Author Correction: Structural insights into µ-opioid receptor activation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Photochemical Fingerprinting Is a Sensitive Probe for the Detection of Synthetic Cannabinoid Receptor Agonists; toward Robust Point-of-Care Detection.

With synthetic cannabinoid receptor agonist (SCRA) use still prevalent across Europe and structurally advanced generations emerging, it is imperative that drug detection methods advance in parallel. SCRAs are a chemically diverse and evolving group, which makes rapid detection challenging. We have previously shown that fluorescence spectral fingerprinting (FSF) has the potential to provide rapid assessment of SCRA presence directly from street material with minimal processing and in saliva. Enhancing the sensitivity and discriminatory ability of this approach has high potential to accelerate the delivery of a point-of-care technology that can be used confidently by a range of stakeholders, from medical to prison staff. We demonstrate that a range of structurally distinct SCRAs are photochemically active and give rise to distinct FSFs after irradiation. To explore this in detail, we have synthesized a model series of compounds which mimic specific structural features of AM-694. Our data show that FSFs are sensitive to chemically conservative changes, with evidence that this relates to shifts in the electronic structure and cross-conjugation. Crucially, we find that the photochemical degradation rate is sensitive to individual structures and gives rise to a specific major product, the mechanism and identification of which we elucidate through density-functional theory (DFT) and time-dependent DFT. We test the potential of our hybrid "photochemical fingerprinting" approach to discriminate SCRAs by demonstrating SCRA detection from a simulated smoking apparatus in saliva. Our study shows the potential of tracking photochemical reactivity via FSFs for enhanced discrimination of SCRAs, with successful integration into a portable device.

Instant Detection of Synthetic Cannabinoids on Physical Matrices, Implemented on a Low-Cost, Ultraportable Device.

Synthetic cannabinoids (SCs) make up a class of novel psychoactive substances (NPS), used predominantly in prisons and homeless communities in the U.K. SCs can have severe side effects, including psychosis, stroke, and seizures, with numerous reported deaths associated with their use. The chemical diversity of SCs presents the major challenge to their detection since approaches relying on specific molecular recognition become outdated almost immediately. Ideally one would have a generic approach to detecting SCs in portable settings. The problem of SC detection is more challenging still because the majority of SCs enter the prison estate adsorbed onto physical matrices such as paper, fabric, or herb materials. That is, regardless of the detection modality used, the necessary extraction step reduces the effectiveness and ability to rapidly screen materials on-site. Herein, we demonstrate a truly instant generic test for SCs, tested against real-world drug seizures. The test is based on two advances. First, we identify a spectrally silent region in the emission spectrum of most physical matrices. Second, the finding that background signals (including from autofluorescence) can be accurately predicted is based on tracking the fraction of absorbed light from the irradiation source. Finally, we demonstrate that the intrinsic fluorescence of a large range of physical substrates can be leveraged to track the presence of other drugs of interest, including the most recent iterations of benzodiazepines and opioids. We demonstrate the implementation of our presumptive test in a portable, pocket-sized device that will find immediate utility in prisons and law enforcement agencies around the world.

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

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

Pleiotropic Effects of Kappa Opioid Receptor-Related Ligands in Non-human Primates.

The kappa opioid receptor (KOR)-related ligands have been demonstrated in preclinical studies for several therapeutic potentials. This chapter highlights (1) how non-human primates (NHP) studies facilitate the research and development of ligands targeting the KOR, (2) effects of the endogenous opioid peptide, dynorphin A-(1-17), and its analogs in NHP, and (3) pleiotropic effects and therapeutic applications of KOR-related ligands. In particular, synthetic ligands targeting the KOR have been extensively studied in NHP in three therapeutic areas, i.e., the treatment for itch, pain, and substance use disorders. As the KORs are widely expressed in the peripheral and central nervous systems, pleiotropic effects of KOR-related ligands, such as discriminative stimulus effects, neuroendocrine effects (e.g., prolactin release and stimulation of hypothalamic-pituitary-adrenal axis), and diuresis, in NHP are discussed. Centrally acting KOR agonists are known to produce adverse effects including dysphoria, hallucination, and sedation. Nonetheless, with strategic advances in medicinal chemistry, three classes of KOR-related agonists, i.e., peripherally restricted KOR agonists, mixed KOR/mu opioid receptor partial agonists, and G protein-biased KOR agonists, warrant additional NHP studies to improve our understanding of their functional efficacy, selectivity, and tolerability. Pharmacological studies in NHP which carry high translational significance will facilitate future development of KOR-based medications.

