Kinetics of the time-dependent inactivation of CYP2D6 in cryopreserved human hepatocytes by methylenedioxymethamphetamine (MDMA).
Methylenedioxymethamphetamine (MDMA) was investigated in cryopreserved human hepatocytes as a time-dependent inactivator (TDI) of CYP2D6 using dextromethorphan (DEX) as a probe substrate. Inhibition kinetic parameters k(inact), the maximal rate of inactivation, and K(I), the inhibitor concentration at half the maximal activation rate, were determined. Time- and concentration-dependent inhibition were confirmed, and the influence of different elements of study design (e.g. cell number, stability of hepatocytes, dilution after preincubation) on estimated kinetic parameters were evaluated. Dilution factors (DF) of 1.2, 5 or total removal of inhibitor (by washing cells after preincubation, WR) resulted in k(inact) and K(I) (+/-S.E.) values of 0.02+/-0.002 min(-1) and 0.88+/-0.31 microM, 0.01+/-0.001 min(-1) and 1.23+/-0.70 microM, and 0.01+/-0.001 min(-1) and 2.10+/-1.32 microM, respectively; indicating that insufficient dilution may lead to overestimation of CYP2D6 inactivation. Accounting for MDMA depletion during the preincubation, corrected K(I) values were significantly lower (0.11+/-0.05 microM, 0.15+/-0.09 microM, 0.24+/-0.16 microM for DF of 1.2, 5, and WR, respectively). Inactivation efficiency in hepatocytes, as measured by k(inact)/K(I), was 10-fold less than that previously reported in human liver microsomes or recombinantly expressed systems. Possible causes for the observed differences between in vitro systems warrant further investigation. These may include differences in metabolic consumption of MDMA in each system, non-specific binding and presence of active efflux in hepatocytes.
Van
Linh M
LM
University of Sheffield, Academic Unit of Clinical Pharmacology, School of Medicine, Sheffield S10 2JF, UK.
Swales
John
J
Hammond
Clare
C
Wilson
Claire
C
Hargreaves
Judith A
JA
Rostami-Hodjegan
Amin
A
D000465:Algorithms; D002452:Cell Count; D002470:Cell Survival / Q000187:drug effects; D015925:Cryopreservation; D019389:Cytochrome P-450 CYP2D6 / Q000378:metabolism; D065690:Cytochrome P-450 CYP2D6 Inhibitors; D003915:Dextromethorphan / Q000378:metabolism; D003917:Dextrorphan / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D018817:N-Methyl-3,4-methylenedioxyamphetamine / Q000378:metabolism / Q000494:pharmacology*; D011802:Quinidine / Q000378:metabolism / Q000494:pharmacology; D013997:Time Factors
D065690:Cytochrome P-450 CYP2D6 Inhibitors; D004791:Enzyme Inhibitors; D003917:Dextrorphan; D003915:Dextromethorphan; D019389:Cytochrome P-450 CYP2D6; D011802:Quinidine; D018817:N-Methyl-3,4-methylenedioxyamphetamine