1521-009X/43/12/1864–1871$25.00 http://dx.doi.org/10.1124/dmd.115.066340 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 43:1864–1871, December 2015 Copyright ª 2015 by The American Society for Pharmacology and Experimental Therapeutics Chiral Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine and its Phase I and II Metabolites following Controlled Administration to Humans

Andrea E. Steuer, Corina Schmidhauser, Yasmin Schmid, Anna Rickli, Matthias E. Liechti, and Thomas Kraemer

Department of Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, University of Zurich, Switzerland (A.E.S, C.S., T.K.); Division of Clinical Pharmacology and Toxicology, Department of Biomedicine and Department of Clinical Research, University Hospital Basel, Switzerland (Y.S., A.R., M.E.L.)

Received July 16, 2015; accepted September 21, 2015 Downloaded from ABSTRACT Generally, pharmacokinetic studies on 3,4-methylenedioxymetham- dihydroxymethamphetamine (DHMA), and sulfate and glucuronide phetamine (MDMA) in blood have been performed after conjugate of 4-hydroxy-3-methoxymethamphetamine (HMMA) were identified, cleavage, without taking into account that phase II metabolites whereas free phase I metabolites were not detected. Stereoselec- represent distinct chemical entities with their own effects and tive differences in Cmax and AUC24 were observed with the following stereoselective pharmacokinetics. The aim of the present study was preferences: R>S for MDMA and DHMA 4-sulfate; S>R for 3,4- dmd.aspetjournals.org to stereoselectively investigate the pharmacokinetics of intact methylenedioxyamphetamine (MDA), DHMA 3-sulfate, and HMMA glucuronide and sulfate metabolites of MDMA in blood plasma after glucuronide; and no preference in Cmax for HMMA sulfate. R/S ratios a controlled single MDMA dose. Plasma samples from 16 healthy were >1 for all analytes after 24 hours, independent of the initial participants receiving 125 mg of MDMA orally in a controlled study chiral preference. These are the first data on chiral pharmacokinet- were analyzed using liquid chromatography–tandem mass spec- ics of MDMA phase II metabolites in human plasma in vivo after troscopy after chiral derivatization. Pharmacokinetic parameters controlled administration. The main human MDMA metabolites were of R-andS-stereoisomers were determined. Sulfates of 3,4- shown to be sulfate and glucuronide conjugates. at ASPET Journals on September 26, 2021

Introduction interaction with dopamine and/or serotonin metabolism, and for- 3,4-Methylenedioxymethamphetamine (MDMA, Ecstasy) is a rec- mation of reactive oxygen species (Carvalho et al., 2012). MDMA reational drug that acts by releasing dopamine, norepinephrine, and metabolites were also discussed to play a role in acute cardiovas- serotonin in the brain from presynaptic terminals via the respec- cular effects observed after MDMA consumption (Schindler et al., tive monoamine transporter (Hysek et al., 2012). In addition to 2014). amphetamine-like stimulant effects, MDMA increases empathy and As shown in Fig. 1, the main metabolic pathways of MDMA prosociality (Rietjens et al., 2012; Hysek et al., 2014) and exhibits observed in humans include O-demethylation to 3,4-dihydroxymeth- some hallucinogenic-like effects (Liechti et al., 2001). However, amphetamine (DHMA) mainly via the cytochrome P450 2D6 also proposed were severe acute poisonings, including tachycardia, (CYP2D6), followed by O-methylation mainly to 4-hydroxy-3- hypertension, hyperthermia, hyponatremia, and serotonin syn- methoxymethamphetamine (HMMA). DHMA is further sulfated by drome (Fallon et al., 1999; Kalant, 2001). Furthermore, MDMA sulfotransferases (SULT) to DHMA 3-sulfate and DHMA 4-sulfate. has been described as exhibiting neurotoxicity to serotonergic HMMA can be further conjugated by UDP-glucuronosyltransferases neurons (Monks et al., 2004; Easton and Marsden, 2006; McCann (UGT) or by SULT. A minor pathway includes the formation of 3,4- et al., 2008). methylenedioxyamphetamine (MDA) by N-demethylation followed MDMA metabolism may be responsible for neurotoxicity, presum- again by O-demethylation, O-methylation, and conjugation (Maurer, ably through the formation of glutathione adducts (Hiramatsu et al., 1996; Maurer et al., 2000; de la Torre et al., 2004). Analysis of urine 1990; Miller et al., 1997; Bai et al., 1999; Capela et al., 2009; Mueller samples after recreational MDMA consumption revealed MDMA and et al., 2009; Antolino-Lobo et al., 2010; Carvalho et al., 2012), primarily phase II metabolites (DHMA 3-sulfate, HMMA 4-sulfate, and HMMA 4-glucuronide) as major excretion products (Schwaninger et al., 2011a). Other metabolites could only be detected at negligible

This work was supported financially by the Emma Louise Kessler Fund of the concentrations. Data on disposition and concentrations of phase II Zurich Institute of Forensic Medicine and the Swiss National Science Foundation metabolites in plasma relative to their unconjugated primary metabo- [320030_149493]. lites are not available so far. Generally, pharmacokinetic studies have dx.doi.org/10.1124/dmd.115.066340. used different conjugate cleavage procedures prior to analysis (Helmlin

ABBREVIATIONS: AUC, Area under the curve; DHMA, 3,4-dihydroxymethamphetamine; HMMA, 4-hydroxy-3-methoxymethamphetamine; LOQ, limit of quantification; MDA, 3,4-methylenedioxyamphetamine; MDMA, 3,4-methylenedioxymethamphetamine; SULT, sulfotransferase; tmax, time of maximum concentration; UGT, UDP-glucuronosyltransferase.