The Buprenorphine Analogue BU10119 Attenuates Drug-Primed and Stress-Induced Cocaine Reinstatement in Mice.

There are no Food and Drug Administration-approved medications for cocaine use disorder, including relapse. The µ-opioid receptor (MOPr) partial agonist buprenorphine alone or in combination with naltrexone has been shown to reduce cocaine-positive urine tests and cocaine seeking in rodents. However, there are concerns over the abuse liability of buprenorphine. Buprenorphine's partial agonist and antagonist activity at the nociception receptor (NOPr) and κ-opioid receptor (KOPr), respectively, may contribute to its ability to inhibit cocaine seeking. Thus, we hypothesized that a buprenorphine derivative that exhibits antagonist activity at MOPr and KOPr with enhanced agonist activity at the NOPr could provide a more effective treatment. Here we compare the pharmacology of buprenorphine and two analogs, BU10119 and BU12004, in assays for antinociception and for cocaine- and stress-primed reinstatement in the conditioned place preference paradigm. In vitro and in vivo assays showed that BU10119 acts as an antagonist at MOPr, KOPr, and δ-opioid receptor (DOPr) and a partial agonist at NOPr, whereas BU12004 showed MOPr partial agonist activity and DOPr, KOPr, and NOPr antagonism. BU10119 and buprenorphine but not BU12004 lessened cocaine-primed reinstatement. In contrast, BU10119, BU12004, and buprenorphine blocked stress-primed reinstatement. The selective NOPr agonist SCH221510 but not naloxone decreased cocaine-primed reinstatement. Together, these findings are consistent with the concept that NOPr agonism contributes to the ability of BU10119 and buprenorphine to attenuate reinstatement of cocaine-conditioned place preference in mice. The findings support the development of buprenorphine analogs lacking MOPr agonism with increased NOPr agonism for relapse prevention to cocaine addiction. SIGNIFICANCE STATEMENT: There are no Food and Drug Administration-approved medications for cocaine use disorder. Buprenorphine has shown promise as a treatment for cocaine relapse prevention; however, there are concerns over the abuse liability of buprenorphine. Here we show a buprenorphine analogue, BU10119, which lacks µ-opioid receptor agonism and inhibits cocaine-primed and stress-primed reinstatement in a conditioned place-preference paradigm. The results suggest the development of BU10119 for the management of relapse to cocaine seeking.

Structural insights into µ-opioid receptor activation.

Activation of the µ-opioid receptor (µOR) is responsible for the efficacy of the most effective analgesics. To shed light on the structural basis for µOR activation, here we report a 2.1 Å X-ray crystal structure of the murine µOR bound to the morphinan agonist BU72 and a G protein mimetic camelid antibody fragment. The BU72-stabilized changes in the µOR binding pocket are subtle and differ from those observed for agonist-bound structures of the ß2-adrenergic receptor (ß2AR) and the M2 muscarinic receptor. Comparison with active ß2AR reveals a common rearrangement in the packing of three conserved amino acids in the core of the µOR, and molecular dynamics simulations illustrate how the ligand-binding pocket is conformationally linked to this conserved triad. Additionally, an extensive polar network between the ligand-binding pocket and the cytoplasmic domains appears to play a similar role in signal propagation for all three G-protein-coupled receptors.

OREX-1019: A Novel Treatment of Opioid Use Disorder and Relapse Prevention.

