Heat-shock protein 90 (Hsp90) promotes opioid-induced anti-nociception by an ERK mitogen-activated protein kinase (MAPK) mechanism in mouse brain.

Recent advances in developing opioid treatments for pain with reduced side effects have focused on the signaling cascades of the µ-opioid receptor (MOR). However, few such signaling targets have been identified for exploitation. To address this need, we explored the role of heat-shock protein 90 (Hsp90) in opioid-induced MOR signaling and pain, which has only been studied in four previous articles. First, in four cell models of MOR signaling, we found that Hsp90 inhibition for 24 h with the inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) had different effects on protein expression and opioid signaling in each line, suggesting that cell models may not be reliable for predicting pharmacology with this protein. We thus developed an in vivo model using CD-1 mice with an intracerebroventricular injection of 17-AAG for 24 h. We found that Hsp90 inhibition strongly blocked morphine-induced anti-nociception in models of post-surgical and HIV neuropathic pain but only slightly blocked anti-nociception in a naive tail-flick model, while enhancing morphine-induced precipitated withdrawal. Seeking a mechanism for these changes, we found that Hsp90 inhibition blocks ERK MAPK activation in the periaqueductal gray and caudal brain stem. We tested these signaling changes by inhibiting ERK in the above-mentioned pain models and found that ERK inhibition could account for all of the changes in anti-nociception induced by Hsp90 inhibition. Taken together, these findings suggest that Hsp90 promotes opioid-induced anti-nociception by an ERK mechanism in mouse brain and that Hsp90 could be a future target for improving the therapeutic index of opioid drugs.

Evaluation of a Postoperative Pain-Like State on Motivated Behavior in Rats: Effects of Plantar Incision on Progressive-Ratio Food-Maintained Responding.

There has been recent interest in characterizing the effects of pain-like states on motivated behaviors in order to quantify how pain modulates goal-directed behavior and the persistence of that behavior. The current set of experiments assessed the effects of an incisional postoperative pain manipulation on food-maintained responding under a progressive-ratio (PR) operant schedule. Independent variables included injury state (plantar incision or anesthesia control) and reinforcer type (grain pellet or sugar pellet); dependent variables were tactile sensory thresholds and response breakpoint. Once responding stabilized on the PR schedule, separate groups of rats received a single ventral hind paw incision or anesthesia (control condition). Incision significantly reduced breakpoints in rats responding for grain, but not sugar. In rats responding for sugar, tactile hypersensitivity recovered within 24 hr, indicating a faster recovery of incision-induced tactile hypersensitivity compared to rats responding for grain, which demonstrated recovery at PD2. The NSAID analgesic, diclofenac (5.6 mg/kg) completely restored incision-depressed PR operant responding and tactile sensitivity at 3 hr following incision. The PR schedule differentiated between sucrose and grain, suggesting that relative reinforcing efficacy may be an important determinant in detecting pain-induced changes in motivated behavior.

K-12 Neuroscience Education Outreach Program: Interactive Activities for Educating Students about Neuroscience.

The University of New England's Center for Excellence in the Neurosciences has developed a successful and growing K-12 outreach program that incorporates undergraduate and graduate/professional students. The program has several goals, including raising awareness about fundamental issues in neuroscience, supplementing science education in area schools and enhancing undergraduate and graduate/professional students' academic knowledge and skill set. The outreach curriculum is centered on core neuroscience themes including: Brain Safety, Neuroanatomy, Drugs of Abuse and Addiction, Neurological and Psychiatric Disorders, and Cognition and Brain Function. For each theme, lesson plans were developed based upon interactive, small-group activities. Additionally, we've organized our themes in a "Grow-up, Grow-out" approach. Grow-up refers to returning to a common theme, increasing in complexity as we revisit students from early elementary through high school. Grow-out refers to integrating other scientific fields into our lessons, such as the chemistry of addiction, the physics of brain injury and neuronal imaging. One of the more successful components of our program is our innovative team-based model of curriculum design. By creating a team of undergraduate, graduate/professional students and faculty, we create a unique multi-level mentoring opportunity that appears to be successful in enhancing undergraduate students' skills and knowledge. Preliminary assessments suggest that undergraduates believe they are enhancing their content knowledge and professional skills through our program. Additionally, we're having a significant, short-term impact on K-12 interest in science. Overall, our program appears to be enhancing the academic experience of our undergraduates and exciting K-12 students about the brain and science in general.

