Designer Benzodiazepines' Activity on Opioid Receptors: A Docking Study.

BACKGROUND: Previous studies have reported that benzodiazepines (BZDs) seem to enhance euphoric and reinforcing properties of opioids in opioid users so that a direct effect on opioid receptors has been postulated, together with a possible synergistic induction of severe side effects due to co use of BDZs and opioids. This is particularly worrisome given the appearance on the market of designer benzodiazepines (DBZDs), whose activity/toxicity profiles are scarcely known. OBJECTIVES: This study aimed to evaluate, through computational studies, the binding affinity (or lack thereof) of 101 DBZDs identified online on the kappa, mu, and delta opioid receptors (K, M, DOR); and to assess whether their mechanism of action could include activation of the latter. METHODS: MOE® was used for the computational studies. Pharmacophore mapping based on strong opioids agonist binders' 3D chemical features was used to filter the DBZDs. Resultant DBZDs were docked into the crystallised 3D active conformation of KOR (PDB6B73), DOR (PDB6PT3) and MOR (PDB5C1M). Co-crystallised ligands and four strong agonists were used as reference compounds. A score (S, Kcal/mol) representative of the predicted binding affinity, and a description of ligand interactions were obtained from MOE®. RESULTS: The docking results, filtered for S < -8.0 and the interaction with the Asp residue, identified five DBZDs as putative binders of the three ORs : ciclotizolam, fluloprazolam, JQ1, Ro 48-6791, and Ro 48-8684. CONCLUSION: It may be inferred that at least some DBZDs may have the potential to activate opioid receptors. This could mediate/increase their anxiolytic, analgesic, and addiction potentials, as well as worsen the side effects associated with opioid co-use.

Effects of dezocine on morphine tolerance and opioid receptor expression in a rat model of bone cancer pain.

BACKGROUND: Clinically, the coadministration of opioids to enhance antinociception and decrease tolerance has attracted increasing research attention. We investigated the effects of dezocine, a mu- and kappa-opioid receptor agonist/antagonist, on morphine tolerance and explored the involvement of opioid receptor expression in a rat model of bone cancer pain. METHODS: Thermal nociceptive thresholds were measured after the subcutaneous injection of morphine (10 mg/kg) alone or combined with dezocine (10 or 1 mg/kg) for 7 consecutive days. Real-time PCR and western blot analysis were used to examine opioid receptor expression in the periaqueductal gray (PAG) and spinal cord. RESULTS: The analgesic effect was significantly decreased after 4 days of morphine administration. We observed that low-dose dezocine significantly attenuated morphine tolerance without reducing the analgesic effect of morphine. Low-dose dezocine coadministration significantly reversed the downregulated expression of mu (MOR) and delta (DOR) opioid receptors in the PAG and the upregulated expression of kappa (KOR) and DOR in the spinal cord induced by morphine. Moreover, low-dose dezocine coadministered with morphine significantly inhibited KOR expression in both the PAG and spinal cord. CONCLUSIONS: The combination of low-dose dezocine with morphine may prevent or delay the development of morphine tolerance in a rat model of bone cancer pain. The regulation of opioid receptor expression in the PAG and spinal cord may be part of the mechanism.

Replacing a Palatable High-Fat Diet with a Low-Fat Alternative Heightens κ-Opioid Receptor Control over Nucleus Accumbens Dopamine.

Diet-induced obesity reduces dopaminergic neurotransmission in the nucleus accumbens (NAc), and stressful weight loss interventions could promote cravings for palatable foods high in fat and sugar that stimulate dopamine. Activation of κ-opioid receptors (KORs) reduces synaptic dopamine, but contribution of KORs to lower dopamine tone after dietary changes is unknown. Therefore, the purpose of this study was to determine the function of KORs in C57BL/6 mice that consumed a 60% high-fat diet (HFD) for six weeks followed by replacement of HFD with a control 10% fat diet for one day or one week. HFD replacement induced voluntary caloric restriction and weight loss. However, fast-scan cyclic voltammetry revealed no differences in baseline dopamine parameters, whereas sex effects were revealed during KOR stimulation. NAc core dopamine release was reduced by KOR agonism after one day of HFD replacement in females but after one week of HFD replacement in males. Further, elevated plus-maze testing revealed no diet effects during HFD replacement on overt anxiety. These results suggest that KORs reduce NAc dopamine tone and increase food-related anxiety during dietary weight loss interventions that could subsequently promote palatable food cravings and inhibit weight loss.