There is an urgent need for new pharmacological treatments for substance use disorders, including opioid use disorder, particularly for use in relapse prevention. A combination of buprenorphine with naltrexone has shown particular promise, with clinical studies indicating a substantial improvement over treatment with naltrexone alone. OREX-1019 (formerly BU10119) is a compound that mimics the pharmacology of the buprenorphine/naltrexone combination. This study evaluated, in rhesus monkeys, the therapeutic potential of OREX-1019 for treating opioid use disorder. Pretreatment with OREX-1019 (0.01-0.3 mg/kg s.c.) dose-dependently decreased responding for the µ opioid receptor agonist remifentanil in rhesus monkeys but did not maintain levels of responding above vehicle when it was available for self-administration. OREX-1019 (0.01-1.0 mg/kg s.c.) also decreased cue- plus heroin-primed reinstatement of extinguished responding in monkeys that self-administered remifentanil but did not alter cue- plus cocaine-primed reinstatement of responding in monkeys that self-administered cocaine. OREX-1019 (0.3 mg/kg s.c.), like naltrexone (0.1 mg/kg s.c.), increased heart rate and blood pressure, produced overt observable signs, and eliminated food-maintained responding in monkeys treated chronically with morphine. These results confirm that OREX-1019 has little or no efficacy at µ opioid receptorsand has low abuse potential, and, combined with promising safety (clean profile vs. other off-target proteins including the hERG (human ether-a-go-go-related gene) K+ channel) and pharmacokinetic data (supporting administration by subcutaneous or sublingual routes, but with low oral bioavailability), suggest it could be a safe and effective alternative to current treatments for opioid use disorders particularly as applied to relapse prevention. SIGNIFICANCE STATEMENT: The novel opioid OREX-1019 potentially provides an improved relapse prevention agent for use in opioid use disorder. The current study demonstrates that in monkeys OREX-1019 is able to inhibit the self-administration of, and cue- plus heroin-primed reinstatement of, responding previously maintained by remifentanil.

Pharmacological Properties of δ-Opioid Receptor-Mediated Behaviors: Agonist Efficacy and Receptor Reserve.

δ-Opioid receptor (δ-receptor) agonists produce antihyperalgesia, antidepressant-like effects, and convulsions in animals. However, the role of agonist efficacy in generating different δ-receptor-mediated behaviors has not been thoroughly investigated. To this end, efficacy requirements for δ-receptor-mediated antihyperalgesia, antidepressant-like effects, and convulsions were evaluated by comparing the effects of the partial agonist BU48 and the full agonist SNC80 and changes in the potency of SNC80 after δ-receptor elimination. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. An antidepressant-like effect was evaluated in the forced swim test. Mice were observed for convulsions after treatment with SNC80 or the δ-opioid receptor partial agonist BU48. Ligand-induced G protein activation was measured by [35S]guanosine 5'-O-[γ-thio]triphosphate binding in mouse forebrain tissue, and δ-receptor number was measured by [3H]D-Pen2,5-enkephalin saturation binding. BU48 produced antidepressant-like effects and convulsions but antagonized SNC80-induced antihyperalgesia and G protein activation. The potency of SNC80 was shifted to the right in δ-receptor heterozygous knockout mice and naltrindole-5'-isothiocyanate-treated mice, and the magnitude of potency shift differed across assays, with the largest shift occurring in the thermal hyperalgesia assay, followed by the forced swim test and then convulsion observation. Naltrindole antagonized these SNC80-induced behaviors with similar potencies, suggesting that these effects are mediated by the same type of δ-receptor. These data suggest that δ-receptor-mediated behaviors display a rank order of efficacy requirement, with antihyperalgesia having the highest requirement, followed by antidepressant-like effects and then convulsions. These findings further our understanding of the pharmacological mechanisms mediating the in vivo effects of δ-opioid receptor agonists. SIGNIFICANCE STATEMENT: δ-Opioid receptor (δ-receptor) agonists produce antihyperalgesia, antidepressant-like effects, and convulsions in animal models. This study evaluates pharmacological properties, specifically the role of agonist efficacy and receptor reserve, underlying these δ-receptor-mediated behaviors. These data suggest that δ-receptor-mediated behaviors display a rank order of efficacy requirement, with antihyperalgesia having the highest requirement, followed by antidepressant-like effects and then convulsions.

Synthetic Cannabinoid Receptor Agonists Detection Using Fluorescence Spectral Fingerprinting.

Synthetic cannabinoid receptor agonists (SCRAs), termed "Spice" or "K2", are molecules that emulate the effects of the active ingredient of marijuana, and they have gained enormous popularity over the past decade. SCRAs are Schedule 1 drugs that are highly prevalent in the U.K. prison system and among homeless populations. SCRAs are highly potent and addictive. With no way to determine the dose/amount at the point-of care, they pose severe health risks to users, including psychosis, stroke, epileptic seizures, and they can kill. SCRAs are chemically diverse, with over a hundred compounds used as recreational drugs. The chemical diversity of SCRA structures presents a challenge in developing detection modalities. Typically, GC-MS is used for chemical identification; however, this cannot be in place in most settings where detection is critical, e.g., in hospital Emergency Departments, in custody suites/prisons, or among homeless communities. Ideally, real time, point-of-care identification of SCRAs is desirable to direct the care pathway of overdoses and provide information for informed consent. Herein, we show that fluorescence spectral fingerprinting can be used to identify the likely presence of SCRAs, as well as provide more specific information on structural class and concentration (∼1 µg mL-1). We demonstrate that that fluorescence spectral fingerprints, combined with numerical modeling, can detect both parent and combusted material, and such fingerprinting is also practical for detecting them in oral fluids. Our proof-of-concept study suggests that, with development, the approach could be useful in a range of capacities, notably in harm reduction for users of Spice/K2.