6ß-N-heterocyclic substituted naltrexamine derivative NAP as a potential lead to develop peripheral mu opioid receptor selective antagonists.

A 6ß-N-heterocyclic substituted naltrexamine derivative, NAP, was proposed as a peripheral mu opioid receptor (MOR) selective antagonist based on the in vitro and in vivo pharmacological and pharmacokinetic studies. To further validate this notion, several functional assays were carried out to fully characterize this compound. In the charcoal gavage and intestinal motility assay in morphine-pelleted mice, when administered 0.3 mg/kg or higher doses up to 3 mg/kg subcutaneously, NAP significantly increased the intestinal motility compared to the saline treatment. The comparative opioid withdrawal precipitation study and the lower locomotor assay demonstrated that NAP showed only marginal intrinsic effect in the central nervous system either given subcutaneously or intravenously: no jumps were witnessed for the tested animals even given up to a dose of 50 mg/kg, while similar noticeable wet-dog shakes only occurred at the dose 50 times of those for naloxone or naltrexone, and significant reduction of the hyper-locomotion only happened at the dose as high as 32 mg/kg. Collectively, these results suggested that NAP may serve as a novel lead to develop peripheral MOR selective antagonist which might possess therapeutic potential for opioid-induced bowel dysfunction (OBD), such as opioid-induced constipation (OIC).

In vivo characterization of MMP-2200, a mixed δ/µ opioid agonist, in mice.

We have previously reported the chemistry and antinociceptive properties of a series of glycosylated enkephalin analogs (glycopeptides) exhibiting approximately equal affinity and efficacy at δ opioid receptors (DORs) and µ opioid receptors (MORs). More detailed pharmacology of the lead glycopeptide MMP-2200 [H2N-Tyr-D-Thr-Gly-Phe-Leu-Ser-(O-ß-D-lactose)-CONH2] is presented. MMP-2200 produced dose-related antinociception in the 55°C tail-flick assay after various routes of administration. The antinociceptive effects of MMP-2200 were blocked by pretreatment with the general opioid antagonist naloxone and partially blocked by the MOR-selective antagonist ß-funaltrexamine and the DOR-selective antagonist naltrindole. The κ opioid receptor antagonist nor-binaltorphimine and the peripherally active opioid antagonist naloxone-methiodide were ineffective in blocking the antinociceptive effects of MMP-2200. At equi-antinociceptive doses, MMP-2200 produced significantly less stimulation of locomotor activity compared with morphine. Repeated administration of equivalent doses of morphine and MMP-2200 (twice daily for 3 days) produced antinociceptive tolerance (~13- and 5-fold rightward shifts, respectively). In acute and chronic physical dependence assays, naloxone precipitated a more severe withdrawal in mice receiving morphine compared with equivalent doses of the glycopeptide. Both morphine and MMP-2200 inhibited respiration and gastrointestinal transit. In summary, MMP-2200 acts as a mixed DOR/MOR agonist in vivo, which may in part account for its high antinociceptive potency after systemic administration, as well as its decreased propensity to produce locomotor stimulation, tolerance, and physical dependence in mice, compared with the MOR-selective agonist morphine. For other measures (e.g., gastrointestinal transit and respiration), the significant MOR component may not allow differentiation from morphine.

6ß-naltrexol, a peripherally selective opioid antagonist that inhibits morphine-induced slowing of gastrointestinal transit: an exploratory study.

