Neural Circuitry Polarization in the Spinal Dorsal Horn (SDH): A Novel Form of Dysregulated Circuitry Plasticity during Pain Pathogenesis.

Pathological pain emerges from nociceptive system dysfunction, resulting in heightened pain circuit activity. Various forms of circuitry plasticity, such as central sensitization, synaptic plasticity, homeostatic plasticity, and excitation/inhibition balance, contribute to the malfunction of neural circuits during pain pathogenesis. Recently, a new form of plasticity in the spinal dorsal horn (SDH), named neural circuit polarization (NCP), was discovered in pain models induced by HIV-1 gp120 and chronic morphine administration. NCP manifests as an increase in excitatory postsynaptic currents (EPSCs) in excitatory neurons and a decrease in EPSCs in inhibitory neurons, presumably facilitating hyperactivation of pain circuits. The expression of NCP is associated with astrogliosis. Ablation of reactive astrocytes or suppression of astrogliosis blocks NCP and, concomitantly, the development of gp120- or morphine-induced pain. In this review, we aim to compare and integrate NCP with other forms of plasticity in pain circuits to improve the understanding of the pathogenic contribution of NCP and its cooperation with other forms of circuitry plasticity during the development of pathological pain.

Microglial exosome miR-124-3p in hippocampus alleviates cognitive impairment induced by postoperative pain in elderly mice.

Cognitive impairment induced by postoperative pain severely deteriorates the rehabilitation outcomes in elderly patients. The present study focused on the relationship between microglial exosome miR-124-3p in hippocampus and cognitive impairment induced by postoperative pain. Cognitive impairment model induced by postoperative pain was constructed by intramedullary nail fixation after tibial fracture. Morphine intraperitoneally was carried out for postoperative analgesia. Morris water maze tests were carried out to evaluate the cognitive impairment, while mRNA levels of neurotrophic factors (BDNF, NG) and neurodegenerative biomarker (VILIP-1) in hippocampus were tested by q-PCR. Transmission electron microscope was used to observe the axon degeneration in hippocampus. The levels of pro-inflammatory factors (TNF-α, IL-1ß, IL-6), the levels of anti-inflammatory factors (Ym, Arg-1, IL-10) and microglia proliferation marker cyclin D1 in hippocampus were measured to evaluate microglia polarization. Bioinformatics analysis was conducted to identify key exosomes while BV-2 microglia overexpressing exosome miR-124-3p was constructed to observe microglia polarization in vitro experiments. Exogenous miR-124-3p-loaded exosomes were injected into hippocampus in vivo. Postoperative pain induced by intramedullary fixation after tibial fracture was confirmed by decreased mechanical and thermal pain thresholds. Postoperative pain induced cognitive impairment, promoted axon demyelination, decreased BDNF, NG and increased VILIP-1 expressions in hippocampus. Postoperative pain also increased pro-inflammatory factors, cyclin D1 and decreased anti-inflammatory factors in hippocampus. However, these changes were all reversed by morphine analgesia. Bioinformatics analysis identified the critical role of exosome miR-124-3p in cognitive impairment, which was confirmed to be down-regulated in hippocampus of postoperative pain mice. BV-2 microglia overexpressing exosome miR-124-3p showed decreased pro-inflammatory factors, cyclin D1 and increased anti-inflammatory factors. In vivo, stereotactic injection of exogenous miR-124-3p into hippocampus decreased pro-inflammatory factors, cyclin D1 and increased anti-inflammatory factors. The cognitive impairment, axon demyelination, decreased BDNF, NG and increased VILIP-1 expressions in hippocampus were all alleviated by exogenous exosome miR-124-3p. Microglial exosome miR-124-3p in hippocampus alleviates cognitive impairment induced by postoperative pain through microglia polarization in elderly mice.

The potential protective effect of melatonin and N-acetylcysteine alone and in combination on opioid-induced testicular dysfunction and degeneration in rat.

