Multiple Drugs Of Abuse Research Articles

Validation of an LC-MS/MS Method for Analysis of 58 Drugs of Abuse in Oral Fluid and Method Comparison with an Established LC-HRMS Method.

Liquid chromatography-mass spectrometry (LC-MS) methods for detection of multiple drugs of abuse (DoA) in oral fluid (OF) samples are being implemented in many clinical routine laboratories. Therefore, there is a need to develop new multi-analyte methods with simple sample pre-treatment and short analysis times. The purpose of this work was to validate a method detecting 58 DoA to be used with two different OF sampling kits, the saliva collection system (SCS) from Greiner Bio-One and Quantisal from Immunalysis, using the same sample pretreatment and analytical method. A set of 110 samples collected with the SCS kit was further compared to an LC-HRMS (high resolution mass spectrometry) method in another laboratory. The method was successfully validated, with precision and accuracy of ≤15% and z-scores of <2 for external controls. Using a sensitive LC-MS/MS instrument, the detection limits were <1 µg/L in neat oral fluid. In the comparative study between the LC-MS/MS and LC-HRMS methods using SCS samples, a good agreement was observed. Discrepancies were limited to lower concentration ranges, attributable to differences in cut-off thresholds between the methods. This work contributes to the development of LC-MS multi-analyte methods for OF samples, which are suitable for clinical routine laboratories.

Rapid screening of drugs in environmental water using metal organic framework/Ti3C2Tx composite coated blade spray-mass spectrometry

Simultaneous rapid screening of multiple drugs of abuse in environmental water facilitates effective monitoring and trend assessments. Herein, a novel porphyrin-based metal organic frameworks modified Ti3C2Tx nanosheets (Cu-TCPP/Ti3C2Tx) composite was prepared and utilized as solid-phase microextraction (SPME) coating for the simultaneous analysis of 21 drugs from water samples. The composite was embedded with matrix-compatible polyacrylonitrile binder to prepare a coated blade with thin and uniform coating layer. Ambient mass spectrometry (MS) technique was used to create a coated blade spray-MS (CBS-MS) method for the quantitative determination of drugs in water samples. High throughput and automated sample preparation were achieved with the use of a Concept 96-well plate system, enabling analysis of 21 drugs of abuse within 1 min per sample, while using only 8 µL of organic solvent for desorption and CBS-MS detection. The developed method showed favorable linearity (R2 ≥ 0.9983) in the range of 0.05 to 10 ng mL−1, low limits of detection (1.5–9.0 ng L-1), sufficient recovery (67.6–133.2%), as well as satisfactory precision (RSDs≤13.5%). This study not only delivers a novel and efficient SPME coating composite, but also demonstrates the excellent performance of a high-throughput, efficient, and green analytical method for determination of drugs in environmental water.

Solvent-free parallel artificial liquid membrane extraction for drugs of abuse in plasma samples using LC-MS/MS

BackgroundParallel artificial liquid membrane extraction (PALME) is a 96-well plate setup variant of liquid-phase microextraction. Basic or acidic analytes are extracted in neutral form from the sample, through a supported liquid membrane (SLM), and into aqueous acceptor. PALME is already considered a green extraction technique, but in the current conceptual work, we sought to make it even greener by replacing the use of organic solvents with essential oils (EO). PALME was combined with LC-MS/MS for analysis of plasma samples and multiple drugs of abuse with toxicological relevance (amphetamines, phenethylamines, synthetic cathinones, designer benzodiazepines, ayahuasca alkaloids, lysergic acid diethylamide, and ketamine). ResultsFourteen EO were compared to organic solvents frequently used in PALME. The EO termed smart & sassy yielded the best analyte recovery for all drugs studied and was thus selected as SLM. Then, factorial screening and Box-Behnken were employed to optimize the technique. The extraction time, concentration of base, sample volume, and percentage of trioctylamine significantly impacted analyte recovery. The optimum values were defined as 120 min, 10 mmol/L of NaOH, 150 μL, and 0%, respectively. Once optimized, validation parameters were 1–100 ng mL-1 as linear range, accuracy ±16.4%, precision >83%, 1 ng mL-1 as limit of quantitation, 0.1–0.75 ng mL-1 as limit of detection, matrix effect <20%, and recovery 20–106%. Additionally, EO purchased from different production batches were tested and achieved acceptable reproducibility. Data were in compliance with requirements set by internationally accepted validation guidelines and the applicability of the technique was proven using authentic samples. SignificanceIn this study, the use of an EO provided a solvent-free sample preparation technique suited to extract different classes of drugs of abuse from plasma samples, dismissing the use of hazardous organic solvents. The method also provided excellent sample clean-up, thus being a simple and efficient tool for toxicological applications that is in agreement with the principles of sustainable chemistry.