Endogenous opioid and cannabinoid systems modulate the muscle pain: A pharmacological study into the peripheral site.

The participation of the peripheral opioid and cannabinoid endogenous systems in modulating muscle pain and inflammation has not been fully explored. Thus, the aim of this study was to investigate the involvement of these endogenous systems during muscular-tissue hyperalgesia induced by inflammation. Hyperalgesia was induced by carrageenan injection into the tibialis anterior muscles of male Wistar rats. We padronized an available Randal-Sellito test adaptation to evaluate nociceptive behavior elicited by mechanical insult in muscles. Western blot analysis was performed to evaluate the expression levels of opioid and cannabinoid receptors in the dorsal root ganglia. The non-selective opioid peptide receptor antagonist (naloxone) and the selective mu opioid receptor MOP (clocinnamox) and kappa opioid receptor KOP (nor-binaltorphimine) antagonists were able to intensify carrageenan-induced muscular hyperalgesia. On the other hand, the selective delta opioid receptor (DOP) antagonist (naltrindole) did not present any effect on nociceptive behavior. Moreover, the selective inhibitor of aminopeptidases (Bestatin) provoked considerable dose-dependent analgesia when intramuscularly injected into the hyperalgesic muscle. The CB1 receptor antagonist (AM251), but not the CB2 receptor antagonist (AM630), intensified muscle hyperalgesia. All irreversible inhibitors of anandamide hydrolase (MAFP), the inhibitor for monoacylglycerol lipase (JZL184) and the anandamide reuptake inhibitor (VDM11) decreased carrageenan-induced hyperalgesia in muscular tissue. Lastly, MOP, KOP and CB1 expression levels in DRG were baseline even after muscular injection with carrageenan. The endogenous opioid and cannabinoid systems participate in peripheral muscle pain control through the activation of MOP, KOP and CB1 receptors.

Shifting motivational states: The effects of nucleus accumbens dopamine and opioid receptor activation on a modified effort-based choice task.

The nucleus accumbens (NAc) is critical for regulating the appetitive and consummatory phases of motivated behavior. These experiments examined the effects of dopamine and opioid receptor manipulations within the NAc during an effort-based choice task that allowed for simultaneous assessment of both phases of motivation. Male Sprague-Dawley rats received bilateral guide cannulas targeting the NAc core and were tested in 1-hr sessions with free access to rat chow and the choice to work for sugar pellets on a progressive ratio 2 (PR2) reinforcement schedule. Individual groups of rats were tested following stimulation or blockade of NAc D1-like or D2-like receptors, stimulation of µ-, δ-, or κ-opioid receptors, or antagonism of opioid receptors. Behavior was examined under ad libitum conditions and following 23-h food restriction. NAc blockade of the D1-like receptors or stimulation of the D2 receptor reduced break point for earning sugar pellets; D2 receptor stimulation also modestly lowered chow intake. NAc µ-opioid receptor stimulation increased intake of the freely-available chow while simultaneously reducing break point for the sugar pellets. In non-restricted conditions, δ-opioid receptor stimulation increased both food intake and breakpoint. There were no effects of stimulating NAc D1 or κ receptors, nor did blocking D2 or opioid receptors affect task behavior. These data support prior literature linking dopamine to appetitive motivational processes, and suggest that µ- and δ-opioid receptors affect food-directed motivation differentially. Specifically, µ-opioid receptors shifted behavior towards consumption, and δ-opioid receptor enhanced both sugar-seeking and consumption of the pabulum chow when animals were not food restricted.

Kappa-opioid receptor-dependent changes in dopamine and anxiety-like or approach-avoidance behavior occur differentially across the nucleus accumbens shell rostro-caudal axis.

