Neuroanatomical, electrophysiological, and morphological characterization of melanin-concentrating hormone cells coexpressing cocaine- and amphetamine-regulated transcript.

Melanin-concentrating hormone (MCH) cells in the hypothalamus regulate fundamental physiological functions like energy balance, sleep, and reproduction. This diversity may be ascribed to the neurochemical heterogeneity among MCH cells. One prominent subpopulation of MCH cells coexpresses cocaine- and amphetamine-regulated transcript (CART), and as MCH and CART can have opposing actions, MCH/CART+ and MCH/CART- cells may differentially modulate behavioral outcomes. However, it is not known if there are differences in the cellular properties underlying their functional differences; thus, we compared the neuroanatomical, electrophysiological, and morphological properties of MCH cells in male and female Mch-cre;L10-Egfp reporter mice. Half of MCH cells expressed CART and were most prominent in the medial hypothalamus. Whole-cell patch-clamp recordings revealed differences in their passive and active membrane properties in a sex-dependent manner. Female MCH/CART+ cells had lower input resistances, but male cells largely differed in their firing properties. All MCH cells increased firing when stimulated, but their firing frequency decreases with sustained stimulation. MCH/CART+ cells showed stronger spike rate adaptation than MCH/CART- cells. The kinetics of excitatory events at MCH cells also differed by cell type, as the rising rate of excitatory events was slower at MCH/CART+ cells. By reconstructing the dendritic arborization of our recorded cells, we found no sex differences, but male MCH/CART+ cells had less dendritic length and fewer branch points. Overall, distinctions in topographical division and cellular properties between MCH cells add to their heterogeneity and help elucidate their response to stimuli or effect on modulating their respective neural networks.

High Intensity Interval Training can Ameliorate Hypothalamic Appetite Regulation in Male Rats with Type 2 Diabetes: The Role of Leptin.

Disruption of leptin (LEP) signaling in the hypothalamus caused by type 2 diabetes (T2D) can impair appetite regulation. The aim of this study was to investigate whether the improvement in appetite regulation induced by high-intensity interval training (HIIT) in rats with T2D can be mediated by LEP signaling. In this study, 20 male Wister rats were randomly assigned to one of four groups: CO (non-type 2 diabetes control), T2D (type 2 diabetes), EX (non-type 2 diabetes exercise), and T2D + EX (type 2 diabetes + exercise).To induce T2D, a combination of a high-fat diet for 2 months and a single dose of streptozotocin (35 mg/kg) was administered. Rats in the EX and T2D + EX groups performed 4-10 intervals of treadmill running at 80-100% of their maximum velocity (Vmax). Homeostatic Model Assessment for Insulin Resistance (HOMA-IR), serum levels of insulin (INS) and LEP (LEPS) as well as hypothalamic expression of LEP receptors (LEP-R), Janus kinase 2 (JAK-2), signal transducer and activator of transcription 3 (STAT-3), neuropeptide Y (NPY), agouti-related protein (AGRP), pro-opiomelanocortin cocaine (POMC), amphetamine-related transcript (CART), suppressor of cytokine signaling (SOCS3), forkhead box protein O1 (FOXO1) were assessed. ANOVA and Tukey post hoc tests were used to compare the results between the groups. The levels of LEPS and INS, as well as the levels of LEP-R, JAK-2, STAT-3, POMC, and CART in the hypothalamus were found to be higher in the T2D + EX group compared to the T2D group. On the other hand, the levels of HOMA-IR, NPY, AGRP, SOCS3, and FOXO1 were lower in the T2D + EX group compared to the T2D group (P < 0.0001). The findings of this study suggest that HIIT may improve appetite regulation in rats with T2D, and LEP signaling may play a crucial role in this improvement. Graphical abstract (leptin signaling in the hypothalamus), Leptin (LEP), Leptin receptor (LEP-R), Janus kinase 2 (JAK2), Signal transducer and activator of transcription 3 (STAT3), expressing Neuropeptide Y (NPY), Agouti-related protein (AGRP), anorexigenic neurons (expressing pro-opiomelanocortin cocaine (POMC), Amphetamine-related transcript (CART), suppressor of cytokine signaling (SOCS3), forkhead box protein O1 (FOXO1).