OBJECTIVE: Opioid-induced constipation is a frequent side effect of opioid pain therapy due to opioid effects on the enteric nervous system, including gastric emptying and fluid absorption. The current exploratory studies were conducted to determine whether the neutral opioid antagonist 6ß-naltrexol, the primary metabolite of naltrexone, could selectively inhibit gastrointestinal opioid effects in human subjects. DESIGN: Volunteers participated in a randomized, double-blind, placebo-controlled, five-way crossover study under an Exploratory Investigational New Drug application. INTERVENTIONS: 6ß-Naltrexol has been reported to act as a neutral antagonist with peripheral selectivity in opioid-naïve and opioid-dependent systems in vitro and in vivo. SUBJECTS: Ten healthy, opioid-naïve male volunteers were enrolled in the study. OUTCOME MEASURES: Oral-cecal transit time was measured using the lactulose-hydrogen breath test. For central nervous system effects, analgesia was evaluated using a cold pressor test, and pupil size was measured. Blood samples were collected over 36 hours for pharmacokinetic analyses. RESULTS: The mean terminal plasma elimination half-life of 6ß-naltrexol was 11.1±2.4 hours. 6ß-Naltrexol potently blocked morphine-induced slowing of gastrointestinal transit, with a median effective dose (ED(50) ) of ~3 mg. In contrast, no effect was observed with 6ß-naltrexol doses up to 20 mg on morphine-induced analgesia or pupil constriction. Intravenous 6ß-naltrexol infusion over 30 minutes was well-tolerated up to the highest dose tested. CONCLUSIONS: 6ß-Naltrexol acts as a potent, peripherally selective opioid antagonist. The compound was well-tolerated in this study and may have clinical potential in the therapy of peripheral opioid effects such as opioid-induced constipation.

Comprehensive somatosensory and neurological phenotyping of NCS1 knockout mice.

Neuronal calcium sensor 1 (NCS1) regulates a wide range of cellular functions throughout the mammalian nervous systems. Altered NCS1 expression is associated with neurodevelopmental and neurodegenerative diseases. Previous studies focused on affective and cognitive behaviors in NCS1 knockout (KO) mice, but little is known about the physiological and pathological states associated with the loss of NCS1 in the peripheral nervous system. We previously reported that NCS1 expression was reduced following paclitaxel-induced peripheral neuropathy. Here, we comprehensively investigated the phenotypes of NCS1-KO mice through a battery of behavioral tests examining both central and peripheral nervous systems. Generally, only mild differences were observed in thermal sensation and memory acquisition between NCS1-WT and -KO male mice, but not in female mice. No differences were observed in motor performance, affective behaviors, and hearing in both sexes. These results suggest that NCS1 plays a modulatory role in sensory perceptions and cognition, particularly in male mice. NCS1 has been proposed as a pharmacological target for various diseases. Therefore, the sex-specific effects of NCS1 loss may be of clinical interest. As we examined a constitutive KO model, future studies focusing on various conditional KO models will further elucidate the precise physiological significance of NCS1.

Identification of a novel "almost neutral" micro-opioid receptor antagonist in CHO cells expressing the cloned human mu-opioid receptor.