Long-Lasting Effects of Methocinnamox on Opioid Self-Administration in Rhesus Monkeys.

Opioid abuse remains a serious public health challenge, despite the availability of medications that are effective in some patients (naltrexone, buprenorphine, and methadone). This study explored the potential of a pseudoirreversible mu-opioid receptor antagonist [methocinnamox (MCAM)] as a treatment for opioid abuse by examining its capacity to attenuate the reinforcing effects of mu-opioid receptor agonists in rhesus monkeys. In one experiment, monkeys responded for heroin (n = 5) or cocaine (n = 4) under a fixed-ratio schedule. Another group (n = 3) worked under a choice procedure with one alternative delivering food and the other alternative delivering the mu-opioid receptor agonist remifentanil. A third group (n = 4) responded for food and physiologic parameters were measured via telemetry. The effects of MCAM were determined in all experiments and, in some cases, were compared with those of naltrexone. When given immediately before sessions, naltrexone dose-dependently decreased responding for heroin and decreased choice of remifentanil while increasing choice of food, with responding returning to baseline levels 1 day after naltrexone injection. MCAM also decreased responding for heroin and decreased choice of remifentanil while increasing choice of food; however, opioid-maintained responding remained decreased for several days after treatment. Doses of MCAM that significantly decreased opioid-maintained responding did not decrease responding for cocaine or food. MCAM did not impact heart rate, blood pressure, body temperature, or activity at doses that decreased opioid self-administration. Because MCAM selectively attenuates opioid self-administration for prolonged periods, this novel drug could be a safe and effective alternative to currently available treatments for opioid abuse.

Reversal and Prevention of the Respiratory-Depressant Effects of Heroin by the Novel µ-Opioid Receptor Antagonist Methocinnamox in Rhesus Monkeys.

One consequence of the ongoing opioid epidemic is a large number of overdose deaths. Naloxone reverses opioid-induced respiratory depression; however, its short duration of action limits the protection it can provide. Methocinnamox (MCAM) is a novel opioid receptor antagonist with a long duration of action. This study examined the ability of MCAM to prevent and reverse the respiratory-depressant effects (minute volume [VE]) of heroin in five monkeys. MCAM (0.32 mg/kg) was given before heroin to determine whether it prevents respiratory depression; heroin dose-effect curves were generated 1, 2, 4, and 8 days later, and these effects were compared with those of naltrexone (0.032 mg/kg). Heroin dose dependently decreased VE MCAM and naltrexone prevented respiratory depression, shifting the heroin dose-effect curve rightward at least 10-fold. MCAM, but not naltrexone, attenuated these effects of heroin for 4 days. MCAM (0.1-0.32 mg/kg) was given 30 minutes after heroin to determine whether it reverses respiratory depression; heroin dose-effect curves were generated 1, 2, 4, 8, and 16 days later, and these effects were compared with those of naloxone (0.0032-0.1 mg/kg). MCAM and naloxone reversed respiratory depression within 30 minutes, although only MCAM antagonized heroin on subsequent days. Thus, MCAM prevents and reverses respiratory depression, the potentially lethal effect of heroin, longer than opioid receptor antagonists currently in use. Because of its sustained effects, MCAM might provide more effective rescue from and protection against the fatal respiratory-depressant effects of opioids, thereby improving treatment of opioid overdose.

Methocinnamox Produces Long-Lasting Antagonism of the Behavioral Effects of µ-Opioid Receptor Agonists but Not Prolonged Precipitated Withdrawal in Rats.