What We Have Gained from Ibogaine: α3β4 Nicotinic Acetylcholine Receptor Inhibitors as Treatments for Substance Use Disorders.

For decades, ibogaine─the main psychoactive alkaloid found in Tabernanthe iboga─has been investigated as a possible treatment for substance use disorders (SUDs) due to its purported ability to interrupt the addictive properties of multiple drugs of abuse. Of the numerous pharmacological actions of ibogaine and its derivatives, the inhibition of α3β4 nicotinic acetylcholine receptors (nAChRs), represents a probable mechanism of action for their apparent anti-addictive activity. In this Perspective, we examine several classes of compounds that have been discovered and developed to target α3β4 nAChRs. Specifically, by focusing on compounds that have proven efficacious in pre-clinical models of drug abuse and have been evaluated clinically, we highlight the promising potential of the α3β4 nAChRs as viable targets to treat a wide array of SUDs. Additionally, we discuss the challenges faced by the existing classes of α3β4 nAChR ligands that must be overcome to develop them into therapeutic treatments.

Application of the Evidence MultiSTAT benchtop analyser to the fast (under 30 minutes), simultaneous, semi-quantitative determination of 29 drugs of abuse from one urine sample

Biochip array technology allows for simultaneous screening of multiple drugs of abuse from a single human specimen and applied to the fully automated benchtop analyser Evidence MultiSTAT, provides rapid test results. Current technology compares the signal output of a sample to that of a cut-off, reporting a qualitative test result. Further advancement of the Evidence MultiSTAT technology allows a semi-quantitative result to be generated. Embedded software, preloaded with multiple calibration curves for each drug target, selects an optimal calibration curve by running two adjuster levels. Additionally, the software allows a user defined cut-off concentration to be selected enabling two samples to be analysed in parallel in under 30 minutes. The MultiSTAT DOA ToxPlex Urine array reports semi-quantitative test results for 29 drug targets simultaneously from a single sample. Simultaneous competitive chemiluminescent immunoassays ( n = 29) were employed. The assays were applied to the Evidence MultiSTAT biochip analyser, which processes a self-contained cartridge comprising all the components required for the assays. The system software selects the optimal calibration for each drug target from the database and processes and reports the multiple semi-quantitative results generated. The Graphical User Interface (GUI) presents the sample concentration for the 29 drug targets, the user defined cut-off concentration and whether the sample is classified as positive or negative in comparison to the selected cut-off. Inter-Assay Precision and Recovery performance were evaluated; three samples (−50%, cut-off and +50% samples) were assayed in triplicate per run for a total of 5 runs, n = 15. Resulting CVs were < 20% and recovery to target was within 70–130% for all 29 assays, confirming the suitability of the new semi-quantitative functionality. Application of the fully automated Evidence MultiSTAT allows for fast (under 30 minutes) semi-quantitative determination of 29 drug targets. The multiple results generated are displayed in an easy to visualise tabulated format and the system has the capacity to store and archive the results produced, which greatly aids accurate drug screening.

