HIV-1 and opiates modulate miRNA profiles in extracellular vesicles.

Opiate abuse increases the risk of HIV transmission and exacerbates HIV neuropathology by increasing inflammation and modulating immune cell function. Exosomal EVs(xEV) contain miRNAs that may be differentially expressed due to HIV infection or opiate abuse. Here we develop a preliminary exosomal-miRNA biomarker profile of HIV-infected PBMCs in the context of opiate use. PBMCs infected with HIV were treated with increasing dosages of morphine for 72 hours, the culture supernatants were collected, and the exosomes isolated using differential centrifugation. Exosomal miRNAs were extracted, expression levels determined via Nanostring multiplexed microRNA arrays, and analyzed with Webgestalt. The effect of the exosomes on neuronal function was determined by measuring calcium. Preliminary findings show that HIV-1 infection altered the miRNA profile of PBMC-derived EVs concurrently with opiate exposure. MicroRNA, hsa-miR-1246 was up-regulated 12-fold in the presence of morphine, relative to uninfected control. PBMCs infected with HIV-1 MN, an X4-tropic HIV-1 strain and exposed to morphine, displayed a trend which suggests potential synergistic effects between HIV-1 infection and morphine exposure promoting an increase in viral replication. Dose-dependent differences were observed in miRNA expression as a result of opiate exposure. The xEVs derived from PBMCs exposed to morphine or HIV modulated neuronal cell function. SH-SY5Y cells, treated with xEVs derived from ART-treated PBMCs, exhibited increased viability while for SH-SY5Ys exposed to xEVs derived from HIV-1 infected PBMCs viability was decreased compared to the untreated control. Exposing SH-SY5Y to xEVs derived from HIV-infected PBMCs resulted in significant decrease in calcium signaling, relative to treatment with xEVs derived from uninfected PBMCs. Overall, HIV-1 and morphine induced differential miRNA expression in PBMC-derived exosomes, potentially identifying mechanisms of action or novel therapeutic targets involved in opiate use disorder, HIV neuropathology, TNF signaling pathway, NF-κB signaling pathway, autophagy, and apoptosis in context of HIV infection.

Effects of sodium oxybate on hypocretin/orexin and locus coeruleus neurons.

Long-term use of sodium oxybate (SXB), (also called gamma-hydroxybutyrate [GHB]) attenuates the cataplexy and sleepiness of human narcolepsy. We had previously found that chronic opiate usage in humans and long-term opiate administration to mice significantly increased the number of detected hypocretin/orexin (Hcrt) neurons, decreased their size, and increased Hcrt level in the hypothalamus. We also found that opiates significantly decreased cataplexy in human narcoleptics as well as in narcoleptic mice and that cessation of locus coeruleus neuronal activity preceded and was tightly linked to cataplectic attacks in narcoleptic dogs. We tested the hypothesis that SXB produces changes similar to opiates and now report that chronic SXB administration significantly increased the size of Hcrt neurons, the reverse of what we had seen with opiates in humans and mice. Levels of Hcrt in the hypothalamus were nonsignificantly lower, in contrast to the significant increase in hypothalamic Hcrt level after opiates. SXB decreased tyrosine hydroxylase levels in the locus coeruleus, the major descending projection of the hypocretin system, also the reverse of what we saw with opioids. Therefore despite some similar effects on narcoleptic symptomatology, SXB does not produce anatomical changes similar to those elicited by opiates. Analysis of changes in other links in the cataplexy pathway might further illuminate SXB's mechanism of action on narcolepsy.

Add-On Selective Estrogen Receptor Modulators for Methadone Maintenance Treatment.