1864 Plasma Pharmacokinetics of Chiral MDMA Metabolites 1865 et al., 1996; Fallon et al., 1999; Segura et al., 2001; Pizarro et al., R/S-DHMA sulfates, R/S-HMMA 4-sulfate, and diastereomers of HMMA 2002; Kolbrich et al., 2008; Shen et al., 2013) only allowing quantifi- 4-glucuronides were synthesized as described (Schwaninger et al., 2009, 2011c). cation of sum values for unconjugated and conjugated metabolites. Water was purified with a Millipore filtration unit and acetonitrile and methanol of However, the different conjugates represent individual chemical entities high-performance liquid chromatography grade were obtained from Fluka (Buchs, with their own potential risks of adverse effects, toxicity, or drug Switzerland). All other chemicals used were from Merck (Zug, Switzerland) and of the highest grade available. interactions. Controlled Oral MDMA Administration. We used plasma samples from Chemically, MDMA possesses a chiral center with pharmacody- a double-blind, placebo-controlled, crossover study with four experimental test namic and pharmacokinetic differences for the R-and S-enantiomers sessions (placebo-placebo, bupropion–placebo, placebo-MDMA, and bupropion- (Fallon et al., 1999; Kalant, 2001; Kraemer and Maurer, 2002; Peters MDMA) that were performed in a counterbalanced order according to a Latin et al., 2003; Pizarro et al., 2004; Peters et al., 2005). Whereas the square randomization design as described in detail by Schmid et al. (2015). The S-MDMA enantiomer primarily causes the described stimulating clinical study was conducted at the University Hospital of Basel in accordance effects, R-MDMA produces more hallucinogenic effects (de la Torre with the Declaration of Helsinki and International Conference on Harmonization et al., 2004). Elimination of S-MDMA was shown to be faster Guidelines in Good Clinical Practice and with approval by the Ethics Commit- compared with the R-enantiomer (Fallon et al., 1999; Kalant, 2001; tee of the Canton of Basel, Switzerland, and the Swiss Agency for Therapeu- Kraemer and Maurer, 2002; Peters et al., 2003, 2005; Pizarro et al., tic Products (Swissmedic). The study was registered at ClinicalTrials.gov (NCT01771874). All subjects provided written informed consent and were 2004) most probably owing to stereoselective metabolism that has paid for their participation. been extensively studied in vitro (Meyer et al., 2008; Meyer and

Plasma samples from 16 healthy Caucasian subjects (eight men and eight Downloaded from Maurer, 2009; Schwaninger et al., 2009, 2011b). As not only women, age 20–27, body mass index of 22.7 6 2.1 kg/m2) from the placebo- stereoselectivity of MDMA itself but also of its primary metabolite MDMA session were analyzed in the Institute of Forensic Pharmacology and DHMA is discussed in terms of (neuro-)toxicity (Felim et al., 2010; Toxicology Zurich as described in Sample Preparation and Analysis following. Martinez et al., 2012), the further metabolic fate of DHMA and Placebo was administered at 8:00 AM and MDMA (125 mg p.o.) at 10:00 AM. resulting stereoselectivities of metabolites is of interest. Comprehen- Blood samples were collected at 0, 0.33, 0.67, 1, 1.5, 2, 2.5, 3, 4, 6, 8, and sive controlled pharmacokinetic studies on the stereoselectivity of 24 hours after MDMA administration. MDMA and all relevant metabolites—including phase II metabolites— Sample Preparation and Analysis. Blood plasma samples were analyzed dmd.aspetjournals.org are still missing but are needed to understand the entire stereoselective using a Thermo Fisher Ultimate 3000 UHPLC system coupled to an ABSciex disposition of MDMA and its differences in pharmacological effects and QTRAP 5500 in positive electrospray ionization (ESI) mode. Chromatography kinetics. was performed on a Phenomenex Kinetex C18 column after chiral derivatization with Marfey’s reagent (N-(2,4-dinitro-5-fluorophenyl) L-valinamide) as de- Therefore, the aim of this study was first to assess the extent of scribed in detail by Steuer et al. (2015). Briefly, to 200 ml plasma mixed with glucuronidation and sulfation of DHMA and HMMA in blood plasma 20 ml of the internal standard (IS) mixture (MDMA-d5, MDA-d5, pholedrine, and second to characterize plasma pharmacokinetics of MDMA and dihydroxybenzylamine, 2.5 mM each), 1000 ml of acetonitrile were added; the phase I and II metabolites stereoselectively, following controlled oral mixture was shaken and centrifuged (10,000g, 5 minutes). An aliquot of 1000 ml at ASPET Journals on September 26, 2021 MDMA administration to humans. was transferred into an autosampler vial, 50 ml of formic acid were added and the mixture was evaporated to dryness under a gentle stream of nitrogen at 40C. The residue was dissolved in 100 ml of carbonate buffer (1 M, pH 9). N-(2,4-dinitro- Materials and Methods 5-fluorophenyl) L-valinamide DNPV (100 ml, 0.3% in acetone) was added, and Hydrochlorides of racemic MDA, HMA, MDMA, HMMA, and DHMA the mixture was left in a heating block for 30 minute at 50C. Afterward the and methanolic solutions (1 mg/ml) of MDA-d5 and MDMA-d5 were obtained reaction was stopped by the addition of 20 ml of 1 M HCl. Finally, 80 mlof from Lipomed (Arlesheim, Switzerland). 4-Hydroxymethamphetamine, 3,4- a mixture of mobile phases A and B (1:1, v/v) were added, and aliquots of 10 ml dihydroxybenzylamine and the derivatization reagent N-(2,4-dinitro-5- of this solution were injected into the liquid chromatography–tandem mass fluorophenyl) L-valinamide were from Sigma-Aldrich (Buchs, Switzerland). spectroscopy system. Full derivatization was observed with no peaks for

Fig. 1. Main metabolic pathways of MDMA. The asterisk marks the chiral center. 1866 Steuer et al. underivatized analytes left and the formed diastereomers were baseline- separated from all analytes except for DHMA. The method was fully validated, including selectivity, recovery, matrix effects, bias and imprecision, stabilities, and limit of quantification (LOQ) and criteria were fulfilled and LOQs sufficient for the expected plasma concentrations of all analytes except for DHMA (Steuer et al., 2015). All blood plasma samples of a previous study (Schmid et al., 2015) were stored at –20C prior to analysis. Data on stability of MDMA and its phase I metabolites were previously published and no instability could be observed up to 6 months (Clauwaert et al., 2001). Sufficient stability of phase II metabolites under these storage conditions for 24 months and over at least three freeze/thaw cycles were shown during method validation (Steuer et al., 2015). Aliquots of plasma analyzed in the present pharmacokinetic study were thawed at a maximum of two times. Data Correlation. For data correlation to previous racemic data (Schmid et al., 2015), R-andS-stereoisomers of all analytes were summed up. MDMA, MDA, and total HMMA (sum of glucuronide and sulfate), HMMA sulfate, and HMMA glucuronide were compared with concentrations obtained after conjugate cleavage and racemic analysis (Schmid et al., 2015) using

Spearman correlations in GraphPad Prism 6.0 (GraphPad Software, La Jolla, Downloaded from CA). Detectability of MDMA and Metabolites. For evaluation of racemic MDMA or MDMA metabolites, amounts of R- and S-stereoisomers were summed up. Percentages of MDMA or metabolites at different time points were given as the amount of each analyte (racemate) relative to the sum of all analytes (racemates) detected, set to 100%.

