opioid receptors (Ki values in nM, mu:delta:kappa = 0.073:0.13:0.30) in rodent brain membranes, with complete loss of mu-receptor selectivity compared to 14-OMO. In guinea-pig ileum and mouse vas deferens bioassays, POMO was a highly efficacious and full agonist, being more potent than 14-OMO. In the [35S]GTPγS binding assays with membranes from CHO cells expressing human opioid receptors, POMO was a potent mu/delta-receptor full agonist and a kappa-receptor partial agonist. In vivo, POMO was highly effective in acute thermal nociception (hot-plate test, AD50 = 0.7 nmol/kg) in mice after subcutaneous administration, with over 70- and 9000-fold increased potency than 14-OMO and morphine, respectively. POMO-induced antinociception is mediated through the activation of the mu-opioid receptor, and it does not involve delta- and kappa-opioid receptors. In the charcoal test, POMO produced fourfold less inhibition of the gastrointestinal transit than 14-OMO and morphine. In summary, POMO emerges as a new potent mixed mu/delta/kappa-opioid receptor agonist with reduced liability to cause constipation at antinociceptive doses.

Differential Tolerance to FTY720-Induced Antinociception in Acute Thermal and Nerve Injury Mouse Pain Models: Role of Sphingosine-1-Phosphate Receptor Adaptation.

The immunomodulatory prodrug 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), which acts as an agonist for sphingosine-1-phosphate (S1P) receptors (S1PR) when phosphorylated, is proposed as a novel pain therapeutic. In this study, we assessed FTY720-mediated antinociception in the radiant heat tail-flick test and in the chronic constriction injury (CCI) model of neuropathic pain in mice. FTY720 produced antinociception and antiallodynia, respectively, and these effects were dose-dependent and mimicked by the S1PR1-selective agonist CYM-5442. Repeated administration of FTY720 for 1 week produced tolerance to acute thermal antinociception, but not to antiallodynia in the CCI model. S1PR-stimulated [35S]GTPγS autoradiography revealed apparent desensitization of G protein activation by S1P or the S1PR1 agonist 5-[4-phenyl-5-(trifluoromethyl)-2-thienyl]-3-[3-(trifluoromethyl)phenyl]-1,2,4-oxadiazole (SEW-2871) throughout the brain. Similar results were seen in spinal cord membranes, whereby the Emax value of S1PR-stimulated [35S]GTPγS binding was greatly reduced in repeated FTY720-treated mice. These results suggest that S1PR1 is a primary target of FTY720 in alleviating both acute thermal nociception and chronic neuropathic nociception. Furthermore, the finding that tolerance develops to antinociception in the tail-flick test but not in chronic neuropathic pain suggests a differential mechanism of FTY720 action between these models. The observation that repeated FTY720 administration led to desensitized S1PR1 signaling throughout the central nervous system suggests the possibility that S1PR1 activation drives the acute thermal antinociceptive effects, whereas S1PR1 desensitization mediates the following: 1) tolerance to thermal antinociceptive actions of FTY720 and 2) the persistent antiallodynic effects of FTY720 in neuropathic pain by producing functional antagonism of pronociceptive S1PR1 signaling.

Negative modulation of spinal κ-opioid receptor-mediated antinociception by the µ-opioid receptor at selective doses of (-)-pentazocine.

The mixed-action κ-opioid receptor (KOR) agonist, pentazocine, binds to both KOR and the µ-opioid receptor (MOR). Racemic (±)-pentazocine and (−)-pentazocine, each administered systemically, have been shown to produce antinociception in various animal models. In contrast, racemic (±)-pentazocine failed to produce antinociception when administered intrathecally (i.t.). However, whether spinal activation of KOR and MOR by (−)-pentazocine produces antinociception and the relative contribution of KOR and MOR in mediating antinociception remain unknown. Hence, we investigated whether i.t. (−)-pentazocine produces dose-dependent modulation of acute thermal nociception. Drugs were administered intrathecally in Sprague-Dawley rats and tail flick latency was recorded. Pentazocine produced a significant antinociceptive effect that was mediated by KOR and/or MOR at differential doses. MOR blockade restored the antinociceptive effect of an ineffective dose and prolonged the duration of an effective dose of pentazocine. Hence, spinal KOR and MOR mediated the effect of pentazocine. This study provides evidence that spinal MOR negatively modulates the KOR-mediated antinociceptive effect of i.t. pentazocine.

Synthesis and Structure-Activity Relationships of LP1 Derivatives: N-Methyl-N-phenylethylamino Analogues as Novel MOR Agonists.