A novel µ-opioid receptor antagonist, methocinnamox (MCAM), attenuates some abuse-related and toxic effects of opioids. This study further characterized the pharmacology of MCAM in separate groups of rats using procedures to examine antinociception, gastrointestinal motility, and withdrawal in morphine-dependent rats. Antinociceptive effects of opioid receptor agonists were measured before and after MCAM (1-10 mg/kg) using warm water tail withdrawal and sensitivity to mechanical stimulation in inflamed paws (complete Freund's adjuvant). Before MCAM, morphine, fentanyl, and the κ-opioid receptor agonist spiradoline dose dependently increased tail-withdrawal latency from 50°C water; MCAM attenuated the antinociceptive effects of morphine and fentanyl, but not spiradoline. Morphine increased sensitivity to mechanical stimulation and decreased gastrointestinal motility, and MCAM blocked both effects. These antagonist effects of 10 mg/kg MCAM were persistent, lasting for 2 weeks or longer. Withdrawal emerged after discontinuation of morphine treatment or administration of 10 mg/kg MCAM or 17.8 mg/kg naloxone; other than the day of antagonist administration when withdrawal signs were greater in rats that received antagonist compared with rats that received vehicle, there was no difference among groups in directly observable withdrawal signs or decreased body weight. These results confirm that MCAM is a selective µ-opioid receptor antagonist with an exceptionally long duration of action, likely due to pseudoirreversible binding. Despite its sustained antagonist effects, the duration of withdrawal precipitated by MCAM is not different from that precipitated by naloxone, suggesting that the long duration of antagonism provided by MCAM could be particularly effective for treating opioid abuse and overdose. SIGNIFICANCE STATEMENT: The opioid receptor antagonist MCAM attenuates some abuse-related and toxic effects of opioids. This study demonstrates that MCAM selectively antagonizes multiple effects mediated by µ-opioid receptor agonists for 2 weeks or longer, and like naloxone, MCAM precipitates withdrawal in morphine-dependent rats. Despite this persistent antagonism, withdrawal signs precipitated by MCAM are not significantly different from signs precipitated by naloxone or occurring after discontinuation of morphine, suggesting that using MCAM for opioid abuse or overdose would not produce sustained withdrawal.

Simultaneous Transdermal Delivery of Buprenorphine Hydrochloride and Naltrexone Hydrochloride by Iontophoresis.

The opioids buprenorphine hydrochloride (BUP) and naltrexone hydrochloride (NTX) show promise as a combination treatment for addiction, but no means of delivering the two compounds in one medicine currently exist. In this paper, we report sufficient input rates of both these drugs from one iontophoretic transdermal drug delivery system. Experiments were performed using dermatomed pig skin mounted in glass side-bi-side cells. BUP and NTX were iontophoretically delivered together from the anode using direct constant current from Ag/AgCl electrodes. The transdermal drug fluxes and the masses of drugs in both the stratum corneum and the underlying epidermis/dermis were measured. The apparent electroosmotic flow was quantified using a neutral marker (acetaminophen). The effects of donor composition (drug concentration/molar fraction and pH), current density and profile, and the choice of receptor solution were assessed. Iontophoresis dramatically increased the flux of both drugs compared to passive control values. Target fluxes (calculated from literature clearance values and required therapeutic plasma concentrations) were greatly exceeded for NTX and were met for BUP. The latter accumulated in the skin and suppressed electroosmotic flow, inhibiting both its own flux and that of NTX. NTX, in turn, negatively influenced the flux of BUP via co-ion competition. Lowering current density by increasing the delivery area resulted in increased electroosmotic flow but did not significantly affect current-normalized drug fluxes. Delivering the drugs from both electrodes and reversing the polarity for every 2 h did not increase the flux of either compound. In summary, during iontophoresis, BUP and NTX inhibited each other's flux by two distinct mechanisms. While the more complex behavior of BUP complicates the optimization of this drug combination, iontophoresis nevertheless appears to be a feasible approach for the controlled codelivery of NTX and BUP through the skin.

BU10038 as a safe opioid analgesic with fewer side-effects after systemic and intrathecal administration in primates.