Methadone (Met) is the most common treatment for opioid addiction. Although Met is effective for treatment of opioid dependence, sexual dysfunctions and infertility have been reported as a major problem in patients under Met treatment. The present study aimed to evaluate the effect of melatonin and N-acetylcysteine (N) on morphine and Met-induced oxidative stress, apoptosis, suppression of blood sexual hormones, impairment in sperm parameters, and sexual dysfunction. Adult male Wistar rats (n = 66) were randomly divided into 11 equal groups (n = 6) as follows: control, sham, morphine, Met, Met+N, Met+ melatonin, Met+melatonin+N, morphine+ Met, morphine+Met+ melatonin, morphine+Met+N, and morphine+Met+ melatonin+N groups. On day 56 post-treatment, the blood was collected from the tail and the serum levels of sex hormones were evaluated, then the rats were sacrificed, and their bilateral testes and epididymis were retrieved for histological, immunohistochemical, molecular, testicular tissue stress oxidative status, and sperm parameters assays. Exposure to morphine, Met, and shift of morphine to Met resulted in testicular degeneration that can be attributed to generating the stress oxidative-induced- apoptotic testicular cell death and impairing spermatogenesis. Melatonin and N alone and particularly, in combination with each other improved testicular degeneration, sex hormone suppression, and testicular function mediated by increasing the testicular antioxidant capacity and inhibition of the apoptosis pathway. It's suggested that oral administration of antioxidants may be an effective treatment for attenuating some opioid-related testicular dysfunction and degeneration.

P-glycoprotein (MDR1/ABCB1) Restricts Brain Penetration of the Main Active Heroin Metabolites 6-monoacetylmorphine (6-MAM) and Morphine in Mice.

BACKGROUND & PURPOSE: Heroin (diacetylmorphine; diamorphine) is a highly addictive opioid prodrug. Heroin prescription is possible in some countries for chronic, treatment-refractory opioid-dependent patients and as a potent analgesic for specific indications. We aimed to study the pharmacokinetic interactions of heroin and its main pharmacodynamically active metabolites, 6-monoacetylmorphine (6-MAM) and morphine, with the multidrug efflux transporters P-glycoprotein/ABCB1 and BCRP/ABCG2 using wild-type, Abcb1a/1b and Abcb1a/1b;Abcg2 knockout mice. METHODS & RESULTS: Upon subcutaneous (s.c.) heroin administration, its blood levels decreased quickly, making it challenging to detect heroin even shortly after dosing. 6-MAM was the predominant active metabolite present in blood and most tissues. At 10 and 30 min after heroin administration, 6-MAM and morphine brain accumulation were increased about 2-fold when mouse (m)Abcb1a/1b and mAbcg2 were ablated. Fifteen minutes after direct s.c. administration of an equimolar dose of 6-MAM, we observed good intrinsic brain penetration of 6-MAM in wild-type mice. Still, mAbcb1 limited brain accumulation of 6-MAM and morphine without affecting their blood exposure, and possibly mediated their direct intestinal excretion. A minor contribution of mAbcg2 to these effects could not be excluded. CONCLUSIONS: We show that mAbcb1a/1b can limit 6-MAM and morphine brain exposure. Pharmacodynamic behavioral/postural observations, while non-quantitative, supported moderately increased brain levels of 6-MAM and morphine in the knockout mouse strains. Variation in ABCB1 activity due to genetic polymorphisms or environmental factors (e.g., drug interactions) might affect 6-MAM/morphine exposure in individuals, but only to a limited extent.

Opium alkaloids, biosynthesis, pharmacology and association with cancer occurrence.

Papaver somniferum L. (Family: Papaveraceae) is a species well known for its diverse alkaloids (100 different benzylisoquinoline alkaloids (BIAs)). L-tyrosine serves as a precursor of several specific metabolites like BIAs. It has been used as an antitussive and potent analgesic to alleviate mild to extreme pain since ancient times. The extraction of pharmaceutically important alkaloids like morphine and codeine from poppy plant reflects the need for the most suitable and standard methods. Several analytical and extraction techniques have been reported in open literature for morphine, codeine and other important alkaloids which play a vital function in drug development and drug discovery. Many studies suggest that opioids are also responsible for adverse effects or secondary complications like dependence and withdrawal. In recent years, opium consumption and addiction are the most important risk factors. Many evidence-based reviews suggest that opium consumption is directly linked or acts as a risk factor for different cancers. In this review, we highlight significant efforts related to research which have been done over the past 5 decades and the complete information on Papaver somniferum including its phytochemistry, pharmacological actions, biosynthetic pathways and analytical techniques of opium alkaloid extraction and the link between opium consumption and cancer-related updates.

