Enantiomeric Metabolic Interactions and Stereoselective Human Methadone Metabolism

Abstract

Methadone is administered as a racemate, although opioid activity resides in the R-enantiomer. Methadone disposition is stereoselective, with considerable unexplained variability in clearance and plasma R/S ratios. N-Demethylation of methadone in vitro is predominantly mediated by cytochrome P450 CYP3A4 and CYP2B6 and somewhat by CYP2C19. This investigation evaluated stereoselectivity, models, and kinetic parameters for methadone N-demethylation by recombinant CYP2B6, CYP3A4, and CYP2C19, and the potential for interactions between enantiomers during racemate metabolism. CYP2B6 metabolism was stereoselective. CYP2C19 was less active, and stereoselectivity was opposite that for CYP2B6. CYP3A4 was not stereoselective. With all three isoforms, enantiomer N-dealkylation rates in the racemate were lower than those of (R)-(6-dimethyamino-4,4-diphenyl-heptan-3-one) hydrochloride (R-methadone) or (S)-(6-dimethyamino-4,4-diphenyl-heptan-3-one) hydrochloride (S-methadone) alone, suggesting an enantiomeric interaction and mutual metabolic inhibition. For CYP2B6, the interaction between enantiomers was stereoselective, with S-methadone as a more potent inhibitor of R-methadone N-demethylation than R-of S-methadone. In contrast, enantiomer interactions were not stereoselective with CYP2C19 or CYP3A4. For all three cytochromes P450, methadone N-demethylation was best described by two-site enzyme models with competitive inhibition. There were minor model differences between cytochromes P450 to account for stereoselectivity of metabolism and enantiomeric interactions. Changes in plasma R/S methadone ratios observed after rifampin or troleandomycin pretreatment in humans in vivo were successfully predicted by CYP2B6-but not CYP3A4-catalyzed methadone N-demethylation. CYP2B6 is a predominant catalyst of stereoselective methadone metabolism in vitro. In vivo, CYP2B6 may be a major determinant of methadone metabolism and disposition, and CYP2B6 activity and stereoselective metabolic interactions may confer variability in methadone disposition.The chiral opioid methadone, a mainstay in the treatment of opiate addiction and acute and chronic pain, is used clinically as a racemate, although opioid activity resides in the R-enantiomer. Methadone disposition is stereoselective; there is considerable interindividual variability in clearance (up to 100-fold) and plasma R/S ratios (4-fold), and methadone clearance is subject to numerous drug interactions (Plummer et al., 1988;Eap et al., 2002). Nevertheless, the mechanism of stereoselective clearance and metabolism and interindividual variability remains poorly understood. Methadone elimination is due mainly to metabolic clearance (Anggard et al., 1975). The main pathway of methadone metabolism is N-demethylation to inactive 2-ethylidine-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP). A minor reductive route to normethadol is not cytochrome P450-mediated. The significant correlation between methadone clearance and the plasma EDDP/methadone area under the curve ratios suggest that N-dem...

Differences in Methadone Metabolism by CYP2B6 Variants

“…Assessed over broader methadone concentrations, in vitro intrinsic clearances for CYP2B6.6 and CYP2B6.9 were significantly lower than for CYP2B6.1. Stereoselectivity of methadone metabolism was maintained in all CYP2B6 variants, with S-methadone N-demethylation 2-fold greater than that of R-methadone, similar to that with CYP2B6.1 (Gerber et al, 2004;Totah et al, 2007Totah et al, , 2008Chang et al, 2011;Gadel et al, 2013). Lower rates of CYP2B6.6-catalyzed methadone Ndemethylation in the present investigation, in which insect cells were transfected with individual virus constructs for CYP2B6, P450 reductase, and cytochrome b 5 , were also seen in an insect cell system transfected with a single virus containing all three proteins (Gadel et al, 2013).…”

“…EDDP formation by CYP2B6 variants was compared by analysis of variance. EDDP formation versus substrate concentration data were analyzed by nonlinear regression analysis (SigmaPlot 12.5; Systat, San Jose, CA) evaluating a single-enzyme Michaelis-Menten, AdairPauling, or substrate (or product inhibition) model as described previously (Totah et al, 2007(Totah et al, , 2008Gadel et al, 2013). The choice of model was based on whether the Eadie-Hofstee plots were linear or nonlinear and the goodness of fit regression diagnostics.…”

“…Both methadone clearance and N-demethylation are stereoselective. Methadone N-demethylation in vitro, by human liver microsomes and by expressed cytochrome P450s (P450s), is catalyzed most efficiently by CYP2B6 and CYP3A4, and only CYP2B6 N-demethylates methadone stereoselectively (S.R) (Gerber et al, 2004;Kharasch et al, 2004;Totah et al, 2007Totah et al, , 2008Chang et al, 2011;Gadel et al, 2013).…”

Cytochrome P4503A Does Not Mediate the Interaction between Methadone and Ritonavir-Lopinavir

“…Methadone clearance was doubled by efavirenz and rifampin, which induce CYP2B6, as well as CYP3A4, and by ritonavir, which induces CYP2B6 while inhibiting CYP3A (Kharasch et al, 2004a(Kharasch et al, , 2008a. Increasing the activity of CYP2B6, which metabolizes methadone stereoselectively (Gerber et al, 2004;Kharasch et al, 2004a;Totah et al, 2007Totah et al, , 2008Chang et al, 2011), increased plasma R/S methadone ratios (Kharasch et al, 2004a(Kharasch et al, , 2008a(Kharasch et al, ,c, 2012bTotah et al, 2008), whereas inhibiting CYP3A, which metabolizes methadone nonstereoselectively, did not (Kharasch et al, 2009a(Kharasch et al, ,b, 2012a. CYP2B6 inhibition by ticlopidine increased methadone enantiomers AUC and reduced methadone N-demethylation and clearance (Kharasch and Stubbert, 2013).…”

“…CYP2B6 catalyzes methadone N-demethylation in vitro as avidly as CYP3A4 and CYP2B6 but not CYP3A4 is stereoselective (Gerber et al, 2004;Kharasch et al, 2004a;Totah et al, 2007Totah et al, , 2008Chang et al, 2011). Methadone clearance was doubled by efavirenz and rifampin, which induce CYP2B6, as well as CYP3A4, and by ritonavir, which induces CYP2B6 while inhibiting CYP3A (Kharasch et al, 2004a(Kharasch et al, , 2008a.…”