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Clonazolam is a designer triazolobenzodiazepine first synthesized in 1971 and is primarily used for its anxiolytic and sedative effects. It became a drug of misuse in 2012 and is known for its high potency and long duration of effect. Previous studies of nitrobenzodiazepines, such as nitrazepam, clonazepam, and flunitrazepam, as well as their metabolites, have demonstrated that bacterial species native to the gastrointestinal tract and active during postmortem (PM) decomposition are capable of affecting positivity and compound-to-metabolite ratios. Further studies have not been performed with clonazolam; however, it possesses the nitro functional group necessary for this biotransformation. To understand whether clonazolam may be similarly affected, PM cases (n = 288) and driving under the influence of drugs (DUID, n = 54) cases, positive for 8-aminoclonazolam reported by NMS Laboratories from 2020 to 2023, were selected for inclusion in this study. Concentrations of clonazolam and 8-aminoclonazolam were evaluated, and concurrent identification of parent drugs and their metabolites occurred less frequently in PM cases (n = 1, 0.30% of cases) than in DUID cases (n = 21, 38% of cases). The clonazolam concentration in one PM case was 13 ng/mL. In DUID cases, the median clonazolam concentration was 4.0 ng/mL and ranged from 2.0 to 10 ng/mL. 8-Aminoclonazolam had median concentrations of 13 and 19 ng/mL, with ranges 2.0-580 and 2.8-59 ng/mL for PM and DUID cases, respectively. Due to the ever-changing landscape of the designer benzodiazepine market, in vitro studies of PM microbial biotransformation of clonazolam are unavailable. The data reported herein provide valuable information in the absence of such studies and represent an alternative method of investigating this phenomenon as a potential cause of parent nitrobenzodiazepine to metabolite conversion., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

The types and quantities of new psychoactive substances synthesized based on structural modifications have increased rapidly in recent years and pose a great challenge to clinical and forensic laboratories. N-benzyl derivatives of phenethylamines, 25B-NBOH, 25E-NBOH, 25H-NBOH, and 25iP-NBOMe have begun to flow into the black market and have caused several poisoning cases and even fatal cases. The aim of this study was to avoid false negative results by detecting the parent drug and its metabolites to extend the detection window in biological matrices and provide basic data for the simultaneous determination of illegal drugs and metabolites in forensic and emergency cases. To facilitate the comparison of metabolic characteristics, we divided the four compounds into two groups of types, 25X-NBOH and 25X-NBOMe. The in vitro phase I and phase II metabolism of these four compounds was investigated by incubating 10 mg mL -1 pooled human liver microsomes with co-substrates for 180 min at 37 ℃, and then analyzing the reaction mixture using ultrahigh-performance liquid chromatography-quadrupole/electrostatic field orbitrap mass spectrometry. In total, 70 metabolites were obtained for the four compounds. The major biotransformations were O-demethylation, hydroxylation, dehydrogenation, N-dehydroxybenzyl, N-demethoxybenzyl, oxidate transformation to ketone and carboxylate, glucuronidation, and their combination reactions. We recommended the major metabolites with high peak area ratio as biomarkers, B2-1 (56.61%), B2-2 (17.43%) and B6 (17.78%) for 25B-NBOH, E2-1 (42.81%), E2-2 (34.90%) and E8-2 (10.18%) for 25E-NBOH, H5 (49.28%), H2-1 (21.54%), and H1 (18.37%) for 25H-NBOH, P3-1 (10.94%), P3-2 (33.18%), P3-3 (14.85%) and P12-2 (23.00%) for 25iP-NBOMe. This is a study to evaluate their metabolic characteristics in detail. Comparative analysis of the N-benzyl derivatives of phenethylamines provided basic data for elucidating their pharmacology and toxicity. Timely analysis of the metabolic profiles of compounds with abuse potential will facilitate the early development of regulatory measures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

G-protein-coupled receptors (GPCRs) transduce diverse signals into the cell by coupling to one or several Gα subtypes. Of the 16 Gα subtypes in human cells, Gα 12 and Gα 13 belong to the G 12 subfamily and are reported to be functionally different. Notably, certain GPCRs display selective coupling to either Gα 12 or Gα 13 , highlighting their significance in various cellular contexts. However, the structural basis underlying this selectivity remains unclear. Here, using a Gα 12 -coupled designer receptor exclusively activated by designer drugs (DREADD; G 12 D) as a model system, we identified residues in the α5 helix and the receptor that collaboratively determine Gα 12 -vs-Gα 13 selectivity. Residue-swapping experiments showed that G 12 D distinguishes differences between Gα 12 and Gα 13 in the positions G.H5.09 and G.H5.23 in the α5 helix. Molecular dynamics simulations observed that I378 G.H5.23 in Gα 12 interacts with N103 2.39 , S169 3.53 and Y176 34.53 in G 12 D, while H364 G.H5.09 in Gα 12 interact with Q264 5.71 in G 12 D. Screening of mutations at these positions in G 12 D identified G 12 D mutants that enhanced coupling with Gα 12 and to an even greater extent with Gα 13 . Combined mutations, most notably the dual Y176 34.53 H and Q264 5.71 R mutant, further enhanced Gα 12 / 13 coupling, thereby serving as a potential Gα 12/13 -DREADD. Such novel Gα 12/13 -DREADD may be useful in future efforts to develop drugs that target Gα 12/13 signaling as well as to identify their therapeutic indications., (© 2024. The Author(s).)

Abstract: Clonazolam is a derivative of the Xanax active ingredient, alprazolam. Classified as a designer benzodiazepine, clonazolam availability has been rising because of its circulation on illegal internet drug markets and marginal cost in comparison to its parent analogs. Clonazolam's accessibility encourages abuse prevalence and use of designer benzodiazepines. In our case, a 14-year-old male was found unresponsive the morning after ingesting multiple tablets believed to be Xanax. Toxicology testing indicated 140 ng/mL of 8-aminoclonazolam, a clonazolam metabolite, in the decedent's system. Alprazolam was not identified. Pathological analysis determined cerebral and respiratory depression to be the mechanism of death, resulting from acute clonazolam intoxication. This case presents the first death induced by clonazolam alone. Current literature identifies a gap in designer benzodiazepine confirmatory testing and a lack of awareness within the forensic and medical communities. Knowledge of designer benzodiazepines is needed to better understand their potency and to help prevent future intoxications. We present this case to aid in the recognition of novel benzodiazepines by medical examiners and coroners, to encourage their consideration in suspected Xanax and other substance related investigations, and to be aware of the capabilities of toxicological testing to improve novel benzodiazepine identification and subsequent interpretation., Competing Interests: The authors report no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) have become a premier neuroscience research tool for enabling reversible manipulations of cellular activity following experimenter-controlled delivery of a DREADD-specific ligand. However, several DREADD ligands, e.g., clozapine-N-oxide (CNO), have metabolic and off-target effects that may confound experimental findings. New DREADD ligands aim to reduce metabolic and potential off-target effects while maintaining strong efficacy for the designer receptors. Recently a novel DREADD ligand, deschloroclozapine (DCZ), was shown to induce chemogenetic-mediated cellular and behavioral effects in mice and monkeys without detectable side effects. The goal of the present study was to examine the effectiveness of systemic DCZ for DREADD-based chemogenetic manipulations in behavioral and slice electrophysiological applications in rats. We demonstrate that a relatively low dose of DCZ (0.1 mg/kg) supports excitatory DREADD-mediated cFos induction, DREADD-mediated inhibition of a central amygdala-dependent behavior, and DREADD-mediated inhibition of neuronal activity in a slice electrophysiology preparation. In addition, we show that this dose of DCZ does not alter gross locomotor activity or induce a place preference/aversion in control rats without DREADD expression. Together, our findings support the use of systemic DCZ for DREADD-based manipulaations in rats, and provide evidence that DCZ is a superior alternative to CNO., (© 2022. The Author(s).)