A reduced complexity cross between BALB/c substrains identifies Zhx2 as a candidate gene underlying oxycodone metabolite brain concentration and state‐dependent learning of opioid reward

Understanding the pharmacokinetic profile of an opioid drug is vital to therapeutic success, and mutations in human PK genes can drastically alter therapeutic efficacy of opioids. We observed that at 30 min post‐oxycodone administration (1.25 mg/kg, i.p.) BALB/cJ mice showed a higher whole brain concentration of oxycodone, and female specific increase in noroxycodone, and oxymorphone compared to BALB/cByJ. This observation could explain the sex‐specific increase in oxycodone state‐dependent conditioned place preference in BALB/cJ female mice. To potentially link behavioral differences with PK differences, we conducted quantitative trait locus (QTL) mapping of whole brain oxycodone and metabolite concentrations in a reduced complexity cross (RCC). Because BALB/cJ and BALB/cByJ substrains differ by ~8,500 SNPs/indels, large genetic loci identified in an F2 cross are offset by a dramatic reduction in potentially causal variants. QTL mapping in 133 BALB/cJ x BALB/cByJ F2 mice (68F, 65M) revealed a single QTL on chromosome 15 associated with brain oxymorphone concentration that explained 29% of the phenotypic variance in females. Oxymorphone is a full agonist at the mu opioid receptor, with 8x the potency of oxycodone, and likely contributes to oxycodone addictive properties. Hippocampal and striatal cis‐eQTL analysis revealed genetically regulated expression of Zhx2, a transcriptional inhibitor known to harbor a private BALB/cJ retroviral insertion that dramatically reduces protein expression and leads to sex specific dysregulation of CYP450 genes within the liver. Whole brain mass spectroscopy proteomics in the parental strains corroborated these eQTL findings. We hypothesize that decreased Zhx2 expression leads to increased CYP450 expression, increased brain oxymorphone, and increased oxycodone‐induced behaviors. Interestingly, human GWAS of nicotine consumption identified a nominal association (10^‐7) with ZHX2, indicating that this transcriptional repressor could influence metabolism of multiple drugs of abuse.

Determination of multiple drugs of abuse in human urine using dispersive liquid–liquid microextraction and capillary electrophoresis with PDA detection

A new method was developed for pre-concentration and determination of multiple drugs of abuse in human urine using dispersive liquid–liquid microextraction (DLLME) and capillary electrophoresis (CE) with photodiode array detection. The method was based on the formation of tiny droplets of an organic extractant in the prepared sample solution using water-immiscible organic solvent (chloroform) dissolved in water-miscible organic dispersive solvent (isopropyl alcohol). The organic phase, which extracted eight drugs of abuse from the prepared urine solution, was separated by centrifugation. The sedimented phase was transferred into a small volume CE auto-sampler vial with 10 µL of 1% HCl methanol solution and evaporated to dryness. The residue was reconstituted in lidocaine hydrochloride (internal standard) aqueous solution and introduced by electrokinetic injection into CE. Under the optimum conditions, acceptable linear relationship was observed in the range of 3.0–500 ng/mL with the correlation coefficient ( r ) of 0.9982–0.9994 for spiked urine samples. The limit of detection (LOD) (S/N = 3) was estimated to be 1.0 ng/mL. A recovery of 75.7%–90.6% was obtained for spiked samples. The mean relative error (MRE) was within ±7.0% and the relative standard deviation (RSD) was less than 6.9%. The proposed DLLME-CE procedure offers an alternative analytical approach for the sensitive detection of drugs of abuse in real urine samples. Key points: The dispersive liquid-liquid microextraction (DLLME) was involved for the determination of drugs in urine with capillary electrophoresis with photodiode array detection (CE-PDA). Good linearity, sensitivity, recovery and precision were achieved. The proposed method was eco-friendly with microliter scale solvent consumption.

CaMKII Modulates Diacylglycerol Lipase-α Activity in the Rat Nucleus Accumbens after Incubation of Cocaine Craving.