Pharmacokinetic Analysis. Concentration maxima (Cmax), time of maxi- dmd.aspetjournals.org mum concentration (tmax), area under the concentration-time curve 0–24 hours (AUC24), AUC0–infinity (AUC‘), and terminal elimination half-life (t1/2) were calculated for all analytes using noncompartmental analysis with PK solutions 2.0 software (Summit Research Services, Montrose, CO). The time interval after dosing for the first positive sample was designated as the time of first detection

(tonset). Statistical data comparison between pharmacokinetic parameters of R- and S-stereoisomers was performed using a nonparametric Wilcoxon matched pairs signed rank test, as most parameters were not normally distributed at ASPET Journals on September 26, 2021 according to D’Agostino and Pearson normality tests. Gender differences in kinetic parameters were compared using a nonparametric Mann-Whitney test (confidence interval 95%). All statistical calculations were performed with GraphPad Prism (confidence interval 95%).

Results Comparison with Previous Data. A comparison of the concen- trations previously obtained after enzymatic cleavage (study A) (Schmid et al., 2015) with the sum of the two stereoisomers in the present study (study B) showed good correlation for MDMA (Spearman correlation coefficient r=0.942) and MDA (r=0.939). Total HMMA concentrations (HMMA sulfate + glucuronide) were higher in the present study, but good correlation was observed between HMMA from study A with HMMA glucuronide from study B (r=0.96), but not with HMMA sulfate from study B (r= 0.86) (Fig. 2). Detectability of MDMA and Metabolites. Of all plasma samples post-MDMA dose, 98% were positive for MDMA and 90% for MDA. Unconjugated DHMA could not be detected in any of the analyzed plasma samples, whereas DHMA 3-sulfate and DHMA 4-sulfate were found in 99% of the samples. Free HMMA was only detectable in traces Fig. 2. Total HMMA concentrations (mM) determined after enzymatic cleavage in far below the LOQ of the method, but HMMA sulfate could be seen in a previous study (x axis) and sum of intact stereoisomers of HMMA glucuronide and 100% and HMMA glucuronide in 99% of all plasma samples. Mean HMMA sulfate (mM) (y-axis) (A); sum of intact stereoisomers of HMMA sulfate (mM) concentrations of MDMA and each MDMA metabolite at different (B); and sum of intact stereoisomers of HMMA glucuronide (mM) (C). Lines given in 2 times are given in Fig. 3. Immediately after dosing (0.33 hours) until 1 bold print represent deviations from unity of +/ 30%. hour postdose, DHMA 3-sulfate and HMMA sulfate represent the most abundant metabolites. In contrast, MDMA reached much lower metabolites but reached comparable concentrations 4 hours postdose. concentrations at the earliest time point but steadily increased until 2 MDA and DHMA 4-sulfate represented less abundant metabolites. hours, then it represented the most abundant compound in plasma. Chiral Pharmacokinetic Analysis. Plasma concentration time HMMA glucuronide increased more slowly compared with sulfate profiles for R-andS-stereoisomers and the corresponding R/S ratios Plasma Pharmacokinetics of Chiral MDMA Metabolites 1867

