Short Communication The Role of Human Hepatic Cytochrome P450 Isozymes in the Metabolism of Racemic 3,4-Methylenedioxyethylamphetamine and Its Single Enantiomers

The 3,4-methylenedioxy-methamphetamine (MDMA)-related designer drug 3,4-methylenedioxyethylamphetamine (MDEA, Eve) is a chiral compound that is mainly metabolized by N-deethylation and demethylenation during phase I metabolism. The involvement of several cytochrome P450 (P450) isozymes in these metabolic steps has been demonstrated by inhibition assays using human liver microsomes. However, a comprehensive study on the involvement of all relevant human P450s has not been published yet. In addition, the chirality of this drug was not considered in these in vitro studies. The aim of the present work was first to elucidate the contribution of the relevant human P450 isozymes in the demethylenation as well as in the N-dealkylation of racemic MDEA and its single enantiomers and secondly to compare these findings with recently published data concerning the enantioselective metabolism of MDMA. Racemic MDEA and its single enantiomers were incubated using heterologously expressed human P450s, and the corresponding metabolites dihydroxyethylamphetamine and methylenedioxyamphetamine were determined by gas chromatography-mass spectrometry after chiral derivatization with S-heptafluorobutyrylprolyl chloride. The highest contributions to both metabolic steps as calculated from the enzyme kinetic data were obtained for CYP3A4 and CYP2D6 at substrate concentrations corresponding to plasma concentrations of recreational users after intake of racemic MDEA. Both metabolic reactions were found to be enantioselective with a general preference for the S-enantiomers, which was particularly pronounced in the case of CYP2C19. In conclusion, different pharmacokinetic properties of MDEA enantiomers observed in vivo are therefore partially caused by P450dependent enantioselective metabolism. 3,4-Methylenedioxyethylamphetamine (MDEA, Eve) is chemically and pharmacologically related to 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy, Adam) (Freudenmann and Spitzer, 2004). The MDEA enantiomers have different pharmacokinetic properties: S-MDEA, which produces elevated mood and impairment in conceptually driven cognition, and R-MDEA, which produces increased depression and enhanced visual feature processing. Concerning chronic toxicity, data from animal experiments strongly suggest that both MDMA and MDEA can cause irreversible damage to serotoninergic nerve terminals in the central nervous system (Kalant, 2001; de la Torre et al., 2004; Freudenmann and Spitzer, 2004; Monks et al., 2004; Easton and Marsden, 2006). However, most in vitro and animal studies indicate that compared with MDMA, the neurotoxic potential of MDEA is lower (Kalant, 2001; Freudenmann and Spitzer, 2004; Monks et al., 2004). More recently, we have published studies concerning the involvement of human cytochrome P450 (P450) in the metabolism of racemic MDMA as well as the metabolites of its enantiomers (Meyer et al., 2008). In this MDMA study, we were able to show that CYP2C19 (in addition to CYP2D6) is the most enantioselective P450 isozyme enzyme toward the two main metabolic steps, namely N-demethylation and demethylenation, with a preference for the S-enantiomer. These results could in part explain the different pharmacokinetics of Rand S-MDMA in vivo. As shown in Fig. 1, in vivo and in vitro studies showed that the main metabolic steps of MDEA are the same as those of MDMA, namely N-deethylation and demethylenation (Maurer et al., 2000). Later, MDEA was investigated concerning enantioselective pharmacokinetics in vivo (Brunnenberg and Kovar, 2001; Buechler et al., 2003), and the plasma half-life of R-MDEA was found to be longer than that of S-MDEA. Accordingly, the plasma concentrations of the S-enantiomers of the main metabolites N-ethyl4-hydroxy-3-methoxyamphetamine and 3,4-methylenedioxyamphetamine (MDA) were much higher than those of the R-enantiomers. Enantioselective pharmacokinetics of MDEA resulting in higher plasma concentrations of R-MDEA were also confirmed by other authors (Spitzer et al., 2001; Meyer et al., 2002; Peters et al., 2003). Enantioselective metabolism is the most likely explanation for the enantioselective pharmacokinetics of MDEA. Previously published data from inhibition studies and with recombinant P450 (Kreth et al., 2000; Maurer et al., 2000) indicated that CYP1A2, 2B6, 2D6, and 3A4 are involved in the N-deethylation and/or demethylenation of MDEA but did not provide any information about the possible enantioselectivity of these metabolic steps. Hence, no systematic enzyme kinetic data are available for (enantioselective) MDEA metabolism by Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.026203. □S The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. ABBREVIATIONS: MDEA, 3,4-methylenedioxyethylamphetamine; MDMA, 3,4-methylenedioxymethamphetamine; P450, cytochrome P450; MDA, 3,4-methylenedioxyamphetamine; CL, clearance; MAB3A4, monoclonal antibody inhibitory to 3A4. 