Neural circuit engagement within the nucleus accumbens (NAc) shell is implicated in the regulation of both negative and positive affect. Classically, the dynorphin/kappa opioid receptor (KOR) system in the NAc was believed to promote aversion, while dopamine was viewed as interacting with reward behavior, and KOR activation was known to inhibit dopamine release. Recently, however, both the KOR and dopamine systems have, separately, been shown to have differential effects across the rostro-caudal axis of the NAc shell on hedonic responses. Whether or not this is due to interactions between KORs and dopamine, and if it extends to anxiety-like or approach-avoidance behaviors, remains to be determined. In this study, we examined in rats the relationship between the KOR and dopamine systems in both the rostral and caudal NAc shell using ex vivo fast scan cyclic voltammetry and the impact of KOR activation on affective behavior using exploration-based tasks. We report here that activation of KORs in the caudal NAc shell significantly inhibits dopamine release, stimulates rearing behavior in a novel environment, increases anxiety-like or avoidance behavior, and reduces locomotor activity. In contrast, activation of KORs in the rostral NAc shell inhibits dopamine release to a lesser extent and instead reduces anxiety-like behavior or increases approach behavior. Taken together, these results indicate that there is heterogeneity across the rostro-caudal axis of the NAc shell in the effects of KOR stimulation on affective behaviors, and they suggest that this might be due to differences in KOR control over dopamine release.

Pharmacological and phosphoproteomic approaches to roles of protein kinase C in kappa opioid receptor-mediated effects in mice.

Kappa opioid receptor (KOR) agonists possess adverse dysphoric and psychotomimetic effects, thus limiting their applications as non-addictive anti-pruritic and analgesic agents. Here, we showed that protein kinase C (PKC) inhibition preserved the beneficial antinociceptive and antipruritic effects of KOR agonists, but attenuated the adverse condition placed aversion (CPA), sedation, and motor incoordination in mice. Using a large-scale mass spectrometry-based phosphoproteomics of KOR-mediated signaling in the mouse brain, we observed PKC-dependent modulation of G protein-coupled receptor kinases and Wnt pathways at 5 min; stress signaling, cytoskeleton, mTOR signaling and receptor phosphorylation, including cannabinoid receptor CB1 at 30 min. We further demonstrated that inhibition of CB1 attenuated KOR-mediated CPA. Our results demonstrated the feasibility of in vivo biochemical dissection of signaling pathways that lead to side effects.

Alcohol drinking alters stress response to predator odor via BNST kappa opioid receptor signaling in male mice.

Maladaptive responses to stress are a hallmark of alcohol use disorder, but the mechanisms that underlie this are not well characterized. Here, we show that kappa opioid receptor signaling in the bed nucleus of the stria terminalis (BNST) is a critical molecular substrate underlying abnormal stress responses to predator odor following heavy alcohol drinking. Exposure to predator odor during protracted withdrawal from intermittent alcohol drinking resulted in enhanced prefrontal cortex (PFC)-driven excitation of prodynorphin-containing neurons in the BNST. Furthermore, deletion of prodynorphin in the BNST and chemogenetic inhibition of the PFC-BNST pathway restored abnormal responses to predator odor in alcohol-exposed mice. These findings suggest that increased corticolimbic drive may promote abnormal stress behavioral responses to predator odor during protracted withdrawal. Various nodes of this PFC-BNST dynorphin-related circuit may serve as potential targets for potential therapeutic mediation as well as biomarkers of negative responses to stress following heavy alcohol drinking.

The connection between stress and alcohol use is highly complex. On one hand, there is the idea of having a drink to 'steady the nerves'. On the other hand, in alcoholics, abnormal responses to stress often accompany heavy drinking. In this case, it remains unknown whether stress cause excessive drinking, or vice versa. Areas of the brain that normally help respond to stress work differently in long-term, heavy drinkers. One example is a structure called the bed nucleus of the stria terminalis (BNST), which is over-active in anxiety disorders and is also associated with some of the symptoms of alcohol withdrawal. The mechanism behind both problems is thought to be a specific 'signaling system' that is activated by a small molecule called dynorphin. Previous research into the effects of dynorphin was performed either in the context of alcoholism or of anxiety disorders, but it was not known if there was a connection between the two. Therefore, Hwa et al. wanted to determine how prolonged alcohol use might affect responses to stress, and whether dynorphin signaling plays a role. To model long-term alcohol use in the laboratory, a group of mice was given free access to alcohol every other day, ensuring that they developed the mouse equivalent of a drinking habit. After six weeks, these 'heavy drinkers' went through a period of abstinence, mimicking alcohol withdrawal. Then, the mice were stressed by exposing them to a chemical that smelled like a fox, one of the mice's predators in the wild. When mice smell predators, they normally respond by fleeing from the area and digging up debris to defend itself. As expected, the control mice in this study, which did not drink alcohol, did just that. In contrast, the heavy drinkers largely ignored the predator scent by not digging and even spent time hanging around the area that smelled like the predator. Blocking dynorphin-induced signaling in the alcoholic mice, either using a drug or by deleting the gene that codes for dynorphin, reset the stress response to normal, allowing these mice to avoid the predator and dig as normal. Furthermore, measuring the electrical activity in the brain revealed that the BNST was abnormally active in alcohol-drinking mice, driven by signals from another part of the brain, the prefrontal cortex. This reveals part of the circuitry in the brain responsible for the connection between alcohol withdrawal and the stress response. These results shed new light on the biological mechanisms underpinning the relationship between alcohol use and stress. In the future, these could be used to determine why heavy drinking can overlap with anxiety disorders, or to develop new treatments that would help recovering alcoholics cope better with stress.