Methadone maintenance treatment (MMT) remains the cornerstone for the management of opiate abuse. However, MMT can be associated with complex factors, including complications during the tolerance phase, the inability of some patients to maintain treatment effects during the tapering or abstinence phases, and the development of methadone dependence. Previous studies have revealed a sex disparity in MMT efficacy, showing that women undergoing MMT experiencing an increase in psychological symptoms compared with men and suggesting a link between disparate responses and the effects of estrogen signaling on methadone metabolism. More specifically, estradiol levels are positively associated with MMT dosing, and the expression of a single-nucleotide polymorphism (SNP) associated with estrogen receptor (ER) regulation is also associated with MMT dosing. In addition to performing mechanistic dissections of estrogen signaling in the presence of methadone, past studies have also proposed the targeting of estrogen signaling during MMT. The present report provides an overview of the relevant literature regarding sex effects, including differences in sex hormones and their potential impacts on MMT regimens. Moreover, this article provides a pharmacological perspective on the targeting of estrogen signals through the use of selective ER modulators (SERMs) during MMT. Preliminary preclinical experiments were also performed to evaluate the potential effects of targeting estrogen signaling with tamoxifen on methadone metabolism.

[The chemoreactomic analysis of the central mechanisms of action of non-steroidal anti-inflammatory drugs]. / Khemoreaktomnyi analiz tsentral'nykh mekhanizmov nesteroidnykh protivovospalitel'nykh preparatov.

AIM: To perform a chemoreactome modeling of the pharmacological central effects of 4 non-steroidal anti-inflammatory drugs (NSAIDs): dexketoprofen, ketoprofen, aceclofenac, lornoxicam. MATERIAL AND METHODS: An analysis of the pharmacological spectrum of the central action of dexketoprofen, ketoprofen, aceclofenac and lornoxicam was based on the chemoinformatic approach, which compared drug-likeness properties with public and commercial software. RESULTS: The effectiveness of NSAIDs is related to the inhibition of cannabinoid receptors CB-1, the vanilloid receptor TRPV1, NMDA and AMPA receptors and of the GABA reuptake transporter, with dexketoprofen being the most effective inhibitor. The safety of the central effects of NSAID is due to weak interactions of the NSAIDs studied with opioid, adrenergic, serotonin and dopamine receptors. Chemoreactome modeling made it possible to compare the particulars of the effects of the studied NSAIDs on experimental pain and cramps. CONCLUSION: Inhibition of CB-1, TRPV1, NMDA, AMPA, GABA transporter by the NSAID molecules corresponds to a decrease in the intensity of nociceptive signals. A weak intervention of the studied NSAIDs in opioid, adrenergic, serotonin and dopaminergic neurotransmission corresponds to a decrease in the central side-effects of NSAIDs and to a lessened antagonism of these NSAIDs towards exogenous and endogenous opioids.

Generalized seizures evoked by nucleus accumbens stimulation induced an opiate-mediated suppression of psychosis relevant behaviors.

We have previously demonstrated that kindling of the nucleus accumbens (NAc) induced psychosis relevant behaviors only after one, but not after five, stage-5 seizures, suggesting that five stage-5 NAc-evoked seizures antagonized psychosis relevant behaviors in rats. We hypothesized that brain opioid receptors are responsible for seizure-induced reduction of psychosis relevant behaviors in NAc kindled rats. Rats received NAc kindling until a stage-4 seizure was induced, after which naloxone, a non-specific opioid receptor antagonist, at dose of 1 or 10 mg/kg i.p., or saline (0.3 mL) i.p., was injected 15 min before each kindled seizure. Duration of afterdischarge (AD) was not significantly different among naloxone- and saline-treated groups. However, duration of postictal behavioral depression induced by a stage-5 seizure was significantly shorter in 10 mg/kg naloxone-treated than saline-treated rats, for long (>36 s) AD duration. When tested 3-4 days after five stage-5 seizures, 10 mg/kg naloxone-treated rats, as compared to saline-treated rats, showed a statistically significant loss of gating of hippocampal auditory evoked potentials, and significant reduction of startle response amplitude, but non-significant differences in prepulse inhibition and methamphetamine-induced locomotion. It is inferred that stage-5 seizures, by releasing endogenous opiates, contribute to postictal behavioral depression, and some long-term seizure-induced antipsychotic effects.

