Identification of Cytochrome P450 Enzymes Involved in the Metabolism of the New Designer Drug 4′-Methyl-α-pyrrolidinobutyrophenone

Abstract

ABSTRACT:The involvement of human hepatic cytochrome P450 (P450) isoenzymes in the metabolism of the new designer drug 4-methyl-␣-pyrrolidinobutyrophenone (MPBP) to 4-(hydroxymethyl)-␣-pyrrolidinobutyrophenone (HO-MPBP) was studied using insect cell microsomes with cDNA-expressed human P450s and human liver microsomes (HLM). Incubation samples were analyzed by liquid chromatography-mass spectrometry. Only CYP2D6, CYP2C19, and CYP1A2 were capable of catalyzing MPBP 4-hydroxylation. According to the relative activity factor approach, these enzymes accounted for 54, 30, and 16% of net clearance. At 1 M MPBP, the chemical inhibitors quinidine (CYP2D6), fluconazole (CYP2C19), and ␣-naphthoflavone (CYP1A2) reduced metabolite formation in pooled HLM by 83, 53, and 47%, respectively, and at 50 M MPBP by 41, 47, and 45%, respectively. In experiments with HLM from CYP2D6 and CYP2C19 poor metabolizers, HO-MPBP formation was found to be 78 and 79% lower in comparison with pooled HLM, respectively. From these data, it can be concluded that polymorphically expressed CYP2D6 is mainly responsible for MPBP hydroxylation.The cytochrome P450 (P450) family accounts for more than 90% of oxidative metabolic reactions of xenobiotics (Guengerich, 2005). The involvement of particular P450 enzymes in the biotransformation of a new chemical entity is usually thoroughly investigated before it can be marketed in order to assess the risk of increased side effects in poor metabolizer subjects and of drug-drug or drug-food interactions. However, such data are typically acquired for substances intended for therapeutic use, not for drugs of the illicit market.1-(4-Methylphenyl)-2-pyrrolidin-1-ylbutan-1-one [4Ј-methyl-␣-pyrrolidinobutyrophenone (MPBP)] is a new designer drug of the pyrrolidinophenone type. Together with 4Ј-methyl-␣-pyrrolidinobutyrophenone (Roesner et al., 1999;Springer et al., 2003c), 4Ј-methyl-␣-pyrrolidinopropiophenone (MPPP) (Roesner et al., 1999;Springer et al., 2002;Springer et al., 2003c) However, they may be expected to be very similar to those of pyrovalerone (4Ј-methyl-␣-pyrrolidinovalerophenone) due to their close structural relation to this drug. Pyrovalerone is a psychostimulant that acts by releasing dopamine and norepinephrine from the respective nerve terminals (Fauquet et al., 1976;Servin et al., 1978) and inhibits the reuptake of these neurotransmitters (Meltzer et al., 2006). The latter was also shown for PVP. In comparison with amphetamine, pyrovalerone reportedly has similar psychostimulant effects but less influence on motor function in animals and humans (Stille et al., 1963;Holliday et al., 1964;Heimann and Lukacs, 1965). Pyrovalerone had been studied as a therapeutic drug (Heinmann and Vetter, 1965;Goldberg et al., 1973;Soderholm et al., 1976) but was withdrawn from the market and scheduled as a controlled substance after reports of its intravenous abuse (Deniker et al., 1975). A similar pharmacological profile of the pyrrolidinophenones would clearly be in line with their abuse as stimulant designer drug...

In Vitro, in Vivo and In Silico Metabolic Profiling of α-Pyrrolidinopentiothiophenone, A Novel Thiophene Stimulant

“…Characterizing NPS metabolic pathways is imperative for PK profiling and understanding PD effects. The metabolic pathways of other α-pyrrolidinophenones, including MDPV, α-PVP, α-PBP, α-PPP, 4-methyl-α-pyrrolidonophene, 4-methoxy-α-pyrrolidinophenone and 4-methyl-α-pyrrolidinohexiophenone were previously investigated in human liver microsomes (HLM) and rat urine [17][18][19][20][21][22][23][24][25][26][27]. In a recent review of pyrrolidinophenones' pharmacology, Zaitsu et al summarized the main metabolites to include ketone reduction to the corresponding alcohol and oxidation to the 2'-oxo metabolites [28].…”

From Known Knowns to Known Unknowns: Predicting in Vivo Drug Metabolites

“…In addition to these, CYP-activity phenotyped liver preparations are used to reflect the formation of the metabolites, so that metabolite formation correlating with certain CYP isoform activity in the livers suggests high involvement of that particular CYP isoform. A large number of studies for identification of the CYP enzyme responsible for metabolism of some particular compound are reported and some of the most recent ones are cited here [93][94][95][96][97]. Similarly, articles describing the role of analytical techniques in the identification of UDP-glucuronosyltransferase enzyme isoforms responsible for glucuronide conjugation have been published [98][99][100].…”

Designer drug 2,5-dimethoxy-4-methyl-amphetamine (DOM, STP): Involvement of the cytochrome P450 isoenzymes in formation of its main metabolite and detection of the latter in rat urine as proof of a drug intake using gas chromatography–mass spectrometry

“…[21][22][23][24]. The mixtures (final volume: 50 l, n = 2) consisted of 90 mM phosphate buffer (pH 7.4; for CYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) or Tris buffer (pH 7.4; for CYP2A6 and CYP2C9), 5 mM Mg 2+ , 5 mM isocitrate, 1.2 mM NADP + , 0.5 U/ml isocitrate dehydrogenase, 200 U/ml superoxide dismutase, 50 pmol/ml of CYP, and substrate at 37 • C. The substrates were added after dilution of 250 mM methanolic stock solutions in respective buffer.…”