Multiple Drugs Of Abuse Research Articles

Neuroproteomics and its applications in research on nicotine and other drugs of abuse

The rapidly growing field of neuroproteomics is able to track changes in protein expression and protein modifications underlying various physiological conditions, including the neural diseases related to drug addiction. Thus, it presents great promise in characterizing protein function, biochemical pathways, and networks to understand the mechanisms underlying drug dependence. In this article, we first provide an overview of proteomics technologies and bioinformatics tools available to analyze proteomics data. Then we summarize the recent applications of proteomics to profile the protein expression pattern in animal or human brain tissues after the administration of nicotine, alcohol, amphetamine, butorphanol, cocaine, and morphine. By comparing the protein expression profiles in response to chronic nicotine exposure with those appearing in response to treatment with other drugs of abuse, we identified three biological processes that appears to be regulated by multiple drugs of abuse: energy metabolism, oxidative stress response, and protein degradation and modification. Such similarity indicates that despite the obvious differences among their chemical properties and the receptors with which they interact, different substances of abuse may cause some similar changes in cellular activities and biological processes in neurons.

Simultaneous Determination of Multiple Drugs of Abuse and Relevant Metabolites in Urine by LC-MS-MS

A method was developed for the quantitative analysis of 30 drugs of abuse and their metabolites in urine, including opiates, barbiturates, amphetamines, cocaine, cannabinoids, phencyclidine, methadone, and benzodiazepines. This method uses solid-phase extraction (SPE) on an Oasis HLB column followed by liquid chromatography-tandem mass spectrometry. Analytes were quantified by multiple reaction monitoring with the deuterated analogues as internal standards, using an atmospheric pressure ionization-electrospray interface. The method was validated by examining specificity, precision, accuracy, linearity, recovery, reproducibility, and detection limits. The limits of detection ranged from 9 pg/mL to 2.29 ng/mL in urine depending on the analyte. The SPE procedure was automated on a RapidTrace workstation to increase analytical throughput, and the results obtained via automated SPE were compared to those obtained by manual SPE to examine carryover effect, precision, accuracy, recovery, and reproducibility. To evaluate method performance, 108 urine samples were collected anonymously and tested for the presence of these drugs.

Usefulness of multiple opiate and amphetamine analysis of hair segments under methadone therapy using LC-APCI-MS-MS

A detailed procedure for simultaneous analysis of morphine, codeine, 6-monoacetylmorphine, amphetamine, methadone, and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in human hair segments by liquid chromatography (LC)-atmospheric pressure chemical ionization (APCI)-tandem mass spectrometry (MS-MS) was established. Hair samples were pulverized and extracted with methanol. The blank hair obtained from healthy subjects showed no interfering impurity peaks. Good linearity was obtained for all compounds in the range of 0.2–20 ng/mg. Accuracy and precision data were also satisfactory. Using the established method, the opiates, amphetamine, methadone, and EDDP in hair segments were measured for 20 patients undergoing methadone therapy. Complete abstinence was achieved by only 6 of the 20 patients. Other patients failed to abstain from opiate(s) and/or amphetamine. Our data show that the present hair analysis of multiple drugs of abuse by LC-APCI-MS-MS is useful for monitoring the success or failure of methadone therapy.

Development of a capillary electrophoresis method for the screening of human urine for multiple drugs of abuse

A new capillary electrophoresis (CE) method was developed and validated for the screening of human urine for nineteen drugs of abuse. In order to achieve sufficient separation, the electrolyte composition was modified using β-cyclodextrin (β-CD) and organic solvents. To process each sample, a sequential injection-solid-phase extraction (SI-SPE) system was constructed. Using this device, matrix clean-up, extraction, and preconcentration of analytes were performed onto a C 18 cartridge. Optimal separation and detection were obtained using a background electrolyte consisting of 100 mM phosphate adjusted to pH 6.0, with 20 mM β-CD, 5% acetonitrile and 20% isopropanol. Electrokinetic injection was performed at 5 kV for 10 s, separation voltage was 25 kV and column temperature was set to 25 °C. The separation was carried out in a 67.0 cm × 50 μm fused-silica capillary with UV detection at 214 nm. The combination of SI-SPE and sample stacking showed significant sensitivity enhancement with limits of detection in the range of 5–30 ng ml −1. A validation study showed good reproducibility of both migration time (RSD = 0.003–0.088%) and peak area (RSD = 0.54–4.8%). Overall, this automated and miniaturized SI-SPE system provides a rapid, sensitive, and robust procedure for analysis; as well as minimizes sample and solvent consumption.