A central role for glial CCR5 in directing the neuropathological interactions of HIV-1 Tat and opiates.

BACKGROUND: The collective cognitive and motor deficits known as HIV-associated neurocognitive disorders (HAND) remain high even among HIV+ individuals whose antiretroviral therapy is optimized. HAND is worsened in the context of opiate abuse. The mechanism of exacerbation remains unclear but likely involves chronic immune activation of glial cells resulting from persistent, low-level exposure to the virus and viral proteins. We tested whether signaling through C-C chemokine receptor type 5 (CCR5) contributes to neurotoxic interactions between HIV-1 transactivator of transcription (Tat) and opiates and explored potential mechanisms. METHODS: Neuronal survival was tracked in neuronal and glial co-cultures over 72 h of treatment with HIV-1 Tat ± morphine using cells from CCR5-deficient and wild-type mice exposed to the CCR5 antagonist maraviroc or exogenously-added BDNF (analyzed by repeated measures ANOVA). Intracellular calcium changes in response to Tat ± morphine ± maraviroc were assessed by ratiometric Fura-2 imaging (analyzed by repeated measures ANOVA). Release of brain-derived neurotrophic factor (BDNF) and its precursor proBDNF from CCR5-deficient and wild-type glia was measured by ELISA (analyzed by two-way ANOVA). Levels of CCR5 and µ-opioid receptor (MOR) were measured by immunoblotting (analyzed by Student's t test). RESULTS: HIV-1 Tat induces neurotoxicity, which is greatly exacerbated by morphine in wild-type cultures expressing CCR5. Loss of CCR5 from glia (but not neurons) eliminated neurotoxicity due to Tat and morphine interactions. Unexpectedly, when CCR5 was lost from glia, morphine appeared to entirely protect neurons from Tat-induced toxicity. Maraviroc pre-treatment similarly eliminated neurotoxicity and attenuated neuronal increases in [Ca2+]i caused by Tat ± morphine. proBDNF/BDNF ratios were increased in conditioned media from Tat ± morphine-treated wild-type glia compared to CCR5-deficient glia. Exogenous BDNF treatments mimicked the pro-survival effect of glial CCR5 deficiency against Tat ± morphine. CONCLUSIONS: Our results suggest a critical role for glial CCR5 in mediating neurotoxic effects of HIV-1 Tat and morphine interactions on neurons. A shift in the proBDNF/BDNF ratio that favors neurotrophic support may occur when glial CCR5 signaling is blocked. Some neuroprotection occurred only in the presence of morphine, suggesting that loss of CCR5 may fundamentally change signaling through the MOR in glia.

Exposure to opiates in male adolescent rats alters pain perception in the male offspring.

During the past decades, the use/misuse of opioids has increased dramatically among adolescent population. It is now well acknowledged that various morphological and physiological changes occur in the brain during adolescence. During this critical period, brain development and maturation could be affected by several factors including stress, drug abuse, nutritional status, etc. Although studies on transgenerational effects of substances such as alcohol, nicotine, and cocaine have focused on both paternal and maternal drug exposure, most reports on transgenerational effects of morphine are restricted to maternal exposure. Thus, in this study, we aimed to investigate the transgenerational effect of paternal morphine exposure during adolescence on pain perception and antinociceptive effect of morphine in rat offspring. Male rats received escalating doses of morphine for 10 days during postnatal days 31-40. Twenty days after the last morphine injection, male rats were mated with intact female rats, and then behavioral tests were conducted on the male offspring on postnatal day 60. Pain perception and morphine antinociception were evaluated using the formalin test. Our results demonstrated that morphine-sired and saline-sired animals differed in the interphase and phase 2 of the formalin test. These findings indicate a significant transgenerational effect of paternal morphine exposure on pain-related behaviors in rat offspring.

Insights into the link between drug use and criminality: Lifetime offending of criminally-active opiate users.