The basal (constitutive) activity of G protein-coupled receptors allows for the measurement of inverse agonist activity. Some competitive antagonists turn into inverse agonists under conditions where receptors are constitutively active. In contrast, neutral antagonists have no inverse agonist activity, and they block both agonist and inverse agonist activity. The mu-opioid receptor (MOR) demonstrates detectable constitutive activity only after a state of dependence is produced by chronic treatment with a MOR agonist. We therefore sought to identify novel MOR inverse agonists and novel neutral MOR antagonists in both untreated and agonist-treated MOR cells. CHO cells expressing the cloned human mu receptor (hMOR-CHO cells) were incubated for 20 h with medium (control) or 10 microM (2S,4aR,6aR,7R,9S,10aS,10bR)-9-(benzoyloxy)-2-(3-furanyl)dodecahydro-6a,10b-dimethyl-4,10-dioxo-2H-naphtho-[2,1-c]pyran-7-carboxylic acid methyl ester (herkinorin, HERK). HERK treatment generates a high degree of basal signaling and enhances the ability to detect inverse agonists. [(35)S]-GTP-gamma-S assays were conducted using established methods. We screened 21 MOR "antagonists" using membranes prepared from HERK-treated hMOR-CHO cells. All antagonists, including CTAP and 6beta-naltrexol, were inverse agonists. However, LTC-274 ((-)-3-cyclopropylmethyl-2,3,4,4alpha,5,6,7,7alpha-octahydro-1H-benzofuro[3,2-e]isoquinolin-9-ol)) showed the lowest efficacy as an inverse agonist, and, at concentrations less than 5 nM, had minimal effects on basal [(35)S]-GTP-gamma-S binding. Other efforts in this study identified KC-2-009 ((+)-3-((1R,5S)-2-((Z)-3-phenylallyl)-2-azabicyclo[3.3.1]nonan-5-yl)phenol hydrochloride) as an inverse agonist at untreated MOR cells. In HERK-treated cells, KC-2-009 had the highest efficacy as an inverse agonist. In summary, we identified a novel and selective MOR inverse agonist (KC-2-009) and a novel MOR antagonist (LTC-274) that shows the least inverse agonist activity among 21 MOR antagonists. LTC-274 is a promising lead compound for developing a true MOR neutral antagonist.

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.

RATIONALE AND OBJECTIVES: 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). RESULTS: 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; 10-32 mg kg-1, i.v.) and morphine (1-10; 1-3.2 mg kg-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 (1-18 mg kg-1, i.v.) had similar efficacy to gabapentin (10-56 mg kg-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 (32 mg kg-1, s.c.) and then decrease (56 mg kg-1, s.c.) in minute volume (MV) whereas morphine (3.2-32 mg kg-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. CONCLUSIONS: 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.

Synthesis and Structure-Activity Relationships of 5'-Aryl-14-alkoxypyridomorphinans: Identification of a µ Opioid Receptor Agonist/δ Opioid Receptor Antagonist Ligand with Systemic Antinociceptive Activity and Diminished Opioid Side Effects.

We previously identified a pyridomorphinan (6, SRI-22138) possessing a 4-chlorophenyl substituent at the 5'-position on the pyridine and a 3-phenylpropoxy at the 14-position of the morphinan as a mixed µ opioid receptor (MOR) agonist and δ/κ opioid receptor (DOR/KOR) antagonist with potent antinociceptive activity and diminished tolerance and dependence in rodents. Structural variations at the 5'- and 14-positions of this molecule gave insights into the structure-activity relationships for binding and functional activity. Subtle structural changes exerted significant influence, particularly on the ability of the compounds to function as agonists at the MOR. In vivo evaluation identified compound 20 (SRI-39067) as a MOR agonist/DOR antagonist that produced systemically active potent antinociceptive activity in tail-flick assay in mice, with diminished tolerance, dependence/withdrawal, reward liability, and respiratory depression versus morphine. These results support the hypothesis that mixed MOR agonist/DOR antagonist ligands may emerge as novel opioid analgesics with reduced side effects.

Design, synthesis, and characterization of 6beta-naltrexol analogs, and their selectivity for in vitro opioid receptor subtypes.

