Opioid Pharmacology Research Articles

Potential Contribution of 7‐Hydroxymitragynine, a Metabolite of the Primary Kratom (Mitragyna Speciosa) Alkaloid Mitragynine, to the μ‐Opioid Activity of Mitragynine in Rats

Mitragyna speciosa (kratom) has been used as a treatment for opioid use disorder in Southeast Asia. The primary kratom alkaloid mitragynine has received much attention due to its opioid pharmacology. One of the in vivo metabolites of mitragynine, 7‐hydroxymitragynine, is a more potent μ‐opioid receptor agonist than mitragynine; this has led to the hypothesis that mitragynine is a prodrug that exerts opioid agonist activity, in part, through metabolic conversion to 7‐hydroxymitragynine. Here, we investigated this hypothesis by comparing mitragynine and 7‐hydroxymitragynine for 1) in vitro binding affinity ([3H]DAMGO) and efficacy (GTPgS) at the μ‐opioid receptor, 2) plasma levels following p.o. mitragynine using a UPLC‐MS/MS method, and 3) potency and effectiveness to produce acute antinociception in the hotplate assay at 52° C. In the hotplate assay, the i.v. route of administration was used to minimize the conversion of mitragynine to 7‐hydroxymitragynine in order to directly compare the pharmacology of the two alkaloids. The binding affinity of 7‐hydroxymitragynine at the human μ‐opioid receptor (Ki=78 nM) was 22‐fold lower than morphine and 9.0‐fold higher than mitragynine. The % maximum GTPgS stimulations of 7‐hydroxymitragynine and morphine at the μ‐opioid receptor were 45% (up to 30 μM) and 90% (up to 30 μM), respectively. In contrast, mitragynine (up to 100 μM) did not significantly stimulate GTPgS. Following p.o. administration of mitragynine (HCl salt, 55 mg/kg), the Cmax value of 7‐hydroxymitragynine (85 ng/mL) was 14‐fold less than that of mitragynine. The Tmax values of 7‐hydroxymitragynine and mitragynine were 30 and 84 minutes, respectively. The % metabolite ratio [(AUC7‐hydroxymitragynine/AUCmitragynine)*100] was 8%. The % maximum possible effects (MPE, ED50 values in mg/kg, i.v.) of 7‐hydroxymitragynine (up to 0.1 mg/kg), morphine (up to 1.0 mg/kg) and mitragynine (up to 17.8 mg/kg) were 100% (1.9), 100% (5.1) and 34% (>17.8). In addition, 32 mg/kg mitragynine was lethal. Finally, drug discrimination was used as a pharmacologically selective measure of μ‐opioid receptor agonism in vivo. In rats discriminating morphine (3.2 mg/kg, i.p.) from vehicle, the discriminative stimulus effects of mitragynine were assessed 90 minutes after p.o. administration to correspond to its Tmax. Mitragynine (up to 178 mg/kg) produced 76% morphine‐lever responding (ED50=51 mg/kg). Though the conversion rate of 7‐hydroxymitragynine from p.o. mitragynine is low, 7‐hydroxymitragynine is a more potent and efficacious μ‐opioid receptor agonist than mitragynine, suggesting that conversion to this metabolite may contribute to the in vivo μ‐opioid activity of mitragynine.Support or Funding InformationSupported by NIDA grants DA23205 and DA48353.

A Concise Review of Concepts in Opioid Pharmacology up to the Discovery of Endogenous Opioids.

This brief review covers concepts in opioid pharmacology that were promoted during the period leading up to the establishment of the International Narcotics Research Conference (INRC) in the early 1970s and the discovery of endogenous opioid peptides in 1975. The founders of INRC, meeting together during the International Union of Pharmacology meeting in Basel in 1969, recognized that the time was ripe for the creation of an international society that would provide a venue for the discussion of research across disciplines in this rapidly expanding area of science. The emphasis here is on studies leading to the demonstration that specific receptors for morphine-like analgesics exist, the search for endogenous ligands for these receptors, and early attempts to elucidate the mechanisms underlying opiate drug tolerance, dependence, and addiction. SIGNIFICANCE STATEMENT: Research on opioids in the 20th century was driven by the search for nonaddicting analgesics. This review discusses the development of the "analgesic" receptor concept, the demonstration that such receptors existed, and the search for an endogenous ligand. Conceptual models were proposed to explain tolerance to the actions of opiate drugs and the development of dependence and addiction. This review explains these models and indicates how they foreshadowed more recent discoveries on the acute and chronic actions of opiate drugs.

