european journal of pharmaceutical sciences 31 ( 2 0 0 7 ) 53–61 available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/ejps Kinetics of the time-dependent inactivation of CYP2D6 in cryopreserved human hepatocytes by methylenedioxymethamphetamine (MDMA) Linh M. Van a , John Swales b , Clare Hammond b , Claire Wilson b , Judith A. Hargreaves b , Amin Rostami-Hodjegan a,c,∗ a University of Sheffield, Academic Unit of Clinical Pharmacology, School of Medicine, Sheffield S10 2JF, UK b AstraZeneca, DMPK, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK c Simcyp Limited, Blades Enterprise Centre, Sheffield S2 4SU, UK article info Article history: Received 8 November 2006 Received in revised form 4 February 2007 Accepted 15 February 2007 Published on line 20 February 2007 Keywords: Metabolic drug–drug interactions Mechanism-based inhibition CYP2D6 inhibition MDMA abstract Methylenedioxymethamphetamine (MDMA) was investigated in cryopreserved human hep- atocytes 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 preincuba- tion) 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 M, 0.01 ± 0.001 min -1 and 1.23 ± 0.70 M, and 0.01 ± 0.001 min -1 and 2.10 ± 1.32 M, 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 M, 0.15 ± 0.09 M, 0.24 ± 0.16 M 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 micro- somes 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. © 2007 Published by Elsevier B.V. ∗ Corresponding author at: Academic Unit of Clinical Pharmacology, University of Sheffield, Floor M, Royal Hallamshire Hospital, Sheffield S10 2JF, UK. Tel.: +44 114 271 2156; fax: +44 114 226 8986. E-mail address: A.Rostami@sheffield.ac.uk (A. Rostami-Hodjegan). Abbreviations: CYP2D6, cytochrome P450 2D6; DEX, dextromethorphan; DF, dilution factor; DOR, dextrorphan; HLM, human liver microsome; k inact , maximum inactivation rate; K I , apparent inactivation constant; k obs , observed rate; LC–MS/MS, tandem mass spec- trometry; LEV, levallorphan; MBI, mechanism-based inhibition; MDMA, 3,4-methylenedioxymethamphetamine; MRM, multiple reaction monitoring; rCYP, recombinant CYP450; TDI, time-dependent inactivation; WR, wash and removal of inhibitor after centrifugation 0928-0987/$ – see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.ejps.2007.02.005