Since the mu opioid receptor (MOR) is known to be involved in the therapeutically relevant pathways leading to the manifestation of pain and addiction, we are currently studying the specific structural characteristics that promote antagonism at the MOR. The opiates 6beta-naltrexol and 6beta-naltrexamide function as neutral antagonists in in vitro and in vivo systems previously exposed to morphine, and are under investigation as improved treatments for narcotic dependence. In this research, we synthesized and characterized carbamate and sulfonate ester derivates of 6beta-naltrexol that do not contain a protic group at C(6), and evaluated these compounds for opioid receptor affinity. In vitro receptor subtype (mu, kappa, and delta opioid receptors) binding data of the carbamate and sulfonate derivatives is reported. All four compounds synthesized exhibited affinity for the MOR better than the standard 6beta-naltrexol HCl. Based on K(i) data, the order of MOR affinity is as follows: 9>13>14>10>6beta-naltrexol HCl. Carbamate 9 and tosylate 13 displayed subnanomolar affinity for the MOR, while 10 was the most mu-selective compound synthesized. In conclusion, our data indicate that the absence of a hydrogen-bond donor on the C(6) oxygen enhances rather than impedes the in vitro affinity of naltrexol derivatives for the MOR. Additionally, data also suggest that increasing the bulk around C(6) may allow control of subtype selectivity within these compound series.

Behavioral pharmacology of the mu/delta opioid glycopeptide MMP2200 in rhesus monkeys.

H(2)N-Tyr-D-Thr-Gly-Phe-Leu-Ser-(O-beta-D-lactose)-CONH(2) (MMP2200) is a novel glycopeptide opioid agonist with similar affinities for mu and delta receptors. Glycosylation promoted brain penetration and production of centrally mediated behavioral effects in mice; however, it is unknown whether the magnitude of enhanced brain penetration is sufficient to permit central mediation of drug effects and production of synergistic mu/delta antinociceptive interactions after systemic administration in primates. To address this issue, the present study compared the effects of MMP2200 and the mu-agonist morphine in four behavioral procedures in rhesus monkeys. In an assay of thermal nociception, morphine (1.0-5.6 mg/kg) produced dose-dependent antinociception, whereas MMP2200 (10-56 mg/kg) was ineffective. In an assay of capsaicin-induced thermal allodynia, both morphine (0.01-1.0 mg/kg) and MMP2200 (0.032-3.2 mg/kg) produced dose-dependent antiallodynic effects. MMP2200-induced antiallodynia was blocked by the moderately mu-selective antagonist naltrexone (0.01 mg/kg), the delta-selective antagonist naltrindole (1.0 mg/kg), and the peripherally selective opioid antagonist quaternary naltrexone (0.32 mg/kg). In an assay of schedule-controlled behavior, both morphine (0.01-1.0 mg/kg) and MMP2200 (10-56 mg/kg) decreased response rates. Morphine effects were antagonized by naltrexone (0.001-0.01 mg/kg); however, the effects of MMP2200 were not antagonized by either naltrexone (0.01 mg/kg) or naltrindole (1.0 mg/kg). In an assay of drug self-administration, morphine (0.0032-0.32 mg/kg/injection) produced reinforcing effects, whereas MMP2200 (0.032-0.32 mg/kg/injection) did not. These results suggest that systemically administered MMP2200 acted as a peripheral, mu/delta-opioid agonist with limited distribution to the central nervous system in rhesus monkeys. These results also suggest the existence of species differences in the pharmacokinetics and brain penetration of glycopeptides.

Peripherally Acting µ-Opioid Receptor Antagonists for the Treatment of Opioid-Related Side Effects: Mechanism of Action and Clinical Implications.

Opioid receptors are distributed throughout the central and peripheral nervous systems and on many nonneuronal cells. Therefore, opioid administration induces effects beyond analgesia. In the enteric nervous system (ENS), stimulation of µ-opioid receptors triggers several inhibitory responses that can culminate in opioid-induced bowel dysfunction (OBD) and its most common side effect, opioid-induced constipation (OIC). OIC negatively affects patients' quality of life (QOL), ability to work, and pain management. Although laxatives are a common first-line OIC therapy, most have limited efficacy and do not directly antagonize opioid effects on the ENS. Peripherally acting µ-opioid receptor antagonists (PAMORAs) with limited ability to cross the blood-brain barrier have been developed. The PAMORAs approved by the U S Food and Drug Administration for OIC are subcutaneous and oral methylnaltrexone, oral naloxegol, and oral naldemedine. Although questions of cost-effectiveness and relative efficacy versus laxatives remain, PAMORAs can mitigate OIC and improve patient QOL. PAMORAS may also have applications beyond OIC, including reducing the increased cardiac risk or potential tumorigenic effects of opioids. This review discusses the burden of OIC and OBD, reviews the mechanism of action of new OIC therapies, and highlights other potential opioid-related side effects mediated by peripheral opioid receptors in the context of new OIC therapies.

