In vitro Metabolismus synthetischer Cannabinoide und deren Nachweis in vivo

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The detection of synthetic cannabinoid abuse via urine screenings requires knowledge of their metabolism, major metabolites and urinary targets, recommendable and suitable for imple-mentation in an existing LC-MS/MS method. The targets identified within this dissertation were implemented as part of a routine screening LC-ESI-MS/MS method for urinary synthetic can-nabinoid metabolites, having been developed and published in parallel to this dissertation.
In the absence of urine samples from proven users of synthetic cannabinoids that could be used to elucidate their in vivo metabolism, various in vitro assays are used to identify potential metabolites. These in vitro assays not only possess the potential to cover a broad range of metabolites as well as the ability to perform in vitro – in vivo extrapolations, but can also be used to investigate the influence of CYP isoenzyme polymorphisms on their metabolism path-ways, along with the influence of co-consumption of other xenobiotics.
The individual metabolism studies focused on phase I-metabolism investigations but refrained from phase II-metabolism investigations, as urine samples are commonly treated with β-glucuronidase prior to clinical and toxicological analysis or doping controls.
The main objective was to investigate the phase I-metabolism of the synthetic cannabinoids THJ-018 and THJ-2201 (5F-THJ-018) as well as EG-018 and EG-2201 (5F-EG-018), using different in vitro approaches. Phase I metabolic pathways were examined by means of two microsomal assays (pHLM and CYP) and a fungal model (C. elegans). In addition, the poten-tial applicability and limitations of the in vitro – in vivo investigations carried out using those assays were evaluated by screening urine samples from proven THJ-018, THJ-2201 and EG-018 users as well as by comparison with the literature. In vivo metabolites and in vitro generated metabolites were analysed and evaluated via LC-ESI-MS/MS and LC-HRMS anal-ysis, following extraction from urine and incubation samples.
A further objective was to study the post-mortem distribution of the synthetic cannabinoid MDMB-CHMICA and its metabolites in different post-mortem samples, in order to find suitable matrices for further studies in the field of post-mortem toxicology. The aim being to facilitate the interpretation of comparable cases and to make appropriate recommendations when iden-tifying synthetic cannabinoid intoxication.
The first article covers the in vitro and in vivo phase I-metabolism investigations of THJ-018 and THJ-2201 by pHLM and CYP incubations, and urine screening, using extractive workup (SPE) and LC-HRMS. For THJ-018, 33 different in vitro metabolites were detected, while 46 different in vitro metabolites were observed for THJ-2201. Following the comparison with the results obtained in vivo, the most suitable urinary targets were identified as the dehydrogenat-ed THJ-018 metabolite M07.1 and the THJ-2201 metabolite F01.1 with a monohydroxylation of the naphthyl ring.
The second article comprises the phase I-metabolism of the synthetic cannabinoids EG-018 and EG-2201. This study further incorporated a fungal model, C. elegans, and LLE for extrac-tion. Metabolite identification was performed via LC-MS/MS. The pHLM assay gave rise to the identification of 15 different EG-018 and 21 different EC 2201 in vitro metabolites. Following the screening of urine samples, the EG-018 metabolite M1.2 (monohydroxylated at the pentyl side chain), and the two EG-2201 metabolites F1.1 and F1.2 (monohydroxylated at the 5F-pentyl side chain), were identified as appropriate to be implemented in the routine LC-ESI-MS/MS method.
With regard to the CYP dependent metabolism of THJ-018 and THJ-2201 as well as EG-018 and EG-2201, a significant contribution was observed to the microsomal metabolism originat-ing from the CYP isoenzymes 2C19, 3A4 and 3A5. For THJ-018 and THJ-2201, the CYP iso-enzyme 2B6 also took part in the metabolism, but not in the metabolism of EG-018 and EG-2201. By contrast, the CYP isoenzymes 2D6 and 2C9 possess a higher impact on EG-018 and EG-2201 metabolism in comparison to THJ-018 and THJ-2201. Overall, the results were in good agreement with previously published data on both the CYP dependent metabolism of phytocannabinoids and synthetic cannabinoids.