Kappa opioid receptors internalization is protective against oxygen-glucose deprivation through ß-arrestin activation and Akt-mediated signaling pathway.

Hypoxia induces reversible κ-opioid receptor (KOR) internalization similar to the internalization that is induced by KOR agonists. In the current study, we demonstrate that this KOR internalization is a protective mechanism via the ß-arrestin specific pathway in an oxygen-glucose deprivation (OGD) model. Mouse neuroblastoma Neuro2A cells were stably transfected with mouse KOR-tdTomato fusion protein (N2A-mKOR-tdT cells). Various concentrations of salvinorin A (SA), a highly selective KOR agonist, were given in the presence and absence of norbinaltorphimine (norBNI), which is a KOR antagonist, or Dyngo-4a (internalization inhibitor) or API-2 (Akt/Protein kinase B signaling inhibitor-2). Various concentrations of SA and RB-64 (22-thiocyanatosalvinorin A, selective for the G protein signaling pathway) were administered both in normoxic and hypoxic conditions. Autophagosomes and ultrastructural components of cells were observed using transmission electron microscopy (TEM). Cell viability, severity of cell injury, and levels of proteins related to the Akt signaling pathway were evaluated using live cell counting (by Cell Counting Kit-8), the lactic acid dehydrogenase (LDH) release rate, and Western blot analysis, respectively. SA promoted cell survival and attenuated OGD-induced cell injury. The Akt signaling pathway is activated by SA. KOR internalization, when blocked by norBNI or Dyngo-4a, increased LDH release and decreased cell viability under OGD. Treatment with SA significantly inhibited autophagy, and the effects of SA on autophagy were reversed by API-2 pretreatment. RB-64 in a low concentration without ß-arrestin recruitment did not reduce LDH release and increase cell viability as observed with SA. KOR internalization through ß-arrestin activation is a protective mechanism against OGD. The Akt pathway might play a critical role in modulating these protective effects by inhibiting autophagy.

Potential therapeutic treatments of cancer-induced bone pain.

PURPOSE OF REVIEW: The treatment of cancer-induced bone pain (CIBP) has been proven ineffective and relies heavily on opioids, the target of highly visible criticism for their negative side effects. Alternative therapeutic agents are needed and the last few years have brought promising results, detailed in this review. RECENT FINDINGS: Cysteine/glutamate antiporter system, xc, cannabinoids, kappa opioids, and a ceramide axis have all been shown to have potential as novel therapeutic targets without the negative effects of opioids. SUMMARY: Review of the most recent and promising studies involving CIBP, specifically within murine models. Cancer pain has been reported by 30-50% of all cancer patients and even more in late stages, however the standard of care is not effective to treat CIBP. The complicated and chronic nature of this type of pain response renders over the counter analgesics and opioids largely ineffective as well as difficult to use due to unwanted side effects. Preclinical studies have been standardized and replicated while novel treatments have been explored utilizing various alternative receptor pathways: cysteine/glutamate antiporter system, xc, cannabinoid type 1 receptor, kappa opioids, and a ceramide axis sphingosine-1-phosphate/sphingosine-1-phosphate receptor 1.

Dynorphin and its role in alcohol use disorder.