Relapse is a major challenge to the treatment of substance use disorders. A progressive increase in cue-induced drug craving, termed incubation of craving, is observed after withdrawal from multiple drugs of abuse in humans and rodents. Incubation of cocaine craving involves the strengthening of excitatory synapses onto nucleus accumbens (NAc) medium spiny neurons via postsynaptic accumulation of high-conductance Ca2+-permeable AMPA receptors. This enhances reactivity to drug-associated cues and is required for the expression of incubation. Additionally, incubation of cocaine craving is associated with loss of the synaptic depression normally triggered by stimulation of metabotropic glutamate receptor 5 (mGlu5), leading to endocannabinoid production, and expressed presynaptically via cannabinoid receptor 1 activation. Previous studies have found alterations in mGlu5 and Homer proteins associated with the loss of this synaptic depression. Here we conducted coimmunoprecipitation studies to investigate associations of diacylglycerol lipase-α (DGL), which catalyzes formation of the endocannabinoid 2-arachidonylglycerol (2-AG), with mGlu5 and Homer proteins. Although these interactions were unchanged in the NAc core at incubation-relevant withdrawal times, the association of DGL with total and phosphorylated Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) and CaMKIIβ was increased. This would be predicted, based on other studies, to inhibit DGL activity and therefore 2-AG production. This was confirmed by measuring DGL enzymatic activity. However, the magnitude of DGL inhibition did not correlate with the magnitude of incubation of craving for individual rats. These results suggest that CaMKII contributes to the loss of mGlu5-dependent synaptic depression after incubation, but the functional significance of this loss remains unclear.

Investigating the Mechanism of Sodium Binding to SERT Using Direct Simulations.

The serotonin transporter (SERT) terminates neurotransmission by transporting serotonin from the synapse into the pre-synaptic nerve terminal. Altered SERT function leads to several neurological diseases including depression, anxiety, mood disorders, and attention deficit hyperactivity disorders (ADHD). Accordingly SERT is the target for their pharmacological treatments, but also targeted by multiple drugs of abuse. Transport of serotonin by SERT is energized by the transmembrane electrochemical gradient of sodium. We used extensive molecular dynamics simulations to investigate the process of sodium binding to SERT, which is the first step in the transport cycle that leads to serotonin uptake. Comparing data from 51 independent simulations, we find a remarkably well-defined path for sodium entry and could identify two transient binding sites, while observing binding kinetics that are comparable to experimental data. Importantly, the structure and dynamics of the sodium binding sites indicate that sodium binding is accompanied by an induced-fit mechanism that leads to new conformations and reduces local dynamics.

Increased NMDA Receptor Function during Protracted Withdrawal from Chronic Intermittent Ethanol Exposure

Functional dysregulation of the glutamatergic receptor system during withdrawal from chronic drug exposure is a primary driver of drug craving and relapse. Animal models of psychostimulant use have demonstrated dynamic alterations in both AMPA and NMDA receptor function and expression that contribute to drug seeking behavior. Regulation of these receptor systems can begin as early as a few days following cessation of drug use and are persistently expressed into long term withdrawal (>60d). Short term withdrawal (24hr) from chronic exposure to ethanol also induces functional dysregulation of AMPA and NMDA receptors suggesting that similar mechanisms may be regulating drug craving and relapse behaviors across drugs of abuse. To this end, we investigated NMDA receptor mediated synaptic function during protracted withdrawal (>35d) from chronic intermittent ethanol (CIE) exposure using whole cell patch clamp electrophysiology. We focused on the basolateral amygdala (BLA), as glutamatergic signaling in this region is robustly modulated by short term (24h) withdrawal from CIE (10d, 12hr/day) and regulates anxiety like behavior expressed during withdrawal. Adolescent rats exposed to repeated cycles of CIE (12hr/day, 4d on/3d off, 3 cycles) demonstrated increased functional contributions of NMDA receptors in male but not female animals in comparison to sex matched control animals exposed to room air (CON). These functional increases occurred at resting (-80mV) and depolarized (+40mV) membrane potentials. Psychostimulant studies showing similar increases at resting potentials (-80mV) have implicated GluN3-containing NMDA receptors as the primary mechanism driving this effect. Pharmacological studies using the GluN2B antagonist ifenprodil, suggest that increased NMDAR contributions in male animals is mediated through GluN2B containing receptors. Interestingly, ifenprodil antagonized the increased responses at both resting and depolarized membrane potentials. These results mirror increased NMDA receptor function found during protracted withdrawal from cocaine self-administration suggesting possible common mechanisms underlying aberrant synaptic function during withdrawal from multiple drugs of abuse. Ongoing studies are focused on elucidating the specific contributions of NMDA receptor subunits (GluN2B/GluN3) to the overall functional increases found and exploring sex differences in NMDA receptor regulation.