Heroin and its metabolites: relevance to heroin use disorder.

Heroin is an opioid agonist commonly abused for its rewarding effects. Since its synthesis at the end of the nineteenth century, its popularity as a recreational drug has ebbed and flowed. In the last three decades, heroin use has increased again, and yet the pharmacology of heroin is still poorly understood. After entering the body, heroin is rapidly deacetylated to 6-monoacetylmorphine (6-MAM), which is then deacetylated to morphine. Thus, drug addiction literature has long settled on the notion that heroin is little more than a pro-drug. In contrast to these former views, we will argue for a more complex interplay among heroin and its active metabolites: 6-MAM, morphine, and morphine-6-glucuronide (M6G). In particular, we propose that the complex temporal pattern of heroin effects results from the sequential, only partially overlapping, actions not only of 6-MAM, morphine, and M6G, but also of heroin per se, which, therefore, should not be seen as a mere brain-delivery system for its active metabolites. We will first review the literature concerning the pharmacokinetics and pharmacodynamics of heroin and its metabolites, then examine their neural and behavioral effects, and finally discuss the possible implications of these data for a better understanding of opioid reward and heroin addiction. By so doing we hope to highlight research topics to be investigated by future clinical and pre-clinical studies.

Effects of intrathecally administered interferon α on chronic constriction injury model rats' mechanical pain threshold and G protein expression in the spinal cord.

INTRODUCTION: The aim of the study was to explore the analgesic mechanism of effects of intrathecally administered interferon a (IFN-a) on chronic constriction injury (CCI) model rats. MATERIAL AND METHODS: 24 rats were divided into 6 groups, with 4 rats in each group, including the negative control group (Group N, no operation or treatment), the sham operation group (Group S, only the left sciatic nerve of the rats was exposed without ligation, 0.9% NaCl was intrathecally administered), and four experimental groups (CCI model was established first and then different drugs were intrathecally administered respectively), including 0.9% NaCl (Group C), IFN-a (Group CI), morphine (Group CM), and IFN-a combined with morphine (Group CIM). The mRNA levels of G proteins in both the spinal cord and dorsal root ganglia (DRG), as well as the content of amino acid and chemokine (C-X-C motif) ligand 6 (CXCL-6) in the cerebrospinal fluid were measured and analysed in each group. RESULTS: Intrathecal administration of IFN-a increased the mechanical pain threshold in CCI rats (33.32 ±1.36 vs. 21.08 ±1.59, p < 0.001), achieving the effect comparable to that of morphine (33.32 ±1.36 vs. 32.44 ±3.18, p > 0.05), increased the mRNA expression level of Gi protein (0.62 ±0.04 vs. 0.49 ±0.05, p = 0.006), and decreased the mRNA expression level of Gs protein in the spinal cord (1.80 ±0.16 vs. 2.06 ±0.15, p = 0.035) and DRG (2.11 ±0.10 vs. 2.79 ±0.13, p < 0.001). The intrathecal administration of both IFN-a and morphine can reduce the glutamate content in the cerebrospinal fluid (261.55 ±38.12 vs. 347.70 ±40.69, p = 0.012), but without any statistically significant difference in the content of CXCL-6 across all groups ( p > 0.05). CONCLUSIONS: Intrathecal injection of IFN-a improved the mechanical pain threshold in CCI rats, so we inferred that intrathecal administration of IFN-a had analgesic effects on neuropathic pain, possibly related to the activation of G-proteincoupled µ receptors in the spinal cord and the inhibition of glutamate release.

6-Monoacetylmorphine-antibody distribution in tissues from heroin-related death cases: An experimental study to investigate the distributive response.

Heroin, a semisynthetic opioid drug synthesized from morphine, is the 3,6-diacetyl ester of morphine (diacetylmorphine). The post-mortem diagnosis of heroin-related death could be an issue and usually rely on a combination of investigations, including the autopsy, histological and toxicological analysis. We conducted the present study to evaluate the correlation between the heroin concentration in biological fluids (peripheral blood, bile and urine) and the post-mortem anti-6-MAM antibody expression in various tissues (brain, heart, lung, liver and kidney) using immunohistochemical staining. A quantitative analysis of the immunohistochemical reaction was carried out. 45 cases of heroin-related death investigated at the Forensic Pathology Institutes of the University of Rome, Foggia and Pisa were included. The control group was composed of 15 cases of death due to other causes, without brain lesions and negative toxicological analysis for drugs. We found a positive immunohistochemical reaction in different organs and it was related to the timing of heroin metabolization. No reaction was found in the control group. Our findings show that immunohistochemistry can be a valuable tool for the post-mortem diagnosis of acute heroin abuse. A better understanding of the timing of heroin's metabolism can be useful in the forensic field and for future therapeutic applications.

