Monitoring metabolism of synthetic cannabinoid 4F-MDMB-BINACA via high-resolution mass spectrometry assessed in cultured hepatoma cell line, fungus, liver microsomes and confirmed using urine samples

Abstract

Purpose A tert-leucinate derivative synthetic cannabinoid, methyl (2S)-2-([1-(4-fluorobutyl)-1H-indazole-3-carbonyl]amino)-3,3-dimethylbutanoate (4F-MDMB-BINACA, 4F-MDMB-BUTINACA or 4F-ADB) is known to adversely impact health. This study aimed to evaluate the suitability of three different modes of monitoring metabolism: HepG2 liver cells, fungus Cunninghamella elegans (C. elegans) and pooled human liver microsomes (HLM) for comparison with human in-vivo metabolism in identifying suitable urinary marker(s) for 4F-MDMB-BINACA intake. Methods Tentative structure elucidation of in-vitro metabolites was performed on HepG2, C. elegans and HLM using liquid chromatography–tandem mass spectrometry and high-resolution mass spectrometry analysis. In-vivo metabolites obtained from twenty authentic human urine samples were analysed using liquid chromatography–Orbitrap mass spectrometry. Results Incubation with HepG2, C. elegans and HLM yielded nine, twenty-three and seventeen metabolites of 4F-MDMB-BINACA, respectively, formed via ester hydrolysis, hydroxylation, carboxylation, dehydrogenation, oxidative defluorination, carbonylation or reaction combinations. Phase II metabolites of glucosidation and sulfation were also exclusively identified using C. elegans model. Eight in-vivo metabolites tentatively identified were mainly products of ester hydrolysis with or without additional dehydrogenation, N-dealkylation, monohydroxylation and oxidative defluorination with further oxidation to butanoic acid. Metabolites with intact terminal methyl ester moiety, i.e., oxidative defluorination with further oxidation to butanoic acid, were also tentatively identified. Conclusions The in-vitro models presented proved useful in the exhaustive metabolism studies. Despite limitations, HepG2 identified the major 4F-MDMB-BINACA ester hydrolysis metabolite, and C. elegans demonstrated the capacity to produce a wide variety of metabolites. Both C. elegans and HLM produced all the in-vivo metabolites. Ester hydrolysis and ester hydrolysis plus dehydrogenation 4F-MDMB-BINACA metabolites were recommended as urinary markers for 4F-MDMB-BINACA intake.

Recent trends in drugs of abuse metabolism studies for mass spectrometry–based analytical screening procedures

“…However, it should be considered that all cell culture experiments require special laboratory equipment as well as trained personnel. Unfortunately, HepG2 cells are known for their poor gene expression of several CYP enzymes, which might explain the low number of publications in the context of NPS metabolism studies [ 5 , 20 , 21 ]. HepaRG cells are more frequently used to identify metabolites of NPS, probably due to the fact that HepaRG cells show gene expression levels for metabolizing enzymes comparable to PHH.…”

“…C. elegans is known to have enzymatic activity for both phase I and II reactions with some similarities in CYP enzyme expression but equal differences especially in phase II enzymes compared to humans. Watanabe et al and Leong et al described that C. elegans was able to generate both phase I and II metabolites in case of four out of five synthetic cannabinoids [ 14 , 21 ]. By contrast, Grafinger et al detected only phase I metabolites of four investigated synthetic tryptamines in fungi biomass and growth medium used [ 15 ].…”

“…Concerning the cellular systems in general, the metabolites generated by HepG2 cells provided the smallest overlap with human data [ 5 , 21 ]. Metabolites formed by the fungus C. elegans showed a moderate or good agreement with confirmed human biomarkers [ 14 , 21 ].…”

“…Concerning the cellular systems in general, the metabolites generated by HepG2 cells provided the smallest overlap with human data [ 5 , 21 ]. Metabolites formed by the fungus C. elegans showed a moderate or good agreement with confirmed human biomarkers [ 14 , 21 ]. The metabolic fate of the synthetic cannabinoid 5F-MDMB-P7AICA was extensively investigated in different in vitro and in vivo model systems as well as in human biosamples [ 26 , 27 , 29 ].…”

Induction of Liver Size Reduction in Zebrafish Larvae by the Emerging Synthetic Cannabinoid 4F-MDMB-BINACA and Its Impact on Drug Metabolism

“…4F-MDMB-BINACA was chosen for this study because it is an emerging and highly potent new SC for which a high number of seizure cases were reported in 2019 by the European Union Early Warning System [31,32]. Therefore, 4F-MDMB-BINACA has been controlled under Schedule II of the Convention on Psychotropic Substances of 1971, designed as a United Nations treaty, which came into force on 3 November 2020 [33,34].…”