Fig. 3. Concentrations of MDMA and metabolites (mean of 16 participants) at different time points after ingestion of 125 mg MDMA-HCl. Downloaded from over time of MDMA, MDA, DHMA 3-sulfate, DHMA 4-sulfate, Gender differences were observed particularly for R- and S-MDMA HMMA sulfate, and HMMA glucuronide are shown in Fig. 4. with significantly higher Cmax and AUC24 values in females compared Stereoselective differences were noted for all analytes except for with males (p , 0.001), but no differences were observed in other HMMA sulfate in the first 6 hours after MDMA administration. Higher pharmacokinetic parameters. By contrast, males showed higher Cmax dmd.aspetjournals.org concentrations of S-stereoisomers were observed for MDA, DHMA and AUC24 values for S-HMMA sulfate and S-HMMA glucuronide 3-sulfate, and HMMA glucuronide up to 8 hours after drug adminis- (p,0.05), but no significant differences for the respective R-stereoisomers tration, whereas MDMA and DHMA 4-sulfate showed the opposite or in either DHMA sulfate isomers. stereoselectivity. After 24 hours, all analytes increased to R/S . 1 independent of the initial stereo-preference. R/S ratios of MDMA increased over the entire time period studied, while ratios of all Discussion metabolites were rather constant in the first 8 hours postdose, but Comparing results to the previous achiral analysis after enzymatic rapidly increased between 8 and 24 hours. Table 1 summarizes the cleavage (Schmid et al., 2015) showed good correlation for MDMA and at ASPET Journals on September 26, 2021 calculated pharmacokinetic parameters for all R- and S-stereoisomers. MDA, whereas the sum value for HMMA was significantly higher in The elimination half-life and AUC‘ could not be determined for the present study. However, comparing HMMA sulfate and HMMA R-MDA as the terminal elimination phase was not reached within the glucuronide separately, good correlation was observed for HMMA and 24 hours of sample collection. Median times of first detection for MDMA, HMMA glucuronide but not for HMMA and HMMA sulfate (Fig. 2). DHMA 3-sulfate, DHMA 4-sulfate, HMMA sulfate, and HMMA glucu- This finding can be explained by the fact that enzymatic cleavage often ronide were 0.33 hours for both R-andS- stereoisomers. Only MDA was results in insufficient cleavage of sulfates (Segura et al., 2001). Similar detected considerably later, with median values, of 0.8 and 0.7 hours results were obtained in preanalytic studies (data not shown) with for R- and S-MDA, respectively. After 24 hours both stereoisomers of sulfate cleavage efficiency for Helix pomatia of only 15% despite MDMA and of all metabolites were still detectable. declared sulfatase activity. Statistically significant differences in Cmax between the two Free DHMA could not be detected in any plasma sample as stereoisomers could be seen for all analytes except for HMMA sulfate, reported previously (Segura et al., 2001). Owing to rather high LOQs while AUC24 values were significantly different for all stereoisomers (Steuer et al., 2015) presence of low DHMA amounts cannot be (Table 1). The highest median Cmax and AUC24 was obtained for excluded. However, free HMMA also was not detectable (LOQ R-MDMA, followed by S-MDMA, S-DHMA 3-sulfate, and R-HMMA 0.0025 mM per enantiomer). By contrast, high concentrations of sulfate. Median Cmax and AUC24 of MDA and DHMA 4-sulfate were DHMA 3-sulfate and HMMA sulfate were observed. For HMMA, lower by a factor of approximately 10. High intersubject variability also the respective glucuronide was present in abundant concen- was observed for MDMA and all metabolites, especially for HMMA trations. In initial experiments, glucuronides of DHMA were not glucuronide. In particular, one participant showed much higher HMMA detected in blood, which is in line with previous findings (Schwaninger glucuronide Cmax and AUC values, compared with all other participants et al., 2012). Consistently, Segura et al. (2001), using different (Fig. 5), but comparable kinetics for MDMA and all other metabolites. cleavage procedures, concluded that DHMA glucuronides were MDMA tmax was reached after 2.7 and 1.9 hours for R- and minor metabolites. S-enantiomer, respectively. MDA had the longest tmax of all metabolites Immediately after dosing, DHMA 3-sulfate and HMMA sulfate analyzed, being significantly shorter for S-MDA compared with presented as the most abundant analytes (Fig. 3). DHMA 4-sulfate R-MDA. In contrast, sulfates reached their maximum concentrations represented only a minor metabolite, indicating regioselective sulfation faster, after about 2 hours with no significant differences between the in position 3 as previously shown in vitro (Schwaninger et al., 2011c). two enantiomers. HMMA glucuronide diastereomers also did not differ HMMA glucuronide increased more slowly but reached comparable in their time to reach maximum concentrations but peaked later than amounts to sulfates after 4 hours. Previous in vitro data assumed that MDMA and sulfate metabolites. Elimination half-lives were signif- sulfation should outweigh glucuronidation for HMMA. In vivo, icantly longer for R-stereoisomers of all analytes with median half- sulfation initially predominated, changing to comparable amounts of lives of R-stereoisomers at least 1.5 times those of S-stereoisomers both conjugates after approximately 3 hours, explainable by differences (Table 1). in enzymatic affinity and capacity observed in vitro. While SULTs 1868 Steuer et al. Downloaded from dmd.aspetjournals.org

Fig. 4. Chiral concentration-time profiles (left panel) and respective R/S ratios over time (right panel) of MDMA (A), MDA (B), DHMA 3-sulfate (C), DHMA 4-sulfate (D), HMMA sulfate (E), and HMMA glucuronide (F). Solid lines represent R, broken lines S-stereoisomer. Dotted lines in the right panels symbolize theoretically equal amounts of R- and S-stereoisomers. Data are mean + S.E.M. in 16 subjects. at ASPET Journals on September 26, 2021

show much higher affinity for HMMA, they are easily saturated, and the in heart rate in rats compared with MDMA (Schindler et al., 2014). higher capacity UGTs can take over (Schwaninger et al., 2009, 2011a, However, as our and other results (Segura et al., 2001) suggest, DHMA 2011c). Consequently, the phase II metabolites should be the main is not present in blood unconjugated in relevant concentrations. metabolites in plasma. This finding is of interest, e.g., in terms of Half an hour after MDMA consumption all participants were potential toxic effects of MDMA metabolites. Specifically, cardiovas- positive for both stereoisomers of MDMA and metabolites. MDA was cular effects were demonstrated for DHMA with even greater increases only detected later. After 24 hours both stereoisomers of all analytes Plasma Pharmacokinetics of Chiral MDMA Metabolites 1869

were still detectable. Therefore, the simple presence of a particular metabolite does not aid in distinguishing between recent or earlier 9.4) 26.6) 11.6) 9.4) 14.0) 7.6) 6.1) – – – – – – – MDMA intake. No conclusion on duration of detectability of MDMA phase II metabolites in plasma was possible from the current data owing to the limited blood collection time. Other (h) * *** ***

1/2 studies were performed over 48 (Pizarro et al., 2004) or even 143 t

36) 6.9 (5.2 (Kolbrich et al., 2008) hours with HMMA still being positive after 61.1) 5.8 (4.7 46.7) 8.3 (6.3 40.6) 6.9 (5.7 61.1) 5.8 (4.7 – 14.4) 4.4 (3.5 – – – – – 95 hours. Various racemic pharmacokinetic studies on MDMA and some metabolites after controlled administration are available (Kolbrich et al., 2008; Shen et al., 2013; Farre et al., 2015). The following discussion will mainly focus on enantioselective effects. Generally, enantiomeric MDMA and MDA pharmacokinetics in 15.5) 15.8 (6.9 1.5) 20.6 (7.6 15.5) 15.8 (6.9 1.5) n.d. 8.6 (5.0 – – 12.7) 21.2 (9.1 – – 6.8) 16.5 (12.1 9.4) 7.7 (5.7 – – – the present study were comparable to those published by others ) 1

– (Pizarro et al., 2004). Among the conjugates, highest concen-

Mh trations were observed for S-DHMA 3-sulfate and S-HMMA m (

** glucuronide followed by R-andS-HMMA sulfate. Median MDA n.s. *** total and DHMA 4-sulfate were approximately 10 times lower. Metab- Downloaded from 16.6) 4.1 (0.84 6.4) 0.75 (0.30 10.1) 5.6 (2.1 16.6) 4.1 (0.84 – – – – 14.1) 4.1 (1.5 19.0) 5.9 (2.5 AUC – – olism was suspected to be the main cause of the stereoselective disposition (Fallon et al., 1999; Pizarro et al., 2004; Peters et al., -stereoisomers

