Mechanism-based inactivation of cytochrome P450 2B6 by methadone through destruction of prosthetic heme.
Methadone is a μ-opioid receptor agonist widely used in the treatment of narcotic addiction and chronic pain conditions. Methadone is metabolized predominantly in the liver by cytochromes P450 to its pharmacologically inactive primary metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine. Initial in vitro data suggested that CYP3A4 is the major isoform responsible for the in vivo clearance of methadone in humans. However, recent clinical data have indicated that CYP2B6 is actually the major isoform responsible for methadone metabolism and clearance in vivo. In this study, methadone was shown to act as a mechanism-based inactivator of CYP2B6. Methadone inactivates CYP2B6 in a time-, concentration-, and NADPH-dependent manner with a K(I) = 10.0 μM and k(inact) = 0.027 min⁻¹. The loss of CYP2B6 activity in the presence of methadone and NADPH occurred with concomitant loss of the reduced CO spectrum of the P450. Moreover, there was good correlation between the loss of CYP2B6 activity and the loss of the CO-binding spectrum. High-performance liquid chromatography analysis of the native heme of the inactivated CYP2B6 demonstrated that approximately 75% loss of heme was accompanied by comparable inactivation of CYP2B6. Liquid chromatography-mass spectrometry analysis did not reveal the formation of a protein adduct during the inactivation. The evidence strongly suggests that destruction of prosthetic heme is the underlying mechanism leading to the inactivation of CYP2B6 by methadone.
Department of Pharmacology, University of Michigan, Ann Arbor, Michigan, USA.
Hemali T
HT
Amunugama
Haoming
H
Zhang
Paul F
PF
Hollenberg
D000701:Analgesics, Opioid / Q000494:pharmacology*; D001189:Aryl Hydrocarbon Hydroxylases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001711:Biotransformation; D002248:Carbon Monoxide / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D065702:Cytochrome P-450 CYP2B6; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006418:Heme / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008691:Methadone / Q000494:pharmacology*; D009249:NADP / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
D000701:Analgesics, Opioid; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006418:Heme; D009249:NADP; D002248:Carbon Monoxide; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D008691:Methadone