Investigation on the Enantioselectivity of the Sulfation of the Methylenedioxymethamphetamine Metabolites 3,4-Dihydroxymethamphetamine and 4-Hydroxy-3-Methoxymethamphetamine using the Substrate-Depletion Approach

Abstract

ABSTRACT:Different pharmacokinetic properties are known for the two enantiomers of the entactogen 3,4-methylendioxy-methamphetamine (MDMA), most likely due to enantioselective metabolism. The aim of the present work was 1) the investigation of the main sulfotransferases (SULT) isoenzymes involved in the sulfation of the main MDMA phase I metabolites 3,4-dihydroxymethamphetamine (DHMA) and 4-hydroxy-3-methoxymethamphetamine (HMMA) and 2) the evaluation of a possible enantioselectivity of this phase II metabolic step. Therefore, racemic DHMA and HMMA were incubated with heterologously expressed SULTs, and quantification of the sulfates by liquid chromatography-high-resolution mass spectrometry was conducted. Because separation of DHMA and HMMA sulfate could not be achieved by liquid chromatography, enantioselective kinetic parameters were determined using the substratedepletion approach with enantioselective quantification of substrate consumption by gas chromatography-negative ion chemical ionization mass spectrometry. SULT1A1 and SULT1A3 catalyzed sulfation of DHMA, and SULT1A3 and SULT1E1 catalyzed sulfation of HMMA. SULT1A1 and SULT1E1 revealed classic Michaelis-Menten kinetics, whereas SULT1A3 kinetics showed deviation from the typical Michaelis-Menten kinetics, resulting in a concentrationdependent self-inhibition. SULT1A3 showed the highest affinity and capacity of the SULT isoforms. Marked enantioselectivity could be observed for S-DHMA sulfation by SULT1A3 and in human liver cytosol, whereas no differences were observed for HMMA sulfation. Finally, comparison of K m and V max values calculated using achiral product formation and chiral substrate depletion showed good correlation within 2-fold of each other. In conclusion, preferences for S-enantiomers were observed for DHMA sulfation, but not for HMMA sulfation. IntroductionThe chiral compound R, R,propane-2-amine], also known as ecstasy, is known as a very popular drug of abuse, but it is also associated with damage of serotonergic neurons (Kalant, 2001;Monks et al., 2004;Easton and Marsden, 2006). Metabolism of MDMA may play a role in this neurotoxicity (Miller et al., 1997;Bai et al., 1999;Mueller et al., 2009). As shown in Fig. 1, one major pathway of MDMA includes cytochrome P450 (P450)-catalyzed O-demethylation to 3,4-dihydroxymethamphetamine (DHMA), followed by O-methylation by catechol-O-methyltransferase (COMT) mainly to 4-hydroxy-3-methoxymethamphetamine (HMMA) and conjugation of DHMA and HMMA by sulfotransferases (SULTs) (Maurer, 1996;Maurer et al., 2000;. In urine samples of recreational MDMA users, more than 90% of DHMA and HMMA are excreted as conjugates, with sulfates present in higher concentrations (Shima et al., 2008; A. E. Schwaninger, M. R. Meyer, A. J. Barnes, E. A. Kolbrich-Spargo, D. A. Gorelick, R. S. Goodwin, M. A. Huestis, and H. H. Maurer, manuscript in preparation). For the two enantiomers of MDMA, differences in their dose-response curves and in their in vivo kinetics were observed (Fallon et al., 1999;Kalant, 2001;Kraemer a...

Stereoselective urinary MDMA (ecstasy) and metabolites excretion kinetics following controlled MDMA administration to humans

“…4. Both pathways revealed preferences for S -enantiomers in vitro [26,28], which could explain the higher C max for the remaining R DHMA. HMMA conjugation to HMMA sulfate in vitro showed no enantioselectivity [28], in line with the in vivo excretion data at C max .…”

“…Enantioselective metabolism is the most likely explanation for different MDMA enantiomer pharmacokinetics in humans. I n vitro experiments mediated by CYP, COMT, SULT and UGT enzymes documented preferential metabolism of S -enantiomers [19,26-28]. …”

Chiral Plasma Pharmacokinetics of 3,4-Methylenedioxymethamphetamine and its Phase I and II Metabolites following Controlled Administration to Humans

“…Median MDA and DHMA 4-sulfate were approximately 10 times lower. Metabolism was suspected to be the main cause of the stereoselective disposition (Fallon et al, 1999;Pizarro et al, 2004;Peters et al, 2005), and in vitro experiments revealed preferences for the formation of MDA, DHMA, DHMA sulfate, HMMA, and HMMA glucuronide, whereas HMMA sulfation was not enantioselective (Meyer et al, 2008;Meyer and Maurer, 2009;Schwaninger et al, 2009Schwaninger et al, , 2011b. These findings are in line with the chiral pharmacokinetic analysis showing higher C max for the S-stereoisomer of MDA, DHMA 3-sulfate, and HMMA glucuronide, higher C max for R-MDMA and R-DHMA 4-sulfate, and no significant difference in HMMA sulfate.…”

“…Whereas the S-MDMA enantiomer primarily causes the described stimulating effects, R-MDMA produces more hallucinogenic effects . Elimination of S-MDMA was shown to be faster compared with the R-enantiomer (Fallon et al, 1999;Kalant, 2001;Kraemer and Maurer, 2002;Peters et al, 2003Peters et al, , 2005Pizarro et al, 2004) most probably owing to stereoselective metabolism that has been extensively studied in vitro (Meyer et al, 2008;Meyer and Maurer, 2009;Schwaninger et al, 2009Schwaninger et al, , 2011b. As not only stereoselectivity of MDMA itself but also of its primary metabolite DHMA is discussed in terms of (neuro-)toxicity (Felim et al, 2010;Martinez et al, 2012), the further metabolic fate of DHMA and resulting stereoselectivities of metabolites is of interest.…”

Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane

“…The cytochrome P450s (P450s) are monooxygenases that mediate the metabolism of the majority of lipophilic drugs in clinical use (Rendic and Guengerich, 2015). The importance of non-P450 drug-metabolizing enzymes has also been highlighted, such as flavin-containing monooxygenases (Usmani et al, 2012), monoamine oxidases (Inoue et al, 1999), aldehyde oxidase (Hutzler et al, 2013), epoxide hydrolases (EHs) (Fretland and Omiecinski, 2000), and conjugating enzymes such as glutathione S-transferases (Wu and Dong, 2012), sulfotransferases (Schwaninger et al, 2011), and UDP-glucuronosyltransferases (Radominska-Pandya et al, 1999). Some of these non-P450 enzymes are not commercially available and thus assessment of their contribution to the biotransformation of new chemical entities can be a challenge in drug discovery.…”