S 2005), and in vitro experiments revealed preferences for the 8.7 (2.3 formation of MDA, DHMA, DHMA sulfate, HMMA, and HMMA - and

R glucuronide, whereas HMMA sulfation was not enantioselective 14.7) 3.9 (0.51 1.0) 1.7 (0.46 14.7) 3.9 (0.51 1.1) n.d. 0.76 (0.36 – – 11.2) 3.5 (0.94 – – 6.4) 9.0) 10.8 (4.7 (Meyer et al., 2008; Meyer and Maurer, 2009; Schwaninger et al., – – – dmd.aspetjournals.org )

1 2009, 2011b). These findings are in line with the chiral pharma- – cokinetic analysis showing higher Cmax for the S-stereoisomer of Mh m ( MDA, DHMA 3-sulfate, and HMMA glucuronide, higher Cmax ** *** *** 24h

– for R-MDMA and R-DHMA 4-sulfate, and no significant differ- 0 10.6) 3.9 (0.75 10.6) 3.9 (0.75 2.9) 0.63 (0.23 4.5) 5.2 (1.9 0.5) 0.61 (0.29 ence in HMMA sulfate. Pizarro et al., (2004) found no differ- – 15.2) 5.5 (2.4 – 8.5) 3.6 (1.3 AUC – – ences in enantioselectivity of HMMA, however, after cleavage of conjugates.

In contrast to Cmax values, significant differences in AUC24 at ASPET Journals on September 26, 2021 were observed for all analytes including HMMA sulfate with TABLE 1 2.7) 5.2 (1.2 4.9) 1.7 (0.29 – 2.7) 0.80 (0.38 – 2.5) 1.9 (0.49 4.9) 1.7 (0.29 6.1) 0.25 (0.13 – 3.6) 9.6 (4.2 – – – – – – – higher AUC24 for R-enantiomer. The reason for that lies most probably in the increased concentrations of R-HMMA sulfate relative to its S-enantiomer after 8 hours (Fig. 4). One explanation (h) might be that the elimination half-life for S-HMMA sulfate is n.s. n.s. n.s. max t significantly shorter compared with its R-enantiomer, resulting in 5.8) 1.8 (0.9 9.1) 2.6 (1.5 4.8) 1.8 (0.9 6.4) 1.7 (1.0 9.1) 2.6 (1.5 24.0) 4.4 (3.7 4.6) 1.9 (1.3 – – – – – – – higher concentrations of R-HMMA sulfate with increasing time after dosing. However, elimination half-lives for S-stereoisomers are significantly shorter for all analytes. Still, HMMA glucuronide or DHMA 3-sulfate, for example, had significantly higher AUC values of S-isomers. The conjugates should represent the final 1.4) 3.4 (1.7 0.095) 1.9 (1.2 1.4) 3.4 (1.7 0.061) 8.0 (6.0 0.70) 1.6 (1.2 1.1) 2.1 (0.9 0.95) 2.7 (1.8

0.0. metabolites. That leads to the conclusion that stereoselective

, formation for DHMA 3-sulfate and HMMA glucuronide out- p weighs stereoselective elimination. Concerning MDMA, mainly

M) metabolism to DHMA followed to S-DHMA sulfate and HMMA m 0.01, *** ( Chiral pharmacokinetic data of MDMA and its metabolites and statistical comparison of ** n.s.

, should be responsible for the faster elimination half-life of the *** max p

C S-enantiomer. Summation of AUC24 values of MDMA and all 0.62) 0.34 (0.12 – 0.70) 0.29 (0.054 – 0.17) 0.037 (0.017 – 0.31) 0.49 (0.15 – 0.70) 0.29 (0.054 – 0.032) 0.038 (0.018 – – – – – – – 1.2) 0.56 (0.29 – analyzed metabolites for R-andS-stereoisomers, respectively, – 0.05, ** m RSRSRSRSRS result in mean values of 18.9 M/h for both stereoisomers. ,

p Metabolites exist more as S (14.0 versus 10.2 mM/h), and MDMA more as R-stereoisomers (Table 1). Gender differences were observed, especially in both MDMA enantiomers being significantly higher in females, in line with previous racemic data (Kolbrich et al., 2008). Likewise, HMMA sulfate and glucuronide isomers tended to be lower in females, but significant effects were only present for S-stereoisomers. Mean (SD) 0.31 (0.15) 0.36 (0.16) 2.0 (1.2) 1.8 (0.6) 4.8 (2.0) 3.7 (1.5) 8.3 (3.6) 4.0 (1.6) 18.2 (6.0) 6.6 (0.7) Mean (SD) 0.18 (0.18) 0.41 (0.38) 3.9 (1.9) 3.1 (1.0) 2.8 (2.7) 4.4 (3.7) 4.8 (4.1) 4.7 (3.8 21.6 (14.7) 6.3 (1.2) Mean (SD) 0.071 (0.047) 0.050 (0.024) 2.2 (1.0) 1.7 (0.5) 1.1 (0.72) 0.59 (0.26) 2.2 (1.6) 0.76 (0.36) 21.2 (11.4) 10.1 (5.3) Mean (SD) 0.13 (0.083) 0.53 (0.26) 2.5 (1.7) 1.6 (0.4) 2.1 (1.1) 5.6 (2.8) 4.3 (2.6) 6.3 (3.2) 21.8 (7.7) 7.2 (1.4) Mean (SD) 0.014 (0.006) 0.041 (0.011) 13.6 (8.3) 4.5 (0.7) 0.25 (0.09) 0.62 (0.17) n.d. 0.78 (0.27) n.d. 9.5 (2.5) Mean (SD) 0.72 (0.20) 0.59 (0.17) 2.9 (0.7) 2.1 (0.7) 9.3 (2.7) 5.3 (1.7) 11.2 (4.0) 5.5 (1.7) 8.6 (2.6) 4.6 (0.7) Median (Range) 0.31 (0.074 Median (Range) 0.10 (0.016 Median (Range) 0.052 (0.029 Median (Range) 0.11 (0.029 Median (Range) 0.10 (0.016 Median (Range) 0.014 (0.006 Median (Range) 0.70 (0.36 Most probably, MDMA metabolism is more prominent in the first hours after ingestion whereas excretion dominates later. That can also be seen when looking at R/S ratios over time. Independent of the initial ratios, increases to values above 1 for all metabolites was n.s., not significant; n.d., not determined; * HMMA G HMMA S DHMA 4S DHMA 3S *** n.s. *** ** *** MDA *** *** *** n.d. n.d. MDMA *** *** *** *** *** Analyte observed between 8 and 24 hours. Generally, initial stereoisomer 1870 Steuer et al.

