Elucidation of the Phase I and Phase II metabolic pathways of (±)-4’-methylmethcathinone (4-MMC) and (±)-4’-(trifluoromethyl) methcathinone (4-TFMMC) in cryopreserved human hepatocytes using LC-MS and LC-MS2

Abstract

The study investigates the in vitro metabolism of 4-MMC using cryopreserved human hepatocytes to characterize the associated Phase I and II metabolites. 4-MMC was incubated with human hepatocytes, and the reaction mixture was analyzed using a zwitterionic hydrophilic interaction (ZIC®-HILIC) column with LC-MS and LC-MS² techniques. The metabolism of 4-MMC resulted in the identification of 14 metabolites, which were structurally characterized based on accurate mass analyses and LC-MS² fragmentation patterns. The primary metabolic pathways for 4-MMC were identified as (i) oxidation of the 4’-methyl group and (ii) reduction of the β-keto moiety. Additionally, the biotransformation of a modified 4’-trifluoromethyl derivative (4-TFMMC) was studied, revealing significant differences in metabolism compared to 4-MMC. Notably, 4-TFMMC was found to be more extensively metabolized than mephedrone. Recent studies have indicated that the enzyme CYP450 (CYP2D6) plays a crucial role in the metabolic process, as it is responsible for Phase I metabolism. Consequently, the activities and expression of this drug-metabolizing enzyme were analyzed in the Life Technologies laboratory using cryopreserved human hepatocytes. A comparison of enzyme activities and expression from the two donors, alongside data from 196 other donors, revealed that hepatocytes obtained from these two donors exhibited intermediate CYP2D6 enzyme activity. Key pharmacokinetic parameters for both drugs were calculated, with biological half-lives (t½) found to be 693.0 minutes for 4-MMC and 216.6 minutes for 4-TFMMC. This data could contribute to a better understanding of in vivo metabolism in humans.

Keywords: 4-MMC, 4-TFMMC, Metabolism, HILIC, Mass Spectrometry.