The Role of Human UDP-Glucuronyltransferases on the Formation of the Methylenedioxymethamphetamine (Ecstasy) Phase II Metabolites R- and S-3-Methoxymethamphetamine 4-O-Glucuronides

Different pharmacokinetic properties have been observed for the two enantiomers of the entactogen 3,4-methylendioxy-methamphetamine, most probably a result of enantioselective metabolism. The aim of the present work was to study the involvement of human UDP-glucuronyltransferase (UGT) isoforms in the glucuronidation of the enantiomers of its major metabolite 4-hydroxy-3-methoxymethamphetamine (HMMA). First, the reference standards of R- and S-HMMA-O-glucuronide were synthesized semipreparatively using the enzymes of rat liver microsomes, followed by isolation with semipreparative high-performance liquid chromatography and identification using mass spectrometry and NMR. Racemic HMMA was then incubated using heterologously expressed human UGTs and pooled human liver microsomes (HLMs), and the glucuronides were quantified by liquid chromatography-linear ion trap-mass spectrometry. UGT1A1, UGT1A3, UGT1A8, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17 were involved in the glucuronidation of HMMA. UGT2B15, UGT2B17, and HLM revealed classic Michaelis-Menten kinetics, whereas UGT1A9 and UGT2B7 showed sigmoidal curves and the respective Eadie-Hofstee plots indicated autoactivation kinetics. UGT2B15 showed the highest affinity and activity. UGT2B15, UGT2B17, and HLMs were not considerably enantioselective but showed slight preferences for S-HMMA. Marked enantioselectivity could only be observed for UGT1A9 with respect to the S-enantiomer and for UGT2B7 with respect to the R-enantiomer. In conclusion, the O-glucuronidation of HMMA in vivo should not be expected to be enantioselective, and the different pharmacokinetic properties may not be caused directly by glucuronidation.