0090-9556/09/3706-1152–1156$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 6 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 26203/3473370 DMD 37:1152–1156, 2009 Printed in U.S.A. 1152 http://dmd.aspetjournals.org/content/suppl/2009/03/19/dmd.108.026203.DC1 Supplemental material to this article can be found at: at A PE T Jornals on D ecem er 2, 2017 dm d.aspurnals.org D ow nladed from recombinant isozymes. However, kinetic data for each involved isozyme could help to explain the differences in MDMA/MDEA metabolism as well as the different pharmacokinetics of the enantiomers in vivo. Therefore, the aim of the present study was to obtain enantioselective enzyme kinetic data of N-deethylation and demethylenation of MDEA by the 10 P450s most relevant in human drug metabolism and to compare these data with the kinetics of MDMA to reveal and explain the differences in enantioselective metabolism. Materials and Methods In principle, materials and methods were the same as described previously (Meyer et al., 2008). Therefore, only the differences are mentioned in the following section. Racemic hydrochlorides of MDEA ( 98.5% purity) and N-ethyl-3,4-dihydroxyamphetamine were obtained from Lipomed (Bad Saeckingen, Germany) and ReseaChem (Burgdorf, Switzerland), respectively. The MDEA enantiomers (98% purity, each) were prepared in the author’s laboratory by enantioseparation as described below. Separation of Racemic MDEA and High-Performance Liquid Chromatography Conditions. Racemic MDEA was separated in aliquots (100 l) of an aqueous stock solution (5 mg/ml, 30 mg in total) using a mobile phase composition of 0.1 M ammonium acetate buffer (pH 6.5)/acetonitrile 85:15 (v/v), a flow rate of 3 ml/min, and UV detection at 263 nm. The cyclodextrin column was mounted into a freezer at 8°C. The fractions containing the separated enantiomers were collected, and the enantiomers were isolated from the aqueous part by liquid/liquid extraction at pH 9 using ethyl acetate (three times using 150 ml each). The extracts were evaporated to dryness using a Rotavapor (Büchi, Essen, Germany) under reduced pressure and reconstituted in 1.0 ml of HCl (0.01 M). Thereafter, the concentrations of the MDEA enantiomers in the resulting solution were determined. The recovery was approximately 75% per enantiomer. Initial Screening Studies and Kinetic Studies. Incubations were performed with 50 M R,S-MDEA, R-MDEA, and S-MDEA. Kinetic constants of N-deethylation (expressed as MDA formation) or demethylenation (expressed as DHEA formation) were derived from incubations with an incubation time of 20 min and a P450 concentration of 40 pmol/ml (N-deethylation) and 30 pmol/ml (demethylenation). The substrate concentrations shown in Supplemental Table S1 were used. Calculation of enzyme kinetic constants was similar to the previously published method (Meyer et al., 2008). In brief, the Michaelis-Menten equation (eq. 1) was used to calculate apparent Km and Vmax values for singleenzyme systems. Eadie-Hofstee plots were used to check for biphasic kinetics. If the Eadie-Hofstee plot indicated biphasic kinetics, eq. 1 and the alternative eq. 2 for a two-enzyme model (Clarke, 1998) were applied to the respective data. For eq. 2, CLint,2 represents the intrinsic clearance or Vmax/Km of the low-affinity component (Clarke, 1998). If eq. 2 was found to fit the data significantly better (F test, P 0.05), biphasic kinetics were assumed. V Vmax [S] Km [S] (1) V Vmax,1 [S] Km,1 [S] CLint,2 S] (2) The relative activity factor approach (Crespi and Miller, 1999; Venkatakrishnan et al., 2000; Grime and Riley, 2006) was used to account for differences in functional levels of redox partners between the two enzyme sources. The corrected activities (contributions), the percentages of net clearance by a particular P450 at a certain substrate concentration, can be calculated according to eq. 3: clearanceenyzme[%] contributionenyzme contributionenzyme 100 (3) Inhibition Studies with Chemical Inhibitors or the MAB3A4. The effect of 3 M quinidine and 6 M omeprazole on (R,S-)DHEA formation was assessed in incubations containing 0.5 mg of human liver microsome protein/ml and 1 M or 5 M R,S-MDEA, R-MDEA, or S-MDEA (n 6 each). Control incubations contained none of these chemical inhibitors. Significance of inhibition was tested by one-tailed unpaired t test using GraphPad Prism 3.02 software (GraphPad Software Inc., San Diego, CA). The effect of MAB inhibitory to CYP3A4 DHEA formation was assessed in the same way as described for MDMA (Meyer et al., 2008). Sample Preparation. Sample preparation and metabolite quantification were also performed according to Meyer et al. (2008) with the following modifications: For detection of (R,S-)DHEA and dihydroxybenzylamine, the gas chromatography conditions were as follows: splitless injection mode; column, 5% phenyl methyl siloxane [HP-5MS; 30 m 0.25 mm (i.d.); 250-nm film thickness]; injection port temperature, 280°C; carrier gas, helium; flow rate, 1 ml/min; column temperature, 150°C increased to 250°C at 40°C/min, to 290°C at 2°C/min, and finally to 310°C. For quantification, the following target ions (m/z) were used in the selected-ion monitoring mode: m/z 437 for MDA-d5, 432 for MDA, 430 for dihydroxybenzylamine, and 780 for DHEA. Results and Discussion The applied gas chromatography-mass spectrometry conditions provided separation of DHEA enantiomers (Supplemental Figure S1), and the chosen target ions were selective for the analytes under these conditions as proven with blank samples (control microsomes without FIG. 1. The two main metabolic steps of Rand S-MDEA leading to the formation of the corresponding enantiomers of dihydroxyethylamphetamine (DHEA) and MDA. 1153 ROLE OF HUMAN P450s IN MDEA ENANTIOMERS METABOLISM at A PE T Jornals on D ecem er 2, 2017 dm d.aspurnals.org D ow nladed from