The Impact of Experimental Design on Assessing Mechanism-based Inactivation of CYP2D6 by MDMA (Ecstasy)

MDMA (3-4-methylenedioxymethamphetamine, commonly known as Ecstasy) is a potent mechanism-based inhibitor (MBI) of cytochrome P450 2D6 (CYP2D6), causing quasi-irreversible inhibition of the enzyme in vitro. An evaluation of the in vivo implications of this phenomenon depends on the accuracy of the estimates of the parameters that define the inhibition in vitro, namely kinact (the maximal inhibition rate) and KI (the inactivation constant). These values are determined in two steps, pre-incubation of the enzyme with the inhibitor (enzyme inactivation), followed by dilution and further incubation to measure residual enzyme activity with a probe substrate. The aim of this study was to assess the impact of different dilutions and probe substrate concentrations on the estimates of kinact and KI using recombinantly expressed CYP2D6. Enzyme activity was measured by the conversion of dextromethorphan (DEX) to dextrorphan (DOR). Dilution factors of 1.25, 2, 5, 10, 25 and 50 (DEX at 30 µM) gave mean (± SE) values of kinact (min-1) of 0.20 ± 0.06, 0.21 ± 0.05, 0.31 ± 0.06, 0.37 ± 0.11, 0.51 ± 0.10 and 0.58 ± 0.08, respectively, and KI (µM) values (after correction for non-specific microsomal binding) of 2.22 ± 1.90, 2.80 ± 1.34, 5.78 ± 2.07, 6.36 ± 2.93, 3.99 ± 1.57 and 4.86 ± 1.37, respectively. Accordingly, high (e.g. 50 fold) and low (e.g. 1.25 fold) dilutions were associated with statistically significant differences in kinetic values (p <0.05). Varying DEX concentration (10-100 µM) was not associated with significant changes in kinact and KI values when a five-fold dilution was used (with the exception of a lower KI at 10 µM DEX). High dilution was also shown to reduce non-specific microsomal binding of MDMA. The changes in the two kinetic parameters were dependent on the experimental procedure and shown to be unlikely to have a material influence on the maximum inhibition of CYP2D6 expected in vivo after typical recreational doses of MDMA (50-100 mg), since the potency of inhibition was high. The different values of the kinetic parameters were predicted to have a marginal influence on the time for recovery of enzyme activity following re-synthesis of CYP2D6.

Author(s): Linh Van, Jiansong Yang, Martin Lennard, Amir Heydari, Karen Rowland Yeo, Jonathan Hargreaves, Geoffrey Tucker, Amin Rostami-Hodjegan

Year: November 1, 2006