Fig. 5. Concentration-time profiles (left panel) and HMMA glucuronide/HMMA sulfate ratio (right panel) of participant 1 (bold print) versus mean + S.E.M. of2–15 participants (gray box), revealing obvious higher concentration of HMMA glucuronide and increased HMMA glucuronide/HMMA sulfate ratios. Solid lines represent R, broken lines S-stereoisomer (left panel) or HMMA glucuronide/HMMA sulfate ratios of R- and S-stereoisomers, respectively. Downloaded from preferences (0–6 hours) of the metabolites mimicked those observed in this observation but seems to be of lower relevance considering in vitro in vitro experiments. Only MDMA showed steady increases in R/S ratio enzyme activities. over the time period. Variation of MDMA R/S ratios based on time The presented study bears some limitations. Only one relatively high postdose, were used to estimate MDMA ingestion time (Fallon et al., MDMA dose was administered that could already have resulted in self- 1999). In urine, better accuracy for such estimations was obtained inhibition of CYP2D6 and possibly altered metabolism. CYP2D6 dmd.aspetjournals.org through metabolite ratios (Schwaninger et al., 2012). However, in blood enzyme status was not taken into account and sampling was only plasma only MDMA R/S ratio follows a steady trend. Metabolites might performed over 24 hours. be useful for a rough estimation of whether intake occurred in the last 6 hours or later, as, for example, DHMA 3-sulfate, HMMA glucuro- Acknowledgments nide, and MDA had R/S ratios ,1 in the first 6 hours and switched to . The authors thank Dr. Markus Baumgartner and Michael Poetzsch for their ratios 1 after 24 hours. support and discussion.

The primary metabolic step in MDMA deactivation is the CYP2D6- at ASPET Journals on September 26, 2021 catalyzed O-demethylation. With CYP2D6 being polymorphically expressed, substantial interindividual differences in MDMA and Authorship Contributions metabolite plasma concentrations are expected. Furthermore, MDMA Participated in research design: Steuer, Kraemer, Liechti. Conducted experiments: Steuer, Schmidhauser. is a mechanism-based inhibitor of CYP2D6 resulting in an irreversible Performed data analysis: Steuer, Schmidhauser. inhibition of its own metabolism in high or after repeated doses (de la Wrote or contributed to the writing of the manuscript: Steuer, Kraemer, Torre et al., 2000a, 2000b; Shen et al., 2013; Farre et al., 2015). The Liechti. dose of MDMA in this study corresponds to a common recreational dose. All metabolites except for MDA are secondarily or even tertiarily formed metabolites from the initial CYP2D6 step. As in vitro experi- References ments suggest that only CYP2D6 contributes relevantly to enantio- Antolino-Lobo I, Meulenbelt J, Nijmeijer SM, Scherpenisse P, van den Berg M, and van Duursen MBM (2010) Differential roles of phase I and phase II enzymes in 3,4-methylendioxymethamphetamine- selectivity of the primary metabolic step (Meyer et al., 2008), differences induced cytotoxicity. Drug Metab Dispos 38:1105–1112. in CYP2D6 phenotype and/or its inhibition by other xenobiotics or from Bai F, Lau SS, and Monks TJ (1999) Glutathione and N-acetylcysteine conjugates of alpha- methyldopamine produce serotonergic neurotoxicity: possible role in methylenedioxyamphetamine- self-inhibition probably result in changed enantioselectivity. mediated neurotoxicity. Chem Res Toxicol 12:1150–1157. Substantial interindividual variation was observed among the Capela JP, Carmo H, Remião F, Bastos ML, Meisel A, and Carvalho F (2009) Molecular and cellular mechanisms of ecstasy-induced neurotoxicity: an overview. Mol Neurobiol 39: participants. In particular, one participant showed considerably higher 210–271. HMMA glucuronide concentrations (Fig. 5). This cannot be an effect Carvalho M, Carmo H, Costa VM, Capela JP, Pontes H, Remião F, Carvalho F, and Bastos MdeL only of altered CYP2D6 status, as no influence on MDMA and the other (2012) Toxicity of amphetamines: an update. Arch Toxicol 86:1167–1231. Clauwaert KM, Van Bocxlaer JF, and De Leenheer AP (2001) Stability study of the designer 2D6-dependently formed metabolites was observable. Various UGT drugs “MDA, MDMA and MDEA” in water, serum, whole blood, and urine under various isoenzymes are able to catalyze HMMA glucuronidation in vitro storage temperatures. Forensic Sci Int 124:36–42. de la Torre R, Farré M, Ortuño J, Mas M, Brenneisen R, Roset PN, Segura J, and Camí J (2000a) (Schwaninger et al., 2009; Shoda et al., 2009). Only UGT2B15 showed Non-linear pharmacokinetics of MDMA (‘ecstasy’) in humans. Br J Clin Pharmacol 49: adequate enzymatic activity in an initial activity screening (Shoda et al., 104–109. de la Torre R, Farré M, Roset PN, Lopez CH, Mas M, Ortuño J, Menoyo E, Pizarro N, Segura J, 2009). However, this cannot prove general involvement in human liver and Cami J (2000b) Pharmacology of MDMA in humans. Ann N Y Acad Sci 914:225–237. at physiologic HMMA concentrations. Schwaninger et al. (2009) de la Torre R, Farré M, Roset PN, Pizarro N, Abanades S, Segura M, Segura J, and Camí J (2004) Human pharmacology of MDMA: pharmacokinetics, metabolism, and disposition. review Ther performed enzyme kinetic studies also in human liver microsomes and Drug Monit 26:137–144. for various UGT isoenzymes, including UGT1A9, UGT2B7, UGT2B15, Easton N and Marsden CA (2006) Ecstasy: are animal data consistent between species and can they translate to humans? review J Psychopharmacol 20:194–210. and UGT2B17 also coming to the conclusion that UGT2B15 showed the Fallon JK, Kicman AT, Henry JA, Milligan PJ, Cowan DA, and Hutt AJ (1999) Stereospecific highest activity. UGT2B15 is polymorphically expressed (Guillemette, analysis and enantiomeric disposition of 3, 4-methylenedioxymethamphetamine (Ecstasy) in humans. Clin Chem 45:1058–1069. 2003) but phenotypic outcome is still controversially discussed. Neverthe- Farre M, Tomillero A, Perez-Mana C, Yubero S, Papaseit E, Roset PN, Pujadas M, Torrens M, less, this could be a probable explanation for the elevated glucuronide Cami J, and de la Torre R (2015) Human pharmacology of 3,4-methylenedioxymethamphet- amine (MDMA, ecstasy) after repeated doses taken 4h apart Human pharmacology of concentrations in this participant. Further genetic differences in other MDMA after repeated doses taken 4h apart. Eur Neuropsychopharmacol DOI: 10.1016/ UGTs such as UGT2B7 (Guillemette, 2003) might also contribute to j.euroneuro.2015.05.007. [published ahead of print]. Plasma Pharmacokinetics of Chiral MDMA Metabolites 1871