Pharmacokinetics and transplacental transfer of codeine and codeine metabolites from Papaver somniferum L.

ETHNOPHARMACOLOGICAL RELEVANCE: Papaveris Pericarpium, which is the dried husk of Papaver somniferum L., has been used as a phytomedicine to relieve cough, diarrhea and pain. The alkaloid codeine contained therein via biotransformation converts to morphine and potentially produces addictive and toxic effects. Due to the healthy concern for a pregnant woman, our hypothesis is that codeine and its metabolites can penetrate the placental barrier to reach the foetus and amniotic fluid, and these processes may be modulated by the transporter. AIM OF THE STUDY: Because codeine is also considered a prodrug of morphine, it has a good analgesic effect. It is often used by pregnant women but may expose the foetus to the risk of morphine harm. The aim of this study is to investigate the metabolic rate, distribution and transplacental transfer mechanism of codeine and its metabolites morphine and morphine-3-glucuronide (M3G) in pregnant rats and to assess the risk of medication for pregnant women. MATERIALS AND METHODS: Ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) combined with a microdialysis system was developed to monitor codeine, morphine and M3G in multiple sites of maternal blood, placenta, foetus and amniotic fluid after codeine administration. A compartmental model was used to calculate the pharmacokinetic parameters of codeine in blood after codeine administration (10 mg/kg, i.v.). The area under the concentration (AUC) ratio of AUCmetabolite/AUCcodeine and AUCtissue/AUCblood was used to represent the metabolic biotransformation ratio and the drug from blood-to-tissue transfer ratio, respectively. RESULTS: The pharmacokinetic results demonstrated that codeine fit well with a two-compartment model and went through rapid metabolism to morphine and M3G in pregnant rats after codeine administration (10 mg/kg, i.v.). The biotransformation ratios of AUCmorphine/AUCcodeine, AUCM3G/AUCmorphine and AUCM3G/AUCcodeine were 0.12 ± 0.03, 54.45 ± 20.61 and 6.53 ± 2.47, respectively, after codeine administration (10 mg/kg, i.v.), which suggested that codeine was easily metabolized into M3G through morphine. The tissue distribution results demonstrated that all of the analytes penetrated into the foetus through the placenta; however, the blood-to-tissue transfer ratio (AUCtissue/AUCblood) of morphine and M3G was relatively lower than that of codeine after codeine administration (10 mg/kg, i.v.), which suggested that the blood-placenta barrier blocks the penetration of morphine and M3G into the foetus. Thus, the tissue transfer of morphine in the placenta and foetus was significantly enhanced by treatment with corticosterone, an inhibitor of organic cation transporter (OCT). CONCLUSION: Based on microdialysis coupled to a validated UHPLC-MS/MS system, the pharmacokinetics and metabolic biotransformation of codeine and its metabolites were analyzed and clarified. The potential mechanism of morphine placental transfer was modulated by OCT transporters.

Prefrontal Cortex Response to Prenatal Insult and Postnatal Opioid Exposure.

The influence of proinflammatory challenges, such as maternal immune activation (MIA) or postnatal exposure to drugs of abuse, on brain molecular pathways has been reported. On the other hand, the simultaneous effects of MIA and drugs of abuse have been less studied and sometimes offered inconsistent results. The effects of morphine exposure on a pig model of viral-elicited MIA were characterized in the prefrontal cortex of males and females using RNA-sequencing and gene network analysis. Interacting and main effects of morphine, MIA, and sex were detected in approximately 2000 genes (false discovery rate-adjusted p-value < 0.05). Among the enriched molecular categories (false discovery rate-adjusted p-value < 0.05 and −1.5 > normalized enrichment score > 1.5) were the cell adhesion molecule pathways associated with inflammation and neuronal development and the long-term depression pathway associated with synaptic strength. Gene networks that integrate gene connectivity and expression profiles displayed the impact of morphine-by-MIA interaction effects on the pathways. The cell adhesion molecules and long-term depression networks presented an antagonistic effect between morphine and MIA. The differential expression between the double-challenged group and the baseline saline-treated Controls was less extreme than the individual challenges. The previous findings advance the knowledge about the effects of prenatal MIA and postnatal morphine exposure on the prefrontal cortex pathways.