BACKGROUND: We test whether the offending trajectory of those who test positive for opiates is greater than test-negative controls and whether the relationship is constant both prior to, and post, opiate initiation. We consider whether these relationships differ according to gender and offence type. METHODS: The study provides an analysis of historical offending records in adults linked to test results for opiate and cocaine metabolites. Those testing positive for opiates were linked to treatment records to retrieve data on age of opiate initiation. Rate ratios (RR) were calculated to compare opiate positive testers to opiate and cocaine negative controls, separately by gender and adjusting for age and birth cohort. Age of opiate initiation was included in a second model as a time-dependent variable. Within-subject clustering was accounted for using generalised estimating equations. RESULTS: Opiate-positive cases had higher rates of offending than test-negative controls, both prior to, and post, opiate initiation. Initiation of opiate use increased the RR by 16% for males but doubled it for females. The RR increase in non-serious acquisitive crime was greater than that seen in serious crime. For males only, opiate initiation narrowed the difference in violent offending rate between cases and controls. A larger offending increase was associated with opiate initiation in female, compared to male, users. CONCLUSIONS: For most crime categories, the difference between groups is exacerbated by opiate initiation. The findings indicate that opiate prevention initiatives might be effective in reducing offending, particularly among females.

Supramolecular Sensors for Opiates and Their Metabolites.

The present study highlights a sensing approach for opiates using acyclic cucurbituril (aCBs) sensors comprising four glycouril units terminated on both ends with naphthalene fluorophore walls. The connectivity between the glycourils and naphthalene rings largely defines the opening size of the cucurbituril cavity and its diameter. The large hydrophobic binding cavity is flexible and is able to adapt to guests of various size and topology. The recognition event between the aCBs and guests results in modification of the fluorescence of the terminal walls, a fluorescence response that can be used to sense the drugs of abuse morphine, heroin, and oxycodone as well as their metabolites. Molecular dynamics is employed to understand the nature of the binding interactions. A simple three sensor cross-reactive array enables the determination of drugs and their metabolites in water with high fidelity and low error. Quantitative experiments performed in urine using a new three-way calibration model allows for determination of drugs and their metabolites using one sensor from a single fluorescence reading.

Anaesthesia in patients with liver disease.

PURPOSE OF REVIEW: The purpose of this review is to summarize the most recent up to date research data and recommendations regarding anaesthetic management of patients with liver disease undergoing surgery. The incidence of chronic liver disease (CLD) continues to rise and perioperative mortality and morbidity remains unacceptably high in this group. Meticulous preoperative assessment and carefully planned anaesthetic management are vital in improving outcomes in patients with liver disease undergoing surgery. RECENT FINDINGS: The presence of cirrhosis is associated with a significantly increased risk of postoperative morbidity and mortality in patients undergoing elective surgery. The Child--Pugh--Turcotte scale and model for end-stage liver disease (MELD) score remain the most commonly applied scoring systems in preoperative risk assessment, but new MELD-based indices and novel scoring systems might offer better prognostic value. Propofol and new inhalational agents (sevoflurane, desflurane) are recommended hypnotic agents. The titration of opiates in the perioperative period is recommended because of their altered metabolism in patients with liver disease. Perioperative management should include close haemodynamic monitoring and admission to a critical care area should be considered. SUMMARY: Patients with liver disease undergoing anaesthesia pose significant challenges and advanced planning and preparation are required in order to improve perioperative outcomes in this group. VIDEO ABSTRACT: http://links.lww.com/COAN/A43.

Productive infection of human neural progenitor cells by R5 tropic HIV-1: opiate co-exposure heightens infectivity and functional vulnerability.