Mate calling alters expression of neuropeptide, cocaine- and amphetamine- regulated transcript (CART) in the brain of male frog Microhyla nilphamariensis.

Croaking is a unique component of reproductive behaviour in amphibians which plays a key role in intraspecies communication and mate evaluation. While gonadal hormones are known to induce croaking, central regulation of sound production is less studied. Croaking is a dramatic, transient activity that sets apart an animal from non-croaking individuals. Herein, we aim at examining the profile of the neuropeptide cocaine- and amphetamine-regulated transcript (CART) in actively croaking and non-croaking frog Microhyla nilphamariensis. In anurans, this peptide is widely expressed in the areas inclusive of acoustical nuclei as well as areas relevant to reproduction. CART immunoreactivity was far more in the preoptic area (POA), anteroventral tegmentum (AV), ventral hypothalamus (vHy), pineal (P) and pituitary gland of croaking frog compared to non-croaking animals. On similar lines, tissue fragments collected from the mid region of the brain inclusive of POA, vHy, AV, pineal and pituitary gland of croaking frog showed upregulation of CART mRNA. However, CART immunoreactivity in the neuronal perikarya of raphe (Ra) was completely abolished during croaking activity. The data suggest that CART signaling in the brain may be an important player in mediating croaking behaviour in the frog.

Umbilical Cord Collection and Drug Testing to Estimate Prenatal Substance Exposure in Utah.

OBJECTIVE: Our primary objective was to estimate statewide prenatal substance exposure based on umbilical cord sampling. Our secondary objectives were to compare prevalence of prenatal substance exposure across urban, rural, and frontier regions, and to compare contemporary findings to those previously reported. METHODS: We performed a cross-sectional prevalence study of prenatal substance exposure, as determined by umbilical cord positivity for 49 drugs and drug metabolites, through the use of qualitative liquid chromatography-tandem mass spectrometry. All labor and delivery units in Utah (N=45) were invited to participate. Based on a 2010 study using similar methodology, we calculated that a sample size of at least 1,600 cords would have 90% power to detect 33% higher rate of umbilical cords testing positive for any substance. Deidentified umbilical cords were collected from consecutive deliveries at participating hospitals. Prevalence of prenatal substance exposure was estimated statewide and by rurality using weighted analysis. RESULTS: From November 2020 to November 2021, 1,748 cords (urban n=988, rural n=384, frontier n=376) were collected from 37 hospitals, representing 92% of hospitals that conduct 91% of births in the state. More than 99% of cords (n=1,739) yielded results. Statewide, 9.9% (95% CI 8.1-11.7%) were positive for at least one substance, most commonly opioids (7.0%, 95% CI 5.5-8.5%), followed by cannabinoid (11-nor-9-carboxy-delta-9-tetrahydrocannabinol [THC-COOH]) (2.5%, 95% CI 1.6-3.4%), amphetamines (0.9%, 95% CI 0.4-1.5), benzodiazepines (0.5%, 95% CI 0.1-0.9%), alcohol (0.4%, 95% CI 0.1-0.7%), and cocaine (0.1%, 95% CI 0-0.3%). Cord positivity was similar by rurality (urban=10.3%, 95% CI 8.3-12.3%, rural=7.1%, 95% CI 3.5-10.7%, frontier=9.2%, 95% CI 6.2-12.2%, P=.31) and did not differ by substance type. Compared with a previous study, prenatal exposure to any substance (6.8 vs 9.9%, P=.01), opioids (4.7 vs 7.0% vs 4.7%, P=.03), amphetamines (0.1 vs 0.9%, P=.01) and THC-COOH (0.5 vs 2.5%, P<.001) increased. CONCLUSION: Prenatal substance exposure was detected in nearly 1 in 10 births statewide.

The nitrosoamphetamine metabolite is accommodated in the active site of human hemoglobin: Spectroscopy and crystal structure.