Sex-dependent effects of an Hnrnph1 mutation on fentanyl addiction-relevant behaviors but not antinociception in mice.

Opioid Use Disorder (OUD) and opioid-related deaths remain a major public health concern in the United States. Both environmental and genetic factors influence risk for OUD. We previously identified Hnrnph1 as a quantitative trait gene underlying the stimulant, rewarding, and reinforcing properties of methamphetamine. Prior work shows that hnRNP H1, the RNA-binding protein encoded by Hnrnph1, post-transcriptionally regulates Oprm1 (mu opioid receptor gene)-the primary molecular target for the therapeutic and addictive properties of opioids. Because genetic variants can exert pleiotropic effects on behaviors induced by multiple drugs of abuse, in the current study, we tested the hypothesis that Hnrnph1 mutants would show reduced behavioral sensitivity to the mu opioid receptor agonist fentanyl. Hnrnph1 mutants showed reduced sensitivity to fentanyl-induced locomotor activity, along with a female-specific reduction in, and a male-specific induction of, locomotor sensitization following three, daily injections (0.2 mg/kg, i.p.). Hnrnph1 mutants also required a higher dose of fentanyl to exhibit opioid reward as measured via conditioned place preference (CPP). Male Hnrnph1 mutants showed reduced fentanyl reinforcement. Hnrnph1 mutants also showed reduced sucrose motivation, suggesting a reward deficit. No genotypic differences were observed in baseline thermal nociception, fentanyl-induced antinociception, physical or negative affective signs of opioid dependence, or in sensorimotor gating. In the context of our prior work, these findings suggest that Hnrnph1 dysfunction exerts a selective role in reducing the addiction liability to drugs of abuse (opioids and psychostimulants), which could provide new biological pathways to improve their therapeutic profiles.

Buprenorphine Analogs to Treat Relapse

Substance use disorders (SUDs) – the overuse and dependence on at least one substance – have a lifetime prevalence of ~10% in the U.S. While buprenorphine (BUP) treatment reduces relapse rates, it is only marginally effective, as 70% of SUD patients relapse within a year. Due to BUP’s marginal effectiveness and abuse potential, there is a need to improve SUD therapies that prevent relapse to multiple drugs of abuse after exposure to stress, drug‐associated context and drug priming. Here we evaluate BUP and two analogs for their ability to inhibit cocaine‐ and stress‐induced relapse using a conditioned place preference (CPP) paradigm. Pharmacologically, BUP is a potent (sub nM) partial agonist at the m‐opioid receptor (MOR) with potent antagonist activity at the k‐(KOR), and d‐opioid receptors (DOR). In vitro BUP can act as a weak (mM) partial agonist at the nociceptin/OrphaninFQ (NOP) receptor with uncertain in vivo consequences. We measured in vitro potency and efficacy using [35S]GTPgS functional assays in CHO cells separately expressing DOR, KOR, MOR or NOP. Since NOP receptor agonists reduce relapse in animal models, we compared BU10119 with NOP agonist activity (EC50 = 120 nM ± 20; EMAX = 44% ± 5%) or BU12004 with NOP antagonist activity (KB = 190 nM ±30) to BUP (EC50 = 1300 nM ± 320; EMAX = 39% ± 7%). Furthermore, BU12004 and BU10119 were MOR antagonists (KB = 0.42 nM ± 0.08 and KB = 1.1 nM ± 0.2, respectively) with less abuse liability than the MOR partial agonist BUP (EC50 = 0.79 nM ± 0.31; EMAX = 34% ± 6%). BUP, BU10119, and BU12004 were all potent antagonists at DOR and KOR. BU10119 (1.0 mg/kg) attenuated cocaine‐primed reinstatement in CPP, but 10 mg/kg BU12004 did not. BUP trended toward reduced drug‐primed reinstatement. The in vivo rank order of BU10119 &gt;BUP &gt;BU12004 matches the rank order for NOP agonism in vitro. Additionally, 1.0 mg/kg of either BU10119 or BU12004 blocked swim stress‐primed reinstatement, ostensibly due to their KOR antagonist activity. Taken together, BU10119 provides a promising lead to treat SUDs with better effectiveness and reduced abuse potential compared to BUP.Support or Funding InformationSupported by NIDA R01 DA07315 and 035316.