Felim A, Herrera G, Neudörffer A, Blanco M, O’Connor JE, and Largeron M (2010) Synthesis MDA and its application to samples from a controlled study with MDMA. Clin Chem 51: and in vitro cytotoxicity profile of the R-enantiomer of 3,4-dihydroxymethamphetamine 1811–1822. (R-(-)-HHMA): comparison with related catecholamines. Chem Res Toxicol 23:211–219. Pizarro N, Ortuño J, Farré M, Hernández-López C, Pujadas M, Llebaria A, Joglar J, Roset PN, Guillemette C (2003) Pharmacogenomics of human UDP-glucuronosyltransferase enzymes. Mas M, and Segura J, et al. (2002) Determination of MDMA and its metabolites in blood and Pharmacogenomics J 3:136–158. urine by gas chromatography-mass spectrometry and analysis of enantiomers by capillary Helmlin HJ, Bracher K, Bourquin D, Vonlanthen D, and Brenneisen R (1996) Analysis of electrophoresis. J Anal Toxicol 26:157–165. 3,4-methylenedioxymethamphetamine (MDMA) and its metabolites in plasma and urine Pizarro N, Farré M, Pujadas M, Peiró AM, Roset PN, Joglar J, and de la Torre R (2004) by HPLC-DAD and GC-MS. see comments J Anal Toxicol 20:432–440. Stereochemical analysis of 3,4-methylenedioxymethamphetamine and its main metabolites in Hiramatsu M, Kumagai Y, Unger SE, and Cho AK (1990) Metabolism of methylenediox- human samples including the catechol-type metabolite (3,4-dihydroxymethamphetamine). ymethamphetamine: formation of dihydroxymethamphetamine and a quinone identified as its Drug Metab Dispos 32:1001–1007. glutathione adduct. J Pharmacol Exp Ther 254:521–527. Pizarro N, Ortuño J, Farré M, Hernández-López C, Pujadas M, Llebaria A, Joglar J, Roset PN, Hysek CM, Simmler LD, Nicola VG, Vischer N, Donzelli M, Krähenbühl S, Grouzmann E, Mas M, and Segura J, et al. (2002) Determination of MDMA and its metabolites in blood and Huwyler J, Hoener MC, and Liechti ME (2012) Duloxetine inhibits effects of MDMA (“ec- urine by gas chromatography-mass spectrometry and analysis of enantiomers by capillary stasy”) in vitro and in humans in a randomized placebo-controlled laboratory study. PLoS One electrophoresis. J Anal Toxicol 26:157–165. 7:e36476. Rietjens SJ, Hondebrink L, Westerink RH, and Meulenbelt J (2012) Pharmacokinetics and Hysek CM, Schmid Y, Simmler LD, Domes G, Heinrichs M, Eisenegger C, Preller KH, Quednow pharmacodynamics of 3,4-methylenedioxymethamphetamine (MDMA): interindividual dif- BB, and Liechti ME (2014) MDMA enhances emotional empathy and prosocial behavior. Soc ferences due to polymorphisms and drug-drug interactions. Crit Rev Toxicol 42:854–876. Cogn Affect Neurosci 9:1645–1652. Schindler CW, Thorndike EB, Blough BE, Tella SR, Goldberg SR, and Baumann MH (2014) Kalant H (2001) The pharmacology and toxicology of “ecstasy” (MDMA) and related drugs. Effects of 3,4-methylenedioxymethamphetamine (MDMA) and its main metabolites on car- review CMAJ 165:917–928. diovascular function in conscious rats. Br J Pharmacol 171:83–91. Kolbrich EA, Goodwin RS, Gorelick DA, Hayes RJ, Stein EA, and Huestis MA (2008) Plasma Schmid Y, Rickli A, Schaffner A, Duthaler U, Grouzmann E, Hysek CM, and Liechti ME (2015) pharmacokinetics of 3,4-methylenedioxymethamphetamine after controlled oral administration Interactions between bupropion and 3,4-methylenedioxymethamphetamine in healthy subjects. to young adults. Ther Drug Monit 30:320–332. J Pharmacol Exp Ther 353:102–111. Kraemer T and Maurer HH (2002) Toxicokinetics of amphetamines: metabolism and tox- Schwaninger AE, Meyer MR, Zapp J, and Maurer HH (2009) The role of human UDP- icokinetic data of designer drugs, amphetamine, methamphetamine, and their N-alkyl deriva- glucuronyltransferases on the formation of the methylenedioxymethamphetamine (ecstasy) Downloaded from tives. review Ther Drug Monit 24:277–289. phase II metabolites R- and S-3-methoxymethamphetamine 4-O-glucuronides. Drug Metab Liechti ME, Geyer MA, Hell D, and Vollenweider FX (2001) Effects of MDMA (ecstasy) on Dispos 37:2212–2220. prepulse inhibition and habituation of startle in humans after pretreatment with citalopram, Schwaninger AE, Meyer MR, Barnes AJ, Kolbrich-Spargo EA, Gorelick DA, Goodwin RS, haloperidol, or ketanserin. Neuropsychopharmacology 24:240–252. Huestis MA, and Maurer HH (2011a) Urinary excretion kinetics of 3,4-methylenedioxy- Martinez CM, Neudörffer A, and Largeron M (2012) A convenient biomimetic synthesis of methamphetamine (MDMA, ecstasy) and its phase I and phase II metabolites in humans optically active putative neurotoxic metabolites of MDMA (“ecstasy”) from R-(-)- and following controlled MDMA administration. Clin Chem 57:1748–1756. S-(+)-N-methyl-a-methyldopamine precursors. Org Biomol Chem 10:3739–3748. Schwaninger