Amphetamine-based (Amph) drugs are metabolized in humans to their hydroxylamine (AmphNHOH) and nitroso (AmphNO) derivatives. The latter metabolites are known to bind to the Fe centers of cytochrome P450 and other heme enzymes to inhibit their activities. Although these AmphNHOH/AmphNO metabolites are present in vivo, their interactions with the blood protein hemoglobin (Hb) and the muscle protein (Mb) have been largely discounted due to a perception that the relatively small heme active sites of Hb and Mb will not be able to accommodate the large AmphNO group. We report the 2.15 Å resolution X-ray crystal structure of the AmphNO adduct of adult human hemoglobin as the Hb [α-FeIII(H2O)][ß-FeII(AmphNO)] derivative. We show that the binding of AmphNO to the ß subunit is enabled by an E helix movement and stabilization of ligand binding by H-bonding with the distal His63 residue. We also observe an AmphNHOH group in the Xe2 pocket in close proximity to the α heme site in this derivative. Additionally, UV-vis spectroscopy was used to characterize this and related wt and mutant Mb adducts. Importantly, our X-ray crystal structure of this Hb-nitrosoamphetamine complex represents the first crystal structure of a wild-type heme protein adduct of any amphetamine metabolite. Our results provide a framework for further studies of AmphNHOH/AmphNO interactions with Hb and Mb as viable processes that potentially contribute to the overall biological inorganic chemistry of amphetamine drugs.

Application of a chiral high-performance liquid chromatography-tandem mass spectrometry method for the determination of 13 related amphetamine-type stimulants to forensic samples: Interpretative hypotheses.

Interpretation of amphetamine-type stimulant (ATS) findings in urine samples can be challenging without chiral information. We present a sensitive enantioselective high-performance liquid chromatography-tandem mass spectrometry method for the quantification of (R)-amphetamine, (S)-amphetamine, (R)-methamphetamine, (S)-methamphetamine, (1R,2R)-pseudoephedrine, (1S,2S)-pseudoephedrine, (1R,2S)-ephedrine, (1S,2R)-ephedrine, (1R,2S)-norephedrine, (1S,2R)-norephedrine, (R)-cathinone, (S)-cathinone, and (1S,2S)-norpseudoephedrine (cathine) in urine. The method was successfully applied to more than 100 authentic urine samples from forensic casework. In addition, samples from a controlled self-administration of (1S,2S)-pseudoephedrine (Rinoral, 1200 mg within 6 days) were analyzed. The results strengthen the hypothesis that (1R,2S)-norephedrine is a minor metabolite of amphetamine and methamphetamine. We suggest cathine and (1S,2R)-norephedrine as minor metabolites of amphetamine racemate in humans. Small methamphetamine concentrations detected in samples with high concentrations of amphetamine could result from a metabolic formation by methylation of amphetamine although in samples with an (R)/(S) ratio for methamphetamine < 1 an additional (previous) (S)-methamphetamine consumption seems likely. Our data suggest that even amphetamine concentrations exceeding methamphetamine concentrations in urine can be caused by the biotransformation of methamphetamine to amphetamine as long as no (R)-amphetamine is detected. However, without chiral information, such findings might be (falsely) assumed as a co-consumption of both substances. Cathinone enantiomers detected in urine samples with high amphetamine concentrations can be interpreted as metabolites of amphetamine. In addition, the results of the self-administration study revealed that both cathinone enantiomers are minor metabolites of (1S,2S)-pseudoephedrine, which is the active ingredient of various medicines used for cold. The enantioselective analysis is a powerful tool to avoid the misinterpretation of ATS findings in urine samples.

Para-Halogenation of Amphetamine and Methcathinone Increases the Mitochondrial Toxicity in Undifferentiated and Differentiated SH-SY5Y Cells.

Halogenation of amphetamines and methcathinones has become a common method to obtain novel psychoactive substances (NPS) also called "legal highs". The para-halogenated derivatives of amphetamine and methcathinone are available over the internet and have entered the illicit drug market but studies on their potential neurotoxic effects are rare. The primary aim of this study was to explore the neurotoxicity of amphetamine, methcathinone and their para-halogenated derivatives 4-fluoroamphetamine (4-FA), 4-chloroamphetamine (PCA), 4-fluoromethcathinone (4-FMC), and 4-chloromethcathinone (4-CMC) in undifferentiated and differentiated SH-SY5Y cells. We found that 4-FA, PCA, and 4-CMC were cytotoxic (decrease in cellular ATP and plasma membrane damage) for both cell types, whereby differentiated cells were less sensitive. IC50 values for cellular ATP depletion were in the range of 1.4 mM for 4-FA, 0.4 mM for PCA and 1.4 mM for 4-CMC. The rank of cytotoxicity observed for the para-substituents was chloride > fluoride > hydrogen for both amphetamines and cathinones. Each of 4-FA, PCA and 4-CMC decreased the mitochondrial membrane potential in both cell types, and PCA and 4-CMC impaired the function of the electron transport chain of mitochondria in SH-SY5Y cells. 4-FA, PCA, and 4-CMC increased the ROS level and PCA and 4-CMC induced apoptosis by the endogenous pathway. In conclusion, para-halogenation of amphetamine and methcathinone increases their neurotoxic properties due to the impairment of mitochondrial function and induction of apoptosis. Although the cytotoxic concentrations were higher than those needed for pharmacological activity, the current findings may be important regarding the uncontrolled recreational use of these compounds.