Probes for narcotic receptor mediated phenomena. 34. Synthesis and structure-activity relationships of a potent mu-agonist delta-antagonist and an exceedingly potent antinociceptive in the enantiomeric C9-substituted 5-(3-hydroxyphenyl)-N-phenylethylmorphan series.

Both of the enantiomers of 5-(3-hydroxyphenyl)-N-phenylethylmorphan with C9alpha-methyl, C9-methylene, C9-keto, and C9alpha- and C9beta-hydroxy substituents were synthesized and pharmacologically evaluated. Three of the 10 compounds, (1R,5R,9S)-(-)-9-hydroxy-5-(3-hydroxyphenyl-2-phenylethyl-2-azabicyclo[3.3.1]nonane ((1R,5R,9S)-(-)-10), (1R,5S)-(+)-5-(3-hydroxyphenyl)-9-methylene-2-phenethyl-2-azabicyclo[3.3.1]nonane ((1R,5S)-(+)-14), and (1R,5S,9R)-(-)-5-(3-hydroxyphenyl)-9-methyl-2-phenethyl-2-azabicyclo[3.3.1]nonane ((1R,5S,9R)-(+)-15) had subnanomolar affinity at mu-opioid receptors (Ki = 0.19, 0.19, and 0.63 nM, respectively). The (1R,5S)-(+)-14 was found to be a mu-opioid agonist and a mu-, delta-, and kappa-antagonist in [35S]GTP-gamma-S assays and was approximately 50 times more potent than morphine in a number of acute and subchronic pain assays, including thermal and visceral models of nociception. The (1R,5R,9S)-(-)-10 compound with a C9-hydroxy substituent axially oriented to the piperidine ring (C9beta-hydroxy) was a mu-agonist about 500 times more potent than morphine. In the single-dose suppression assay, it was greater than 1000 times more potent than morphine. It is the most potent known phenylmorphan antinociceptive. The molecular structures of these compounds were energy minimized with density functional theory at the B3LYP/6-31G* level and then overlaid onto (1R,5R,9S)-(-)-10 using the heavy atoms in the morphan moiety as a common docking point. Based on modeling, the spatial arrangement of the protonated nitrogen atom and the 9beta-OH substituent in (1R,5R,9S)-(-)-10 may facilitate the alignment of a putative water chain enabling proton transfer to a nearby proton acceptor group in the mu-opioid receptor.

Long-term morphine delivery via slow release morphine pellets or osmotic pumps: Plasma concentration, analgesia, and naloxone-precipitated withdrawal.

AIMS: Slow-release morphine sulfate pellets and osmotic pumps are common routes of chronic morphine delivery in mouse models, but direct comparisons of these drug delivery systems are lacking. In this study, we assessed the efficacy of slow-release pellets versus osmotic pumps in delivering morphine to adult mice. MAIN METHODS: Male C57BL/6NCr mice (8weeksold) were implanted subcutaneously with slow-release pellets (25mg morphine sulfate) or osmotic pumps (64mg/mL, 1.0µL/h). Plasma morphine concentrations were quantified via LC-MS/MS, analgesic efficacy was determined by tail flick assay, and dependence was assessed with naloxone-precipitated withdrawal behaviors (jumping) and physiological effects (excretion, weight loss). KEY FINDINGS: Morphine pellets delivered significantly higher plasma drug concentrations compared to osmotic pumps, which were limited by the solubility of the morphine sulfate and pump volume/flow rate. Within 96h post-implantation, plasma morphine concentrations were indistinguishable in pellet vs. pump-treated samples. While osmotic pump did not have an antinociceptive effect in the tail flick assay, pumps and pellets induced comparable dependence symptoms (naloxone-precipitated jumping behavior) from 24-72h post-implantation. SIGNIFICANCE: In this study, we compared slow-release morphine pellets to osmotic minipumps for morphine delivery in mice. We found that osmotic pumps and subcutaneous morphine sulfate pellets yielded significantly different pharmacokinetics over a 7-day period, and as a result significantly different antinociceptive efficacy. Nonetheless, both delivery methods induced dependence as measured by naloxone-precipitated withdrawal.

