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In vitro metabolic profiling of synthetic cannabinoids by pooled human liver microsomes, cytochrome P450 isoenzymes, and Cunninghamella elegans and their detection in urine samples

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Abstract

As synthetic cannabinoids are extensively metabolized, there is an urgent need for data on which metabolites can be used for successful urine screening. This study examines the in vitro metabolism of EG-018 and its 5F-analogue EG-2201 by means of comparing three different in vitro models: pooled human liver microsomes, cytochrome P450 isoenzymes, and a fungal approach utilizing the filamentous fungus Cunninghamella elegans LENDNER, which is known for its ability to mimic human biotransformation of xenobiotics. In addition, this study includes the screening of two authentic urine samples from individuals with proven EG-018 consumption, for the evaluation of in vitro–in vivo extrapolations made in the study. Incubation with pooled human liver microsomes yielded 15 metabolites of EG-018 belonging to six different metabolite subgroups, and 21 metabolites of EG-2201 belonging to seven different metabolite subgroups, respectively. Incubation with cytochrome P450 isoenzymes incubation yielded a further three EG-018 and five EG-2201 metabolites. With reference to their summed metabolite peak abundancies, the isoenzymes CYP2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5 were shown to contribute most to the microsomal metabolism of EG-018 and EG-2201. CYP2B6 was shown to make the lowest contribution, by far. As the phase I metabolism of both synthetic cannabinoids was shown to be distributed over a substantial number of different cytochrome P450 isoenzymes, it was concluded that it is likely to not be significantly affected by co-consumption of other drugs. Although fungal incubation with Cunninghamella elegans yielded an additional three EG-018 and four EG-2201 metabolites not observed after microsomal incubation, metabolites generated by Cunninghamella elegans were in good correlation with those generated by microsomal incubations. The fungal model demonstrated its ability to be an independent in vitro model in synthetic cannabinoid metabolism research. The three tested in vitro models enable sufficient predictive in vitro–in vivo extrapolations, comparable to those obtained from hepatocyte incubation published in the literature. In addition, with regard to the screening of authentic urine samples and comparison with the literature, one monohydroxylated EG-018 metabolite and two monohydroxylated EG-2201 metabolites can be recommended as urinary targets, on the basis of the tested in vitro models.

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Acknowledgments

Very special thanks are directed to June Mercer-Chalmers-Bender for English language editing and idea sharing.

Funding

This work was funded by the German Federal Ministry for Economic Affairs and Energy and the Central innovation program for medium-sized companies, respectively (grant number KF2429613MD3). Last but not least, thanks are directed to the German Union against Alcohol and Drugs in Road Transport (Bund gegen Alkohol und Drogen im Straßenverkehr; BADS) for additional financial support.

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Authors and Affiliations

  1. Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Melatengürtel 60/62, 50823, Cologne, Germany

    Franziska Gaunitz, Patrick Dahm & Katja Mercer-Chalmers-Bender

  2. Institute of Forensic Medicine, Forensic Toxicology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg, Freiburg, Germany

    Lukas Mogler

  3. Hermann Staudinger Graduate School, University of Freiburg, Hebelstraße 27, 79104, Freiburg, Germany

    Lukas Mogler

  4. Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany

    Andreas Thomas & Mario Thevis

  5. Institute of Forensic Medicine, University of Basel, Health Department Basel-Stadt, Pestalozzistrasse 22, 4056, Basel, Switzerland

    Katja Mercer-Chalmers-Bender

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  1. Franziska Gaunitz
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Correspondence to Katja Mercer-Chalmers-Bender.

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Research involving human participants or animals

This article does not contain any studies with human participants or animals performed by any of the authors. The urine samples were collected during routine screening for metabolites of synthetic cannabinoids between January 2016 and February 2018 at the Institute of Forensic Medicine in Freiburg. According to German law, ethical approval is not required when samples are gathered for the purposes of routine screening. Analysis of the urine samples was carried out with the consent of the respective test persons. Data have been anonymized and are not traceable to the sample donors. All procedures performed were in accordance and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Gaunitz, F., Dahm, P., Mogler, L. et al. In vitro metabolic profiling of synthetic cannabinoids by pooled human liver microsomes, cytochrome P450 isoenzymes, and Cunninghamella elegans and their detection in urine samples. Anal Bioanal Chem 411, 3561–3579 (2019). https://doi.org/10.1007/s00216-019-01837-8

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