BACKGROUND: The marked increase in mis-use of prescription opioids has greatly affected our society. One potential solution is to develop improved analgesics which have agonist action at both mu opioid peptide (MOP) and nociceptin/orphanin FQ peptide (NOP) receptors. BU10038 is a recently identified bifunctional MOP/NOP partial agonist. The aim of this study was to determine the functional profile of systemic or spinal delivery of BU10038 in primates after acute and chronic administration. METHODS: A series of behavioural and physiological assays have been established specifically to reflect the therapeutic (analgesia) and side-effects (abuse potential, respiratory depression, itch, physical dependence, and tolerance) of opioid analgesics in rhesus monkeys. RESULTS: After systemic administration, BU10038 (0.001-0.01 mg kg-1) dose-dependently produced long-lasting antinociceptive and antihypersensitive effects. Unlike the MOP agonist oxycodone, BU10038 lacked reinforcing effects (i.e. little or no abuse liability), and BU10038 did not compromise the physiological functions of primates including respiration, cardiovascular activities, and body temperature at antinociceptive doses and a 10-30-fold higher dose (0.01-0.1 mg kg-1). After intrathecal administration, BU10038 (3 µg) exerted morphine-comparable antinociception and antihypersensitivity without itch scratching responses. Unlike morphine, BU10038 did not cause the development of physical dependence and tolerance after repeated and chronic administration. CONCLUSIONS: These in vivo findings demonstrate the translational potential of bifunctional MOP/NOP receptor agonists such as BU10038 as a safe, non-addictive analgesic with fewer side-effects in primates. This study strongly supports that bifunctional MOP/NOP agonists may provide improved analgesics and an alternative solution for the ongoing prescription opioid crisis.

A novel orvinol analog, BU08028, as a safe opioid analgesic without abuse liability in primates.

Despite the critical need, no previous research has substantiated safe opioid analgesics without abuse liability in primates. Recent advances in medicinal chemistry have led to the development of ligands with mixed mu opioid peptide (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor agonist activity to achieve this objective. BU08028 is a novel orvinol analog that displays a similar binding profile to buprenorphine with improved affinity and efficacy at NOP receptors. The aim of this preclinical study was to establish the functional profile of BU08028 in monkeys using clinically used MOP receptor agonists for side-by-side comparisons in various well-honed behavioral and physiological assays. Systemic BU08028 (0.001-0.01 mg/kg) produced potent long-lasting (i.e., >24 h) antinociceptive and antiallodynic effects, which were blocked by MOP or NOP receptor antagonists. More importantly, the reinforcing strength of BU08028 was significantly lower than that of cocaine, remifentanil, or buprenorphine in monkeys responding under a progressive-ratio schedule of drug self-administration. Unlike MOP receptor agonists, BU08028 at antinociceptive doses and ∼10- to 30-fold higher doses did not cause respiratory depression or cardiovascular adverse events as measured by telemetry devices. After repeated administration, the monkeys developed acute physical dependence on morphine, as manifested by precipitated withdrawal signs, such as increased respiratory rate, heart rate, and blood pressure. In contrast, monkeys did not show physical dependence on BU08028. These in vivo findings in primates not only document the efficacy and tolerability profile of bifunctional MOP/NOP receptor agonists, but also provide a means of translating such ligands into therapies as safe and potentially abuse-free opioid analgesics.

Role of G Protein-Coupled Receptor Kinases 2 and 3 in µ-Opioid Receptor Desensitization and Internalization.

There is ongoing debate about the role of G protein-coupled receptor kinases (GRKs) in agonist-induced desensitization of the µ-opioid receptor (MOPr) in brain neurons. In the present paper, we have used a novel membrane-permeable, small-molecule inhibitor of GRK2 and GRK3, Takeda compound 101 (Cmpd101; 3-[[[4-methyl-5-(4-pyridyl)-4H-1,2,4-triazole-3-yl] methyl] amino]-N-[2-(trifuoromethyl) benzyl] benzamidehydrochloride), to study the involvement of GRK2/3 in acute agonist-induced MOPr desensitization. We observed that Cmpd101 inhibits the desensitization of the G protein-activated inwardly-rectifying potassium current evoked by receptor-saturating concentrations of methionine-enkephalin (Met-Enk), [d-Ala(2), N-MePhe(4), Gly-ol(5)]-enkephalin (DAMGO), endomorphin-2, and morphine in rat and mouse locus coeruleus (LC) neurons. In LC neurons from GRK3 knockout mice, Met-Enk-induced desensitization was unaffected, implying a role for GRK2 in MOPr desensitization. Quantitative analysis of the loss of functional MOPrs following acute agonist exposure revealed that Cmpd101 only partially reversed MOPr desensitization. Inhibition of extracellular signal-regulated kinase 1/2, protein kinase C, c-Jun N-terminal kinase, or GRK5 did not inhibit the Cmpd101-insensitive component of desensitization. In HEK 293 cells, Cmpd101 produced almost complete inhibition of DAMGO-induced MOPr phosphorylation at Ser(375), arrestin translocation, and MOPr internalization. Our data demonstrate a role for GRK2 (and potentially also GRK3) in agonist-induced MOPr desensitization in the LC, but leave open the possibility that another, as yet unidentified, mechanism of desensitization also exists.

