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Anesth Analg. 2013 Jul;117(1):52-60. doi: 10.1213/ANE.0b013e3182918252. Epub 2013 Jun 3.

Mechanism of autoinduction of methadone N-demethylation in human hepatocytes.

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Department of Anesthesiology, Washington University in St. Louis, 660 S Euclid Ave., Campus Box 8054, St. Louis, MO 63110-1093, USA.



There is considerable interindividual and intraindividual variability in methadone metabolism and clearance. Methadone dosing is particularly challenging during initiation of therapy, because of time-dependent increases in hepatic clearance (autoinduction). Although methadone N-demethylation is catalyzed in vitro by cytochrome P4502B6 (CYP2B6) and CYP3A4, and clearance in vivo depends on CYP2B6, mechanism(s) of autoinduction are incompletely understood. In this investigation, we determined mechanism(s) of methadone autoinduction using human hepatocytes.


Fresh human hepatocytes were exposed to 0.1 to 10 µM methadone for 72 hours. Cells were washed and methadone N-demethylation assessed. CYP2B6, CYP3A4, and CYP3A5 messenger RNA (mRNA), protein expression (by gel-free high-performance liquid chromatography mass spectrometry) and catalytic activity (bupropion hydroxylation and alfentanil dealkylation for CYP2B6 and CYP3A4/5, respectively) were measured. Mechanisms of CYP induction were characterized using pregnane X receptor and constitutive androstane receptor reporter gene assays.


Methadone (10 µM) increased methadone N-demethylation 2-fold, CYP2B6 and CYP3A4 mRNA 3-fold, and protein expression 2-fold. CYP3A5 mRNA was unchanged. CYP2B6 and CYP3A4/5 activities increased 2-fold. Induction by methadone enantiomers (R-methadone versus S-methadone) did not differ. Induction was relatively weak compared with maximum induction by phenobarbital and rifampin. Lower methadone concentrations had smaller effects. Methadone was an agonist for the pregnane X receptor but not the constitutive androstane receptor.


Methadone caused concentration-dependent autoinduction of methadone N-demethylation in human hepatocytes, related to induction of CYP2B6 and CYP3A4 mRNA expression, protein expression, and catalytic activity. Induction was related to pregnane X receptor but not constitutive androstane receptor activation. These in vitro findings provide mechanistic insights into clinical autoinduction of methadone metabolism and clearance.

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