Biased Agonism as an Emerging Strategy in the Search for Better Opioid Analgesics.

Morphine and related drugs that act through activating opioid receptors are the most effective analgesics for the relief of severe pain. They have been used for decades, despite the range of unwanted side effects that they produce, as no alternative has been found so far. The major goal of opioid research is to understand the mechanism of action of opioid receptor agonists and to improve the therapeutic utility of opioid drugs. In the search for safer and more potent analgesics, analogs with mixed opioid receptor profile gained a lot of interest. However, recently the concept of biased agonism, that highlights the fact that some ligands are able to differentially activate receptor downstream pathways, became a new approach in the design of novel drug candidates for clinical application. In this review, we summarize current knowledge on the development of opioid ligands of peptide and nonpeptide structure, showing how much opioid pharmacology evolved in recent years.

Opioid Pharmacology under the Microscope.

The powerful analgesic effects of opioid drugs have captivated the interest of physicians and scientists for millennia, and the ability of opioid drugs to produce serious undesired effects has been recognized for a similar period of time (Kieffer and Evans, 2009). Many of these develop progressively with prolonged or repeated drug use and then persist, motivating particular interest in understanding how opioid drugs initiate adaptive or maladaptive modifications in neural function or regulation. Exciting advances have been made over the past several years in elucidating drug-induced changes at molecular, cellular, and physiologic scales of analysis. The present review will highlight some recent cellular studies that we believe bridge across scales and will focus on optical imaging approaches that put opioid drug action "under the microscope." SIGNIFICANCE STATEMENT: Opioid receptors are major pharmacological targets, but their signaling at the cellular level results from a complex interplay between pharmacology, regulation, subcellular localization, and membrane trafficking. This minireview discusses recent advances in understanding the cellular biology of opioid receptors, emphasizing particular topics discussed at the 50th anniversary of the International Narcotics Research Conference. Our goal is to highlight distinct signaling and regulatory properties emerging from the cellular biology of opioid receptors and discuss potential relevance to therapeutics.

Translating Opioid Pharmacology From Bench to Bedside, and Back

Translating Opioid Pharmacology From Bench to Bedside, and Back

A Classroom Activity to Increase Student Pharmacists Confidence in Dealing with the Opioid Epidemic

Objective. To implement and assess the impact of a hybrid flipped-classroom activity designed to increase the motivation and confidence of Doctor of Pharmacy (PharmD) students in addressing the opioid crisis. Methods. Third-professional year student pharmacists were provided with reading material developed by federal agencies and professional pharmacy organizations, as well as Georgia-specific information covering medical amnesty and local resources for opioid-overdose prevention prior to class. They then attended a four-hour classroom session that included hearing a lecture on opioid pharmacology and opioid overdose, viewing training videos, and engaging in extensive discussion. The students voluntarily completed pre- and post-intervention assessments regarding opioid abuse and opioid overdose prevention. Results. Seventy of the 107 third-year students enrolled in the course completed the pre-intervention assessment (65% response rate), and 33 of the 70 completed the post-intervention assessment (47% retention rate). The students exhibited a high baseline motivation to assist in combating the opioid crises, but less confidence in their ability to intervene. Significant increases were seen in areas related to student confidence on the post-intervention assessment. Fewer changes were seen in areas related to student motivation. Conclusion. A "hybrid" flipped classroom activity increased the confidence of student pharmacists in their understanding of the physical and adverse effects of opioids and the application of reversal agents. Increased confidence may support increased intervention.

Many medical students applying for surgical residency feel inadequately prepared to prescribe post-operative opioids

BackgroundLittle is known regarding medical school curricular variability regarding safe prescribing of post-operative opioids for students entering surgical residency. MethodsSurveys were administered to general surgery residency interviewees at an accredited academic institution for 2018–2019 application season. Responses were anonymously recorded using web-based software on an electronic tablet. Descriptive statistics were evaluated using proportions and medians with interquartile range. ResultsOf 103 eligible, 90 (87.4%) interviewees participated. Although 96.7% of students reported opioid pharmacology during medical school, 35.6% reported their curriculum did not include educational material on acute pain management. While 91.1% felt their curriculum adequately covered opioid related adverse events, 34.4% felt adequately prepared to prescribe post-operative opioids to surgical patients. ConclusionStudents entering surgical residency from US medical schools have variable exposures to opioid related educational content and many students feel their medical education inadequately prepared them for prescribing postoperative opioids.