Simultaneous analysis of 29 synthetic cannabinoids and metabolites, amphetamines, and cannabinoids in human whole blood by liquid chromatography-tandem mass spectrometry - A New Zealand perspective of use in 2018.

We describe the validation of a method for the simultaneous analysis of 29 synthetic cannabinoids (SCs) and metabolites, 4 amphetamines, and 2 cannabinoids in human whole blood. This method enables one analysis to cover what previously required multiple analyses for these classic and novel drugs-of-abuse with diverse physicochemical properties. The scope of targeted analytes was based on the most prevalent drugs-of-abuse and SCs encountered at the New Zealand border in 2017 and included parent compounds and metabolites belonging to the indole and indazole carboxamide, quinolinyl indole carboxylate, and naphthoylindole classifications. Samples were prepared by supported-liquid-extraction (SLE) followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis with positive electrospray ionization (ESI). The method was validated with respect to selectivity, matrix effects, process efficiency, sensitivity, repeatability, extract stability, and carryover for qualitative confirmation. Linearity as well as accuracy and precision data at target decision concentrations were also evaluated. The limits of detection and confirmation ranged from 0.1 to 6.0 ng/mL and 1.0 to 6.0 ng/mL, respectively. The described method was successfully applied to the analysis of 564 ante- and post-mortem blood samples in 2018. There were 132 cases (23%) with positive findings of at least one SC, with the five most commonly detected SCs being AMB-FUBINACA and/or acid (61%), 5F-ADB and/or acid (40%), ADB-FUBINACA (11%), 5F-MDMB-PICA acid (6%), and MDMB-FUBINACA acid (6%). The results also demonstrate the predominant presence of metabolites at higher levels than the unchanged parent SCs in blood, highlighting the need to maintain forensic screening methods capable of the simultaneous detection of both parent compounds and metabolites.

Simultaneous determination of 75 abuse drugs including amphetamines, benzodiazepines, cocaine, opioids, piperazines, zolpidem and metabolites in human hair samples using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometric method for the simultaneous determination of 75 abuse drugs and metabolites, including 19 benzodiazepines, 19 amphetamines, two opiates, eight opioids, cocaine, lysergic acid diethylamide, zolpidem, three piperazines and 21 metabolites in human hair samples, was developed and validated. Ten-milligram hair samples were decontaminated, pulverized using a ball mill, extracted with 1 mL of methanol spiked with 28 deuterated internal standards in an ultrasonic bath for 60 min at 50°C, and purified with Q-sep dispersive solid-phase extraction tubes. The purified extracts were evaporated to dryness and the residue was dissolved in 0.1 mL of 10% methanol. The 75 analytes were analyzed on an Acquity HSS T3 column using gradient elution of methanol and 0.1% formic acid and quantified in multiple reaction monitoring mode with positive electrospray ionization. Calibration curves were linear (r ≥ 0.9951) from the lower limit of quantitation (2-200 pg/mg depending on the drug) to 2000 pg/mg. The coefficients of variation and accuracy for intra- and inter-assay analysis at three QC levels were 4.3-12.9% and 89.2-109.1%, respectively. The overall mean recovery ranged from 87.1 to 105.3%. This method was successfully applied to the analysis of 11 forensic hair samples obtained from drug abusers.

Recent advances in microextraction procedures for determination of amphetamines in biological samples.

