Inactivation of CYP2D6 by Methylenedioxymethamphetamine in Different Recombinant Expression Systems

Recombinantly expressed CYP450 systems (rCYPs) are often used to screen for irreversible/quasi-irreversible enzyme inhibitors during drug development. The concentration- and time-dependent inactivation of CYP2D6 by methylenedioxymethamphetamine (MDMA) was compared in three different rCYP2D6 systems (yeast microsomes, Supersomes™ and Bactosomes™) under the conditions of the most commonly used protocols in assessing mechanism-based inactivation (MBI). MDMA (2-20µM) was pre-incubated with enzyme for 0, 2.5 and 5min followed by a five-fold dilution and further incubation with dextromethorpan (DEX) (50µM). The formation of dextrorphan (DOR) from DEX was used as a specific marker of CYP2D6 activity. Concentration- and time-dependent inactivation of CYP2D6 by MDMA was observed with each rCYP system. However, the apparent kinetic parameters for MBI (k_inact, the maximum inactivation rate constant and K_I, the inhibitor concentration associated with half maximal rate of inactivation) were significantly greater (p<0.05) for Bactosomes™ (0.95 ± 0.33min^-1), 42.9 ± 20.1µM) than those found using yeast microsomes (0.28 ± 0.04min^-1, 2.86 ±.18µM) and Supersomes™ (0.38 ± 0.05min^-1), 3.66 ± 0.10µM). After correction for depletion of MDMA during pre-incubation, (k_inact and K_I values determined using Bactosomes™ decreased significantly but remained higher than for the other rCYP systems (p<0.05). Substantial metabolism of DOR after its formation from DEX was also observed using Supersomes™ and Bactosomes™. Sub-optimal study design when investigating MBI may compromise the quantitative characterization of inhibitory characteristics using some rCYP systems.