Biased agonism of clinically approved μ-opioid receptor agonists and TRV130 is not controlled by binding and signaling kinetics

Binding and signaling kinetics have previously proven important in validation of biased agonism at GPCRs. Here we provide a comprehensive kinetic pharmacological comparison of clinically relevant μ-opioid receptor agonists, including the novel biased agonist oliceridine (TRV130) which is in clinical trial for pain management. We demonstrate that the bias profile observed for the selected agonists is not time-dependent and that agonists with dramatic differences in their binding kinetic properties can display the same degree of bias. Binding kinetics analyses demonstrate that buprenorphine has 18-fold higher receptor residence time than oliceridine. This is thus the largest pharmacodynamic difference between the clinically approved drug buprenorphine and the clinical candidate oliceridine, since their bias profiles are similar. Further, we provide the first pharmacological characterization of (S)-TRV130 demonstrating that it has a similar pharmacological profile as the (R)-form, oliceridine, but displays 90-fold lower potency than the (R)-form. This difference is driven by a significantly slower association rate. Finally, we show that the selected agonists are differentially affected by G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) expression. GRK2 and GRK5 overexpression greatly increased μ-opioid receptor internalization induced by morphine, but only had modest effects on buprenorphine and oliceridine-induced internalization. Overall, our data reveal that the clinically available drug buprenorphine displays a similar pharmacological bias profile in vitro compared to the clinical candidate drug oliceridine and that this bias is independent of binding kinetics suggesting a mechanism driven by receptor-conformations.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Opioids, the pharmacist and the junior doctor: reducing prescribing error

AbstractAimThis pharmacy and medicine collaborative project investigated the effectiveness of teaching and learning strategies to enhance opioid prescribing by junior doctors in their first year of practice. A key strategy tested was that of pharmacists engaging with junior doctors on the ward to coach and develop their prescribing skills.Data SourcesAn evaluation research framework was implemented that went beyond collecting reaction and self‐reported learning to include impact on patient outcomes. Pre‐ and postintervention prescribing audits were undertaken to identify changes in prescribing practice over the period of the intervention.ResultsLearner reaction feedback was extremely positive for all facets of the intervention. Coaching by the ward pharmacists was recognised and valued. As predicted, there was no significant change in knowledge of the pharmacology and use of opioids but there was a reduction of dose errors, from 54% pre‐intervention to 7% post‐intervention.ConclusionWhile this is a small targeted intervention, we appear to have made a contribution to patient safety through enhanced prescribing practice. The programme has highlighted the potential for further development of the medical–pharmacy relationship as a true teaching and learning partnership. The context of a hospital ward offers unparalleled opportunities to promote learning and develop team‐based understandings.

BPR1M97, a dual mu opioid receptor/nociceptin-orphanin FQ peptide receptor agonist, produces potent antinociceptive effects with safer properties than morphine

There is unmet need to design an analgesic with fewer side effects for severe pain management. Although traditional opioids are the most effective painkillers, they are accompanied by severe adverse responses, such as respiratory depression, constipation symptoms, tolerance, withdrawal, and addiction. We indicated BPR1M97 as a dual mu opioid receptor (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor full agonist and investigated the pharmacology of BPR1M97 in multiple animal models. In vitro studies on BPR1M97 were assessed using cyclic-adenosine monophosphate production, β-arrestin, internalization, and membrane potential assays. In vivo studies were characterized using the tail-flick, tail-clip, lung functional, heart functional, acetone drop, von Frey hair, charcoal meal, glass bead, locomotor activity, conditioned place preference (CPP) and naloxone precipitation tests. BPR1M97 elicited full agonist properties for all cell-based assays tested in MOP-expressing cells. However, it acted as a G protein-biased agonist for NOP. BPR1M97 initiated faster antinociceptive effects at 10 min after subcutaneous injection and elicited better analgesia in cancer-induced pain than morphine. Unlike morphine, BPR1M97 caused less respiratory, cardiovascular, and gastrointestinal dysfunction. In addition, BPR1M97 decreased global activity and induced less withdrawal jumping precipitated by naloxone. Thus, BPR1M97 could serve as a novel small molecule dual receptor agonist for antinociception with fewer side effects than morphine.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Targeting MOR-mGluR5 heteromers reduces bone cancer pain by activating MOR and inhibiting mGluR5

