title: 4-Methoxy-α-PVP: in silico prediction, metabolic stability, and metabolite identification by human hepatocyte incubation and high-resolution mass spectrometry.
abstract: Novel psychoactive substances are continuously developed to circumvent legislative and regulatory efforts. A new synthetic cathinone, 4-methoxy-α-PVP, was identified for the first time in illegal products; however, the metabolism of this compound is not known. Complete metabolic profiles are needed for these novel psychoactive substances to enable identification of their intake and to link adverse effects to the causative agent. This study assessed 4-methoxy-α-PVP metabolic stability with human liver microsomes (HLMs) and identified its metabolites after HLM and hepatocyte incubations followed by high-resolution mass spectrometry (HRMS). A Thermo QExactive high-resolution mass spectrometer (HRMS) was used with full scan data-dependent mass spectrometry, with (1) and without (2) an inclusion list of predicted metabolite, and with full scan and all-ion fragmentation (3) to identify potential unexpected metabolites. In silico predictions were performed and compared to in vitro results. Scans were thoroughly mined with different data processing algorithms using WebMetabase (Molecular Discovery). 4-Methoxy-α-PVP exhibited a long half-life of 79.7 min in HLM, with an intrinsic clearance of 8.7 µL min-1mg-1. In addition, this compound is predicted to be a low-clearance drug with an estimated human hepatic clearance of 8.2 mL min-1kg-1. Eleven 4-methoxy-α-PVP metabolites were identified, generated byO-demethylation, hydroxylation, oxidation, ketone reduction,N-dealkylation, and glucuronidation. The most dominant metabolite in HLM and human hepatocyte samples was 4-hydroxy-α-PVP, also predicted as the #1 in silico metabolite, and is suggested to be a suitable analytical target in addition to the parent compound.
authors.affiliation: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Suite 200 Room 05A-721, Baltimore, MD 21224 USA ; Program in Toxicology, University of Maryland Baltimore, Baltimore, MD USA.
authors.forename: Kayla N
authors.initials: KN
authors.lastname: Ellefsen
authors.affiliation: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Suite 200 Room 05A-721, Baltimore, MD 21224 USA.
authors.forename: Ariane
authors.initials: A
authors.lastname: Wohlfarth
authors.affiliation: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Suite 200 Room 05A-721, Baltimore, MD 21224 USA.
authors.forename: Madeleine J
authors.initials: MJ
authors.lastname: Swortwood
authors.affiliation: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Suite 200 Room 05A-721, Baltimore, MD 21224 USA.
authors.forename: Xingxing
authors.initials: X
authors.lastname: Diao
authors.affiliation: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Suite 200 Room 05A-721, Baltimore, MD 21224 USA ; Department of Sciences, John Jay College of Criminal Justice, City University of New York, New York, NY USA.
authors.forename: Marta
authors.initials: M
authors.lastname: Concheiro
authors.affiliation: Chemistry and Drug Metabolism, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, 251 Bayview Boulevard, Suite 200 Room 05A-721, Baltimore, MD 21224 USA.
authors.forename: Marilyn A
authors.initials: MA
authors.lastname: Huestis
keywords: 4-Methoxy-α-PVP; Human hepatocytes; Human liver microsomes; In silico prediction; Novel psychoactive substances; Synthetic cathinones