Amphetamine and its related derivatives have stimulant and hallucinogenic properties. Illegal use of these drugs is an increasing global problem resulting in significant public health and legal problems. Deaths have been reported after intake of these drugs due to overdose. It is important to determine the type and concentration of illicit drugs in biological samples. These compounds are found in complex matrices at low concentration levels. The microextraction techniques are dominant sample preparation procedure and they are widely accepted as the most labor-intensive part of the bioanalytical process. For this purpose, a survey of recent published advances in microextraction procedures for quantification of amphetamines in biological samples found in the different databases from 2008 to date will be conducted.

Pharmacokinetics of selegiline, R-methamphetamine, R-amphetamine, and desmethylselegiline in oral fluid after a single oral administration of selegiline.

BACKGROUND: Chiral analysis is a crucial way to differentiate selegiline (SG) intake from drug abuse. Oral fluid (OF) has been successfully used as an alternative matrix for blood testing in several pharmacokinetic studies. OBJECTIVE: The aim of this study is to describe the pharmacokinetics of SG and its main metabolites in OF after a single oral administration of SG which is meaningful for results interpretation in forensic analysis. METHODS: Ten milligrams of SG were orally administered to 8 volunteers, and OF samples were collected for up to 96 hours by having participants spit into polypropylene tubes without stimulation. These samples were submitted to liquid-liquid extraction before analysis by liquid chromatography-tandem mass spectrometry operating in positive ion multiple-reaction monitoring mode. RESULTS AND CONCLUSIONS: After oral administration, each analyte could be detected in OF specimens from all volunteers with an initial detection time of 0.50 hours. The Cmax values of SG, R-MA, R-AM and DM-SG were 50.93-992.67 ng/mL, 29.78-653.64 ng/mL, 8.22-150.15 ng/mL, and 4.34-16.25 ng/mL, respectively, at 0.5 hours, 1-11 hours, 1.5-11 hours, and 0.5-6 hours post dose. The times when the compounds were last determined in OF were 5-24 hours for SG, 52-96 hours for R-MA, 31-96 hours for R-AM, and 13-31 hours for DM-SG after oral administration. There is a period of time in OF in which only MA and AM are present without SG and DM-SG after a single dose of SG. The pharmacokinetic data could provide supplementary interpretation for OF tests in forensic science and drug treatment programs.

Nano liquid chromatography-high-resolution mass spectrometry for the identification of metabolites of the two new psychoactive substances N-(ortho-methoxybenzyl)-3,4-dimethoxyamphetamine and N-(ortho-methoxybenzyl)-4-methylmethamphetamine.

Among the emerging new psychoactive substances (NPS), compounds carrying an N-ortho-methoxybenzyl substituent, the so-called NBOMes, represented a highly potent group of new hallucinogens. Recently, 3,4-dimethoxyamphetamine (3,4-DMA)-NBOMe and 4-methylmethamphetamine (4-MMA)-NBOMe occurred, but no data on their pharmacokinetics were available. According to other NBOMes, they are expected to be extensively metabolized. For detection and identification of their phase I and II metabolites, nano liquid chromatography coupled to high resolution tandem mass spectrometry (nanoLC-HRMS/MS) was used. Rat urine was prepared by simple dilution and incubation mixtures with pooled human liver S9 fraction by precipitation. Furthermore, the results concerning detectability using the new nanoLC approach were compared to those obtained by conventional ultra-high performance LC (UHPLC). In addition, the detectability of the compounds by standard urine screening approaches (SUSAs) routinely used by the authors with UHPLC-HRMS/MS, LC-MSn, and GC-MS was tested. Both NBOMes were extensively metabolized mainly by O-demethylation and conjugation with glucuronic acid (3,4-DMA-NBOMe) or oxidation of the tolyl group to the corresponding carboxylic acid (4-MMA-NBOMe). The developed nanoLC-HRMS/MS approach was successfully applied for identification of 38 3,4-DMA-NBOMe metabolites and 33 4-MMA-NBOMe metabolites confirming its detection power. Furthermore, the solvent saving nanoLC system showed comparable results to the UHPLC-HRMS/MS approach. In addition, an intake of an estimated low common user's dose of the compounds was detectable by all SUSAs only via their metabolites. Suggested targets for urine screening procedures were O-demethyl- and O,O-bis-demethyl-3,4-DMA-NBOMe and their glucuronides and carboxy-4-MMA-NBOMe and its glucuronide and N-demethyl-carboxy-4-MMA-NBOMe.