We have investigated the metabolic profile of N-ethyl heptedrone, a new designer synthetic stimulant drug, by using data independent acquisition mass spectrometry. Phase I and phase II metabolism was studied by in vitro models, followed by liquid-chromatography coupled to mass spectrometry, to characterize and pre-select the most diagnostic markers of intake. N-ethyl heptedrone was incubated in the presence of pooled human liver microsomes. The contribution of individual enzymatic isoforms in the formation of the phase I and phase II metabolites was further investigated by using human recombinant cDNA-expressed cytochrome P450 enzymesand uridine 5'-diphospho glucuronosyltransferases. The analytical workflow consisted of liquid-liquid extraction with tert-butyl-methyl-ether at alkaline pH, performed before (to investigate the phase I metabolic profile) and after (to investigate the glucuronidation profile) enzymatic hydrolysis. The separation, identification, and determination of the compounds formed in the in vitro experiments were carried out by using liquid chromatography coupled to either high- or low-resolution mass spectrometry. Data independent acquisition method, namely sequential window acquisition of all theoretical fragment-ion spectra (SWATH®) and product ion scan were selected for high-resolution mass spectrometry, whereas multiple reaction monitoring was used for low-resolution mass spectrometry. Thirteen phase-I metabolites were isolated, formed from reactions being catalyzed mainly by CYP1A2, CYP2C9, CYP2C19 and CYP2D6 and, to a lesser degree, by CYP3A4 and CYP3A5. The phase I biotransformation pathways included hydroxylation in different positions, reduction of the ketone group, carbonylation, N-dealkylation, and combinations of the above. Most of the hydroxylated metabolites underwent conjugation reactions to form the corresponding glucurono-conjugated metabolites. Based on our in vitro observation, the metabolic products resulting from reduction of the keto group, N-dealkylation and hydroxylation of the aliphatic chain appear to be the most diagnostic target analytes to be selected as markers of exposure to N-ethyl heptedrone., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The emerging market of new psychoactive substances (NPSs) is a global-scale phenomenon, and their identification in biological samples is challenging because of the lack of information about their metabolism and pharmacokinetic. In this study, we performed in silico metabolic pathway prediction and in vivo metabolism experiments, in order to identify the main metabolites of mephtetramine (MTTA), an NPS found in seizures since 2013. MetaSite™ software was used for in silico metabolism predictions and subsequently the presence of metabolites in the blood, urine, and hair of mice after MTTA administration was verified. The biological samples were analyzed by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) using a benchtop Orbitrap instrument. This confirmed the concordance between software prediction and experimental results in biological samples. The metabolites were identified by their accurate masses and fragmentation patterns. LC-HRMS analysis identified the dehydrogenated and demethylated-dehydrogenated metabolites, together with unmodified MTTA in the blood samples. Besides unmodified MTTA, 10 main metabolites were detected in urine. In hair samples, only demethyl MTTA was detected along with MTTA. The combination of Metasite™ prediction and in vivo experiment was a powerful tool for studying MTTA metabolism. This approach enabled the development of the analytical method for the detection of MTTA and its main metabolites in biological samples. The development of analytical methods for the identification of new drugs and their main metabolites is extremely useful for the detection of NPS in biological specimens. Indeed, high throughput methods are precious to uncover the actual extent of use of NPS and their toxicity., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

DREADDs (Designer Receptors Exclusively Activated by a Designer Drug) are designer G protein-coupled receptors (GPCRs) that are widely used in the neuroscience field to modulate neuronal activity. In this review, we will focus on DREADD studies carried out with genetically engineered mice aimed at elucidating signaling pathways important for maintaining proper glucose and energy homeostasis. The availability of muscarinic receptor-based DREADDs endowed with selectivity for one of the four major classes of heterotrimeric G proteins (G s , G i , G q , and G 12 ) has been instrumental in dissecting the physiological and pathophysiological roles of distinct G protein signaling pathways in metabolically important cell types. The novel insights gained from this work should inform the development of novel classes of drugs useful for the treatment of several metabolic disorders including type 2 diabetes and obesity., (© Published 2021. This article is a U.S. Government work and is in the public domain in the USA.)

Behavioral analyses using mice chemogenetically manipulated by designer receptors exclusively activated by designer drugs (DREADDs) are powerful tools to elucidate neural functions. Here, we describe the detailed protocols for stereotaxic surgery, adeno-associated virus (AAV)-mediated introduction to Gq-DREADDs in mice, and for behavioral testing and analyses related to anxiety, risk assessment, and burying behaviors. A series of these tests are useful in evaluating animal anxiety and their defensive response patterns to potential threats. For complete details on the use and execution of this protocol, please refer to Horii-Hayashi et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The use of designer benzodiazepines appears to be increasing in many countries, but data concerning blood concentrations are scarce, making interpretation of concentrations difficult. The aim of this study was to report blood concentrations of clonazolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam and phenazepam and to investigate the relationship between blood concentrations and impairment. The concentration data are from blood samples collected from living cases (apprehended drivers and other drug offences) and medico-legal autopsies. The blood samples were analysed for the seven designer benzodiazepines mentioned above by ultra high performance liquid chromatography-tandem mass spectrometry. Positive cases from between 1 June 2016 and 30 September 2019 were included. Blood concentrations and the conclusion from a clinical test of impairment (when available) are reported. The presented seven benzodiazepines were detected in a total of 575 cases, where 554 of these cases concerned apprehended drivers or other criminal offenders. The number of findings and the median (range) concentrations were as follows: clonazolam, n = 22, 0.0041 mg/L (0.0017-0.053 mg/L); diclazepam, n = 334, 0.0096 mg/L (0.0016-0.25 mg/L); etizolam, n = 40, 0.054 mg/L (0.015-0.30 mg/L); flualprazolam, n = 10, 0.0080 mg/L (0.0033-0.056 mg/L); flubromazepam, n = 5, 0.037 mg/L (0.0070-0.70 mg/L); flubromazolam, n = 20, 0.0056 mg/L (0.0004-0.036 mg/L); and phenazepam, n = 138, 0.022 mg/L (0.0018-0.85 mg/L). A designer benzodiazepine was the only drug detected with relevance for impairment in 25 of the 554 living cases. The physician concluded with impairment in 19 of the 25 cases. Most of the concentrations in these cases were relatively similar to or higher than the median reported concentrations. The most frequent other drugs detected were amphetamine, tetrahydrocannabinol, clonazepam and methamphetamine. The presented blood concentrations can be helpful with the interpretation of cases involving one or more of these seven benzodiazepines. The results indicate that concentrations commonly observed in forensic cases are associated with impairment., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Association of Forensic Toxicologists, Inc.)

Among the increasing number of new psychoactive substances, 3',4'-methylenedioxy-α-pyrrolidinohexanophenone (MDPHP) belongs to the group of synthetic cathinones, which are the derivatives of the naturally occurring compound cathinone, the main psychoactive ingredient in the khat plant. Currently, only limited data are available for MDPHP, and no information is available on its human metabolism. We describe the toxicological investigation of nine cases associated with the use of MDPHP during the period February-June 2019. Serum MDPHP concentrations showed a high variability ranging from 3.3 to 140 ng/mL (mean 30.3 ng/mL and median 16 ng/mL). Intoxication symptoms of the described cases could not be explained by the abuse of MDPHP alone because in all cases the co-consumption of other psychotropic drugs with frequent occurrence of opiates and benzodiazepines could be verified. Therefore, the patients showed different clinical symptoms, including aggressive behaviour, delayed physical response, loss of consciousness and coma. Liquid chromatography-high-resolution mass spectrometry was successfully used to investigate the human in vivo metabolism of MDPHP using authentic human urine samples. The metabolism data for MDPHP were further substantiated by the analysis of human urine using gas chromatography-mass spectrometry (GC-MS, a widely used systematic toxicological analysis method appropriate for the toxicological detection of MDPHP intake), which revealed the presence of seven phase I metabolites and three phase II metabolites as glucuronides. GC-MS spectral data for MDPHP and metabolites are provided. The identified metabolite pattern corroborates the principal metabolic pathways of α-pyrrolidinophenones in humans., (© 2020 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Hemapolin (2α,3α-epithio-17α-methyl-5α-androstan-17β-ol) is a designer steroid that is an ingredient in several "dietary" and "nutritional" supplements available online. As an unusual chemical modification to the steroid A-ring could allow this compound to pass through antidoping screens undetected, the metabolism of hemapolin was investigated by an in vivo equine drug administration study coupled with GC-MS analysis. Following administration of synthetically prepared hemapolin to a thoroughbred horse, madol (17α-methyl-5α-androst-2-en-17β-ol), reduced and dihydroxylated madol (17α-methyl-5α-androstane-2β,3α,17β-triol), and the isomeric enone metabolites 17β-hydroxy-17α-methyl-5α-androst-3-en-2-one and 17β-hydroxy-17α-methyl-5α-androst-2-en-4-one, were detected and confirmed in equine urine extracts by comparison with a library of synthetically derived reference materials. A number of additional madol derivatives derived from hydroxylation, dihydroxylation, and trihydroxylation were also detected but not fully identified by this approach. A yeast cell-based androgen receptor bioassay of available reference materials showed that hemapolin and many of the metabolites identified by this study were potent activators of the equine androgen receptor. This study reveals the metabolites resulting from the equine administration of the androgen hemapolin that can be incorporated into routine GC-MS antidoping screening and confirmation protocols to detect the illicit use of this agent in equine sports., (© 2020 John Wiley & Sons, Ltd.)