A non-rewarding, non-aversive buprenorphine/naltrexone combination attenuates drug-primed reinstatement to cocaine and morphine in rats in a conditioned place preference paradigm.

Concurrent use of cocaine and heroin is a major public health issue with no effective relapse prevention treatment currently available. To this purpose, a combination of buprenorphine and naltrexone, a mixed very-low efficacy mu-opioid receptor agonist/kappa-opioid receptor antagonist/nociceptin receptor agonist, was investigated. The tail-withdrawal and the conditioned place preference (CPP) assays in adult Sprague Dawley rats were used to show that naltrexone dose-dependently blocked the mu-opioid receptor agonism of buprenorphine. Furthermore, in the CPP assay, a combination of 0.3 mg/kg buprenorphine and 3.0 mg/kg naltrexone was aversive. A combination of 0.3 mg/kg buprenorphine and 1.0 mg/kg naltrexone was neither rewarding nor aversive, but still possessed mu-opioid receptor antagonist properties. In the CPP extinction and reinstatement method, a combination of 0.3 mg/kg buprenorphine and 1.0 mg/kg naltrexone completely blocked drug-primed reinstatement in cocaine-conditioned rats (conditioned with 3 mg/kg cocaine, drug prime was 3 mg/kg cocaine) and attenuated drug-primed reinstatement in morphine-conditioned rats (conditioned with 5 mg/kg morphine, drug prime was 1.25 mg/kg morphine). These data add to the growing evidence that a buprenorphine/naltrexone combination may be protective against relapse in a polydrug abuse situation.

Palladium-mediated oxidative carbonylation reactions for the synthesis of (11) C-radiolabelled ureas.

Palladium(II)-mediated oxidative carbonylation reactions have been used to synthesize (11) C-radiolabelled ureas via the coupling of amines with [(11) C]carbon monoxide, in a one-pot process. Following trapping of (11) CO in a solution of copper(I) tris(3,5-dimethylpyrazolyl)borate, homocoupling reactions of primary aliphatic amines proceed in the presence of Pd(PPh3 )2 Cl2 to give the corresponding N,N-disubstituted [(11) C]ureas. Secondary amines do not produce the corresponding N,N,N,N-tetrasubsituted [(11) C]ureas under these conditions. This difference in reactivity allows for the formation of unsymmetrical N,N',N'-trisubstituted [(11) C]ureas using a mixture of a primary amine and a reactive secondary amine. The potential use of this method in positron emission tomography (PET) was demonstrated by the synthesis of the M1 muscarinic acetylcholine receptor radiotracer, [(11) C-carbonyl]GSK1034702.

Effects of sex and hydration status on kappa opioid receptor-mediated diuresis in rats.

Understanding the function of the kappa opioid receptor (KOP) is crucial for the development of novel therapeutic interventions that target KOP for the treatment of pain, stress-related disorders and other indications. Activation of KOP produces diuretic effects in rodents and man. Sex is a vital factor to consider when assessing drug response in pre-clinical and clinical studies. In this study, the diuretic effect of the KOP agonist, U50488 (1-10 mg/kg), was investigated in both adult female and male Wistar rats that were either normally hydrated or water-loaded. The KOP antagonist norbinaltorphimine (norBNI, 10 mg/kg) was administered 24 h prior to U50488 to confirm the involvement of KOP. U50488 elicited a significant diuretic response at doses ≥ 3 mg/kg in both female and male rats independent of hydration status. U50488 diuretic effects were inhibited by norBNI pre-administration. Water-loading reduced data variability for urine volume in males, but not in females, compared with normally hydrated rats. Sex differences were also evident in U50488 eliciting a significant increase in sodium and potassium ion excretion only in males. This may suggest different mechanisms of U50488 diuretic action in males where renal excretion mechanisms are directly affected more than in females.