Pain is among the most common symptoms in cancer and approximately 90% of patients experience end-stage cancer pain. The management of cancer pain is challenging due to the significant side effects associated with opioids, and novel therapeutic approaches are needed. MMG22 is a bivalent ligand containing MOR agonist and mGluR5 antagonist pharmacophores joined by a 22-atom spacer. MMG22 exhibited extraordinary analgesia following intrathecal administration in a mouse model of bone cancer pain. Here, we assessed the effectiveness of systemic administration of MMG22 in reducing cancer pain and evaluated whether MMG22 displays side effects associated with opioids. Fibrosarcoma cells were injected into and around the calcaneus bone in C3H mice. Mechanical hyperalgesia was defined as an increase in the paw withdrawal frequencies (PWFs) evoked by application of a von Frey monofilament (3.9 mN bending force) applied to the plantar surface of the hind pawSubcutaneous (s.c.), intramuscular (i.m.), and oral (p.o.) administration of MMG22 produced robust dose-dependent antihyperalgesia, whose ED50 was orders of magnitude lower than morphine. Moreover, the ED50 for MMG22 decreased with disease progression. Importantly, s.c. administration of MMG22 did not produce acute (24 h) or long-term (9 days) tolerance, was not rewarding (conditioned place preference test), and did not produce naloxone-induced precipitated withdrawal or alter motor function. A possible mechanism of action of MMG22 is discussed in terms of inhibition of spinal NMDAR via antagonism of its co-receptor, mGluR5, and concomitant activation of neuronal MOR. We suggest that MMG22 may be a powerful alternative to traditional opioids for managing cancer pain.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Interactions of benzodiazepines with heroin: Respiratory depression, temperature effects, and behavior

Benzodiazepines are important therapeutic drugs, but they are often abused and co-abused with opioids. Clinical evidence suggests that benzodiazepines can inhibit respiration, and when combined with the respiratory-depressive effects of opioids, may increase likelihood of death. In this study we used oxygen sensors coupled with high-speed amperometry and multi-site thermorecording to examine how intravenous (iv) midazolam, a potent benzodiazepine, modulates the brain hypoxic and temperature effects of iv heroin in freely-moving rats. Oxygen levels and brain temperature were assessed with high temporal resolution in the nucleus accumbens (NAc), an important structure in the motivational-reinforcement circuit. When administered alone, midazolam (2 mg/kg) modestly decreased NAc temperature but had no evident effects on oxygen levels in this structure. In contrast, heroin (0.4 mg/kg) induced a strong decrease in NAc oxygen that was followed by a weaker, rebound-like oxygen increase. Midazolam pretreatment did not affect heroin-induced brain hypoxia but potentiated the initial hypothermia induced by heroin. However, co-administration of these drugs potentiated the heroin-induced oxygen decrease and enhanced heroin-induced brain hypothermia. Co-administration of heroin and midazolam also resulted in enhanced locomotor inhibition and loss of motor control. This effect caused some rats to collapse, resulting in nose and mouth occlusion, which caused a secondary hypoxic phase. These results could have important implications for human drug users, as the combined use of benzodiazepines with potent opioids not only results in sustained brain hypoxia but creates conditions of loss of motor control which could result in asphyxia and death.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Relevance of CYP2B6 and CYP2D6 genotypes to methadone pharmacokinetics and response in the OPAL study.

Our study aimed to evaluate the impacts of the cytochrome P450 (CYP) 2B6-G516T and CYP2D6 genetic polymorphisms on pharmacokinetic and clinical parameters in patients receiving methadone maintenance treatment. Opioid PhArmacoLogy (OPAL) was a clinical survey of the sociodemographic characteristics, history and consequences of pathology associated with methadone maintenance treatment response and current addictive comorbidities. A subgroup of 72 methadone patients was genotyped. When comparing the three CYP2B6 genotype groups, the methadone (R)- and (S)-methadone enantiomer concentrations/doses (concentrations relative to doses) were different (P = .029, P = .0019). The CYP2D6 phenotypes did not seem to be relevant with regard to methadone levels. On multivariate analysis, neither the CYP2B6 genotype nor the CYP2D6 phenotype explained the (R)-methadone concentration/dose values (P= .92; P = .86); the (S)-methadone concentration/dose values (P = .052; P = .95 [although there was a difference between the TT group and GT and GG groups {P = .019}]); or opiate cessation (P = .12; P = .90). The genotyping of CYP2B6 G516T could be an interesting tool to explore methadone intervariability.