LC-high resolution-MS/MS for identification of 69 metabolites of the new psychoactive substance 1-(4-ethylphenyl-)-N-[(2-methoxyphenyl)methyl] propane-2-amine (4-EA-NBOMe) in rat urine and human liver S9 incubates and comparison of its screening power with further MS techniques.

4-EA-NBOMe (N-(2-methoxybenzyl)-4-ethylamphetamine, 1-(4-ethylphenyl-)-N-[(2-methoxyphenyl)methyl]propane-2-amine) is an amphetamine-derived new psychoactive substance (NPS) of the N-methoxybenzyl (NBOMe) group first seized by German custom authorities. In contrast to the phenethylamine NBOMes, studies on the pharmacological, toxicological, or metabolic properties are not yet published. The aims of the presented work were the use of LC-HR-MS/MS for identification of the phase I and II metabolites of 4-EA-NBOMe in rat urine and pooled human S9 fraction (pS9) incubations, to compare metabolite formation in both models, to identify involved monooxygenases, and to elucidate its detectability in standard urine screening approaches (SUSAs) using GC-MS, LC-MSn, and LC-HR-MS/MS. 4-EA-NBOMe was mainly metabolized by oxidation of the ethyl group to phenyl acetaldehyde, to benzoic acid, or to phenylacetic acid, by hydroxylation, and all combined with O-demethylation as well as by glucuronidation and sulfation of the main phase I metabolites in rats. With the exception of the oxidation to benzoic acid, all main metabolic reactions could be confirmed in the incubations with pS9. In total, 36 phase I and 33 phase II metabolites could be identified. Monooxygenase activity screenings revealed the general involvement of cytochrome-P450 (CYP) 1A2, CYP2B6, and CYP3A4. An intake of 4-EA-NBOMe was detectable only via its metabolites by all SUSAs after low-dose administration. The main targets for both LC-MS screenings should be the phenylacetic acid derivative, the mandelic acid derivative both with and without additional O-demethylation, and, for GC-MS, the hydroxy metabolite after conjugate cleavage.

Traumatic stress affects alcohol­drinking behavior through cocaine­ and amphetamine­regulated transcript 55­102 in the paraventricular nucleus in rats.

Cumulative evidence has suggested an association between stress and alcohol self­administration; however, less is known about the role of traumatic stress in alcohol drinking behavior. It has been reported that cocaine­ and amphetamine­regulated transcript (CART) 55­102 may be involved in mediating stress responses and regulating reward and reinforcement. The aim of the present study was to evaluate the role of CART 55­102 in alcohol drinking behavior of rats in the presence or absence of traumatic stress. Alcohol drinking behavior was examined using the two­bottle choice drinking paradigm (one bottle contained 10% alcohol and the other contained filtered water), which was initiated 1, 3 and 7 days post­trauma (T1, T3 and T7), for 14 days in rats; the control group was initiated from T0. The results indicated that exposure to trauma significantly increased alcohol consumption and preference, particularly drinking from T3. Immunohistochemistry revealed that the lowest level of CART 55­102 immunoreactivity within the paraventricular nucleus (PVN) was exhibited in the T3 group. Additionally, an intra­PVN injection of CART 55­102 attenuated alcohol­drinking behavior in a dose­dependent manner, in the T3 group. Furthermore, the significant increase in circulating adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) concentrations in the T3 group were inhibited by CART 55­102 administration to the PVN, in particular CORT levels were significantly decreased. Positive correlations between alcohol preference and ACTH and CORT levels were also observed. These results indicated that CART 55­102 in the PVN serves an inhibitory role in traumatic stress­induced alcohol drinking behavior, possibly through disturbing hypothalamus­pituitary­adrenal axis hyperactivity.

One-Step Derivatization-Extraction Method for Rapid Analysis of Eleven Amphetamines and Cathinones in Oral Fluid by GC-MS.