Synthetic cannabinoid receptor agonists (SCRAs) represent the most rapidly proliferating class of "designer drugs" or "new psychoactive substances". SCRAs offer unregulated alternatives to cannabis that evade routine drug tests, but their use is increasingly associated with severe toxicity and death worldwide. Little is currently known about SCRA molecular pharmacology, or the mechanisms underpinning their toxicity, although the effects are believed to be primarily mediated by the type 1 cannabinoid receptor (CB 1 ). In this study, we aimed to characterise the signalling profiles of a structurally diverse panel of novel SCRAs at CB 1 . We compare SCRAs to traditional reference cannabinoids CP55,940, WIN55,212-2, and THC. The activity of the SCRAs was assessed in key receptor signalling and regulatory pathways, including cAMP production, translocation of β-arrestin 1 and 2, and receptor internalisation. The activity profiles of the ligands were also evaluated using operational analysis to identify ligand bias. Results revealed that SCRAs activities were relatively balanced in the pathways evaluated (compared to WIN55,212-2), although 5F-CUMYL-P7AICA and XLR-11 possessed partial efficacy in cAMP stimulation and β-arrestin translocation. Notably, the SCRAs showed distinct potency and efficacy profiles compared to THC. In particular, while the majority of SCRAs demonstrated robust β-arrestin translocation, cAMP stimulation, and internalisation, THC failed to elicit high efficacy responses in any of these assays. Further study is required to delineate if these pathways could contribute to SCRA toxicity in humans., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

5-Methoxy-N,N-Diisopropyltryptamine (5-MeO-DIPT) is a designer hallucinogen derived from tryptamine and its use has been banned by many countries. In this study, a qualitative and quantitative method was developed for determining 5-MeO-DIPT in urine by gas chromatography high-resolution mass spectrometry. 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N-isopropyltryptamine (5-MeO-IPT) were identified as 5-MeO-DIPT metabolites in abusers' urine. 5-MeO-DIPT was extracted from urine by liquid-liquid extraction with ethyl acetate under alkaline conditions. The extract was analyzed by GC-Orbitrap-MS in full scan mode with a resolution of 60,000 full width at half maxima (FWHM). The linear range of this method was 2-300 ng/mL with r > 0.99, and the limit of detection was 1 ng/mL. The accuracy and precision were 93-108.7% and 3.1-10.3%, respectively. This method is simple and sensitive. It has been successfully used to detect 5-MeO-DIPT in drug abusers' urine, which showed that the concentrations of 5-MeO-DIPT were between 1 and 2.8 ng/mL. 5-OH-DIPT and 5-MeO-IPT, two urinary major metabolites of 5-MeO-DIPT, were identified in urine samples from 5-MeO-DIPT users. Furthermore, the stability of 5-MeO-DIPT in human urine was investigated. It was discovered that the concentration of 5-MeO-DIPT in urine decreased by 22.8, 33.2 and 38.2% after samples were stored for 24 h at 25°C, 5 days at 4°C and 7 days at 4°C, respectively. And 5-MeO-DIPT in urine were stable after they were stored for 30 days at -20°C. Therefore, it is recommended that urine should be stored under freezing conditions before performing 5-MeO-DIPT analysis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Rationale: The presence of α-pyrrolidinovalerophenone (α-PVP) and its metabolites in urine is evidence of the administration of α-PVP. A toxicological challenge is that the metabolites of α-PVP exhibit amphoteric properties, which make them unsuitable for detection using gas chromatography-mass spectrometry (GC/MS). In the study reported, proper derivatization and sample extraction were essential for improving the sensitivity for GC/MS analysis., Methods: An automated solid-phase extraction (SPE) method has been developed and optimized. The derivatization efficiency was tested using longer reaction time and the addition of polar pyridine into a mixture of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane. Method validation, including linearity, limit of detection, precision, accuracy, and recovery, was evaluated using automatic SPE and GC/MS., Results: The results suggested that adding pyridine to BSTFA (1:1, v/v) significantly improved derivatization efficiency and precision. After optimization, the linear range was from 25 to 1000 ng mL -1 with R 2  > 0.9950. The limit of detection was 5 ng mL -1 for α-PVP and 25 ng mL -1 for OH-α-PVP. The recovery for SPE was over 88%. The inter-day and intra-day precisions were less than 15%. A forensic sample has been found containing α-PVP (67.3 ng mL -1 ) and OH-α-PVP (560.2 ng mL -1 )., Conclusions: This study is the first to validate an auto-SPE-GC/MS method for the quantification and qualification of α-PVP and OH-α-PVP in urine. We have successfully improved the derivatization efficiency and developed a sensitive and semi-automatic approach. This approach is desirable for the detection of synthetic cathinone at trace levels in biological samples., (© 2019 John Wiley & Sons, Ltd.)

The distribution of so-called new psychoactive substances (NPS) as substitute for common drug of abuse was steadily increasing in the last years, but knowledge about their toxicodynamic and toxicokinetic properties is lacking. However, a comprehensive knowledge of their toxicokinetics, particularly their metabolism, is crucial for developing reliable screening procedures and to verify their intake, e.g., in case of intoxications. The aim of this study was therefore to tentatively identify the metabolites of the methylphenidate-derived NPS isopropylphenidate (isopropyl 2-phenyl-2-(2-piperidyl) acetate, IPH), 4-fluoromethylphenidate (methyl 2-(4-fluorophenyl)-2-(piperidin-2-yl) acetate, 4-FMPH) and 3,4-dichloromethylphenidate (methyl 2-(3,4-dichlorophenyl)-2-(piperidin-2-yl) acetate, 3,4-CTMP) using different in vivo and in vitro techniques and ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS/MS). Urine samples of male rats were analyzed, and the transfer to human metabolism was done by using pooled human S9 fraction (pS9), which contains the microsomal fraction of liver homogenisate as well as its cytosol. UHPLC-HRMS/MS analysis of rat urine revealed 17 metabolites for IPH (14 phase I and 3 phase II metabolites), 13 metabolites were found for 4-FMPH (12 phase I metabolites and 1 phase II metabolite) and 7 phase I metabolites and no phase II metabolites were found for 3,4-CTMP. pS9 incubations additionally indicated that all investigated substances were primarily hydrolyzed, resulting in the corresponding carboxy metabolites. Finally, these carboxy metabolites should be used as additional analytical targets besides the parent compounds for comprehensive mass spectrometry-based screening procedures., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The production and consumption of new psychoactive substances (NPSs) has been raising a major concern worldwide. Due to easy access and available information, many NPSs continue to be synthesized with an alarming increase of those available to purchase, despite all the control efforts created. A new analytical method was developed and validated to determine a group of phenethylamines and synthetic cathinones: cathinone, flephedrone, buphedrone, 4-MTA, α-PVP, methylone, 2C-P, ethylone, pentylone, MDPV and bromo-dragonFLY in whole blood. A mixed-mode solid phase extraction was applied to 250 μL of sample, and the extracts were derivatized with fast microwave technique before being analyzed by gas chromatography-mass spectrometry (GC-MS). The validation procedure followed the Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines with parameters that included selectivity, linearity, limits of detection and quantification, intra- and inter-day precision and accuracy, recoveries and stability. The method presented linearity between 5 and 500 ng/mL for cathinone, buphedrone, 4-MTA, methylone, 2C-P and bromo-dragonFLY, 10-500 ng/mL for flephedrone, ethylone, pentylone and MDPV, and 40-500 ng/mL for α-PVP, with determination coefficients above 0.99 for all analytes. Recoveries ranged between 70.3% and 116.6%, and regarding intra- and inter-day precision, the relative mean errors were typically lower than 8.6%. The method was successfully applied to over 100 authentic samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