The highly selective dopamine D3R antagonist, R-VK4-40 attenuates oxycodone reward and augments analgesia in rodents

Prescription opioid abuse is a global crisis. New treatment strategies for pain and opioid use disorders are urgently required. We evaluated the effects of R-VK4-40, a highly selective dopamine (DA) D3 receptor (D3R) antagonist, on the rewarding and analgesic effects of oxycodone, the most commonly abused prescription opioid, in rats and mice. Systemic administration of R-VK4-40 dose-dependently inhibited oxycodone self-administration and shifted oxycodone dose-response curves downward in rats. Pretreatment with R-VK4-40 also dose-dependently lowered break-points for oxycodone under a progressive-ratio schedule. To determine whether a DA-dependent mechanism underlies the impact of D3 antagonism in reducing opioid reward, we used optogenetic approaches to examine intracranial self-stimulation (ICSS) maintained by optical activation of ventral tegmental area (VTA) DA neurons in DAT-Cre mice. Photoactivation of VTA DA in non-drug treated mice produced robust ICSS behavior. Lower doses of oxycodone enhanced, while higher doses inhibited, optical ICSS. Pretreatment with R-VK4-40 blocked oxycodone-enhanced brain-stimulation reward. By itself, R-VK4-40 produced a modest dose-dependent reduction in optical ICSS. Pretreatment with R-VK4-40 did not compromise the antinociceptive effects of oxycodone in rats, and R-VK4-40 alone produced mild antinociceptive effects without altering open-field locomotion or rotarod locomotor performance. Together, these findings suggest R-VK4-40 may permit a lower dose of prescription opioids for pain management, potentially mitigating tolerance and dependence, while diminishing reward potency. Hence, development of R-VK4-40 as a therapy for the treatment of opioid use disorders and/or pain is currently underway.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

The bivalent ligand MCC22 potently attenuates hyperalgesia in a mouse model of cisplatin-evoked neuropathic pain without tolerance or reward

Cisplatin and other widely employed platinum-based anticancer agents produce chemotherapy-induced peripheral neuropathy (CIPN) that often results in pain and hyperalgesia that are difficult to manage. We investigated the efficacy of a novel bivalent ligand, MCC22, for the treatment of pain arising from CIPN. MCC22 consists of mu opioid receptor (MOR) agonist and chemokine receptor 5 (CCR5) antagonist pharmacophores connected through a 22-atom spacer and was designed to target a putative MOR-CCR5 heteromer localized in pain processing areas. Mice received once daily intraperitoneal (i.p.) injections of cisplatin (1 mg/kg) for seven days and behavior testing began 7 days later. Cisplatin produced mechanical hyperalgesia that was decreased dose-dependently by MCC22 given by intrathecal (ED50 = 0.004 pmol) or i.p. (3.07 mg/kg) routes. The decrease in hyperalgesia was associated with decreased inflammatory response by microglia in the spinal cord. Unlike morphine, MCC22 given daily for nine days did not exhibit tolerance to its analgesic effect and its characteristic antihyperalgesic activity was fully retained in morphine-tolerant mice. Furthermore, MCC22 did not alter motor function and did not exhibit rewarding properties. Given the exceptional potency of MCC22 without tolerance or reward, MCC22 has the potential to vastly improve management of chronic pain due to CIPN.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Evidence for nonlinear accumulation of the ultrapotent fentanyl analog, carfentanil, after systemic administration to male rats

