Generation of metabolites by an automated online metabolism method using human liver microsomes with subsequent identification by LC-MS(n), and metabolism of 11 cathinones

Abstract

Human liver microsomes (HLMs) are used to simulate human xenobiotic metabolism in vitro. In forensic and clinical toxicology, HLMs are popularly used to study the metabolism of new designer drugs for example. In this work, we present an automated online extraction system we developed for HLM experiments, which was compared to a classical offline approach. Furthermore, we present studies on the metabolism of 11 cathinones; for eight of these, the metabolism has not previously been reported. Metabolites were identified based on MS(2) and MS(3) scans. Fifty-three substances encompassing various classes of drugs were employed to compare the established offline and the new online methods. The metabolism of each of the following 11 cathinones was studied using the new method: 3,4-methylenedioxy-N-benzylcathinone, benzedrone, butylone, dimethylcathinone, ethylone, flephedrone, methedrone, methylone, methylethylcathinone, naphyrone, and pentylone. The agreement between the offline and the online methods was good; a total of 158 metabolites were identified. Using only the offline method, 156 (98.7%) metabolites were identified, while 151 (95.6%) were identified using only the online method. The metabolic pathways identified for the 11 cathinones included the reduction of the keto group, desalkylation, hydroxylation, and desmethylenation in cathinones containing a methylenedioxy moiety. Our method provides a straightforward approach to identifying metabolites which can then be added to the library utilized by our clinical toxicological screening method. The performance of our method compares well with that of an established offline HLM procedure, but is as automated as possible.

An integrated pharmacokinetic and pharmacodynamic study of a new drug of abuse, methylone, a synthetic cathinone sold as “bath salts”

“…Surprisingly, we have not found the corresponding demethyl compounds of the hydroxylated metabolites but these was also not found by Mueller and Rentsch (2012), which studied Phase I metabolites. The results indicate that the metabolism of methylone contributes significantly to its plasmatic clearance and this contribution is especially evident when the drug is administered orally, as a result of a hepatic first-pass effect, as is suggested by the differential pharmacokinetic profile observed in both routes of administration.…”

“…To our knowledge, all published papers on pharmacokinetics of methylone are of a forensic nature (urinary collected specimens) (Crawse et al, 2012, Kamata et al, 2006 or from in vitro experiments (Mueller and Rentsch, 2012). This study constitutes the first approach to the kinetics of methylone based on in vivo blood sample data from laboratory animals and will be helpful to…”

“…Moreover, we propose that, in rats, methylone is a substrate of an aliphatic hydroxylation process resulting in the corresponding 3'-hydroxy-methylenedioxymethcathinone. Mueller and Rentsch (2012) proposed these methylone metabolites by an automated online metabolism method using human liver microsomes, but this is the first time that 3'-hydroxymethylenedioxymethcathinone metabolite has been identified in vivo. The same authors found a third metabolite corresponding to the reduction of the beta-keto group which we did not detect in rat blood probably due to its low half-life.…”

In Vitro Metabolism and Pharmacokinetic Studies on Methylone

“…Thus, our main focus here is the metabolism and kinetics of methylone, for which studies have been limited until now. Mueller and Rentsch (2012) showed that human liver microsomes (HLMs) metabolize methylone into nor-methylone, dihydro-methylone, and hydroxy-methylone. Figure 1 shows these in vitro-produced metabolites together with metabolites detected in vivo in rat and/or human urine, as determined by Meyer et al (2010) and Kamata et al (2006).…”

Interactions of Cathinone NPS with Human Transporters and Receptors in Transfected Cells

“…Cathinone NPS may also have active metabolites that should be taken into account in more comprehensive pharmacological substance characterizations. For example, β-keto-MDA is a metabolite of methylone (Mueller & Rentsch, 2012) and interacts with monoamine transporters similarly to MDA in in vitro tests (Rickli et al, 2015b). In vitro testing for active metabolites requires knowledge of the metabolic pathway and synthesis of possibly active metabolites or the use of cell systems that contain metabolic enzymes.…”