A simple analytical method was developed for in-matrix derivatization of 11 amphetamine-like molecules. Ethyl chloroformate as the derivatization reagent and ethyl acetate as the extraction solvent were added directly to oral fluid samples at alkaline pH. Samples were analyzed by gas chromatography-mass spectrometry. Ions monitored for quantification were m/z 44, 91 and 116 for amphetamine (AMP); m/z 58, 91 and 130 for methamphetamine (MA); m/z 44, 105 and 116 for 4-methylamphetamine; m/z 44, 116 and 251 for 3,4-methylenedioxyamphetamine; m/z 58, 130 and 265 for 3,4-methylenedioxymethamphetamine (MDMA); m/z 72, 116 and 144 for 3,4-methylenedioxyethylamphetamine; m/z 44, 105 and 116 for cathinone; m/z 58, 105 and 130 for methcathinone; m/z 58, 119 and 130 for mephedrone; m/z 58, 107 and 130 for ephedrine (EPH); m/z 207, 250 and 322 for fenethylline; m/z 48, 92 and 120 for AMP-D5; m/z 62, 92 and 134 for MA-D5; m/z 48, 120 and 256 for MDA-D5; and m/z 62, 134 and 270 for MDMA-D5. The underlined ions were used as quantifier ions. Calibration curves were linear in the concentration range of 2.5-1,000 ng/mL for all analytes except for EPH, which was linear within 5-1,000 ng/mL. Precision and accuracy were less than 12.9% (relative standard deviation) and ±12.8% (bias), respectively, for all analytes. The method was tested in the analysis of oral fluid specimens collected from users.

Vaccine-driven pharmacodynamic dissection and mitigation of fenethylline psychoactivity.

Fenethylline, also known by the trade name Captagon, is a synthetic psychoactive stimulant that has recently been linked to a substance-use disorder and 'pharmacoterrorism' in the Middle East. Although fenethylline shares a common phenethylamine core with other amphetamine-type stimulants, it also incorporates a covalently linked xanthine moiety into its parent structure. These independently active pharmacophores are liberated during metabolism, resulting in the release of a structurally diverse chemical mixture into the central nervous system. Although the psychoactive properties of fenethylline have been reported to differ from those of other synthetic stimulants, the in vivo chemical complexity it manifests upon ingestion has impeded efforts to unambiguously identify the specific species responsible for these effects. Here we develop a 'dissection through vaccination' approach, called DISSECTIV, to mitigate the psychoactive effects of fenethylline and show that its rapid-onset and distinct psychoactive properties are facilitated by functional synergy between theophylline and amphetamine. Our results demonstrate that incremental vaccination against a single chemical species within a multi-component mixture can be used to uncover emergent properties arising from polypharmacological activity. We anticipate that DISSECTIV will be used to expose unidentified active chemical species and resolve pharmacodynamic interactions within other chemically complex systems, such as those found in counterfeit or illegal drug preparations, post-metabolic tissue samples and natural product extracts.

Substance use in individuals with mild to borderline intellectual disability: A comparison between self-report, collateral-report and biomarker analysis.

BACKGROUND AND AIMS: Individuals with mild or borderline intellectual disability (MBID) are at risk of substance use (SU). At present, it is unclear which strategy is the best for assessing SU in individuals with MBID. This study compares three strategies, namely self-report, collateral-report, and biomarker analysis. METHODS AND PROCEDURES: In a sample of 112 participants with MBID from six Dutch facilities providing care to individuals with intellectual disabilities, willingness to participate, SU rates, and agreement between the three strategies were explored. The Substance use and misuse in Intellectual Disability - Questionnaire (SumID-Q; self-report) assesses lifetime use, use in the previous month, and recent use of tobacco, alcohol, cannabis, and stimulants. The Substance use and misuse in Intellectual Disability - Collateral-report questionnaire (SumID-CR; collateral-report) assesses staff members' report of participants' SU over the same reference periods as the SumID-Q. Biomarkers for SU, such as cotinine (metabolite of nicotine), ethanol, tetrahydrocannabinol (THC), and its metabolite THCCOOH, benzoylecgonine (metabolite of cocaine), and amphetamines were assessed in urine, hair, and sweat patches. RESULTS: Willingness to provide biomarker samples was significantly lower compared to willingness to complete the SumID-Q (p<0.001). Most participants reported smoking, drinking alcohol, and using cannabis at least once in their lives, and about a fifth had ever used stimulants. Collateralreported lifetime use was significantly lower. However, self-reported past month and recent SU rates did not differ significantly from the rates from collateral-reports or biomarkers, with the exception of lower alcohol use rates found in biomarker analysis. The agreement between self-report and biomarker analysis was substantial (kappas 0.60-0.89), except for alcohol use (kappa 0.06). Disagreement between SumID-Q and biomarkers concerned mainly over-reporting of the SumID-Q. The agreement between SumID-CR and biomarker analysis was moderate to substantial (kappas 0.48 - 0.88), again with the exception of alcohol (kappa 0.02). CONCLUSIONS AND IMPLICATIONS: In this study, the three strategies that were used to assess SU in individuals with MBID differed significantly in participation rates, but not in SU rates. Several explanations for the better-than-expected performance of self- and collateral-reports are presented. We conclude that for individuals with MBID, self-report combined with collateralreport can be used to assess current SU, and this combination may contribute to collaborative, early intervention efforts to reduce SU and its related harms in this vulnerable group.