The κ-opioid receptor agonist U-69593 prevents cocaine-induced phosphorylation of DARPP-32 at Thr 34 in the rat brain

DARPP-32 (dopamine- and cAMP-regulated phosphoprotein) is a potent endogenous inhibitor of protein phosphatase-1, which plays an important role in dopaminergic transmission. A large body of evidence supports the key role of DARPP-32-dependent signalling in mediating the actions of multiple drugs of abuse, including cocaine, which, when acutely administered, increases the Thr 34 phosphorylation of DARPP-32 in the striatal and cortical areas. In this study, we have examined the contribution of the kappa opioid system to the regulation of DARPP-32 phosphorylation at Thr 34, following acute cocaine administration, in selected rat brain areas. Results showed that a single injection of cocaine induces a significant increase in DARPP-32 phosphorylation at Thr 34 in the hippocampus, caudate putamen and prefrontal cortex. In addition, pretreatment with the kappa opioid receptor agonist U-69593 prevented cocaine effects in all the investigated areas. These data could be considered consistent with the ability of kappa opioid agonists to attenuate many behavioural and neurochemical effects of cocaine.

Transcriptional profiling of C57 and DBA strains of mice in the absence and presence of morphine

BackgroundThe mouse C57BL/6 (C57) and DBA/2J (DBA) inbred strains differ substantially in many aspects of their response to drugs of abuse. The development of microarray analyses represents a genome-wide method for measuring differences across strains, focusing on expression differences. In the current study, we carried out microarray analysis in C57 and DBA mice in the nucleus accumbens of drug-naïve and morphine-treated animals.ResultsWe identified mRNAs with altered expression between the two strains. We validated the mRNA expression changes of several such mRNAs, including Gnb1, which has been observed to be regulated by several drugs of abuse. In addition, we validated alterations in the enzyme activity of one mRNA product, catechol-O-methyltransferase (Comt). Data mining of expression and behavioral data indicates that both Gnb1 and Comt expression correlate with aspects of drug response in C57/DBA recombinant inbred strains. Pathway analysis was carried out to identify pathways showing significant alterations as a result of treatment and/or due to strain differences. These analyses identified axon guidance genes, particularly the semaphorins, as showing altered expression in the presence of morphine, and plasticity genes as showing altered expression across strains. Pathway analysis of genes showing strain by treatment interaction suggest that the phosphatidylinositol signaling pathway may represent an important difference between the strains as related to morphine exposure.ConclusionmRNAs with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse. One such mRNA is Comt and we hypothesize that altered expression of Comt may represent a potential mechanism for regulating the effect of, and response to, multiple substances of abuse. Similarly, a role for Gnb1 in responses to multiple drugs of abuse is supported by expression data from our study and from other studies. Finally, the data support a role for semaphorin signaling in morphine effects, and indicate that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains.

Reflection Impulsivity in Current and Former Substance Users

Chronic drug use is associated with increased impulsivity, risky decision making, and impaired behavioral control, but the underlying mechanisms of this neurocognitive profile remain unclear. We investigated impulsive responding in the context of decision making, using a novel behavioral measure of reflection impulsivity: the tendency to gather and evaluate information before making a decision. The Information Sampling Task was administered to current substance users dependent on amphetamines (n = 24) or opiates (n = 40), former users of amphetamines or opiates abstinent for at least 1 year (n = 24), and non-drug-using control subjects (n = 26). Current users of amphetamines and opiates sampled less information than control subjects and responded at a lower probability of making a correct response. Amphetamine- and opiate-dependent subjects did not differ. Reduced reflection was also apparent in the former substance users, who did not differ from the current users. Questionnaire ratings of impulsivity (on the Barratt Impulsivity Scale, version 11) were also inflated in three groups of substance users but were not significantly correlated with performance on the behavioral task. Reduced reflection is suggested to represent a cognitive marker for substance dependence that does not recover with prolonged abstinence and is associated with multiple drugs of abuse.