Early substance use in the pathway from childhood attention-deficit/hyperactivity disorder (ADHD) to young adult substance use: Evidence of statistical mediation and substance specificity.

This study tested whether early and developmentally atypical substance use mediates risk for adult substance use among children with attention-deficit/hyperactivity disorder (ADHD), and whether that risk is substance-specific. Participants were children with ADHD previously enrolled in a randomized controlled trial (RCT), and a demographically similar non-ADHD group, assessed at 2 through 16 years after the original RCT baseline. Self-reports of heavy drinking, marijuana use, daily smoking, and other illicit drug use were collected at follow-ups to establish atypically early and frequent use. Models estimated statistically mediated effects of childhood ADHD on adult substance use via early substance involvement, with planned comparisons to evaluate substance specificity. Results supported the mediation hypothesis, showing that childhood ADHD was associated with more frequent adult substance use via early substance involvement for marijuana, cigarettes, illicit drugs, and to a lesser extent, alcohol. Mediation was not escalated by comorbid childhood conduct disorder or oppositional defiant disorder except for early use of nonmarijuana illicit drugs. Substance-specificity in the mediational pathway was largely absent except for cigarette use, where ADHD-related early smoking most strongly predicted adult daily smoking. Findings from this study provide new evidence that atypically early substance use associated with childhood ADHD signals important cross-drug vulnerability by early adulthood, but cigarette use at a young age is especially associated with increased risk for habitual (daily) smoking specifically. Efforts to prevent, delay, or reduce substance experimentation should occur early and focus on factors relevant to multiple drugs of abuse in this at-risk population. (PsycINFO Database Record (c) 2020 APA, all rights reserved).

On-site testing of multiple drugs of abuse in urine by a miniature dual-LIT mass spectrometer

There is an increasing need for rapid and on-site detection of emerging drugs of abuse. In this work, we developed a method using a miniature dual-LIT (linear ion trap) mass spectrometer recently developed with comprehensive tandem mass spectrometry analysis capability, for qualitative and quantitative analysis of multiple drugs of abuse. Paper-capillary spray cartridges were used with related workflow established to simplify overall analysis procedure. Quantitation of ketamine and methamphetamine was achieved by beam-type collision-induced dissociation on the miniature dual-LIT mass spectrometer and a linear concentration range of 100–5000 ng/mL was obtained. The system has been applied in analysis of real urine samples from individuals addicted to morphine and methamphetamine use. The changes of the ratio of cocaine to its metabolite benzoylecgonine were also explored to estimate the time of cocaine intaking.

Double-Blind Placebo-Controlled Trial of Galantamine for Methadone-Maintained Individuals With Cocaine Use Disorder: Secondary Analysis of Effects on Illicit Opioid Use.

Concurrent use of cocaine and opioids is a persistent and challenging problem, particularly within methadone maintenance settings, and there are no approved pharmacotherapies for this population. Galantamine, a cholinesterase inhibitor, was found in a randomized clinical trial to reduce cocaine use among methadone-maintained individuals who were also cocaine dependent. Because of the potential of galantamine to reduce multiple drugs of abuse, it may also reduce opioid use. We conducted a secondary analysis of a randomized, double-blind, placebo-controlled trial of 120 methadone-maintained individuals with concurrent cocaine dependence. Participants were randomized to galantamine or placebo in a 12-week trial with a 6-month follow-up (97% of intention to treat sample reached for final follow-up). There was a significant main effect for galantamine over placebo on percent of urine specimens that were negative for opioids, both within treatment (77% for galantamine vs 62% for placebo, F = 5.0, P = 0.027) and through a 6-month follow-up (81% vs 59%, respectively, F = 10.8, P = 0.001). This effect was seen regardless of whether participants used nonprescribed opioids during the baseline period. Galantamine effects were seen early in treatment, with participants in placebo submitting the first opioid-positive urine specimen significantly sooner than participants in galantamine (median day 15 vs 53, Wilcoxon = 5.7, P = 0.02). If these results are supported in future trials, galantamine may hold promise across multiple drugs of abuse, including opioids. (Am J Addict 2019;28:238-245).

