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, Supersomestrade mark and Bactosomestrade mark) under the conditions of the most commonly used protocols in assessing mechanism-based inactivation (MBI). MDMA (2-20microM) was pre-incubated with enzyme for 0, 2.5 and 5min followed by a five-fold dilution and further incubation with dextromethorpan (DEX) (50microM). 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 Bactosomestrade mark (0.95+/-0.33min(-1), 42.9+/-20.1microM) than those found using yeast microsomes (0.28+/-0.04min(-1), 2.86+/-1.18microM) and Supersomestrade mark (0.38+/-0.05min(-1), 3.66+/-0.10microM). After correction for depletion of MDMA during pre-incubation, k(inact) and K(I) values determined using Bactosomestrade mark 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 Supersomestrade mark and Bactosomestrade mark. Sub-optimal study design when investigating MBI may compromise the quantitative characterization of inhibitory characteristics using some rCYP systems.
Van
Linh M
LM
University of Sheffield, Academic Unit of Clinical Pharmacology, University of Sheffield, Sheffield S10 2JF, UK.
Hargreaves
Judith A
JA
Lennard
Martin S
MS
Tucker
Geoffrey T
GT
Rostami-Hodjegan
Amin
A
D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D003915:Dextromethorphan / Q000494:pharmacology; D003917:Dextrorphan / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D003917:Dextrorphan; D003915:Dextromethorphan; D019389:Cytochrome P-450 CYP2D6; D018817:N-Methyl-3,4-methylenedioxyamphetamine