DARPP-32 mediates the actions of multiple drugs of abuse

Drugs of abuse share the ability to enhance dopaminergic neurotransmission in the dorsal and ventral striatum. The action of dopamine is modulated by additional neurotransmitters, including glutamate, serotonin and adenosine. All these neurotransmitters regulate the phosphorylation state of Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa (DARPP-32). Phosphorylation at Thr(34) by protein kinase A converts DARPP-32 into a potent inhibitor of the multifunctional serine/threonine protein phosphatase, PP-1. Phosphorylation at Thr(75) by Cdk5 converts DARPP-32 into an inhibitor of protein kinase A. The state of phosphorylation of DARPP-32 at Thr(34) also depends on the phosphorylation state of Ser(97) and Ser(130), which are phosphorylated by CK2 and CK1, respectively. By virtue of regulation of these 4 phosphorylation sites, and through its ability to modulate the activity of PP-1 and protein kinase A, DARPP-32 plays a key role in integrating a variety of biochemical, electrophysiological, and behavioral responses controlled by dopamine and other neurotransmitters. Importantly, there is now a large body of evidence that supports a key role for DARPP-32-dependent signaling in mediating the actions of multiple drugs of abuse including cocaine, amphetamine, nicotine, caffeine, LSD, PCP, ethanol and morphine.

Determination of multiple drugs of abuse in human urine using capillary electrophoresis with fluorescence detection.

Methods for separation and determination of multiple drugs of abuse in biological fluids using capillary electrophoresis (CE) with native fluorescence and laser-induced fluorescence (LIF) detection are described herein. Using native fluorescence, normorphine, morphine, 6-acetyl morphine (6-AM), and codeine were analyzed by CE without any derivatization procedure and detected at an excitation wavelength of 245 nm with a cut-off emission filter of 320 nm, providing a rapid and simple analysis. The detection limits were in the range of 200 ng/mL. For a highly sensitive analysis, LIF detection was also examined using a two-step precolumn derivatization procedure. In this case, drugs extracted from human urine were first subjected to an N-demethylation reaction involving the use of 1-chloroethyl chloroformate (ACE-Cl) and then derivatized using fluorescein isothiocyanate isomer I (FITC) and analyzed by CE coupled to a LIF detector. Variables affecting this derivatization: yield of demethylation reaction, FITC concentration, reaction time and temperature, were studied. The estimated instrumental detection limits of the FITC derivatives were in the range of 50-100 pg/mL, using LIF detection with excitation and emission wavelengths of 488 nm and 520 nm, respectively. The linearity, reproducibility and reliability of the methods were evaluated. In addition, a comparison of the characteristics for both native fluorescence and LIF detections was also discussed.

Selective neurotoxic effects of nicotine on axons in fasciculus retroflexus further support evidence that this a weak link in brain across multiple drugs of abuse

When administered continuously for several days at relatively low plasma levels, a variety of drugs of abuse with strong dopaminergic actions induce degeneration in axons traveling from the lateral habenula through the sheath of fasciculus retroflexus to midbrain monoaminergic nuclei. With some of these drugs, such as cocaine, this is virtually the only degeneration induced in brain. Nicotine given continuously also selectively induces degeneration in fasciculus retroflexus, but in the other half of the tract: the cholinergic axons running from medial habenula in the core of the tract to the interpeduncular nucleus. Fasciculus retroflexus appears to be a weak link in brain for diverse drugs of abuse when administered incessantly for several days. Alterations in this tract would be predicted to be especially important for the genesis of the symptomatology which develops during drug binges, residual effects of such binges, and the processes underlying relapse.

Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila.

Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown. We present evidence that dopamine plays a role in the responses of Drosophila to cocaine, nicotine or ethanol. We used a startle-induced negative geotaxis assay and a locomotor tracking system to measure the effect of psychostimulants on fly behavior. Using these assays, we show that acute responses to cocaine and nicotine are blunted by pharmacologically induced reductions in dopamine levels. Cocaine and nicotine showed a high degree of synergy in their effects, which is consistent with an action through convergent pathways. In addition, we found that dopamine is involved in the acute locomotor-activating effect, but not the sedating effect, of ethanol. We show that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating specific behavioral responses to cocaine, nicotine or ethanol. We therefore suggest that Drosophila can be used as a genetically tractable model system in which to study the mechanisms underlying behavioral responses to multiple drugs of abuse.

Contingent reinforcement sustains post-detoxification abstinence from multiple drugs: A preliminary study with methadone patients

This study examined the efficacy of a urinalysis-based contingency management program for preventing relapse to abused drugs following a brief residential detoxification. Fourteen methadone maintenance patients who were chronic benzodiazepine users were enrolled in a 7-day inpatient benzodiazepine detoxification and randomly assigned to receive Contingency Management (N=7) or Standard Care (N=7) therapy upon return to outpatient methadone treatment. In the Contingency Management condition, a methadone take-home dose or a US $25 voucher (patient's choice) could be earned for each urine sample submitted on a Monday, Wednesday or Friday that was free of opiates, cocaine and benzodiazepines. Data analysis and interpretation focused on within-group post-hoc differences due to group differences on employment and legal status, potentially confounding baseline variables. Repeated measures analysis of variance showed that Contingency Management patients submitted significantly more drug-free urine samples during the intervention compared to pre-detoxification (p<0.01), whereas no significance changes were observed from pre- to post-detoxification in the Standard Care patients. Employment and legal status of patients may have facilitated response to contingency management procedures, but did not prevent relapse when contingency management procedures were withdrawn. Overall, these preliminary results suggest that abstinence-based contingency management is a promising strategy for preventing relapse to multiple drugs of abuse in a subset of methadone maintenance patients when abstinence has been initiated through brief inpatient treatment.

Risk/Protective Factors Among Addicted Mothers' Offspring: A Replication Study

There are few systematic studies of the school-aged offspring of drug-dependent patients, although this information is useful for planning evidence-based prevention programs. We have completed such a study, which we compare to a similar study independently conducted in 1998. In both studies, both the parent and offspring were assessed blindly and independently by direct diagnostic interviews, and parental assessment of offspring was also obtained. The similarity in design and methods between studies provided an opportunity for replication by reanalysis of data. The major findings are a replication in two independently conducted studies of school-aged offspring of opiate- and/or cocaine-addicted mothers of the high rates of any psychiatric disorder (60% in both studies), major depression (20%, 26%), oppositional defiant disorder (ODD) (18%, 23%), conduct disorder (17%, 9%), attention-deficit/hyperactivity disorder (ADHD) (13%, 8%), and substance abuse (5%, 10%) among offspring. Both studies also found high rates of comorbid alcohol abuse, depression, and multiple drugs of abuse in the mothers. We conclude that efforts to replicate findings by analyses of independently conducted studies are an inexpensive way to test the sturdiness of findings that can provide the empirical basis for preventive efforts. Clinically, the data in both studies suggest that both drug dependence and associated psychopathology should be assessed and treated in opiate addicts with young offspring, and the offspring should be monitored for the development of conduct and mood disorders and substance use.

