Institute of Legal Medicine, Faculty of Medicine, University of Cologne, Melatengürtel 60/62, 50823, Cologne, Germany.
Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933, Cologne, Germany.
Forensic Toxicol. 2022 Jul;40(2):244-262. doi: 10.1007/s11419-021-00606-6. Epub 2021 Dec 30.
Synthetic cannabinoids (SCs), highly metabolized substances, are rarely found unmodified in urine samples. Urine screening relies on SC metabolite detection, requiring metabolism knowledge. Metabolism data can be acquired via in vitro assays, e.g., human hepatocytes, pooled human liver microsomes (pHLM), cytochrome P450 isoforms and a fungal model; or in vivo by screening, e.g., authentic human samples or rat urine. This work describes the comprehensive study of PX-1 and PX-2 in vitro metabolism using three in vitro models. 5F-APP-PICA (PX-1) and 5F-APP-PINACA (PX-2) were studied as they share structural similarity with AM-2201, THJ-2201 and 5F-AB-PINACA, the metabolism of which was described in the literature.
For SC incubation, pHLM, cytochrome P450 isoenzymes and the fungal model Cunninghamella elegans LENDNER (C. elegans) were used. PX-1 and PX-2 in vitro metabolites were revealed comprehensively by liquid chromatography-high-resolution mass spectrometry measurements.
In total, 30 metabolites for PX 1 and 15 for PX-2 were detected. The main metabolites for PX-1 and PX-2 were the amide hydrolyzed metabolites, along with an indole monohydroxylated (for PX-1) and a defluorinated pentyl-monohydroxylated metabolite (for PX-2).
CYP isoforms along with fungal incubation results were in good agreement to those obtained with pHLM incubation. CYP2E1 was responsible for many of the metabolic pathways; particularly for PX-1. This study shows that all three in vitro assays are suitable for predicting metabolic pathways of synthetic cannabinoids. To establish completeness of the PX-1 and PX-2 metabolic pathways, it is not only recommended but also necessary to use different assays.
合成大麻素（SCs）是高度代谢的物质，在尿液样本中很少发现未修饰的形式。尿液筛查依赖于 SC 代谢物的检测，这需要代谢知识。代谢数据可以通过体外测定法获得，例如人肝细胞、人肝微粒体（pHLM）、细胞色素 P450 同工酶和真菌模型；或者通过筛选获得，例如真实的人体样本或大鼠尿液。本研究使用三种体外模型全面研究了 PX-1 和 PX-2 的体外代谢。5F-APPPICA（PX-1）和 5F-APPPINACA（PX-2）被研究，因为它们与 AM-2201、THJ-2201 和 5F-AB-PINACA 结构相似，文献中描述了它们的代谢情况。
对于 SC 孵育，使用了 pHLM、细胞色素 P450 同工酶和真菌模型 Cunninghamella elegans LENDNER（C. elegans）。通过液相色谱-高分辨率质谱测量全面揭示了 PX-1 和 PX-2 的体外代谢物。
共检测到 PX-1 的 30 种代谢物和 PX-2 的 15 种代谢物。PX-1 和 PX-2 的主要代谢物是酰胺水解代谢物，以及吲哚单羟基化代谢物（用于 PX-1）和去氟化戊基单羟基化代谢物（用于 PX-2）。
CYP 同工酶与真菌孵育结果与 pHLM 孵育结果非常一致。CYP2E1 负责许多代谢途径；特别是对于 PX-1。本研究表明，所有三种体外测定法都适用于预测合成大麻素的代谢途径。为了建立 PX-1 和 PX-2 代谢途径的完整性，不仅建议而且有必要使用不同的测定法。
