Toxicokinetic Studies of the Two Stimulants M-ALPHA and N-Methyl-cyclazodone Using In Vitro and In Vivo Tools

Background/Objectives: Synthetic stimulants represent the most prevalent subclass on the new psychoactive substances (NPSs) market. However, the toxicokinetic properties of M-ALPHA, a regioisomer of MDMA and N-methyl-cyclazodone a pemoline derivative, are not yet characterized. Methods: Therefore, this study investigated the metabolism of both NPSs in pooled liver S9 fraction and rat urine, characterized cytochrome P450 (CYP) kinetics and plasma protein binding (PPB), and assessed the CYP inhibition potential of M-ALPHA, using high-performance liquid chromatography coupled to high resolution tandem mass spectrometry (HPLC-HRMS/MS). Results: Four metabolites of M-ALPHA were detected including one phase I and three phase II metabolites, resulting from demethylenation followed by subsequent methylation or glucuronidation. For N-methyl-cyclazodone, one phase I metabolite formed via N-demethylation was identified. The primary enzymes involved in M-ALPHA metabolism were CYP2B6 and CYP2D6. Notably, M-ALPHA inhibited these enzymes to a strong or moderate extent, respectively. In contrast, the metabolism of N-methyl-cyclazodone was primarily mediated by CYP2A6. PPB studies indicated low-to-moderate binding for both compounds, suggesting that significant protein-binding interactions are unlikely. Conclusions: As M-ALPHA only formed metabolites that overlapped with those of MDMA, differing only by minor retention time shifts, reliable HPLC-HRMS/MS-based identification may be challenging in clinical and forensic toxicology settings as well as doping analysis. Furthermore, drug-drug interactions following polydrug use cannot be excluded for either NPS, particularly when co-ingested with other CYP substrates metabolized by the same isoforms.