APPARENT MECHANISM-BASED INHIBITION OF HUMAN CYP2D6 IN VITRO BY PAROXETINE: COMPARISON WITH FLUOXETINE AND QUINIDINE KIRK M. BERTELSEN, KARTHIK VENKATAKRISHNAN, LISA L. VON MOLTKE, R. SCOTT OBACH, AND DAVID J.GREENBLATT Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine (K.M.B., L.L.vM., D.J.G.), and the Division of Clinical Pharmacology, Tufts-New England Medical Center (L.L.vM., D.J.G.), Boston, Massachusetts; and Pfizer Inc., Groton, Connecticut (K.V., R.S.O.) (Received September 3, 2002; accepted November 27, 2002) This article is available online at http://dmd.aspetjournals.org ABSTRACT: Paroxetine, a selective serotonin reuptake inhibitor, is a potent inhibitor of cytochrome P450 2D6 (CYP2D6) activity, but the mech- anism of inhibition is not established. To determine whether pre- incubation affects the inhibition of human liver microsomal dextro- methorphan demethylation activity by paroxetine, we used a two- step incubation scheme in which all of the enzyme assay components, minus substrate, are preincubated with paroxetine. The kinetic parameters of inhibition were also estimated by varying the time of preincubation as well as the concentration of inhibitor. From these data, a Kitz-Wilson plot was constructed, allowing the estimation of both an apparent inactivator concentration required for half-maximal inactivation (K I ) and the maximal rate constant of inactivation (k INACT ) value for this interaction. Preincubation of paroxetine with human liver microsomes caused an approximately 8-fold reduction in the IC 50 value (0.34 versus 2.54 M). Time- dependent inhibition was demonstrated with an apparent K I of 4.85 M and an apparent k INACT value of 0.17 min 1 . Spectral scanning of CYP2D6 with paroxetine yielded an increase in absorbance at 456 nm suggesting paroxetine inactivation of CYP2D6 via the for- mation of a metabolite intermediate complex. This pattern is con- sistent with the metabolism of the methylenedioxy substituent in paroxetine; such substituents may produce mechanism-based in- activation of cytochrome P450 enzymes. In contrast, quinidine and fluoxetine, both of which are inhibitors of CYP2D6 activity, did not exhibit a preincubation-dependent increase in inhibitory potency. These data are consistent with mechanism-based inhibition of CYP2D6 by paroxetine but not by quinidine or fluoxetine. Although CYP2D6 constitutes a relatively minor fraction of the total hepatic P450 1 content (Shimada et al., 1994), the contribution of this isoform is significant due to its role in the metabolism and clearance of many therapeutic agents that target the cardiovascular and central nervous system. In addition, clinically significant poly- morphisms in the CYP2D6 gene have been identified in a variety of populations with altered metabolic activity. The majority of poor metabolizers, or individuals with impaired enzyme function, are ac- counted for by frame shift deletions, substitutions resulting in splicing defects, or gene deletions (van der Weide and Steijns, 1999; Bertils- son et al., 2002). Extensive metabolizers, or individuals with normal enzyme function, are heterozygous or homozygous for the wild-type allele. In vivo clearance of CYP2D6 substrates in poor metabolizers is generally much lower than in extensive metabolizers, leading to higher plasma concentrations and the potential for clinical toxicities with therapeutic doses (Bertilsson et al., 2002). Paroxetine is a selective serotonin reuptake inhibitor with nonlinear kinetics that is both a substrate for and an inhibitor of CYP2D6 (Greenblatt et al., 1999; Belpaire et al., 1998; Otton et al., 1996; Bloomer et al., 1992; Sindrup et al., 1992a,b). Paroxetine is metabo- lized by CYP2D6 via demethylenation of the methylenedioxy group, yielding a catechol metabolite and formic acid (Haddock et al., 1989; Bloomer et al., 1992). Paroxetine inhibits CYP2D6 activity at IC 50 concentrations ranging from 150 nM to 2.0 M, depending on the substrate (Crewe et al., 1992; von Moltke et al., 1995; Fogelman et al., 1999). Although not investigated directly, paroxetine has been re- garded as a competitive, reversible inhibitor of CYP2D6 (Otton et al., 1996). We previously observed that the in vitro K i for paroxetine versus desipramine hydroxylation yielded an underestimate of the degree of desipramine clearance inhibition when desipramine was coadminis- tered with paroxetine in a clinical study (von Moltke et al., 1995; Alderman et al., 1997). We accounted for this discrepancy on the basis of extensive partitioning of paroxetine from plasma into hepatic tissues such that intrahepatic concentrations substantially exceeded total plasma concentrations (von Moltke et al., 1995). Hemeryck et al. (2000, 2001) observed similar discrepancies, in which in vitro inhi- bition of metoprolol hydroxylation by paroxetine yielded an underes- timate of the in vivo inhibition of metoprolol clearance by cotreatment with paroxetine. They suggest that the discrepancy could be explained This work was supported by Grants MH-58435, DA-13209, DK/AI-58496, DA-05258, DA-13834, AG-17880, MH-34223, MH-01237, and RR-00054 from the Department of Health and Human Services. 1 Abbreviations used are: P450, cytochrome P450; MBI, mechanism-based inhibition; MIC, metabolite intermediate complex; k INACT , the maximal rate con- stant of inactivation; K I , the inactivator concentration required for half-maximal inactivation; HLM, human liver microsome; HPLC, high performance liquid chro- matography. Address correspondence to: David J. Greenblatt, MD, Department of Phar- macology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. E-mail: dj.greenblatt@tufts.edu 0090-9556/03/3103-289–293$7.00 DRUG METABOLISM AND DISPOSITION Vol. 31, No. 3 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 923/1044815 DMD 31:289–293, 2003 Printed in U.S.A. 289 at ASPET Journals on October 13, 2017 dmd.aspetjournals.org Downloaded from