According to the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), there were 179 different synthetic cannabinoids reported as of 2017. In the USA, 5F-MDMB-PINACA, or 5F-ADB, accounted for 28% of cannabinoid seizures 2016-2018. The synthetic cannabinoid, 5F-MDMB-PICA, is structurally similar to 5F-MDMB-PINACA with an indole group replacing the indazole. Limited data exist from in vivo or in vitro metabolic studies of these synthetic cannabinoids, so potential metabolites to identify use may be missed. The goals of this study were to (a) investigate 5F-MDMB-PICA and 5F-MDMB-PINACA in vitro metabolism utilizing human hepatocytes; (b) to verify in vitro metabolites by analyzing authentic case specimens; and (c) to identify the potency and efficacy of 5F-MDMB-PICA and 5F-MDMB-PINACA by examining activity at the CB 1 receptor. Biotransformations found in this study included phase I transformations and phase II transformations. A total of 22 5F-MDMB-PICA metabolites (A1 to A22) were identified. From hepatocyte incubations and urine samples, 21 metabolites (B1 to B21) were identified with 3 compounds unique to urine specimens for 5F-MDMB-PINACA. Phase II glucuronides were identified in 5F-MDMB-PICA (n = 3) and 5F-MDMB-PINACA (n = 5). For both compounds, ester hydrolysis and ester hydrolysis in combination with oxidative defluorination were the most prevalent metabolites produced in vitro. Additionally, the conversion of ester hydrolysis with oxidative defluorination to pentanoic acid for the first time was identified for 5F-MDMB-PICA. Therefore, these metabolites would be potentially good biomarkers for screening urine of suspected intoxication of 5F-MDMB-PICA or 5F-MDMB-PINACA. Both 5F-MDMB-PICA and 5F-MDMB-PINACA were acting as full agonists at the CB 1 receptor with higher efficacy and similar potency as JWH-018., (© 2019 John Wiley & Sons, Ltd.)

The aim of this study was to characterize the in vitro and in vivo metabolism of 2-aminoindane (2,3-dihydro-1H-inden-2-amine, 2-AI), and N-methyl-2-aminoindane (N-methyl-2,3-dihydro-1H-inden-2-amine, NM-2-AI) after incubations using pooled human liver microsomes (pHLMs), pooled human liver S9 fraction (pS9), and rat urine after oral administration. After analysis using liquid chromatography coupled to high-resolution mass spectrometry, pHLM incubations revealed that 2-AI was left unmetabolized, while NM-2-AI formed a hydroxylamine and diastereomers of a metabolite formed after hydroxylation in beta position. Incubations using pS9 led to the formation of an acetyl conjugation in the case of 2-AI and merely a hydroxylamine for NM-2-AI. Investigations on rat urine showed that 2-AI was hydroxylated also forming diasteromers as described for NM-2-AI or acetylated similar to incubations using pS9. All hydroxylated metabolites of NM-2-AI except the hydroxylamine were found in rat urine as additional sulfates. Assuming similar patterns in humans, urine screening procedures might be focused on the parent compounds but should also include their metabolites. An activity screening using human recombinant N-acetyl transferase (NAT) isoforms 1 and 2 revealed that 2-AI was acetylated exclusively by NAT2, which is polymorphically expressed., (© 2019 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Due to the risk of new synthetic opioids (NSOs) for human health, the knowledge of their toxicokinetic characteristics is important for clinical and forensic toxicology. U-48800 is an NSO structurally non-related to classical opioids such as morphine or fentanyl and offered for abuse. As toxicokinetic data of U-48800 is not currently available, the aims of this study were to identify the in vitro metabolites of U-48800 in pooled human liver S9 fraction (pS9), to map the isozymes involved in the initial metabolic steps, and to determine further toxicokinetic data such as metabolic stability, including the in vitro half-life (t 1/2 ), and the intrinsic (CL int ) and hepatic clearance (CL h ). Furthermore, drug detectability studies in rat urine should be done using hyphenated mass spectrometry. In total, 13 phase I metabolites and one phase II metabolite were identified. N-Dealkylation, hydroxylation, and their combinations were the predominant metabolic reactions. The isozymes CYP2C19 and CYP3A4 were mainly involved in these initial steps. CYP2C19 poor metabolizers may suffer from an increased U-48800 toxicity. The in vitro t 1/2 and CL int could be rated as moderate, compared to structural related compounds. After administration of an assumed consumer dose to rats, the unchanged parent compound was found only in very low abundance but three metabolites were detected additionally. Due to species differences, metabolites found in rats might be different from those in humans. However, phase I metabolites found in rat urine, the parent compound, and additionally the N-demethyl metabolite should be used as main targets in toxicological urine screening approaches., (© 2019 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Psychoactive substances of the 2C-series are phenethylamine-based designer drugs that can induce psychostimulant and hallucinogenic effects. The so-called 2C-FLY series contains rigidified methoxy groups integrated in a 2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b']difuran core. The aim of the presented work was to investigate the in vivo and in vitro metabolic fate including isoenzyme activities and toxicological detectability of the three new psychoactive substances (NPS) 2C-E-FLY, 2C-EF-FLY, and 2C-T-7-FLY to allow clinical and forensic toxicologists the identification of these novel compounds. Rat urine, after oral administration, and pooled human liver S9 fraction (pS9) incubations were analyzed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS). By performing activity screenings, the human isoenzymes involved were identified and toxicological detectability in rat urine investigated using standard urine screening approaches (SUSAs) based on gas chromatography (GC)-MS, LC-MS n , and LC-HRMS/MS. In total, 32 metabolites were tentatively identified. Main metabolic steps consisted of hydroxylation and N-acetylation. Phase I metabolic reactions were catalyzed by CYP2D6, 3A4, and FMO3 and N-acetylation by NAT1 and NAT2. Methoxyamine was used as a trapping agent for detection of the deaminated metabolite formed by MAO-A and B. Interindividual differences in the metabolism of the 2C-FLY drugs could be caused by polymorphisms of enzymes involved or drug-drug interactions. All three SUSAs were shown to be suitable to detect an intake of these NPS but common metabolites of 2C-E-FLY and 2C-EF-FLY have to be considered during interpretation of analytical findings., (© 2019 The Authors Drug Testing and Analysis Published by John Wiley & Sons Ltd.)

Chemogenetic technologies such as Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) are widely used to remotely control neuronal and non-neuronal signaling. DREADDs exist for most of the canonical G protein-coupled receptor signaling pathways, and provide a synthetic biology platform useful for elucidating the role of neuronal signaling for brain function. Here, a focused review is provided that shows how recent insights obtained from GPCR structural studies inform our understanding of these chemogenetic tools from a structural perspective., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Indole- and indazole-based synthetic cannabinoid receptor agonists (SCRAs), featuring valine or tert-leucine substituents, are commonly abused new psychoactive substances (NPS). A major metabolic pathway for these SCRAs is hydrolysis of the terminal amide or methylester functionalities. Although these hydrolysis products were already detected as main ingredients in some "legal highs," these metabolites are often poorly characterized. Here, we report a systematic investigation of the activity of 7 common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L-valine or L-tert-leucine in direct comparison to their parent compounds. An activity-based cannabinoid receptor 1 (CB 1 ) bio-assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293T cell system. The recruitment of β-arrestin2 to the activated CB 1 (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96-well plate-reader. The major hydrolysis metabolites of 5F-AB-PINACA, ADB-CHMICA, ADB-CHMINACA, ADB-FUBICA, and their methyl- and ethylester derivatives showed no detectable CB 1 activation at concentrations up to 1 μM. On the other hand, metabolites of 5F-ADB-PINACA, AB-CHMINACA, and ADB-FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC 50 values >100 nM). Activity-based characterization of SCRAs and their metabolites at CB 1 may not only allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications, but may also allow application of the concept of "activity equivalents" present in biological fluids or, alternatively, in confiscated materials., (© 2019 John Wiley & Sons, Ltd.)