Pharmacokinetic modeling of morphine and its glucuronides: Comparison of nebulization versus intravenous route in healthy volunteers.

Intravenous (i.v.) morphine is a safe, robust, and recommended treatment for severe pain using the titration principle. Despite its high efficacy, it is impacted by organizational constraints related to venous access. Nebulized (NEB) morphine may represent an alternative for titration but pharmacokinetic (PK) properties of short nebulization using routine devices need evaluation. Twenty-seven healthy volunteers were included to receive NEB or i.v. morphine administration using increasing amounts according to Dixon's reference method. Plasma morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) were quantified. PK modeling and simulations were performed using Monolix. Dixon's method exhibited a significantly higher morphine dose regimen in the NEB group versus the i.v. group (6.2 [5.3-7.1] vs. 3.0 [2.0-4.0] mg, p < 0.001). Morphine, M3G, and M6G dose-normalized exposure were significantly lower in the NEB group versus the i.v. group: morphine (19 [13-23] vs. 1044 [702-1266] µg min/L, p < 0.001), M3G (245 [162-287] vs. 3752 [2487-5165] µg min/L, p < 0.001) and M6G (28 [21-43] vs. 466 [370-723] µg min/L, p < 0.001). The model that best fitted the data consisted in a transit compartment for morphine absorption, three compartments for morphine distribution followed by multiple transit compartments (8.2 and 57.5-min transit time for M3G and M6G, respectively) and a first order elimination for M3G and M6G. Morphine bioavailability in the NEB group was 3.5% using the i.v. group as reference. Administration route and sex significantly influenced morphine and metabolite PKs. This work aimed to evaluate the PKs of NEB morphine compared with the i.v. route. Despite a bioavailability to improve, NEB morphine administration using a routine device is suitable to plan morphine titration.

Deep proteomics and phosphoproteomics reveal novel biological pathways perturbed by morphine, morphine-3-glucuronide and morphine-6-glucuronide in human astrocytes.

Tolerance and hyperalgesia associated with chronic exposure to morphine are major limitations in the clinical management of chronic pain. At a cellular level, neuronal signaling can in part account for these undesired side effects, but unknown mechanisms mediated by central nervous system glial cells are likely also involved. Here we applied data-independent acquisition mass spectrometry to perform a deep proteome and phosphoproteome analysis of how human astrocytes responds to opioid stimulation. We unveil time- and dose-dependent effects induced by morphine and its major active metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide that converging on activation of mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. We also find that especially longer exposure to M3G leads to significant dysregulation of biological pathways linked to extracellular matrix organization, antigen presentation, cell adhesion, and glutamate homeostasis, which are crucial for neuron- and leukocyte-astrocyte interactions.

Functional phenotyping of the CYP2D6 probe drug codeine in the horse.

BACKGROUND: In humans, the drug metabolizing enzyme CYP2D6 is highly polymorphic resulting in substantial differences in the metabolism of drugs including anti-arrhythmics, neuroleptics, and opioids. The objective of this study was to phenotype a population of 100 horses from five different breeds and assess differences in the metabolic activity of the equine CYP2D6 homolog using codeine as a probe drug. Administration of a probe drug is a common method used for patient phenotyping in human medicine, whereby the ratio of parent drug to metabolite (metabolic ratio, MR) can be used to compare relative enzyme function between individuals. A single oral dose of codeine (0.6 mg/kg) was administered and plasma concentrations of codeine and its metabolites were determined using liquid chromatography mass spectrometry. The MR of codeine O-demethylation [(codeine)/(morphine + morphine-3-glucuronide + morphine-6-glucuronide)] was determined using the area under the plasma concentration-time curve extrapolated from time zero to infinity (AUC0-∞) for each analyte and used to group horses into predicted phenotypes (high-, moderate-, and low-MR). RESULTS: The MR of codeine O-demethylation ranged from 0.002 to 0.147 (median 0.018) among all horses. No significant difference in MR was observed between breeds, age, or sex. Of the 100 horses, 11 were classified as high-MR, 72 moderate-MR, and 17 low-MR. Codeine AUC0-∞ and O-demethylation MR were significantly different (p < 0.05) between all three groups. The mean ± SD MR was 0.089 ± 0.027, 0.022 ± 0.011, and 0.0095 ± 0.001 for high-, moderate-, and low-MR groups, respectively. The AUC for the morphine metabolites morphine-3-glucuronide and morphine-6-glucuronide were significantly different between high-and low-MR groups (p < 0.004 and p < 0.006). CONCLUSIONS: The MR calculated from plasma following codeine administration allowed for classification of horses into metabolic phenotypes within a large population. The range of codeine metabolism observed among horses suggests the presence of genetic polymorphisms in CYP2D82 of which codeine is a known substrate. Additional studies including CYP2D82 genotyping of high- and low-MR individuals are necessary to determine the presence of CYP2D polymorphisms and their functional implications with respect to the metabolism of therapeutics.