The current opioid overdose crisis is being exacerbated by illicitly manufactured fentanyl and its analogs. Carfentanil is a fentanyl analog that is 10,000-times more potent than morphine, but limited information is available about its pharmacology. The present study had two aims: 1) to validate a method for quantifying carfentanil and its metabolite norcarfentanil in small-volume samples, and 2) to use the method for examining pharmacodynamic-pharmacokinetic relationships in rats. The analytical method involved liquid-liquid extraction of plasma samples followed by quantitation of carfentanil and norcarfentanil using ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). The method was validated following SWGTOX guidelines, and both analytes displayed limits of detection and quantification at 7.5 and 15 pg/mL, respectively. Male Sprague-Dawley rats fitted with jugular catheters and temperature transponders received subcutaneous carfentanil (1, 3 and 10 μg/kg) or saline. Repeated blood specimens were obtained over 8 h, along with pharmacodynamic measures including core temperature and catalepsy scores. Carfentanil produced dose-related hypothermia and catalepsy that lasted up to 8 h. Carfentanil Cmax occurred at 15 min whereas metabolite Cmax was at 1–2 h. Concentrations of both analytes increased in a dose-related fashion, but area-under-the-curve values were much greater than predicted after 10 μg/kg. Plasma half-life for carfentanil increased at higher doses. Our findings reveal that carfentanil produces marked hypothermia and catalepsy, which is accompanied by nonlinear accumulation of the drug at high doses. We hypothesize that impaired clearance of carfentanil in humans could contribute to life-threatening effects of this ultrapotent opioid agonist.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Announcement of special issue for 2020: The behavioural pharmacology of opioids

The editors of Behavioural Pharmacology are pleased to announce that a Special Issue on The Behavioural Pharmacology of Opioids will be published in the spring of 2020. In each of the last 30 years, Behavioural Pharmacology has published at least one Special Issue, and we were surprised when reviewing this output to see that opioids as such have never been covered (albeit there have been Special Issues on opioid-related issues such as pain and addiction). Considering the current high levels of abuse of both illegal and legal opioids, it is timely to devote a Special Issue to the behavioural pharmacology of opioids. We now invite behavioural pharmacologists working in this area to submit reports of original, unpublished empirical studies, for inclusion in the Special Issue. Suitable submissions could address any area of opioid pharmacology, such as addiction, pain, reward, or drug development. Review papers are also welcome, but as the Special Issue may include a number of invited reviews, potential reviews should be discussed with the Editors at an early stage to avoid duplication. Also, please ask one of the Editors if you are uncertain whether a report of your research would be suitable for inclusion. All papers should be submitted online at http://www.editorialmanager.com/bpharm. Contributors are advised to submit as early as possible, and should aim to do so before the end of August 2019. Later submissions could be accepted, but the later your submission is received, the higher the likelihood that it might miss the publication deadline for the Special Issue. However, all papers – whether destined for hard-copy publication in Special or regular issues – are published online shortly after they are accepted. ALSO: As already announced, a Special Issue will be published in the autumn of 2019 on Behavioural Aspects of Neuroinflammation. We are still accepting submissions: both reviews and empirical studies concerning behavioural causes and consequences of neuroinflammatory phenomena are welcome. Contributors should aim to submit by the end of April 2019 (but we will remain open to later submissions). All papers should be submitted online at http://www.editorialmanager.com/bpharm. The editors welcome correspondence from potential authors, particularly in relation to review papers. If you are planning to prepare a paper for submission, do please let us know. Paul Willner (Editor) Jack Bergman (Associate Editor) Louk Vanderschuren (Associate Editor) Bart Ellenbroek (Reviews Editor)

Characterizing mu Opioid Receptor Splice Variants Using Gene Targeting Rat Models Generated by Easi‐CRISPR

BackgroundA single‐copy mu opioid receptor (OPRM1) gene generates a vast array of mu opioid receptor splice variants through extensive alternative splicing that is conserved from rodent to human. These variants can be divided into two sets: exon 11 (E11) and exon 1 (E1) associated variants. The functional differences between the E1 and E11 sets of variants have been demonstrated using gene targeted mouse models.Rationale/SignificanceWhile mouse models are valuable, rats have many advantages both in behavioral modeling and in vivo manipulation.HypothesisE11‐ and E1‐associated splice variants play important and distinct roles In opioid pharmacology.ResultsWe have generated two conditional Oprm1 knockout (KO) models, E11‐KO and E1‐KO, in Sprague Dawley rats using a novel Easi‐CRISPR approach. Cre‐mediated E11 and E1 deletions was confirmed through breeding a CAG‐Cre rat. Initial behavioral studies showed that in both E11 and E1 KO rats, buprenorphine analgesia was lost. Buprenorphine CPP was significantly reduced in E11 KO rats. Morphine analgesia was completely lost in the E1‐KO rat, but intact in the E11‐KO rat.DiscussionOur results suggest that buprenorphine analgesia is dependent on both E11 and E1‐associated variants, a similar scenario seen in the E11‐KO and E1‐KO mouse models. E11‐associated variants also involved in buprenorphine reward in rat. These conditional Oprm1 KO rat models will provide valuable resources for the research community and would expand our ability explore mu opioid pharmacology with approaches and techniques not feasible in mice.Support or Funding InformationSupported by grants from NIH (DA40858, DA06241, DA042888 and CA08748)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Opioid-like antinociceptive and locomotor effects of emerging fentanyl-related substances