Combined Simulation and Mutation Studies to Elucidate Selectivity of Unsubstituted Amphetamine-like Cathinones at the Dopamine Transporter.

The dopamine and serotonin transporter proteins (DAT, SERT) play a vital role in behavior and mental illness. Although their substrate transport has been studied extensively, the molecular basis of their selectivity is not completely understood yet. In this study, we exploit molecular dynamics simulations combined with mutagenesis studies to shed light on the driving factors for DAT-over-SERT selectivity of a set of cathinones. Results indicate that these compounds can adopt two binding modes of which one is more favorable. In addition, free energy calculations indicated the substrate binding site (S1) as the primary recognition site for these ligands. By simulating DAT with SERT-like mutations, we hypothesize unsubstituted cathinones to bind more favorably to DAT, due to a Val152 offering more space, as compared to the bulkier Ile172 in SERT. This was supported by uptake inhibition measurements, which showed an increase in activity in SERT-I172V.

Reporting Two Fatalities Associated with the Use of 4-Methylethcathinone (4-MEC) and a Review of the Literature.

We report two fatalities that are related to the cathinone 4-methylethcathinone (4-MEC) and review the current knowledge of 4-MEC. Qualitative and quantitative analysis of 4-MEC was performed by validated high performance liquid chromatography-tandem mass spectrometry methods. In the first case a 22-year-old male died in hospital following collapse and seizures after using 4-MEC. Toxicological analysis of postmortem femoral blood revealed the presence of 4-MEC (0.167 mg/L), ethanol (27 mg/100 mL) and paracetamol (5 mg/L). Death was attributed solely to 4-MEC toxicity. The second case involved a 54-year-old man found with a taped plastic bag over his head. Toxicological analysis of postmortem femoral blood revealed the presence of 4-MEC (1.73 mg/L) along with ethanol (229 mg/100 mL), propranolol (0.036 mg/L), venlafaxine (0.284 mg/L) and its metabolite O-desmethylvenlafaxine (0.205 mg/L), and diazepam (<0.005 mg/L) and its metabolite nordiazepam (0.033 mg/L). Death was attributed primarily to asphyxiation. These cases and a review of the current knowledge of 4-MEC pharmacology/toxicology adds to the body of case material for 4-MEC and will assist with interpretation in postmortem toxicology cases in which 4-MEC is detected.

Why p-OMe- and p-Cl-ß-Methylphenethylamines Display Distinct Activities upon MAO-B Binding.

Despite their structural and chemical commonalities, p-chloro-ß-methylphenethylamine and p-methoxy-ß-methylphenethylamine display distinct inhibitory and substrate activities upon MAO-B binding. Density Functional Theory (DFT) quantum chemical calculations reveal that ß-methylation and para-substitution underpin the observed activities sustained by calculated transition state energy barriers, attained conformations and key differences in their interactions in the enzyme's substrate binding site. Although both compounds meet substrate requirements, it is clear that ß-methylation along with the physicochemical features of the para-substituents on the aromatic ring determine the activity of these compounds upon binding to the MAO B-isoform. While data for a larger set of compounds might lend generality to our conclusions, our experimental and theoretical results strongly suggest that the contrasting activities displayed depend on the conformations adopted by these compounds when they bind to the enzyme.