Identification of characteristic heroin metabolites in urine based on data-mining technology and multivariate statistics analysis combined with a targeted verification approach for distinguishing heroin abusers.

A common phenomenon shows that ingestion of opium poppy shell-containing drugs can result in a "false-positive" urinalysis test result for mandatory or workplace heroin abuse screening. Owing to the short detection window (8 h in urine) of the characteristic heroin metabolite 6-monoacetylmorphine (6-MAM) confirmation or exclusion of heroin abusers still presents major challenges for toxicologists. In this work, we developed an ultra-performance liquid chromatography-time-of-flight mass spectrometry method (UPLC-TOF-MS) with online data acquisition and multiple post-data-mining technologies combined with a multivariate statistical and batch validation analysis workflow to assess the characteristic urine metabolites of heroin abusers. Based on the proposed methods, 28 characteristic metabolites were structurally identified, and their fragmentation patterns and metabolite pathways were also summarized. Correlation analysis was used to investigate the internal relationship and similarities among the identified metabolites, and seven representative metabolites were selected as "Target-metabolites". Multi-batch urine of samples of heroin abusers were certified based on the UPLC-MS/MS method for further validation of the practicability of using this method for routine analysis. Overall, the target-metabolites can be utilized as assistant "biomarkers" in workplace or mandatory drug screenings. This approach encourages further studies on the development of the "false-positive" identification system.

Use of a Noninvasive Brain-Penetrating Peptide-Drug Conjugate Strategy to Improve the Delivery of Opioid Pain Relief Medications to the Brain.

The analgesic potency of morphine-6-glucuronide (M6G) has been shown to be 50-fold higher than morphine after intracerebral injection. However, the brain penetration of M6G is significantly lower than morphine, thus limiting its usefulness in pain management. Here, we created new entities by the conjugation of the angiopep-2 peptide (An2) that crosses the blood-brain barrier (BBB) by low-density lipoprotein receptor-related protein 1 receptor-mediated transcytosis with either morphine or M6G. We demonstrated improvement of BBB permeability of these new entities compared with that of unconjugated M6G and morphine. Intravenous or subcutaneous administration of the An2-M6G conjugate exerted greater and more sustained analgesic activity than equivalent doses of either morphine or M6G. Likewise, subcutaneous An2-morphine induced a delayed but prolonged antinociceptive effect. The effects of these conjugates on the gastrointestinal tract motility were also evaluated. An2-morphine significantly reduced the intestinal transit time, whereas An2-M6G exhibited a reduced constipation profile, as compared with an equimolar dose of morphine. In summary, we have developed new brain-penetrant opioid conjugates exhibiting improved analgesia to side effect ratios. These results thus support the use of An2-carrier peptides as an innovative BBB-targeting technology to deliver effective drugs, such as M6G, for pain management. SIGNIFICANCE STATEMENT: The metabolite morphine-6-glucuronide (M6G) does not efficiently cross the blood-brain barrier. The low-density lipoprotein receptor-related protein 1 peptide ligand angiopep-2 may serve as an effective drug delivery system to the brain. Here, we demonstrated that the coupling of M6G to angiopep-2 peptide (An2) improves its brain penetration and significantly increases its analgesic potency. The An2-M6G conjugate has a favorable side effect profile that includes reduction of developing constipation. An2-M6G exhibits a unique pharmacodynamic profile with a better therapeutic window than morphine.