The dynorphin / kappa opioid receptor (KOR) system has been implicated in many aspects that influence neuropsychiatric disorders. Namely, this system modulates neural circuits that primarily regulate reward seeking, motivation processing, stress responsivity, and pain sensitivity, thus affecting the development of substance and alcohol use disorder (AUD). The effects of this system are often bidirectional and depend on projection targets. To date, a majority of the studies focusing on this system have examined the KOR function using agonists and antagonists. Indeed, there are studies that have examined prodynorphin and dynorphin levels by measuring mRNA and tissue content levels; however, static levels of the neuropeptide and its precursor do not explain complete and online function of the peptide as would be explained by measuring dynorphin transmission in real time. New and exciting methods using optogenetics, chemogenetics, genetic sensors, fast scan cyclic voltammetry are now being developed to detect various neuropeptides with a focus on opioid peptides, including dynorphin. In this review we discuss studies that examine dynorphin projections in areas involved in AUD, its functional involvement in AUD and vulnerability to develop AUD at various ages. Moreover, we discuss dynorphin's role in promoting AUD by dysregulation motivation circuits and how advancements in opioid peptide detection will further our understanding.

Ketamine: The final frontier or another depressing end?

Two decades ago, the observation of a rapid and sustained antidepressant response after ketamine administration provided an exciting new avenue in the search for more effective therapeutics for the treatment of clinical depression. Research elucidating the mechanism(s) underlying ketamine's antidepressant properties has led to the development of several hypotheses, including that of disinhibition of excitatory glutamate neurons via blockade of N-methyl-d-aspartate (NMDA) receptors. Although the prominent understanding has been that ketamine's mode of action is mediated solely via the NMDA receptor, this view has been challenged by reports implicating other glutamate receptors such as AMPA, and other neurotransmitter systems such as serotonin and opioids in the antidepressant response. The recent approval of esketamine (Spravato™) for the treatment of depression has sparked a resurgence of interest for a deeper understanding of the mechanism(s) underlying ketamine's actions and safe therapeutic use. This review aims to present our current knowledge on both NMDA and non-NMDA mechanisms implicated in ketamine's response, and addresses the controversy surrounding the antidepressant role and potency of its stereoisomers and metabolites. There is much that remains to be known about our understanding of ketamine's antidepressant properties; and although the arrival of esketamine has been received with great enthusiasm, it is now more important than ever that its mechanisms of action be fully delineated, and both the short- and long-term neurobiological/functional consequences of its treatment be thoroughly characterized.

Estrogens as arbiters of sex-specific and reproductive cycle-dependent opioid analgesic mechanisms.

The organization of estrogenic signaling in the CNS is exceedingly complex. It is comprised of peripherally and centrally synthesized estrogens, and a plethora of types of estrogen receptor that can localize to both the nucleus and the plasma membrane. Moreover, CNS estrogen receptors can exist independent of aromatase (aka estrogen synthase) as well as oligomerize with it, along with a host of other membrane signaling proteins. This ability of CNS estrogen receptors to either to physically pair or exist separately enables locally produced estrogens to act on multiple spatial levels, with a high degree of gradated regulation and plasticity, signaling either in-phase or out-of phase with circulating estrogens. This complexity explains the numerous contradictory findings regarding sex-dependent pain processing and sexually dimorphic opioid antinociception. This review highlights the increasing awareness that estrogens are major endogenous arbiters of both opioid analgesic actions and the mechanisms used to achieve them. This behooves us to understand, and possibly intercede at, the points of intersection of estrogenic signaling and opioid functionality. Factors that integrate estrogenic actions at subcellular, synaptic, and CNS regional levels are likely to be prime drug targets for novel pharmacotherapies designed to modulate CNS estrogen-dependent opioid functionalities and possibly circumvent the current opioid epidemic.

Kappa opioid receptor and oligodendrocyte remyelination.

Adult oligodendrocyte precursor cells (OPCs) maintain the abilities to differentiate and myelinate denuded axons in demyelinating diseases, such as Multiple Sclerosis (MS), albert often inefficiently. Remyelination therapies seek to enhance endogenous remyelination and represent a promising approach to achieve functional and cellular architectural recovery against neuronal deficits. Recent findings indicate that the kappa opioid receptor (KOR), a G-protein coupled receptor (GPCR), plays an important role in regulating oligodendrocyte differentiation and myelination. In this chapter, we reviewed (1) current knowledge of the functional importance of remyelination in demyelination diseases; (2) the opioids that can alter oligodendroglial proliferation and differentiation; (3) the endogenous KOR signaling in regulating oligodendrocyte myelination.