Despite the increasing relevance of synthetic cannabinoids as one of the most important classes within "New Psychoactive Substances", there is still a lack of knowledge concerning their metabolism in humans. Due to the extensive metabolism of synthetic cannabinoids, metabolites are necessarily the best target analytes in urine, posing additional challenges to forensic analysis. The aims of this study were to identify appropriate urinary targets indicating intake of THJ-018 or THJ-2201 as well as to elucidate the most important cytochrome P450 isoenzymes within the metabolism of THJ-018 and THJ-2201 in vitro. For this purpose, the in vitro metabolism of THJ-018 and THJ-2201 was initially established using pooled human liver microsomes. The results obtained were compared to previously published in vitro results as well as to the results of the metabolic profiles from selected recombinant cytochrome P450 isoenzymes and from 23 urine samples from forensic cases. LC-HRMS was used to conduct product ion scans and to examine the metabolite spectra. For THJ-018, 17 different metabolite groups containing 33 different metabolites and isomers were detected after microsomal incubation, with the major metabolic pathways being monohydroxylation at the pentyl chain and of the naphthyl moiety as well as dihydroxylation of both residues. For THJ-2201, 19 different metabolite groups and 46 different metabolites and isomers were observed. The major metabolic pathways were monohydroxylation at the naphthyl moiety and oxidative defluorination. Significant contribution to the in vitro metabolism of THJ-018 and THJ-2201 originated from CYP2B6, CYP2C19, CYP3A4, and CYP3A5. As several cytochrome P450 isoenzymes are involved in the metabolism of these synthetic cannabinoids, a co-consumption with other drugs is unlikely to have an impact on their metabolism.

5F-ADB (methyl 2-{[1-(5-fluoropentyl)-1H-indazole-3-carbonyl] amino}-3,3-dimethylbutanoate) is a frequently abused new synthetic cannabinoid that has been sold since at least the end of 2014 on the drug market and has been classified as an illicit drug in most European countries, as well as Turkey, Japan, and the United States. In this study, the in vitro metabolism of 5F-ADB was investigated by using pooled human liver microsomes (HLMs) assay and liquid chromatography-high-resolution mass spectrometry (LC-HRMS). 5F-ADB (5 μmol/L) was incubated with HLMs for up to 3 hours, and the metabolites were identified using LC-HRMS and software-assisted data mining. The in vivo metabolism was investigated by the analysis of 30 authentic urine samples and was compared to the data received from the in vitro metabolism study. Less than 3.3% of the 5F-ADB parent compound remained after 1 hour of incubation, and no parent drug was detected after 3 hours. We identified 20 metabolites formed via ester hydrolysis, N-dealkylation, oxidative defluorination, hydroxylation, dehydrogenation, further oxidation to N-pentanoic acid and glucuronidation or a combination of these reactions in vitro. In 12 urine samples (n = 30), 5F-ADB was detected as the parent drug. Three of the identified main metabolites 5F-ADB carboxylic acid (M20), monohydroxypentyl-5F-ADB (M17), and carboxypentyl ADB carboxylic acid (M8) were suggested as suitable urinary markers. The screening of 8235 authentic urine samples for identified 5F-ADB metabolites in vitro resulted in 3135 cases of confirmed 5F-ADB consumption (38%)., (© 2019 John Wiley & Sons, Ltd.)

Three quantitative structure-activity relationship (QSAR) models for predicting the affinity of mu-opioid receptor (OR) ligands have been developed. The resulted models, exploiting the accessibility of the QSAR modeling, generate a useful tool for the investigation and identification of unclassified fentanyl-like structures. The models have been built using a set of 115 molecules using Forge as a software, and the quality was confirmed by statistical analysis, resulting in being effective for their predictive and descriptive capabilities. The three different approaches were then combined to produce a consensus model and were exploited to explore the chemical landscape of 3000 fentanyl-like structures, generated by a theoretical scaffold-hopping approach. The findings of this study should facilitate the identification and classification of new OR ligands with fentanyl-like structures.

Tryptamines can occur naturally in plants, mushrooms, microbes, and amphibians. Synthetic tryptamines are sold as new psychoactive substances (NPS) because of their hallucinogenic effects. When it comes to NPS, metabolism studies are of crucial importance, due to the lack of pharmacological and toxicological data. Different approaches can be taken to study in vitro and in vivo metabolism of xenobiotica. The zygomycete fungus Cunninghamella elegans (C. elegans) can be used as a microbial model for the study of drug metabolism. The current study investigated the biotransformation of four naturally occurring and synthetic tryptamines [N,N-Dimethyltryptamine (DMT), 4-hydroxy-N-methyl-N-ethyltryptamine (4-HO-MET), N,N-di allyl-5-methoxy tryptamine (5-MeO-DALT) and 5-methoxy-N-methyl-N-isoporpoyltryptamine (5-MeO-MiPT)] in C. elegans after incubation for 72 hours. Metabolites were identified using liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) with a quadrupole time-of-flight (QqTOF) instrument. Results were compared to already published data on these substances. C. elegans was capable of producing all major biotransformation steps: hydroxylation, N-oxide formation, carboxylation, deamination, and demethylation. On average 63% of phase I metabolites found in the literature could also be detected in C. elegans. Additionally, metabolites specific for C. elegans were identified. Therefore, C. elegans is a suitable complementary model to other in vitro or in vivo methods to study the metabolism of naturally occurring or synthetic tryptamines., (© 2018 John Wiley & Sons, Ltd.)

2-(2,5-Dimethoxy-4-nitrophenyl)-N-(2-methoxybenzyl)ethanamine (25N-NBOMe, 2C-N-NBOMe, NBOMe-2C-N) is a novel synthetic psychoactive substance of the phenethylamine chemical class. A few metabolism studies have been conducted for 25I-NBOMe, 25B-NBOMe, and 25C-NBOMe, and others, whereas 25N-NBOMe metabolism has not been researched. In this study, the in vitro metabolism of 25N-NBOMe was investigated with human liver microsomes, and the reaction mixture was analyzed using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). Formation of 14 metabolites (M1-M14) was yielded with incubation of 25N-NBOMe in human liver microsomes in the presence of NADPH. The metabolites were structurally characterized on the basis of accurate mass analysis and MS/MS fragmentation patterns. The biotransformations included hydroxylation, O-demethylation, N-dealkylation, nitro reduction, dehydrogenation, carbonylation, and combinations thereof. Hydroxyl metabolite was the most abundant compound after the phase I process. These results provide helpful information establishing biomarkers in case of 25N-NBOMe ingestion.

The use of synthetic cannabinoids causes similar effects as Δ 9 -tetrahydrocannabinol and long-term (ab)use can lead to health hazards and fatal intoxications. As most investigated synthetic cannabinoids undergo extensive biotransformation, almost no parent compound can be detected in urine, which hampers forensic investigations. Limited information about the biotransformation products of new synthetic cannabinoids makes the detection of these drugs in various biological matrices challenging. This study aimed to identify the main in vitro biotransformation pathways of 5Cl-THJ-018 and to compare these findings with an authentic urine sample of a 5Cl-THJ-018 user. The synthetic cannabinoid was incubated with pooled human liver microsomes and cytosol to simulate phase I and phase II biotransformations. Resulting extracts were analyzed with liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Three different data analysis workflows were applied to identify biotransformation products. A suspect screening workflow used an in-house database built from literature data and in silico biotransformation predictions. Two non-target screening workflows used a commercially available software and an open-source software for mass spectrometry data processing. A total of 23 in vitro biotransformation products were identified, with hydroxylation, oxidative dechlorination, and dihydrodiol formation pathways as the main phase I reactions. Additionally, five glucuronidated and three sulfated phase II conjugates were identified. The predominant in vivo pathway was through oxidative dechlorination and in total six metabolites of 5Cl-THJ-018 were identified. Biotransformation products both in vitro and in vivo were successfully identified using complementary suspect and non-target screening workflows., (© 2018 John Wiley & Sons, Ltd.)

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 'designer benzodiazepines' monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864)] offered as 'research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC MS/MS), liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4'-Chlorodiazepam biotransformation consisted of N-dealkylation and hydroxylation. It has to be noted that 4'-chlorodiazepam and its metabolites show almost identical LC-MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings., (© 2018 John Wiley & Sons, Ltd.)

