Characterization of the four designer benzodiazepines clonazolam, deschloroetizolam, flubromazolam, and meclonazepam, and identification of their in vitro metabolites

Huppertz, Laura M.; Bisel, Philippe; Westphal, Folker; Franz, Florian; Auwärter, Volker; Moosmann, Bjoern

doi:10.1007/s11419-015-0277-6

Cited by 59 publications

(53 citation statements)

References 15 publications

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“…[7,8] In one reported in vitro study using pooled human liver microsomes (pHLM), one mono and one dihydroxylated metabolite of flubromazolam were tentatively identified, however without assigning the site of the monohydroxylation. [9] The enzymes-mediated flubromazolam metabolism have not been elucidated so far; for other benzodiazepines, e.g.…”

Section: Flubromazolam Was First Reported In Sweden In 2014 and Inclumentioning

confidence: 99%

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples

Noble

Mardal

Holm

et al. 2017

Drug Testing and Analysis

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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 ) 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 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.

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Section: Flubromazolam Was First Reported In Sweden In 2014 and Inclumentioning

confidence: 99%

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples

Noble

Mardal

Holm

et al. 2017

Drug Testing and Analysis

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“…Meclonazepam, also known as (S)-3-methylclonazepam, , is a benzodiazepine developed and patented by Hoffman-La Roche in 1977 (1) and recently introduced as a drug of abuse (2). Benzodiazepines are of forensic interest for several reasons.…”

Section: Introductionmentioning

confidence: 99%

Identifying Metabolites of Meclonazepam by High-Resolution Mass Spectrometry Using Human Liver Microsomes, Hepatocytes, a Mouse Model, and Authentic Urine Samples

Vikingsson

Wohlfarth

Andersson

et al. 2017

AAPS J

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Abstract.Meclonazepam is a benzodiazepine patented in 1977 to treat parasitic worms, which recently appeared as a designer benzodiazepine and drug of abuse. The aim of this study was to identify metabolites suitable as biomarkers of drug intake in urine using highresolution mass spectrometry, authentic urine samples, and different model systems including human liver microsomes, cryopreserved hepatocytes, and a mice model. The main metabolites of meclonazepam found in human urine were amino-meclonazepam and acetamido-meclonazepam; also, minor peaks for meclonazepam were observed in three of four urine samples. These observations are consistent with meclonazepam having a metabolism similar to that of other nitro containing benzodiazepines such as clonazepam, flunitrazepam, and nitrazepam. Both metabolites were produced by the hepatocytes and in the mice model, but the human liver microsomes were only capable of producing minor amounts of the amino metabolite. However, under nitrogen, the amount of aminomeclonazepam produced increased 140 times. This study comprehensively elucidated meclonazepam metabolism and also illustrates that careful selection of in vitro model systems for drug metabolism is needed, always taking into account the expected metabolism of the tested drug.KEY WORDS: benzodiazepine; in vitro metabolism; LC-MS/MS; new psychoactive substance; toxicokinetics.

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“…Studies in animals have shown that nitrazolam can be several times more effective than diazepam as an antagonist of electroshock-induced tonic extensor convulsions. 13,[16][17][18][19] Low doses such as these can be complicated to administer for users when handling bulk materials; variability of the active ingredient in the tablet are also a concern, where both the effects and unintended overdosing raise concerns about incidences of drug-facilitated crime. 13,[16][17][18][19] Low doses such as these can be complicated to administer for users when handling bulk materials; variability of the active ingredient in the tablet are also a concern, where both the effects and unintended overdosing raise concerns about incidences of drug-facilitated crime.…”

Section: The Welsh Emergingmentioning

confidence: 99%

“…Hydrogen atoms were assigned to calculated positions using a riding model with appro- This investigation describes a novel approach for preparing NPS reference standards using a microscale synthesis procedure and polymer-supported reagents. [10][11][12]15,16 The quantitative analysis of clonazolam tablet samples (reported to contain 0.5 mg), using the synthesized material as a reference standard, yielded a value of 0.52 mg clonazolam per tablet (Supporting Information 5). In addition, both clonazolam and nitrazolam were synthesized on a preparative scale using methods that have previously been described in the literature.…”

Section: X-ray Crystallography For Clonazolammentioning

confidence: 99%

An approach to shortening the timeframe between the emergence of new compounds on the drugs market and the availability of reference standards: The microscale syntheses of nitrazolam and clonazolam for use as reference materials, utilizing polymer‐supported reagents

Dowling

Kavanagh

Eckhardt³

et al. 2018

Drug Testing and Analysis

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Nitrazolam and clonazolam are 2 designer benzodiazepines available from Internet retailers. There is growing evidence suggesting that such compounds have the potential to cause severe adverse events. Information about tolerability in humans is scarce but typically, low doses can be difficult to administer for users when handling bulk material. Variability of the active ingredient in tablet formulations can also be of a concern. Customs, toxicology and forensic laboratories are increasingly encountering designer benzodiazepines, both in tablet and powdered form. The unavailability of reference standards can impact on the ability to identify these compounds. Therefore, the need arises for exploring in-house approaches to the preparation of new psychoactive substances (NPS) that can be carried out in a timely manner. The present study was triggered when samples of clonazolam were received in powdered and tablet form at a time when reference material for this drug was commercially unavailable. Therefore, microscale syntheses of clonazolam and its deschloro analog nitrazolam were developed utilizing polymer-supported reagents starting from 2-amino-2'-chloro-5-nitrobenzophenone (clonazolam) and 2-amino-5-nitrobenzophenone (nitrazolam). The final reaction step forming the 1,2,4-triazole ring moiety was performed within a gas chromatography-mass spectrometry (GC-MS) injector. A comparison with a preparative scale synthesis of both benzodiazepine derivatives showed that microscale synthesis might be an attractive option for a forensic laboratory in terms of time and cost savings when compared with traditional methods of synthesis and when qualitative identifications are needed to direct forensic casework. The reaction by-product profiles for both the micro and the preparative scale syntheses are also presented.

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Characterization of the four designer benzodiazepines clonazolam, deschloroetizolam, flubromazolam, and meclonazepam, and identification of their in vitro metabolites

Cited by 59 publications

References 15 publications

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples

In vitro studies on flubromazolam metabolism and detection of its metabolites in authentic forensic samples

Identifying Metabolites of Meclonazepam by High-Resolution Mass Spectrometry Using Human Liver Microsomes, Hepatocytes, a Mouse Model, and Authentic Urine Samples

An approach to shortening the timeframe between the emergence of new compounds on the drugs market and the availability of reference standards: The microscale syntheses of nitrazolam and clonazolam for use as reference materials, utilizing polymer‐supported reagents

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