Oxycodone suppresses the apoptosis of hippocampal neurons induced by oxygen-glucose deprivation/recovery through caspase-dependent and caspase-independent pathways via κ- and δ-opioid receptors in rats.

Cerebral ischemia/reperfusion injury (CIRI) can lead to perioperative neurocognitive disorders (PND) during clinical recanalization procedures in cerebral vessels, principally due to neuronal apoptosis in the hippocampus. Oxycodone appears to be a multiple opioid receptor agonist and exerts intrinsic antinociception activity via κ-opioid receptor (KOR). Recent evidence has revealed that activation of both δ-opioid receptor (DOR) and KOR can provide neuroprotection against CIRI in vivo and in vitro. In our study, we established an oxygen-glucose deprivation/recovery (OGD/R) model with fetal hippocampal neurons and found that oxycodone could induce CIRI tolerance in these neurons, primarily through KOR and DOR. Possible mechanisms might involve the regulatory effect of oxycodone on the MAPK-Bcl2/Bax-caspase-9-caspase-3 pathway, as well as its inhibitory effect on cellular reactive oxygen species (ROS) production and mitochondrial membrane potential activation. Taken together, our findings may indicate a potential method for the prevention and treatment of PND associated with CIRI.

Emerging Treatment Targets for Migraine and Other Headaches.

Migraine is a complex disorder that is characterized by an assortment of neurological and systemic effects. While headache is the most prominent feature of migraine, a host of symptoms affecting many physiological functions are also observed before, during, and after an attack. Furthermore, migraineurs are heterogeneous and have a wide range of responses to migraine therapies. The recent approval of calcitonin gene-related-peptide based therapies has opened up the treatment of migraine and generated a renewed interest in migraine research and discovery. Ongoing advances in migraine research have identified a number of other promising therapeutic targets for this disorder. In this review, we highlight emergent treatments within the following biological systems: pituitary adenylate cyclase activating peptdie, 2 non-mu opioid receptors that have low abuse liability - the delta and kappa opioid receptors, orexin, and nitric oxide-based therapies. Multiple mechanisms have been identified in the induction and maintenance of migraine symptoms; and this divergent set of targets have highly distinct biological effects. Increasing the mechanistic diversity of the migraine tool box will lead to more treatment options and better patient care.

Kappa opioid signaling in the right central amygdala causes hind paw specific loss of diffuse noxious inhibitory controls in experimental neuropathic pain.

Diffuse noxious inhibitory controls (DNICs) is a pain-inhibits-pain phenomenon demonstrated in humans and animals. Diffuse noxious inhibitory control is diminished in many chronic pain states, including neuropathic pain. The efficiency of DNIC has been suggested to prospectively predict both the likelihood of pain chronification and treatment response. Little is known as to why DNIC is dysfunctional in neuropathic pain. Here, we evaluated DNIC in the rat L5/L6 spinal nerve ligation (SNL) model of chronic pain using both behavioral and electrophysiological outcomes. For behavior, nociceptive thresholds were determined using response to noxious paw pressure on both hind paws as the test stimulus before, and after, injection of a conditioning stimulus of capsaicin into the left forepaw. Functionally, the spike firing of spinal wide-dynamic-range neuronal activity was evaluated before and during noxious ear pinch, while stimulating the ipsilateral paw with von Frey hairs of increased bending force. In both assays, the DNIC response was significantly diminished in the ipsilateral (ie, injured) paw of SNL animals. However, behavioral loss of DNIC was not observed on the contralateral (ie, uninjured) paw. Systemic application of nor-binaltorphimine, a kappa opioid antagonist, did not ameliorate SNL-induced hyperalgesia but reversed loss of the behavioral DNIC response. Microinjection of nor-binaltorphimine into the right central amygdala (RCeA) of SNL rats did not affect baseline thresholds but restored DNIC both behaviorally and electrophysiologically. Cumulatively, these data suggest that net enhanced descending facilitations may be mediated by kappa opioid receptor signaling from the right central amygdala to promote diminished DNIC after neuropathy.

Role of mu, but not delta or kappa, opioid receptors in context-induced reinstatement of oxycodone seeking.