Exercise reverses pain-related weight asymmetry and differentially modulates trabecular bone microarchitecture in a rat model of osteoarthritis.

There is great interest in developing and utilizing non-pharmacological/non-invasive forms of therapy for osteoarthritis (OA) pain including exercise and other physical fitness regimens. AIMS: The present experiments determined the effects of prior wheel running on OA-induced weight asymmetry and trabecular bone microarchitecture. MAIN METHODS: Wheel running included 7 or 21days of prior voluntary access to wheels followed by OA induction, followed by 21days post-OA access to wheels. OA was induced with monosodium iodoacetate (MIA), and weight asymmetry was measured using a hind limb weight bearing apparatus. Bone microarchitecture was characterized using ex vivo µCT. KEY FINDINGS: Relative to saline controls, MIA (3.2mg/25µl) produced significant weight asymmetry measured on post-days (PDs) 3, 7, 14, 21 in sedentary rats. Seven days of prior running failed to alter MIA-induced weight asymmetry. In contrast, 21days of prior running resulted in complete reversal of MIA-induced weight asymmetry on all days tested. As a comparator, the opioid agonist morphine (3.2-10mg/kg) dose-dependently reversed weight asymmetry on PDs 3, 7, 14, but was ineffective in later-stage (PD 21) OA. In runners, Cohen's d (effect sizes) for OA vs. controls indicated large increases in bone volume fraction, trabecular number, trabecular thickness, and connective density in lateral compartment, and large decreases in the same parameters in medial compartment. In contrast, effect sizes were small to moderate for sedentary OA vs. SIGNIFICANCE: Results indicate that voluntary exercise may protect against OA pain, the effect varies as a function of prior exercise duration, and is associated with distinct trabecular bone modifications.

Targeting pain-suppressed behaviors in preclinical assays of pain and analgesia: effects of morphine on acetic acid-suppressed feeding in C57BL/6J mice.

UNLABELLED: Pain increases the rate, frequency, or intensity of some behaviors (eg, withdrawal responses) and suppresses other behaviors (eg, feeding). Our laboratories are developing assays to test analgesic drug candidates using measurements of pain-suppressed rather than pain-elicited behaviors. Such assays may model important aspects of clinical pain and provide a means for distinguishing true analgesics from drugs that produce motor impairment. The present study compared effects of the mu opioid analgesic morphine and the nonanalgesic neuroleptic haloperidol on intraperitoneal acetic acid-induced writhing (a pain-elicited behavior) and suppression of feeding behavior (a pain-suppressed behavior). In feeding studies, C57BL/6J mice were given access to a dish containing 8 mL Ensure(trade mark) liquid food (0-100% in water) during daily sessions (7.5-120 min). Levels of consumption were dependent on both Ensure concentration and session duration. Intraperitoneal injection of acetic acid (0.10-0.56%) produced a time- and concentration-dependent decrease in Ensure consumption. Morphine (1 mg/kg) prevented both acid-induced writhing and acid-induced suppression of feeding, whereas the dopamine antagonist haloperidol inhibited writhing without preventing acid-induced suppression of feeding. The effects of morphine were time-dependent, selective for acid-suppressed feeding, and naltrexone-reversible. These results suggest that assays of pain-suppressed behaviors may complement assays of pain-elicited behaviors in preclinical studies of candidate analgesics. PERSPECTIVE: This paper presents a new preclinical strategy for assessing pain and analgesia in mice that is congruent with current methods of pain assessment in the clinic. This strategy may therefore be a useful complement to more traditional procedures for assessing pain and analgesia.