As several CYP isoenzymes were shown to be involved in the metabolism of THJ-018 and THJ-2201 as well as of EG-018 and EG-2201, and no metabolite was formed exclusively by a single CYP isoenzyme, it was concluded that the co-consumption of other xenobiotics does not have a major impact and that clinically relevant interactions are not likely.
However, due to conflicting data in the literature, further comparative studies on the CYP de-pendent metabolism of THJ-018 and THJ-2201, as well as of EG-018 and EG-2201, are nec-essary in order to conclusively evaluate the influence of the co-consumption of other xenobi-otics. For example, complementary studies with the pHLM assay by adding specific CYP in-hibitors or inducers would be a useful tool to conclusively assess the role of the CYP isoen-zymes identified in the metabolism studies on THJ-018 and THJ-2201 as well as on EG-018 and EG-2201.
The third article deals not only with the results of systematic toxicology analysis of post-mortem samples taken from a case of a 27-year-old man, but also with the examination of MDMB-CHMICA and its metabolites. The extraction and quantification of MDMB-CHMICA was carried out by means of LLE, the standard addition method or 3-point-calibrations and LC-MS/MS analysis. Metabolite extraction was carried out by means of SPE, and their identifica-tion was performed via pHLM assay. MDMB-CHMICA was qualitatively identified in all the ex-amined post-mortem samples, including urine. The highest concentration of MDMB-CHMICA was found in brain tissue (5.5 ng/g). Brain tissue was therefore recommended as an appro-priate matrix for synthetic cannabinoid analysis in post-mortem cases. Since no fat tissue sample was taken during the autopsy, no conclusion on its MDMB-CHMICA concentration compared to the other post-mortem samples could be drawn. The concentration found in the femoral blood sample (1.7 ng/mL) was in a toxicologically relevant range compared to previ-ously published data. As the calculated cardiac/femoral blood concentration ratio for MDMB-CHMICA was comparably low, there was no significant evidence for the susceptibility of MDMB-CHMICA to PMR. Qualitative analysis of MDMB-CHMICA metabolites revealed posi-tive results for most post-mortem samples, with the exception of brain, kidney, lung and gas-tric content. Furthermore, the M1.1 metabolites possessing a monohydroxylation at the cy-clohexylmethyl moiety were identified as suitable targets for detection of an MDMB-CHMICA uptake.
Evaluation of the results obtained from the three metabolism studies, the tested in vitro as-says and the fungal model (pHLM and CYP for THJ-018 and THJ-2201; pHLM, CYP and C. elegans for EG-018 and EG-2201; pHLM for MDMB-CHMICA) concluded that the data can be used for the identification of urinary targets, as they enable sufficient predictive in vitro – in vivo extrapolations.
Following the successful completion of the metabolism studies, the identified urinary targets for identification of the synthetic cannabinoids THJ-018 and THJ-2201, EG-018 and EG-2201 as well as MDMB-CHMICA were implemented in the routine LC-MS/MS screening method for urinary synthetic cannabinoid metabolites.
Furthermore, the metabolism studies, particularly those on EG-018, EG-2201 and MDMB-CHMICA, demonstrate that exclusive use of LC-MS/MS is sufficient for detection and identifi-cation of relevant in vivo metabolites of synthetic cannabinoids. However, LC-HRMS provides a useful supplementary tool for separation and identification of structurally related isomeric synthetic cannabinoids (e.g. MDMB-CHMICA and BB-22 (QUCHIC)) or fragment ions.
The results of this dissertation reveal the possibility suitable urinary target metabolites by means of in vitro assays and their integration into a qualitative LC-MS/MS method, which is of particular relevance for laboratories that have no LC-HRMS analysis.
Due to the highly dynamic and rapidly changing NPS market, metabolism studies must be continuously carried out in order to keep the existing screening LC-MS/MS method up to date. Despite the proven suitability of the tested in vitro assays for in vitro – in vivo extrapolations, screening of urine samples from proven synthetic cannabinoid consumption is essential for final verification of in vitro detected metabolites prior to implementing them into an existing methodology.
Original languageGerman
Place of PublicationKöln
PublisherDeutsche Sporthochschule Köln
Number of pages78
Publication statusPublished - 2019

ID: 5245013

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