Designer benzodiazepines have emerged as recreational drugs. They are available via the Internet without control and are found in the form of falsified (fake) medicines. For some of them, limited information concerning their effects, their toxicity, and their detection in bio fluids is available in the literature. For others, nothing has been published, as in the case of flunitrazolam (FNTZ). To gain preliminary data on its elimination parameters in urine and to investigate its metabolism, one of the authors ingested one pink tablet bought on the Internet, after confirming the absence of other compounds and agreement with the labeled dosage (0.25 mg) by nuclear magnetic resonance (NMR). A software algorithm (MetaboLynx, Waters, Milford, MA, USA) was used to predict FNTZ biotransformation and four potential metabolites were proposed: 4-hydroxy-FNTZ, desnitro-FNTZ, 7-amino-FNTZ, and 7-acetamido-FNTZ. Urine samples were collected over 72 hours following oral administration of one tablet. After liquid/liquid extraction at pH 9.5, FNTZ concentrations were determined using ultra performance liquid chromatography-triple quadrupole-mass spectrometry (UPLC-QqQ-MS/MS). FNTZ remained detectable in hydrolyzed urine for 21 hours after ingestion, with concentrations ranging between 1 and 18 ng/mL. About 3% of the initial dose was excreted in urine as total unchanged FNTZ during this period. In vitro experiments (HLM incubations) were performed using ultra performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-QTOF-MS) in order to investigate the potential CYP- and UGT-dependent metabolites where only 7-amino-FNTZ was detected as the only metabolite. However, in the urine specimens, desnitro-FNTZ, 7-acetamido-FNTZ and 7-amino-FNTZ were the main detected compounds. The identification of FNTZ metabolites dramatically improves the detection windows of the drug up to 37 hours., (© 2018 John Wiley & Sons, Ltd.)

Multiple new psychoactive substances (NPS) are released into the recreational drug market each year. One NPS drug class that has become more common in recent years is that of the benzodiazepines (designer benzodiazepines, DBZ). Several metabolism studies have been performed to improve their bioanalytical detection via the best target. These studies have shown the presence of parent glucuronides and, as polymorphisms have been noted for the catalyzing enzymes (UDP-glucuronyltransferases) responsible for glucuronide conjugation reactions, it is important to keep this in mind when interpreting DBZ cases in clinical and/or forensic toxicology. Therefore, the aim of this study was to determine the UDP-glucuronyltransferases (UGTs) responsible for parent compound conjugation of nine DBZ to facilitate interpretation of related cases. Clonazolam, deschloroetizolam, etizolam, flubromazolam, flunitrazolam, metizolam, nifoxipam, nitrazolam, and pyrazolam were incubated with pooled human liver microsomes (pHLM) or 13 different human UGTs. The samples were analyzed using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). Glucuronide conjugates of flunitrazolam and nifoxipam were only detected in pHLM, suggesting that these reactions are performed by dimer complexes of several UGTs or complexes between UGTs and other metabolizing enzymes contained in pHLM. Nitrazolam or pyrazolam glucuronides were not detected. Glucuronidation of clonazolam, deschloroetizolam, etizolam, flubromazolam, and metizolam was catalyzed exclusively by UGT1A4. The conjugation of the majority of the DBZ was performed by the UGT isoform 1A4 for which polymorphisms have been described. This underlines the importance of taking glucuronidation polymorphism into consideration when interpreting intoxication cases., (© 2018 John Wiley & Sons, Ltd.)

Numerous 2,5-dimethoxy-N-benzylphenethylamines (NBOMe), carrying a variety of lipophilic substituents at the 4-position, are potent agonists at 5-hydroxytryptamine (5HT 2A ) receptors and show hallucinogenic effects. The present study investigated the metabolism of 25D-NBOMe, 25E-NBOMe, and 25N-NBOMe using the microsomal model of pooled human liver microsomes (pHLM) and the microbial model of the fungi Cunninghamella elegans (C. elegans). Identification of metabolites was performed using liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) with a quadrupole time-of-flight (QqToF) instrument. In total, 36 25D-NBOMe phase I metabolites, 26 25E-NBOMe phase I metabolites and 24 25N-NBOMe phase I metabolites were detected and identified in pHLM. Furthermore, 14 metabolites of 25D-NBOMe, 11 25E-NBOMe metabolites, and nine 25N-NBOMe metabolites could be found in C. elegans. The main biotransformation steps observed were oxidative deamination, oxidative N-dealkylation also in combination with hydroxylation, oxidative O-demethylation possibly combined with hydroxylation, oxidation of secondary alcohols, mono- and dihydroxylation, oxidation of primary alcohols, and carboxylation of primary alcohols. Additionally, oxidative di-O-demethylation for 25E-NBOMe and reduction of the aromatic nitro group and N-acetylation of the primary aromatic amine for 25N-NBOMe took place. The resulting 25N-NBOMe metabolites were unique for NBOMe compounds. For all NBOMes investigated, the corresponding 2,5-dimethoxyphenethylamine (2C-X) metabolite was detected. This study reports for the first time 25X-NBOMe N-oxide metabolites and hydroxylamine metabolites, which were identified for 25D-NBOMe and 25N-NBOMe and all three investigated NBOMes, respectively. C. elegans was capable of generating all main biotransformation steps observed in pHLM and might therefore be an interesting model for further studies of new psychoactive substances (NPS) metabolism., (© 2018 John Wiley & Sons, Ltd.)

Designer benzodiazepines have recently emerged as a class of new psychoactive substances. These substances are used in recreational settings and as alternatives to prescription benzodiazepines as self-medication for patients suffering from anxiety or other mental disorders. Due to the limited information available on the metabolic fate of these new substances, it is challenging to reliably detect their usage in bioanalytical (e.g. clinical and forensic) settings. The objective of this study was to investigate the in vitro Phase I and Phase II metabolism of the new designer benzodiazepine cloniprazepam and identify potential biomarkers for its detection in human biological fluids. Cloniprazepam was incubated with human liver microsomes and cytosolic fractions to generate both Phase I and II metabolites. The extracts were analysed using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. Identification of the metabolites was performed using two complementary workflows, including a suspect screening based on in silico predictions and a non-targeted screening. A total of nine metabolites were identified, eight Phase I metabolites and one Phase II metabolite, of which five were specific for cloniprazepam. Clonazepam was the major metabolite of cloniprazepam. Hydroxy-cloniprazepam, dihydroxy-cloniprazepam, 3-keto-cloniprazepam, 7-amino-cloniprazepam, hydroxy-clonazepam, 7-amino-clonazepam and 3-hydroxy-7-amino-clonazepam were formed through oxidation, hydroxylation, and/or reduction of the nitro-group. Glucuronidated hydroxy-cloniprazepam was the only Phase II metabolite detected. Five metabolites were specific for cloniprazepam. This study provided a set of human in vitro biotransformation products which can assist specific detection of cloniprazepam consumption in future studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Indole-, indazole-, or azaindole-based synthetic cannabinoids (SCs), bearing a cumyl substituent are a widespread, recreationally used subgroup of new psychoactive substances (NPS). The latest cumyl-derivative, CUMYL-PEGACLONE, emerged in December 2016 on the German drug market. The substance features a novel γ-carboline core structure, which is most likely synthesized to bypass generic legislative approaches to control SCs by prohibiting distinct core structures. Using liquid chromatography-tandem mass spectrometry and liquid chromatography-high resolution mass spectrometry techniques, the main in vivo phase I metabolites of this new substance were detected. A pooled human liver microsome assay was applied to generate in vitro reference spectra of CUMYL-PEGACLONE phase I metabolites. Additionally, 30 urine samples were investigated leading to 22 in vivo metabolites. A metabolite mono-hydroxylated at the γ-carbolinone core system and a metabolite with an additional carbonyl group at the pentyl side chain were evaluated as highly specific and sensitive markers to proof CUMYL-PEGACLONE uptake. Moreover, 3 immunochemical assays commonly used for SC screening in urine were tested for their capability of detecting the new drug but failed due to insufficient cross-reactivity., (Copyright © 2018 John Wiley & Sons, Ltd.)

Clozapine-N-oxide (CNO) has long been the ligand of choice for selectively activating Designer Receptors Exclusively Activated by Designer Drugs (DREADDs). However, recent studies have challenged the long-held assertion that CNO is otherwise pharmacologically inert. The present study aimed to 1) determine whether CNO is reverse-metabolized to its parent compound clozapine in mice (as has recently been reported in rats), and 2) determine whether CNO exerts clozapine-like interoceptive stimulus effects in rats and/or mice. Following administration of 10.0 mg/kg CNO, pharmacokinetic analyses replicated recent reports of back-conversion to clozapine in rats and revealed that this phenomenon also occurs in mice. In rats and mice trained to discriminate 1.25 mg/kg clozapine from vehicle, CNO (1.0-20.0 mg/kg) produced partial substitution for the clozapine stimulus on average, with full substitution being detected in some individual animals of both species at doses frequently used to activate DREADDs. The present demonstration that CNO is converted to clozapine and exerts clozapine-like behavioral effects in both mice and rats further emphasizes the need for appropriate control groups in studies employing DREADDs, and highlights the utility of the drug discrimination procedure as a tool with which to screen the off-target effects of novel DREADD agonists.