The emergence of several fentanyl-related substances in the recreational drug marketplace has resulted in a surge of opioid overdose deaths in the United States. Many of these substances have never been examined in living organisms under controlled conditions. In the present study, seven fentanyl-related substances were tested in adult male Swiss Webster mice for their effects on locomotion and antinociception and compared to those of fentanyl and morphine. In locomotor activity tests, fentanyl (1, 10 mg/kg), morphine (100, 180 mg/kg), isobutyrylfentanyl (10 mg/kg), crotonylfentanyl (10 mg/kg), para-fluorobutyrylfentanyl (10, 100 mg/kg), para-methoxybutyrylfentanyl (10 mg/kg), thiophenefentanyl (100 mg/kg), and benzodioxolefentanyl (0.1 mg/kg) produced significant (p ≤ 0.05) dose-dependent increases in locomotion. Valerylfentanyl, however, was without effects on locomotion up to 100 mg/kg. In warm-water tail-withdrawal tests, all substances produced significant (p ≤ 0.05) dose-dependent increases in antinociception with increasing ED50 values (CI) of isobutyrylfentanyl [0.0768 mg/kg (0.044–0.128)] > fentanyl [0.0800 mg/kg (0.0403–0.164)] > para-methoxybutyrylfentanyl [0.106 mg/kg (0.0516–0.195)] > crotonylfentanyl [0.226 mg/kg (0.176–0.292)] > para-fluorobutyrylfentanyl [0.908 mg/kg (0.459–1.58)] > thiophenefentanyl [4.66 mg/kg (3.65–5.95)] > valerylfentanyl [6.43 mg/kg (3.91–10.5)] > morphine [7.82 mg/kg (5.42–11.0)] > benzodioxolefentanyl [46.3 mg/kg (25.8–83.4)]. Naltrexone (1 mg/kg) increased antinociceptive ED50 values several fold in decreasing magnitudes of isobutyrylfentanyl (233x) > para-methoxybutyrylfentanyl (37.7x) > thiophenefentanyl (34.6x) > valerylfentanyl (11.9x) > para-fluorobutyrylfentanyl (10.9x) > benzodioxolefentanyl (8.42x) > crotonylfentanyl (6.27x) > fentanyl (3.95x) > morphine (1.48x). These findings establish that locomotor and antinociceptive effects of several fentanyl-related substances are similar to those of morphine and fentanyl and are mediated by opioid receptors.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

Role of agonist efficacy in exposure-induced enhancement of mu opioid reward in rats

The abuse potential of opioid analgesics in humans appears to increase rapidly during initial regimens of opioid exposure. Previous work using intracranial self-stimulation (ICSS), a preclinical procedure useful for studying rewarding drug effects in drug-naïve animals, has similarly shown that rewarding effects of mu opioid receptor (MOR) agonists increase rapidly in rats during initial regimens of opioid administration. The goal of the present study was to evaluate the role of MOR agonist efficacy as a determinant in eliciting this trajectory of increased rewarding effects during initial opioid exposure in opioid-naïve rats. Separate groups of adult, male Sprague-Dawley rats responded for electrical brain stimulation using a frequency-rate ICSS procedure and received repeated daily treatment with vehicle or one of five MOR agonists that ranged from low to high efficacy (NAQ, nalbuphine, buprenorphine, fentanyl, methadone). Two additional groups were used to evaluate effects of repeated treatment with non-opioids (the cannabinoid CP55940 or the monoamine releaser amphetamine). Morphine was tested after each repeated treatment. In opioid-naïve rats tested before repeated dosing, MOR agonists produced primarily dose- and efficacy-dependent decreases in ICSS. Following repeated treatment, all MOR agonists except NAQ produced tolerance to opioid-induced rate-decreasing effects and enhanced expression of ICSS facilitation (indicative of opioid reward) by both the repeatedly administered drug and morphine. Repeated treatment with CP55940 and amphetamine produced different effects. Collectively, these results provide evidence to suggest that enhanced expression of opioid reward after initial regimens of opioid exposure has a low requirement for MOR agonist efficacy and is pharmacologically selective.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.