OBJECTIVE: HIV type-1 (HIV-1) causes a spectrum of central nervous system (CNS) complications; many are worsened by opiate co-exposure. Human neural progenitor cells (hNPCs) give rise to all CNS neurons and macroglia. We tested the hypothesis that hNPC maturation and fate are altered by HIV and opiates, contributing to HIV-1-related neuropathology. Reports of hNPC infection remain controversial. We rigorously examined this question, testing whether hNPCs propogated infection, and whether HIV affected hNPCs absent their infection. DESIGN AND METHODS: Primary hNPCs were characterized over multiple passages. Following R5 HIV-1BaL exposure, p24, Nef, and tat assays monitored infection; a serial dilution approach tested infection transfer to naive hNPCs. Bromodeoxyuridine uptake, population doubling time, and immunostaining assessed proliferation and differentiation. Morphine co-exposure assessed opiate interactions. Supernatant from HIV-1BaL-infected PBMCs (HIVsup), HIV-1BaL, and ultraviolet light-inactivated HIVsup were compared to test effects of inflammatory milieu versus virus or infection per se. RESULTS: The hNPCs (CD4/CD8/Iba/CXC3CL1/CD11b) were infectable and could transfer infection to naive hNPCs. Infection was partly blocked by maraviroc, implicating CCR5. HIVsup reduced hNPC proliferation and caused premature differentiation into neurons/astroglia. Effects on proliferation were due to soluble factors/viral proteins, not infection per se. Morphine co-exposure exacerbated certain functional consequences of HIVsup, and sustained the infection of hNPCs. CONCLUSION: hNPCs can be infected and propagate virus in vitro. hNPCs or their progeny may represent an underappreciated viral reservoir. Factors from infected cells alter hNPC proliferation and neural cell maturation, which likely compromises CNS structure and function. Morphine-HIV interactions may worsen dysfunction and sustain infection.

Synthesis and evaluation of aryl-naloxamide opiate analgesics targeting truncated exon 11-associated µ opioid receptor (MOR-1) splice variants.

3-Iodobenzoylnaltrexamide 1 (IBNtxA) is a potent analgesic acting through a novel receptor target that lack many side-effects of traditional opiates composed, in part, of exon 11-associated truncated six transmembrane domain MOR-1 (6TM/E11) splice variants. To better understand the SAR of this drug target, a number of 4,5-epoxymorphinan analogues were synthesized. Results show the importance of a free 3-phenolic group, a phenyl ring at the 6 position, an iodine at the 3'or 4' position of the phenyl ring, and an N-allyl or c-propylmethyl group to maintain high 6TM/E11 affinity and activity. 3-Iodobenzoylnaloxamide 15 (IBNalA) with a N-allyl group displayed lower δ opioid receptor affinity than its naltrexamine analogue, was 10-fold more potent an analgesic than morphine, elicited no respiratory depression or physical dependence, and only limited inhibition of gastrointestinal transit. Thus, the aryl-naloxamide scaffold can generate a potent analgesic acting through the 6TM/E11 sites with advantageous side-effect profile and greater selectivity.

Fragmentation pathways of drugs of abuse and their metabolites based on QTOF MS/MS and MS(E) accurate-mass spectra.

A study of the fragmentation pathways of several classes of drugs of abuse (cannabinoids, ketamine, amphetamine and amphetamine-type stimulants (ATS), cocaine and opiates) and their related substances has been made. The knowledge of the fragmentation is highly useful for specific fragment selection or for recognition of related compounds when developing MS-based analytical methods for the trace-level determination of these compounds in complex matrices. In this work, accurate-mass spectra of selected compounds were obtained using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry, performing both MS/MS and MS(E) experiments. As regards fragmentation behavior, the mass spectra of both approaches were quite similar and were useful to study the fragmentation of the drugs investigated. Accurate-mass spectra of 37 drugs of abuse and related compounds, including metabolites and deuterated analogues, were studied in this work, and structures of fragment ions were proposed. The accurate-mass data obtained allowed to confirm structures and fragmentation pathways previously proposed based on nominal mass measurements, although new insights and structure proposals were achieved in some particular cases, especially for amphetamine and ATS, 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) and opiates.

Neurochemistry of drug action: insights from proton magnetic resonance spectroscopic imaging and their relevance to addiction.