The Role of interleukin‐1 receptor‐associated kinase 4 in drug addiction

Drug addiction remains an unmanageable and costly disease. Apart from neuronal adaptation, growing recognition arises that glial proinflammatory activation importantly contributes to the rewarding effects of multiple drugs of abuse. Recent studies suggest the central importance of Toll‐like receptor 4 (TLR4), a candidate neuroimmune therapeutic target for drug addiction. While Interleukin‐1 receptor associated kinase 4 (IRAK4) plays a crucial role in TLR4 mediated innate immunity, there is no further studies support its functioning in drug addiction. We hypothesized that drug‐taking activates IRAK4 and induces reinforcing effects, and disruption of IRAK4 signaling attenuates the addictive behaviors. In the present study, IRAK4 and IRAK1 phosphorylation after morphine and cocaine self‐administration was evaluated using western blotting. The role of IRAK4 in morphine self‐administration and cue‐induced reinstatement was examined with its inhibitor, PF06650833. Moreover, local pharmacological manipulation was conducted to determine the role of IRAK4 in the nucleus accumbens (NAc) core in the cue‐induced reinstatement of morphine seeking. We found that morphine self‐administration significantly increased the phosphorylation of IRAK4, but not IRAK1, both in NAc and VTA. However, neither cocaine short access nor long access self‐administration had any effect on the phosphorylation of IRAK4. Systemic administration of PF06650833 significantly attenuated cue‐induced reinstatement of morphine seeking without affecting the spontaneous locomotion in rats, and microinjection of PF06650833 into NAc core sufficiently decreased cue‐induced morphine reinstatement. These results demonstrated that modulation of IRAK4 activity regulates the cue‐induced morphine reinstatement, and suggested it as a novel candidate target in treating drug addiction.Support or Funding InformationThis work was supported by NIDA [Grant R21DA040777].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Evaluation of three rapid oral fluid test devices on the screening of multiple drugs of abuse including ketamine

Rapid oral fluid testing (ROFT) devices have been extensively evaluated for their ability to detect common drugs of abuse; however, the performance of such devices on simultaneous screening for ketamine has been scarcely investigated. The present study evaluated three ROFT devices (DrugWipe® 6S, Ora-Check® and SalivaScreen®) on the detection of ketamine, opiates, methamphetamine, cannabis, cocaine and MDMA.A liquid chromatography tandem mass spectrometry (LCMS) assay was firstly established and validated for confirmation analysis of the six types of drugs and/or their metabolites. In the field test, the three ROFT devices were tested on subjects recruited from substance abuse clinics/rehabilitation centre. Oral fluid was also collected using Quantisal® for confirmation analysis.A total of 549 samples were collected in the study. LCMS analysis on 491 samples revealed the following drugs: codeine (55%), morphine (49%), heroin (40%), methamphetamine (35%), THC (8%), ketamine (4%) and cocaine (2%). No MDMA-positive cases were observed.Results showed that the overall specificity and accuracy were satisfactory and met the DRUID standard of >80% for all 3 devices. Ora-Check® had poor sensitivities (ketamine 36%, methamphetamine 63%, opiates 53%, cocaine 60%, THC 0%). DrugWipe® 6S showed good sensitivities in the methamphetamine (83%) and opiates (93%) tests but performed relatively poorly for ketamine (41%), cocaine (43%) and THC (22%). SalivaScreen® also demonstrated good sensitivities in the methamphetamine (83%) and opiates (100%) tests, and had the highest sensitivity for ketamine (76%) and cocaine (71%); however, it failed to detect any of the 28 THC-positive cases. The test completion rate (proportion of tests completed with quality control passed) were: 52% (Ora-Check®), 78% (SalivaScreen®) and 99% (DrugWipe® 6S).