Relapse to non-medical use of prescription opioids often occurs after exposure to places previously associated with drug use. Here, we describe a rat model of context-induced reinstatement of oxycodone seeking after repeated cycles of drug self-administration and extinction-induced abstinence. We also determined the role of mu, delta and kappa opioid receptors (MOR, DOR, KOR) in this reinstatement. We trained rats to self-administer oxycodone for 6 h/day in context A; lever pressing was paired with a discrete cue. Next, we extinguished the lever pressing in the presence of the discrete cue in context B and then tested the rats for reinstatement of oxycodone seeking in both contexts. We retrained rats to self-administer oxycodone in context A, re-extinguished their lever pressing in context B and retested them for reinstatement in both contexts. Prior to testing, we injected the rats with vehicle or antagonists of MOR (naltrexone; 0.5 or 1.0 mg/kg), DOR (naltrindole; 7.5 or 15 mg/kg) or KOR (LY2456302; 5 or 10 mg/kg). We also tested the effect of naltrexone, naltrindole and LY2456302 on oxycodone self-administration under fixed-ratio-1 (FR1) and progressive ratio (PR) reinforcement schedules. We observed context-induced reinstatement of oxycodone seeking after repeated cycles of drug self-administration and extinction. Naltrexone, but not naltrindole or LY2456302, injections decreased this reinstatement. Additionally, naltrexone increased oxycodone self-administration under the FR1 schedule and decreased oxycodone self-administration under the PR schedule; naltrindole and LY2456302 were ineffective. Results demonstrate a critical role of MOR, but not DOR or KOR, in context-induced reinstatement of oxycodone seeking and oxycodone self-administration.

Ethanol and Naltrexone Have Distinct Effects on the Lateral Nano-organization of Mu and Kappa Opioid Receptors in the Plasma Membrane.

The complex spatiotemporal organization of proteins and lipids in the plasma membrane is an important determinant of receptor function. Certain substances, such as ethanol, can penetrate into the hydrophobic regions of the plasma membrane. By altering protein-lipid and protein-protein interactions, these substances can modify the dynamic lateral organization and the function of plasma membrane receptors. To assess changes in plasma membrane receptor organization, we used photoactivated localization microscopy (PALM). This single molecule localization microscopy technique was employed to quantitatively characterize the effects of pharmacologically relevant concentrations of ethanol and naltrexone (an opioid receptor antagonist and medication used to treat alcohol use disorders) on the lateral nano-organization of mu and kappa opioid receptors (MOR and KOR, respectively). Ethanol affected the lateral organization of MOR and KOR similarly: It reduced the size and occupancy of opioid receptor nanodomains and increased the fraction of opioid receptors residing outside of nanodomains. In contrast, naltrexone affected MOR and KOR lateral organization differently. It significantly increased KOR surface density, nanodomain size, and the occupancy of KOR nanodomains. However, naltrexone marginally affected these parameters for MOR. Pretreatment with naltrexone largely protected against ethanol-induced changes in MOR and KOR lateral organization. Based on these data, we propose a putative mechanism of naltrexone action that operates in addition to its canonical antagonistic effect on MOR- and KOR-mediated signaling.

Methylation Products of 6ß- N-Heterocyclic Substituted Naltrexamine Derivatives as Potential Peripheral Opioid Receptor Modulators.

Two 6ß- N-heterocyclic naltrexamine derivatives, NAP and NMP, have been identified as peripherally selective mu opioid receptor (MOR) antagonists. To further enhance the peripheral selectivity of both compounds, the 17-amino group and the nitrogen atom of the pyridine ring in both NAP and NMP were methylated to obtain dMNAP and dMNMP, respectively. Compared with NAP and NMP, the binding affinities of dMNAP and dMNMP shifted to MOR and KOR (kappa opioid receptor) dual selective and they acted as moderate efficacy partial agonists. The results from radioligand binding studies were further confirmed by molecular docking studies. In vivo studies demonstrated that dMNAP and dMNMP did not produce antinociception nor did they antagonize morphine's antinociceptive activity, indicating that these compounds did not act on the central nervous system. Meanwhile, both dMNAP and dMNMP significantly slowed down fecal excretion, which indicated that they were peripherally acting opioid receptor agonists. All together, these results suggested that dMNAP and dMNMP acted as peripheral mu/kappa opioid receptor modulators and may be applicable in the treatment of diarrhea in patients with bowel dysfunction.