Trihexyphenidyl (Artane) Dependence: A Controlled Investigation between Users and Abusers

This controlled study aims to identify the socioclinical factors predisposing psychiatric patients to abuse trihexyphenidyl (artane) and to document the extrapyramidal symptoms in artane abusers and users. Thirty patients (n=30), with mainly two major functional psychoses and who were abusing trihexyphenidyl, were compared with 90 artane user patients (n=90), who were matched for both the diagnosis and treatment. Besides a detailed clinical interview, each patient was assessed by using DSM-IIIR criteria, Brief Psychiatric Rating Scale, Simpson and Angus Scale, and Abnormal Involuntary Movement Scale. The analysis of data showed that, compared with users, trihexyphenidyl abusers were significantly characterized by being unmarried, unemployed, smokers and having past and concurrent history of multiple drug abuse, and genetic loading of mental disorders. Both groups of patients were prescribed antipsychotic drugs and trihexyphenidyl on a longer basis. Besides other socioclinical parameters, premorbid personalities, stressful life events and extrapyramidals, including tardive dyskinesias, we did not differentiate between the two groups. Artane abusers, when compared with users, were significantly characterized by less negative psychopathology. However, other psychopathological domains, in particular, the positive symptoms and depression, did not differentiate between abusers and users. In conclusion, patients having these socioclinical profiles tend to develop trihexyphenidyl abuse. The mental health professionals should not prescribe trihexyphenidyl indiscriminately or for a long time to such patients, who indeed require long-term antipsychotic maintenance medications.

Multiple drugs of abuse in urine detected with a single reagent and fluorescence polarization.

We describe a simple polarization fluoroimmunoassay for the detection of several commonly abused drugs in urine. The single reagent used, prepared by mixing four different antisera and three fluorescein-labeled derivatives, produces a polarization signal that is the average of the individual signals of the derivatives. One adds urine to the pre-mixed reagent, incubates at room temperature for a few minutes, then measures fluorescence polarization. The presence of any of several abused drugs, at concentrations of 1 mg/L or more, noticeably decreases the average signal. Although other combinations are possible, the present assay detects the presence of a cocaine metabolite, amphetamine, and (or) several barbiturates.

THE CAUSATION OF DEATH DURING THE ADMINISTRATION OF CHLOROFORM

<b>32</b> <h3><b>Objectives</b></h3> Studies of dopamine (DA) release to psychostimulant challenge have consistently shown blunting in the striatum across multiple drugs of abuse, but results have been equivocal for cannabis. We examined amphetamine (AMPH) induced DA release in striatal and extra-striatal regions with PET to study cannabis dependent (CD) individuals who used cannabis heavily and no other substances, including nicotine. We used the D₃R-preferring agonist tracer [¹¹C]PHNO. <h3><b>Methods</b></h3> 11 CD, following acute detoxification for 3 days, and 12 healthy controls (HC) underwent 2 PET scans on one day with [¹¹C]PHNO, at baseline and 3 hr following PO AMPH, 0.5 mg/kg. Scans were 2 hr and acquired on an mCT scanner. Each subject9s PET data were registered to a high-resolution T1-weighted MRI image on which ROIs were drawn and transferred to the PET. Data were analyzed using SRTM with cerebellum as reference tissue. ROIs included associative striatum (AST), sensory motor striatum (SMST), limbic striatum (LST), globus pallidus (GP), thalamus (THA) and midbrain (MID). Outcome measures were BP_ND and the % change in BP_ND following AMPH (ΔBP_ND). <h3><b>Results</b></h3> Baseline BP_ND did not differ between groups. Robust decrease of BP_ND was observed in all subjects following AMPH, but the extent of decrease was less in CD than in HC in AST (15% vs 21%, p=.008), SMST (25% vs 32% p=.005), and GP (13% vs. 23%, p=.01). Effect sizes for these differences were 1.4, 1.3 and 1.2, respectively. ΔBP_ND was decreased but not significantly different between groups in LST (26% vs 32%, p=.13 ), MID (28% vs 34%, p=.43), and THA (25% vs %27, p=.85). <h3><b>Conclusions</b></h3> Blunted dopamine transmission in the associative and sensory motor subregions of the striatum is observed in CD with heavy use and no other substance dependencies. This pattern is different from that observed with other substances where the predominant effect is in LST. Future studies should investigate the significance of this pattern for the behavioral effects of cannabis, especially in terms of increasing the vulnerability to psychosis. <h3><b>Research Support</b></h3> R01DA022455