Regulation of glutamate transporter 1 (GLT-1) gene expression by cocaine self-administration and withdrawal

Downregulation of the astroglial glutamate transporter GLT-1 is observed in the nucleus accumbens (NAc) following administration of multiple drugs of abuse. The decrease in GLT-1 protein expression following cocaine self-administration is dependent on both the amount of cocaine self-administered and the length of withdrawal, with longer access to cocaine and longer withdrawal periods leading to greater decreases in GLT-1 protein. However, the mechanism(s) by which cocaine downregulates GLT-1 protein remains unknown. We used qRT-PCR to examine gene expression of GLT-1 splice isoforms (GLT-1A, GLT-1B) in the NAc, prelimbic cortex (PL) and basolateral amygdala (BLA) of rats, following two widely used models of cocaine self-administration: short-access (ShA) self-administration, and the long-access (LgA) self-administration/incubation model. While downregulation of GLT-1 protein is observed following ShA cocaine self-administration and extinction, this model did not lead to a change in GLT-1A or GLT-1B gene expression in any brain region examined. Forced abstinence following ShA cocaine self-administration also was without effect. In contrast, LgA cocaine self-administration and prolonged abstinence significantly decreased GLT-1A gene expression in the NAc and BLA, and significantly decreased GLT-1B gene expression in the PL. No change was observed in NAc GLT-1A gene expression one day after LgA cocaine self-administration, indicating withdrawal-induced decreases in GLT-1A mRNA. In addition, LgA cocaine self-administration and withdrawal induced hypermethylation of the GLT-1 gene in the NAc. These results indicate that a decrease in NAc GLT-1 mRNA is only observed after extended access to cocaine combined with protracted abstinence, and that epigenetic mechanisms likely contribute to this effect.

The medial habenula and interpeduncular nucleus circuitry is critical in addiction, anxiety, and mood regulation.

Abstinence from chronic use of addictive drugs triggers an aversive withdrawal syndrome that compels relapse and deters abstinence. Many features of this syndrome are common across multiple drugs, involving both affective and physical symptoms. Some of the network signaling underlying withdrawal symptoms overlaps with activity that is associated with aversive mood states, including anxiety and depression. Given these shared features, it is not surprising that a particular circuit, the dorsal diencephalic conduction system, and the medial habenula (MHb) and interpeduncular nucleus (IPN), in particular, have been identified as critical to the emergence of aversive states that arise both as a result and, independently, of drug addiction. As the features of this circuit continue to be characterized, the MHb-IPN axis is emerging as a viable target for therapeutics to aid in the treatment of addiction to multiple drugs of abuse as well as mood-associated disorders. This is an article for thespecial issue XVth International Symposium on Cholinergic Mechanisms.

The dual orexin receptor antagonist TCS1102 does not affect reinstatement of nicotine-seeking.

The orexin/hypocretin system is important for appetitive motivation towards multiple drugs of abuse, including nicotine. Both OX1 and OX2 receptors individually have been shown to influence nicotine self-administration and reinstatement. Due to the increasing clinical use of dual orexin receptor antagonists in the treatment of disorders such as insomnia, we examined whether a dual orexin receptor antagonist may also be effective in reducing nicotine seeking. We tested the effect of intracerebroventricular (i.c.v.) administration of the potent and selective dual orexin receptor antagonist TCS1102 on orexin-A-induced food self-administration, nicotine self-administration and reinstatement of nicotine-seeking in rats. Our results show that 30 μg of TCS1102 i.c.v. abolishes orexin-A-induced increases in food self-administration but does not reduce nicotine self-administration. Neither i.c.v. 10 μg nor 30 μg of TCS1102 reduced compound reinstatement after short-term (15 days) self-administration nicotine, but 30 μg transiently reduced cue/nicotine compound reinstatement after chronic self-administration (29 days). These results indicate that TCS1102 has no substantial effect on motivation for nicotine seeking following chronic self-administration and no effect after shorter periods of intake. Orexin receptor antagonists may therefore have little clinical utility against nicotine addiction.