AE, Meyer MR, and Maurer HH (2011b) Investigation on the enantioselectivity of Maurer HH (1996) On the metabolism and the toxicological analysis of methylenedioxy- the sulfation of the methylenedioxymethamphetamine metabolites 3,4-dihydroxymethamphetamine phenylalkylamine designer drugs by gas chromatography-mass spectrometry. Ther Drug Monit and 4-hydroxy-3-methoxymethamphetamine using the substrate-depletion approach. Drug dmd.aspetjournals.org 18:465–470. Metab Dispos 39:1998–2002. Maurer HH, Bickeboeller-Friedrich J, Kraemer T, and Peters FT (2000) Toxicokinetics and Schwaninger AE, Meyer MR, Zapp J, and Maurer HH (2011c) Sulfation of the 3,4-methy- analytical toxicology of amphetamine-derived designer drugs (‘Ecstasy’). Toxicol Lett 112- lenedioxymethamphetamine (MDMA) metabolites 3,4-dihydroxymethamphetamine (DHMA) 113:133–142. and 4-hydroxy-3-methoxymethamphetamine (HMMA) and their capability to inhibit human McCann UD, Szabo Z, Vranesic M, Palermo M, Mathews WB, Ravert HT, Dannals RF, sulfotransferases. Toxicol Lett 202:120–128. and Ricaurte GA (2008) Positron emission tomographic studies of brain dopamine and sero- Schwaninger AE, Meyer MR, Barnes AJ, Kolbrich-Spargo EA, Gorelick DA, Goodwin RS, tonin transporters in abstinent (+/-)3,4-methylenedioxymethamphetamine (“ecstasy”) users: Huestis MA, and Maurer HH (2012) Stereoselective urinary MDMA (ecstasy) and metabolites relationship to cognitive performance. Psychopharmacology (Berl) 200:439–450. excretion kinetics following controlled MDMA administration to humans. Biochem Pharmacol Meyer MR and Maurer HH (2009) Enantioselectivity in the methylation of the catecholic phase I 83:131–138. metabolites of methylenedioxy designer drugs and their capability to inhibit catechol-O- Segura M, Ortuño J, Farré M, McLure JA, Pujadas M, Pizarro N, Llebaria A, Joglar J, Roset PN, – methyltransferase-catalyzed dopamine 3-methylation. Chem Res Toxicol 22:1205 1211. and Segura J, et al. (2001) 3,4-Dihydroxymethamphetamine (HHMA). A major in vivo 3,4- at ASPET Journals on September 26, 2021 Meyer MR, Peters FT, and Maurer HH (2008) The role of human hepatic cytochrome P450 methylenedioxymethamphetamine (MDMA) metabolite in humans. Chem Res Toxicol 14: isozymes in the metabolism of racemic 3,4-methylenedioxy-methamphetamine and its enan- 1203–1208. tiomers. Drug Metab Dispos 36:2345–2354. Shen M, Xiang P, Sun Y, and Shen B (2013) Disappearance of 6-acetylmorphine, morphine and Miller RT, Lau SS, and Monks TJ (1997) 2,5-Bis-(glutathion-S-yl)-alpha-methyldopamine, codeine from human scalp hair after discontinuation of opiate abuse. Forensic Sci Int 227: a putative metabolite of (+/-)-3,4-methylenedioxyamphetamine, decreases brain serotonin 64–68. concentrations. Eur J Pharmacol 323:173–180. Shoda T, Fukuhara K, Goda Y, and Okuda H (2009) 4-Hydroxy-3-methoxymethamphetamine Monks TJ, Jones DC, Bai F, and Lau SS (2004) The role of metabolism in 3,4-(+)-methy- glucuronide as a phase II metabolite of 3,4-methylenedioxymethamphetamine: enzyme-assisted lenedioxyamphetamine and 3,4-(+)-methylenedioxymethamphetamine (ecstasy) toxicity. re- synthesis and involvement of human hepatic uridine 59-diphosphate-glucuronosyltransferase view Ther Drug Monit 26:132–136. 2B15 in the glucuronidation. Chem Pharm Bull (Tokyo) 57:472–475. Mueller M, Yuan J, Felim A, Neudörffer A, Peters FT, Maurer HH, McCann UD, Largeron M, Steuer AE, Schmidhauser C, Liechti ME, and Kraemer T (2015) Development and validation of and Ricaurte GA (2009) Further studies on the role of metabolites in (+/-)-3,4- an LC-MS/MS method after chiral derivatization for the simultaneous stereoselective de- methylenedioxymethamphetamine-induced serotonergic neurotoxicity. Drug Metab Dispos 37: termination of methylenedioxy-methamphetamine (MDMA) and its phase I and II metabolites 2079–2086. in human blood plasma. Drug Test Anal 7:592–602. Peters FT, Samyn N, Kraemer T, de Boeck G, and Maurer HH (2003) Enantioselective de- termination of amphetamine, methamphetamine, MDA and MDMA in plasma samples from clinical toxicology and driving under the influence of drugs cases using GC-NICI-MS. abstract Address correspondence to: Dr. rer. nat. Andrea E. Steuer, Department of Ther Drug Monit 25:539. Forensic Pharmacology and Toxicology, Zurich Institute of Forensic Medicine, Peters FT, Samyn N, Lamers C, Riedel W, Kraemer T, de Boeck G, and Maurer HH (2005) Drug testing in blood: validated negative-ion chemical ionization gas chromatographic-mass spec- University of Zurich, Switzerland. E-mail: [email protected] trometric assay for enantioselective measurement of the designer drugs MDEA, MDMA, and