Proton magnetic resonance spectroscopy ((1)H MRS) is a noninvasive imaging technique that permits measurement of particular compounds or metabolites within the tissue of interest. In the brain, (1)H MRS provides a snapshot of the neurochemical environment within a defined volume of interest. A search of the literature demonstrates the widespread utility of this technique for characterizing tumors, tracking the progress of neurodegenerative disease, and for understanding the neurobiological basis of psychiatric disorders. As of relatively recently, (1)H MRS has found its way into substance abuse research, and it is beginning to become recognized as a valuable complement in the brain imaging toolbox that also contains positron emission tomography, single-photon-emission computed tomography, and functional magnetic resonance imaging. Drug abuse studies using (1)H MRS have identified several biochemical changes in the brain. The most consistent alterations across drug class were reductions in N-acetylaspartate and elevations in myo-inositol, whereas changes in choline, creatine, and amino acid transmitters also were abundant. Together, the studies discussed herein provide evidence that drugs of abuse may have a profound effect on neuronal health, energy metabolism and maintenance, inflammatory processes, cell membrane turnover, and neurotransmission, and these biochemical changes may underlie the neuropathology within brain tissue that subsequently gives rise to the cognitive and behavioral impairments associated with drug addiction.

The detection of various opiates and benzodiazepines by comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry.

A technique using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/TOFMS) is applied to qualitative and quantitative drug testing. Human serum was 'spiked' with known quantities of benzodiazepines and a 'street heroin' mixture including some of the major metabolites and impurities. The sample components were extracted from the matrix by solid-phase extraction (SPE). Constituents containing polar hydroxyl and/or secondary amine groups were derivatised with N-methyl-N-(tert-butyldimethyl)trifluoroacetamide (MTBSTFA) to improve the chromatographic performance. An orthogonal separation of the matrix constituents was achieved by coupling a DB-5ms (5% phenyl) to a BPX50 (50% phenyl) GC column. The eluant was focused onto the second column by a twin-stage cryo-modulator. Rapid 6 s modulation times were achieved by transfer from a 30 m x 0.25 mm (length x internal diameter) to a 2 m x 0.1 mm column. TOFMS with rapid spectral acquisition (< or =500 spectra/s) was employed in the mass range m/z 40-650. A clean mass spectrum was obtained for each analyte using mass spectral deconvolution software. The sensitivity and repeatability of the method were evaluated by the preparation of calibration standards for two benzodiazepines, flunitrazepam and its major metabolite 7-aminoflunitrazepam (7-amino-FN), in the concentration range 5-1000 ng/mL. The limits of detection (LODs) and limits of quantitation (LOQs), calculated by repeat injections (x10) of the lowest standard, were 1.6 and 5.4 ng/mL (flunitrazepam); 2.5 and 8.5 ng/mL (7-amino-FN), respectively. There is scope to extend this protocol to screen a large number of drugs and metabolites stored in a library database.

Integration of GC/EI-MS and GC/NCI-MS for simultaneous quantitative determination of opiates, amphetamines, MDMA, ketamine, and metabolites in human hair.