The synthetic tryptamine 5-methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5-MeO-MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5-MeO-MiPT using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography-mass spectrometry (GC-MS), LC-HRMS/MS, and nuclear magnetic resonance (NMR). 5-MeO-MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5-MeO-MiPT in urine. Seven different in vitro phase I metabolites of 5-MeO-MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5-MeO-MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5-methoxy-N-isopropyltryptamine (5-MeO-NiPT), 5-hydroxy-N-methyl-N-isopropyltryptamine (5-OH-MiPT), 5-methoxy-N-methyl-N-isopropyltryptamine-N-oxide (5-MeO-MiPT-N-oxide), and hydroxy-5-methoxy-N-methyl-N-isopropyltryptamine (OH-5-MeO-MiPT) as biomarkers for the development of new methods for the detection of 5-MeO-MiPT consumption, as they have been present in both blood and urine samples., (Copyright © 2017 John Wiley & Sons, Ltd.)

Over the past ~8 years, hundreds of unregulated new psychoactive substances (NPS) of various chemical categories have been introduced as recreational drugs through mainly open online trade. This study was performed to further investigate the human metabolic pattern of the NPS, or designer benzodiazepines flubromazolam and pyrazolam, and to propose analytical targets for urine drug testing of these substances. The urine samples originated from patient samples confirmed by liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) analysis to contain flubromazolam or pyrazolam. The LC-HRMS/MS system consisted of a YMC-UltraHT Hydrosphere C18 column (YMC, Dinslaken, Germany) coupled to a Thermo Scientific (Waltham, MA, USA) Q Exactive Orbitrap MS operating in positive electrospray mode. The samples were analyzed both with and without enzymatic hydrolysis using β-glucuronidase. Besides the parent compounds, the main urinary excretion products were parent glucuronides, mono-hydroxy metabolites, and mono-hydroxy glucuronides. In samples prepared without hydrolysis, the most common flubromazolam metabolites were 1 of the mono-hydroxy glucuronides and 1 of the parent glucuronides. For pyrazolam, a parent glucuronide was the most common metabolite. These 3 metabolites were detected in all samples and were considered the primary targets for urine drug testing and confirmation of intake. After enzymatic hydrolysis of the urine samples, a 2-19-fold increase in the concentration of flubromazolam was found, highlighting the value of hydrolysis for this analyte. With hydrolysis, the flubromazolam hydroxy metabolites should be used as target metabolites., (Copyright © 2017 John Wiley & Sons, Ltd.)

The orbitofrontal cortex (OFC) is thought to link stimuli and actions with anticipated outcomes in order to sustain flexible behavior in an ever-changing environment. How it retains these associations to guide future behavior is less well-defined. Here we focused on one subregion of this heterogeneous structure, the ventrolateral OFC (VLO). CaMKII-driven inhibitory Gi-coupled designer receptors exclusively activated by designer drugs (DREADDs) were infused and subsequently activated by their ligand Clozapine-N-oxide (CNO) in conjunction with fear extinction training (a form of aversive conditioning) and response-outcome conditioning (a form of appetitive conditioning). Gi-DREADD-mediated inactivation of the VLO during extinction conditioning interfered with fear extinction memory, resulting in sustained freezing when mice were later tested drug-free. Similarly, Gi-DREADD-mediated inactivation in conjunction with response-outcome conditioning caused a later decay in goal-directed responding-that is, mice were unable to select actions based on the likelihood that they would be rewarded in a sustainable manner. By contrast, inhibitory Gi-DREADDs in the basolateral amygdala (BLA) impaired the acquisition of both conditioned fear extinction and response-outcome conditioning, as expected based on prior studies using other inactivation techniques. Meanwhile, DREADD-mediated inhibition of the dorsolateral striatum enhanced response-outcome conditioning, also in line with prior reports. Together, our findings suggest that learning-related neuroplasticity in the VLO may be necessary for memory retention in both appetitive and aversive domains.

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-MS n , 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.

Phenethylamine derivatives are being increasingly exploited for recreational use as "designer" stimulants designed to mimic psychostimulant properties of amphetamine or other illicit substances like 3,4-methylenedioxymethamphetamine (MDMA [ecstasy]). Clandestine operations meticulously design phenethylamines so the user can bypass legal action when detected, as many of these are yet to be regulated by government authorities. Substituted phenethylamines or 2C amines, N-methoxybenzyl derivatives of the corresponding 2C amines commonly known as NBOMe compounds, and cathinones are among the most commonly abused phenethylamines. Current FDA-approved assays used in screening for illicit drug use lack the sensitivity needed to detect designer stimulants making it challenging for toxicologists to accurately identify these compounds. Gas chromatography mass spectrometry (GC-MS) is a sensitive method for identifying designer stimulants. This unit describes and compares two qualitative GC-MS methods for identifying 2C amines, NBOMe compounds, and cathinones in urine. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)

