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.

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.

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.

Structural Requirements for CNS Active Opioid Glycopeptides.

Glycopeptides related to ß-endorphin penetrate the blood-brain barrier (BBB) of mice to produce antinociception. Two series of glycopeptides were assessed for opioid receptor binding affinity. Attempts to alter the mu-selectivity of [D-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin (DAMGO)-related glycopeptides by altering the charged residues of the amphipathic helical address were unsuccessful. A series of pan-agonists was evaluated for antinociceptive activity (55 °C tail flick) in mice. A flexible linker was required to maintain antinociceptive activity. Circular dichroism (CD) in H2O, trifluoroethanol (TFE), and SDS micelles confirmed the importance of the amphipathic helices (11s → 11sG → 11) for antinociception. The glycosylated analogues showed only nascent helices and random coil conformations in H2O. Chemical shift indices (CSI) and nuclear Overhauser effects (NOE) with 600 MHz NMR and CD confirmed helical structures in micelles, which were rationalized by molecular dynamics calculations. Antinociceptive studies with mice confirm that these glycosylated endorphin analogues are potential drug candidates that penetrate the BBB to produce potent central effects.

The mixed-action delta/mu opioid agonist MMP-2200 does not produce conditioned place preference but does maintain drug self-administration in rats, and induces in vitro markers of tolerance and dependence.

Previous work in our laboratories provides preclinical evidence that mixed-action delta/mu receptor glycopeptides have equivalent efficacy for treating pain with reduced side effect profiles compared to widely used mu agonist analgesics such as morphine. This study evaluated the rewarding and reinforcing effects of a lead candidate, mixed-action delta/mu agonist MMP-2200, using a conditioned place preference assay as well as a drug self-administration procedure in rats. In place conditioning studies, rats underwent a 2-week conditioning protocol and were then tested for chamber preference. Rats receiving MMP-2200, at previously determined analgesic doses, could not distinguish between the drug and saline-paired chamber, whereas rats receiving the opioid agonist morphine showed a strong preference for the morphine-paired chamber. In self-administration studies, rats were trained to respond for the high efficacy mu opioid receptor agonist fentanyl on an FR5 schedule of reinforcement. Following complete dose-response determinations for fentanyl, a range of doses of MMP-2200 as well as morphine were tested. Relative to the mu agonist morphine, MMP-2200 maintained a significantly lower number of drug infusions. To begin investigating potential molecular mechanisms for the reduced side effect profile of MMP-2200, we also examined ßarrestin2 (ßarr2) recruitment and chronic MMP-2200 induced cAMP tolerance and super-activation at the human delta and mu receptors in vitro. MMP-2200 efficaciously recruited ßarr2 to both receptors, and induced cAMP tolerance and super-activation equivalent to or greater than morphine at both receptors. The in vivo findings suggest that MMP-2200 may be less reinforcing than morphine but may have some abuse potential. The reduced side effect profile cannot be explained by reduced ßarr2 recruitment or reduced cAMP tolerance and superactivation at the monomeric receptors in vitro.

Identification of opioid ligands possessing mixed micro agonist/delta antagonist activity among pyridomorphinans derived from naloxone, oxymorphone, and hydromorphone [correction of hydropmorphone].

A series of pyridomorphinans derived from naloxone, oxymorphone, and hydromorphone (7a-k) were synthesized and evaluated for binding affinity at the opioid delta, micro, and kappa receptors in brain membranes using radioligand binding assays and for functional activity in vitro using [(35)S]GTP-gamma-S binding assays in brain tissues and bioassays using guinea pig ileum (GPI) and mouse vas deferens (MVD) smooth muscle preparations. The pyridine ring unsubstituted pyridomorphinans possessing the oxymorphone and hydromorphone framework displayed nearly equal binding affinity at the micro and delta receptors. Their affinities at the kappa site were nearly 10-fold less than their binding affinities at the micro and delta sites. Introduction of aryl substituents at the 5'-position on the pyridine ring improved the binding affinity at the delta site while decreasing the binding affinity at the micro site. Nearly all of the ligands possessing an N-methyl group at the17-position with or without a hydroxyl group at the 14-position of the morphinan moiety displayed agonist activity at the micro receptor with varying potencies and efficacies. In the [(35)S]GTP-gamma-S binding assays, most of these pyridomorphinans were devoid of any significant agonist activity at the delta and kappa receptors but displayed moderate to potent antagonist activity at the delta receptors. In antinociceptive evaluations using the warm-water tail-withdrawal assay in mice, the pyridomorphinans produced analgesic effects with varying potencies and efficacies when administered by the intracerebroventricular route. Among the ligands studied, the hydromorphone-derived 4-chlorophenylpyridomorphinan 7h was identified as a ligand possessing a promising profile of mixed micro agonist/delta antagonist activity in vitro and in vivo. In a repeated administration paradigm in which the standard micro agonist morphine produces significant tolerance, repeated administration of the micro agonist/delta antagonist ligand 7h produced no tolerance. These results indicate that appropriate molecular manipulations of the morphinan templates could provide ligands with mixed micro agonist/delta antagonist profiles and such ligands may have the potential of emerging as novel analgesic drugs devoid of tolerance, dependence, and related side effects.