In this paper, the possibility of using a multiple ionization mode approach of GC/MS was developed for the simultaneous hair testing of common drugs of abuse in Asia, including amphetamines (amphetamine, AP; methamphetamine, MA; methylenedioxy amphetamine, MDA; methylenedioxy methamphetamine, MDMA; methylenedioxy ethylamphetamine, MDEA), ketamine (ketamine, K; norketamine, NK), and opiates (morphine, MOR; codeine, COD; 6-acetylmorphine, 6-AM). This strategy integrated the characteristics of gas chromatography-mass spectrometry (GC-MS) using electron impact ionization (EI) and negative chemical ionization (NCI). Hair samples (25 mg) were washed, cut, and incubated overnight at 25 degrees C in methanol-trifluoroacetic acid (methanol-TFA). The samples were extracted by solid phase extraction (SPE) procedure, derivatized using heptafluorobutyric acid anhydride (HFBA) at 70 degrees C for 30 min, and the derivatives analyzed by GC-MS with EI and NCI. The limit of detection (LOD) with GC/EI-MS analysis obtained were 0.03 ng/mg for AP, MA, MDA, MDMA, and MDEA; 0.05 ng/mg for K, NK, MOR, and COD; and 0.08 ng/mg for 6-AM. The LOD of GC/NCI-MS analysis was much lower than GC/EI-MS analysis. The LOD obtained were 30 pg/mg for AP and MDA in GC/EI-MS and 2 pg/mg in GC/NCI-MS. Therefore, the sensitivity of AP and MDA in GC/NCI-MS was improved from 15-fold compared with EI. The sensitivity of AP, MA, MDA, MDMA, MDEA, MOR, and COD was improved from 15- to 60-fold compared with EI. In addition, the sensitivity of 6-AM increased 8-fold through selection of m/z 197 for the quantitative ion. Moreover, K and NK could dramatically improve their sensitivity at 200- and 2000-fold. The integration of GC/EI-MS and GC/NCI-MS can obtain the high sensitivity and complementary results of drugs of abuse in hair. Six hair samples from known drug abusers were examined by this new strategy. These results show that integrating the characteristics of GC/EI-MS and GC/NCI-MS were not only enhancement of the sensitivity but also avoid wrong results and wrong interpretations of correct results.

Method for quantification of opioids and their metabolites in autopsy blood by liquid chromatography-tandem mass spectrometry.

A method using liquid chromatography-electrospray ionization-tandem mass spectrometry was developed and validated for the determination of morphine, codeine, hydromorphone, dihydrocodeine, oxycodone, buprenorphine, and naloxone with their metabolites morphine-3-glucuronide, morphine-6-glucuronide, normorphine, 6-acetylmorphine, 6-acetylcodeine, codeine-6-glucuronide, norcodeine, hydromorphine-3-glucuronide, dihydrocodeine-6-glucuronide, dihydromorphine, dihydromorphine-3-glucuronide, dihydromorphine-6-glucuronide, oxymorphone, norbuprenorphine, buprenorphine-3-glucuronide, norbuprenorphine-3-glucuronide, and naloxone-3-glucuronide in human whole blood. Polar metabolites (glucuronides) and other analytes were extracted by SPE using Bond Elut C18. Chromatographic separation was performed on a Phenomenex Synergi reversed-phase column with gradient elution based on a mobile phase consisting of 10mM ammonium formate adjusted to pH 3 and acetonitrile. Intraday and interday precision for all analytes were between 0.6% and 13.8%, and recoveries were between 80.3% and 101.4%. Calibration curves were linear for all analytes over the concentration range 5-400 ng/mL, and correlation coefficients (R(2)) were better than 0.999. Limits of detection and quantitation were 0.16-1.2 ng/mL and 0.5-4.09 ng/mL, respectively. The method described consolidates previous work on opioids and their metabolites published in the literature and is the first to include the detection of naloxone-3-glucuronide. The method has been applied in routine postmortem cases after opiate overdose with the threefold purpose of providing interpretive information on the cause and type of death (rapid, sub-acute, or delayed death) and to distinguish heroin, morphine, and codeine users.

Brain opioid receptor binding in early abstinence from opioid dependence: positron emission tomography study.

BACKGROUND: Although opioid receptor function in humans is clearly reduced during opioid dependence, what happens to the receptor in early abstinence is not understood. AIMS: This study sought to examine changes in opioid receptor availability in early abstinence from opioid dependence. METHOD: Ten people with opioid dependence who had completed in-patient detoxification and 20 healthy controls underwent [11C]-diprenorphine positron emission tomography. Clinical variables were assessed with structured questionnaires. Opioid receptor binding was characterised as the volume of distribution of [11C]-diprenorphine using a template of predefined brain volumes and an exploratory voxel-by-voxel analysis. RESULTS: Compared with controls, participants with opioid dependence had increased [11C]-diprenorphine binding in the whole brain and in 15 of the 21 a priori regions studied. CONCLUSIONS: This study suggests that opioid receptor binding is increased throughout the brain in early abstinence from dependent opioid use. These data complement the findings in cocaine and alcohol dependence.