The emergence of novel psychoactive substances (NPS) such as hallucinogenic NBOMes (N-methoxybenzyl derivatives of 2C phenethylamines) in the past few years into the recreational drug market has introduced various challenges in forensic analytical toxicology in regard to adequate and timely detection of these compounds. This is especially true in samples from individuals who have experienced severe and fatal intoxications. The aim of this research was to identify the major Phase I metabolites of selected NBOMe compounds to generate a predicted human metabolic pathway of these substances. An in vitro incubation method of pooled human liver microsomes (HLMs) with four (4) NBOMes was used to identify major metabolites. These metabolic products were identified and confirmed from accurate mass findings of samples analyzed by Ultra Performance Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry. The most common biotransformations observed among this group of NBOMes include O-demethylations at the three methoxy groups, hydroxylations and reduction at the amine group. Other metabolic products observed include positional isomers from various hydroxylation possibilities on the benzene ring and alkyl chains, and secondary metabolism resulting in multiple combinations of the reactions. Many of the major metabolites were subsequently identified in authentic human samples of blood and urine from drug users., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Flubromazolam is a triazole benzodiazepine with high potency and long-lasting central nervous system depressant effects; however, limited data about its pharmacokinetics are available. Here, we report in vitro studies of the human flubromazolam metabolism analyzed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). In vitro investigations were carried out in pooled human liver microsomes (pHLM) and recombinant cytochrome P450 (CYP)-enzymes. To confirm those metabolites detected in vitro, authentic samples obtained from two forensic cases were also analyzed by LC-HRMS. Additionally, determination of the unbound fraction of flubromazolam in pHLM and in plasma was performed by equilibrium dialysis with subsequent prediction of its hepatic clearance (CL H ) using well-stirred and parallel-tube models. Additional findings obtained by routine screening methods of these forensic cases are also reported. Studies using incubations with nicotinamide adenine dinucleotide phosphate-fortified pHLM with or without uridine 5'-diphosphoglucuronic acid and incubations with CYP-enzymes identified the main metabolic pathway of flubromazolam as hydroxylation on the α- and/or 4-position mediated by CYP3A4 and CYP3A5, with subsequent glucuronidation of the hydroxylated metabolites as well as of the parent drug. Further, α-hydroxy-flubromazolam and its corresponding glucuronide were detected in vivo together with the N-glucuronide of flubromazolam. The predicted CL H of flubromazolam using the well-stirred and parallel-tube models were 0.42 and 0.43 mL/min/kg, respectively. Based on the data presented here, flubromazolam is primarily metabolized by CYP3A4/5 with a high protein-binding and a predicted low clearance. Analysis of authentic samples suggested that analytical targets for flubromazolam should be the compound itself and α-hydroxy-flubromazolam. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Designer benzodiazepines provide an attractive alternative to prescribed benzodiazepines for abuse purposes as they are readily available via the Internet without control. Metizolam was ordered via the Internet and a 2 mg blue tablet was orally administered to a 54-year-old man. Urine samples were collected over 6 days in polypropylene tubes. After liquid/liquid extraction at pH 9.5, metizolam was analyzed by ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) using a standard method devoted to benzodiazepines, and ions transitions, at m/z 328.9 > 275.0 and 328.9 > 300.0. Metizolam was detectable in hydrolyzed urine during the 46-h period, with concentrations always lower than 11 ng/mL. About 0.3% of the initial dose was excreted in urines as total unchanged metizolam during the first 24 h. The most relevant potential CYP- and UGT-dependent metabolites of metizolam were investigated in vitro using human liver microsome incubation and, subsequently, liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF-MS) analysis. Three mono-hydroxylated metabolites were produced including a hydroxylation compound at the 2-ethyl moiety of metizolam (M1) as quantitatively main metabolite, and a N-hydroxymetiazolam (M2). The structure of the third metabolite (M3) could not be elucidated because of a too low experimental production rate. Two authentic urine samples were analyzed using the same analytical method to search for metabolites of metizolam. M1, together with its glucuronide (M1-Glu), and M2 were observed in urine at the 8 h mark, whereas only M1 and M1-Glu were still detected in urine at 30 h post administration. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Increasing numbers of new psychoactive substances (NPS) among them fentanyl derivatives has been reported by the European monitoring centre for drugs and drug addiction (EMCDDA). Butyrfentanyl is a new fentanyl derivative whose potency ratio was found to be seven compared to morphine and 0.13 compared to fentanyl. Several case reports on butyrfentanyl intoxications have been described. Little is known about its pharmacokinetic properties including its metabolism. However, knowledge of metabolism is essential for analytical detection in clinical and forensic toxicology. Therefore, in vitro and in vivo phase I and phase II metabolites of butyrfentanyl were elucidated combining liquid chromatography with a qTOF high resolution mass spectrometer. Human liver microsomes and recombinant cytochrome P450 enzymes (CYP) were used for in vitro assays. Authentic blood and urine samples from a fatal intoxication case were available for in vivo comparison. Butyrfentanyl was shown to undergo extensive metabolism. Six pathways could be postulated with hydroxylation and N-dealkylation being the major ones in vitro. In vivo, hydroxylation of the butanamide side chain followed by subsequent oxidation to the carboxylic acid represented the major metabolic step in the authentic case. Initial screening experiments with the most relevant CYPs indicated that mainly CYP2D6 and 3A4 were involved in the primary metabolic steps. Altered CYP2D6 and CYP3A4 status might cause a different metabolite pattern, making the inclusion of metabolites of different pathways recommendable when applying targeted screening procedures in clinical and forensic toxicology. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The dimethoxyphenyl-N-((2-methoxyphenyl)methyl)ethanamine (NBOMe) compounds are potent serotonin 5-HT2A receptor agonists and have recently been subject to recreational use due to their hallucinogenic effects. Use of NBOMe compounds has been known since 2011, and several non-fatal and fatal intoxication cases have been reported in the scientific literature. The aim of this study was to determine the importance of the different cytochrome P450 enzymes (CYP) involved in the metabolism of 2-(4-iodo-2,5-dimethoxyphenyl)-N-(2methoxybenzyl)ethanamine (25I-NBOMe) and 2-[[2-(4-iodo-2,5dimethoxyphenyl)ethylamino]methyl]phenol (25I-NBOH) and to characterize the metabolites. The following approaches were used to identify the main enzymes involved in primary metabolism: incubation with a panel of CYP and monoamine oxidase (MAO) enzymes and incubation in pooled human liver microsomes (HLM) with and without specific CYP chemical inhibitors. The study was further substantiated by an evaluation of 25I-NBOMe and 25I-NBOH metabolism in single donor HLM. The metabolism pathways of 25I-NBOMe and 25I-NBOH were NADPHdependent with intrinsic clearance values of (CLint) of 70.1 and 118.7 mL/min/kg, respectively. The biotransformations included hydroxylation, O-demethylation, N-dealkylation, dehydrogenation, and combinations thereof. The most abundant metabolites were all identified by retention time and spectrum matching with synthesized reference standards. The major CYP enzymes involved in the metabolism of 25I-NBOMe and 25INBOH were identified as CYP3A4 and CYP2D6, respectively. The compound 25I-NBOH was also liable to direct glucuronidation, which may diminish the impact of CYP2D6 genetic polymorphism. Users of 25I-NBOMe may be subject to drug-drug interactions (DDI) if 25I-NBOMe is taken with a strong CYP3A4 inhibitor. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Synthetic cannabinoids are manufactured clandestinely with little quality control and are distributed as herbal "spice" for smoking or as bulk compound for mixing with a solvent and inhalation via electronic vaporizers. Intoxication with synthetic cannabinoids has been associated with seizure, excited delirium, coma, kidney damage, and other disorders. The chemical alterations produced by heating these structurally novel compounds for consumption are largely unknown. Here, we show that heating synthetic cannabinoids containing tetramethylcyclopropyl-ring substituents produced thermal degradants with pharmacological activity that varied considerably from their parent compounds. Moreover, these degradants were formed under conditions simulating smoking. Some products of combustion retained high affinity at the cannabinoid 1 (CB 1 ) and CB 2 receptors, were more efficacious than (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) in stimulating CB 1 receptor-mediated guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding, and were potent in producing Δ 9 -tetrahydrocannabinol-like effects in laboratory animals, whereas other compounds had low affinity and efficacy and were devoid of cannabimimetic activity. Degradants that retained affinity and efficacy also substituted in drug discrimination tests for the prototypical synthetic cannabinoid 1-pentyl-3-(1-naphthoyl)indole (JWH-018), and are likely to produce psychotropic effects in humans. Hence, it is important to take into consideration the actual chemical exposures that occur during use of synthetic cannabinoid formulations to better comprehend the relationships between dose and effect., (Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.)

ADB-CHMINACA (MAB-CHMINACA) is a new synthetic cannabinoid with high potency and many reported adverse events and fatalities. The drug is currently scheduled in several countries in Europe and the USA. Analytical methods need to be developed to confirm ADB-CHMINACA intake for clinical and forensic programs. For many synthetic cannabinoids, parent compound is not detectable in biological samples after intake, making the detection of metabolites the only way to prove consumption. Therefore, detection of ADB-CHMINACA metabolites in biological specimens is critical. Since there are currently no published data on ADB-CHMINACA metabolism, we aimed to identify its major metabolites. Cryopreserved human hepatocytes were incubated with 10 μmol/L ADB-CHMINACA for 3 h. Incubations were analyzed with liquid chromatography on a biphenyl column, high resolution tandem mass spectrometry (orbitrap), and metabolite identification software. A reference standard of six commercially available potential metabolites was simultaneously analyzed under the same conditions to allow correct assignment of isomers. We detected ten major metabolites. Biotransformations mainly occurred at the cyclohexylmethyl tail of the compound, as also observed with structural analogs' metabolism. Minor reactions also occurred at the tert-butyl chain. Only two analytical standards of potential metabolites matched an actual metabolite detected in hepatocyte incubations. We recommend A9 (ADB-CHMINACA hydroxycyclohexylmethyl), A4 (ADB-CHMINACA 4″-hydroxycyclohexyl), and A6 (ADB-CHMINACA hydroxycyclohexylmethyl) as metabolite targets to document ADB-CHMINACA intake in clinical and forensic cases. Additionally, these results will guide analytical standard manufacturers to better provide suitable references for further studies on ADB-CHMINACA metabolism.

The novel psychoactive compounds derived from amphetamine have been illegally abused as recreational drugs, some of which are known to be hepatotoxic in humans and experimental animals. The cytotoxic effects and mechanisms of 5-(2-aminopropyl)benzofuran (5-APB) and N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB), both of which are benzofuran analogues of amphetamine, and 3,4-methylenedioxy-N-methamphetamine (MDMA) were studied in freshly isolated rat hepatocytes. 5-MAPB caused not only concentration-dependent (0-4.0 mm) and time-dependent (0-3 h) cell death accompanied by the depletion of cellular ATP and reduced glutathione and protein thiol levels, but also accumulation of oxidized glutathione. Of the other analogues examined at a concentration of 4 mm, 5-MAPB/5-APB-induced cytotoxicity with the production of reactive oxygen species and loss of mitochondrial membrane potential was greater than that induced by MDMA. In isolated rat liver mitochondria, the benzofurans resulted in a greater increase in the rate of state 4 oxygen consumption than did MDMA, with a decrease in the rate of state 3 oxygen consumption. Furthermore, the benzofurans caused more of a rapid mitochondrial swelling dependent on the mitochondrial permeability transition than MDMA. 5-MAPB at a weakly toxic level (1 mm) was metabolized slowly: levels of 5-MAPB and 5-APB were approximately 0.9 mm and 50 μm, respectively, after 3 h incubation. Taken collectively, these results indicate that mitochondria are target organelles for the benzofuran analogues and MDMA, which elicit cytotoxicity through mitochondrial failure, and the onset of cytotoxicity may depend on the initial and/or residual concentrations of 5-MAPB rather than on those of its metabolite 5-APB. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)