Morphine-3-glucuronide causes antinociceptive cross-tolerance to morphine and increases spinal substance P expression.

Morphine-3-glucuronide (M3G), the main metabolite of morphine, has been implicated in the development of tolerance and of opioid-induced hyperalgesia, both limiting the analgesic use of morphine. We evaluated the acute and chronic effects of M3G and morphine as well as development of antinociceptive cross-tolerance between morphine and M3G after intrathecal administration and assessed the expression of pain-associated neurotransmitter substance P in the spinal cord. Sprague-Dawley rats received intrathecal M3G or morphine twice daily for 6 days. Nociception and tactile allodynia were measured with von Frey filaments after acute and chronic treatments. Substance P levels in the dorsal horn of the spinal cord were determined by immunohistochemistry after 4-day treatments. Acute morphine caused antinociception as expected, whereas acute M3G caused tactile allodynia, as did both chronic M3G and morphine. Chronic M3G also induced antinociceptive cross-tolerance to morphine. M3G and morphine increased substance P levels similarly in the nociceptive laminae of the spinal cord. This study shows that chronic intrathecal M3G sensitises animals to mechanical stimulation and elevates substance P levels in the nociceptive laminae of the spinal cord. Chronic M3G also induces antinociceptive cross-tolerance to morphine. Thus, chronic M3G exposure might contribute to morphine-induced tolerance and opioid-induced hyperalgesia.

Lipopolysaccharide and Morphine-3-Glucuronide-Induced Immune Signalling Increases the Expression of Polysialic Acid in PC12 Cells.

Polysialic acid (polySia), a long homopolymer of 2,8-linked sialic acids, is abundant in the embryonic brain and is restricted largely in adult brain to regions that exhibit neurogenesis and structural plasticity. In the central nervous system (CNS), polySia is highly important for cell-cell interactions, differentiation, migration and cytokine responses, which are critical neuronal functions regulating intercellular interactions that underlie immune signalling in the CNS. In recent reports, a metabolite of morphine, morphine-3-glucuronide (M3G), has been shown to cause immune signalling in the CNS. In this study, we compared the effects of neurite growth factor (NGF), lipopolysaccharide (LPS) and M3G exposure on the expression of polySia in PC12 cells using immunocytochemistry and Western blot analysis. PolySia was also extracted from stimulated cell proteins by endo-neuraminidase digestion and quantitated using fluorescent labelling followed by HPLC analysis. PolySia expression was significantly increased following NGF, M3G or LPS stimulation when compared with unstimulated cells or cells exposed to the TLR4 antagonist LPS-RS. Additionally, we analyzed the effects of test agent exposure on cell migration and the oxidative stress response of these cells in the presence and absence of polySia expression on their cell surface. We observed an increase in oxidative stress in cells without polySia as well as following M3G or LPS stimulation. Our study provides evidence that polySia expression in neuronal-like PC12 cells is influenced by M3G and LPS exposure alike, suggestive of a role of TLR4 in triggering these events.

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.

6-Monoacetylmorphine (6-MAM), Not Morphine, Is Responsible for the Rapid Neural Effects Induced by Intravenous Heroin.

Heroin rapidly enters the CNS but is quickly metabolized into 6-monoacetylmorphine (6-MAM) and then morphine. Although morphine is often thought to mediate heroin's neural effects, pharmacokinetic data question this view. To further understand the effects of heroin and its metabolites, oxygen sensors were used to examine changes in nucleus accumbens (NAc) oxygen levels. Heroin, 6-MAM, and morphine were all administered intravenously at two human-relevant doses (0.25 µmol/kg and 0.98 µmol/kg) in freely moving rats. Intravenous heroin induced a biphasic change in NAc oxygen, with a decrease resulting from respiratory depression and an increase resulting from cerebral vasodilation. 6-MAM caused similar but more rapid and slightly weaker effects  than heroin. The stronger response to heroin can be primarily attributed to heroin's permeability and metabolism resulting in more 6-MAM in the brain. Morphine only induced weak increases in NAc oxygen. Therefore, it appears that 6-MAM is the major contributor to acute neural effects induced by iv heroin.