The opioid pharmacological profile of cis-(−)-N-normetazocine derivatives is deeply affected by the nature of their N-substituents. Here, our efforts were focused on the synthesis and pharmacological evaluation of novel derivatives of the lead LP1, a multitarget opioid analgesic compound featuring an N-phenylpropanamido substituent. LP1 derivatives 5a–d and 6a–d were characterized by flexible groups at the N-substituent that allow them to reposition themselves relative to cis-(−)-N-normetazocine nucleus, thus producing different pharmacological profiles at the mu, delta and kappa opioid receptors (MOR, DOR and KOR) in in vitro and in vivo assays. Among the series, compound 5c, with the best in vitro and in vivo profile, resulted a MOR agonist which displays a KiMOR of 6.1 nM in a competitive binding assay, and an IC50 value of 11.5 nM and an Imax of 72% in measurement of cAMP accumulation in HEK293 cells stably expressing MOR, with a slight lower efficacy than LP1. Moreover, in a mouse model of acute thermal nociception, compound 5c, intraperitoneally administered, exhibits naloxone-reversed antinociceptive properties with an ED50 of 4.33 mg/kg. These results expand our understanding of the importance of N-substituent structural variations in the opioid receptor profile of cis-(−)-N-normetazocine derivatives and identify a new MOR agonist useful for the development of novel opioid analgesics for pain treatment.

Leptin status alters buprenorphine-induced antinociception in obese mice with dysfunctional leptin receptors.

Buprenorphine is an opiate used for pain management and to treat opiate addiction. The cytokine leptin can modulate nociception, but the extent to which buprenorphine-induced antinociception varies as a function of leptin signaling has not been characterized. Four congenic mouse lines with phenotypes that include differences in body weight and leptin status were used to test the hypothesis that the antinociceptive effects of buprenorphine vary as function of sex and leptin signaling. Each mouse line was comprised of males (n=12) and females (n=12) for a total of 96 animals. Groups included C57BL/6J (B6) mice (wild type), B6 mice with diet-induced obesity (DIO), obese B6.Cg-Lepob/J (ob/ob) mice lacking leptin, and obese B6.BKS(D)-Leprdb/J (db/db) mice with dysfunctional leptin receptors. The dependent measure was tail flick latency (TFL) in seconds for mouse-initiated tail removal from a warm water bath. Independent variables were intraperitoneal administration of saline (control) or buprenorphine (0.3mg/kg). Within every mouse line, buprenorphine significantly increased TFL relative to saline. Compared to the other mouse lines, db/db mice with dysfunctional leptin receptors had a significantly longer TFL after saline and after buprenorphine. TFL did not vary significantly by body weight or sex. The results provide novel support for the interpretation that acute thermal nociception is associated with altered leptin signaling.

Influence of tramadol on acute thermal and mechanical cutaneous nociception in dogs

ObjectiveThe aim of the study was to evaluate the influence of tramadol on acute nociception in dogs. Study designExperimental, blinded, randomized, crossover study. AnimalsSix healthy laboratory Beagle dogs. MethodsDogs received three treatments intravenously (IV): isotonic saline placebo (P), tramadol 1 mg kg−1 (T1) and tramadol 4 mg kg−1 (T4). Thermal thresholds were determined by ramped contact heat stimulation (0.6 °C second−1) at the lateral thoracic wall. Mechanical thresholds (MT) were measured using a probe containing three blunted pins which were constantly advanced over the radial bone, using a rate of force increase of 0.8 N second−1. Stimulation end points were defined responses (e.g. skin twitch, head turn, repositioning, vocalization) or pre-set cut-out values (55 °C, 20 N). Thresholds were determined before treatment and at predetermined time points up to 24 hours after treatment. At each measurement point, blood was collected for determination of O-desmethyltramadol concentrations. The degree of sedation and behavioural side effects were recorded. Data were analysed by one-way anova and two-way anova for repeated measurements. ResultsThermal nociception was not influenced by drug treatment. Mechanical nociception was significantly increased between P and T1 at 120 and 240 minutes, and between P and T4 at 30, 60, 240 and 420 minutes. T1 and T4 did not differ. O-desmethyltramadol (M1) maximum plasma concentrations (Cmax) were 4.2±0.8 ng mL−1 and 14.3±2.8 ng mL−1 for T1 and T4, respectively. Times to reach maximum plasma concentrations (Tmax) were 27.6±6.3 minutes for T1 and 32.1±7.8 minutes for T4. No sedation occurred. There were signs of nausea and mild to moderate salivation in both groups. Conclusion and clinical relevanceTramadol was metabolized marginally to O-desmethyltramadol and failed to produce clinically relevant acute antinociception. Therefore, the use of tramadol for acute nociceptive pain is questionable in dogs.