FAAH inhibitor OL-135 disrupts contextual, but not auditory, fear conditioning in rats.

Anxiety disorders are among the most prevalent psychological disorders, have significant negative impacts on quality of life and the healthcare system, and yet effective treatments remain elusive. Manipulating the endocannabinoid system has demonstrated potential for treating anxiety, although the side effects of direct manipulations of cannabinoid receptors keeps them from widespread clinical use. Disrupting the degradation enzyme fatty acid amide hydrolase (FAAH) enhances endogenous signaling and may produce similar efficacy without the side effects. The current experiments examine the effects of low (5.6mg/kg) or moderate (10.0mg/kg) doses of OL-135, a FAAH inhibitor, on the acquisition and consolidation of classical fear conditioning, a common model of trauma-induced anxiety. The acquisition of contextual, but not auditory, fear conditioning was disrupted by both doses of OL-135. Shock reactivity was not affected. Due to the additional neural circuitry required for contextual, but not auditory, fear conditioning, these data suggest that endocannabinoid signaling outside the amygdala may be critical for a subset of fearful memories.

17-Cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-(4'-pyridylcarboxamido)morphinan (NAP) Modulating the Mu Opioid Receptor in a Biased Fashion.

Mounting evidence has suggested that G protein-coupled receptors can be stabilized in multiple conformations in response to distinct ligands, which exert discrete functions through selective activation of various downstream signaling events. In accordance with this concept, we report biased signaling of one C6-heterocyclic substituted naltrexamine derivative, namely, 17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6ß-(4'-pyridylcarboxamido)morphinan (NAP) at the mu opioid receptor (MOR). NAP acted as a low efficacy MOR partial agonist in the G protein-mediated [(35)S]GTPγS binding assay, whereas it did not significantly induce calcium flux or ß-arrestin2 recruitment. In contrast, it potently blocked MOR full agonist-induced ß-arrestin2 recruitment and translocation. Additionally, NAP dose-dependently antagonized MOR full agonist-induced intracellular calcium flux and ß-arrestin2 recruitment. Further results in an isolated organ bath preparation confirmed that NAP reversed the morphine-induced reduction in colon motility. Ligand docking and dynamics simulation studies of NAP at the MOR provided more supporting evidence for biased signaling of NAP at an atomic level. Due to the fact that NAP is MOR selective and preferentially distributed peripherally upon systemic administration while ß-arrestin2 is reportedly required for impairment of intestinal motility by morphine, biased antagonism of ß-arrestin2 recruitment by NAP further supports its utility as a treatment for opioid-induced constipation.

Basal opioid receptor activity, neutral antagonists, and therapeutic opportunities.

The mu opioid receptor (MOR, OPRM)--the principal receptor involved in narcotic addiction--has been shown to display basal (spontaneous, constitutive) signaling activity. Interaction with other signaling proteins, such as calmodulin, regulates basal MOR activity. Providing a mechanism for long-lasting regulation, basal MOR activity potentially plays a key role in addiction, in combination with gene regulation and synaptic remodeling. Recent results support a link to physical dependence--one of the main manifestations of addiction to drugs of abuse. The prototypical opioid antagonists, naloxone and naltrexone, were shown to act as inverse agonists in the morphine-dependent state (i.e., they suppress basal MOR signaling) and thereby appear to elicit or contribute to precipitated withdrawal. This affords the opportunity to explore therapeutic applications for neutral antagonists (blocking agonists at MOR without affecting basal activity) with reduced adverse effects. Neutral antagonists are promising drug candidates in the treatment of addiction and overdose, and of peripheral adverse effects of narcotic analgesics.