Abstract
The in vitro metabolism of buprenorphine was investigated to explore new metabolic pathways and identify the cytochromes P450 (P450s) responsible for the formation of these metabolites. The resulting metabolites were identified by liquid chromatography-electrospray ionization-tandem mass spectrometry. In addition to norbuprenorphine, two hydroxylated buprenorphine (M1 and M2) and three hydroxylated norbuprenorphine (M3, M4, and M5) metabolites were produced by human liver microsomes (HLMs), with hydroxylation occurring at the tert-butyl group (M1 and M3) and at unspecified site(s) on the ring moieties (M2, M4, and M5). Time course and other data suggest that buprenorphine is N-dealkylated to form norbuprenorphine, followed by hydroxylation to form M3; buprenorphine is hydroxylated to form M1 and M2, followed by N-dealkylation to form M3 and M4 or M5. The involvement of selected P450s was investigated using cDNA-expressed P450s coupled with scaling models, chemical inhibition, monoclonal antibody (MAb) analysis, and correlation studies. The major enzymes involved in buprenorphine elimination and norbuprenorphine and M1 formation were P450s 3A4, 3A5, 3A7, and 2C8, whereas 3A4, 3A5, and 3A7 produced M3 and M5. Based on MAb analysis and chemical inhibition, the contribution of 2C8 was higher in HLMs with higher 2C8 activity, whereas 3A4/5 played a more important role in HLMs with higher 3A4/5 activity. Examination of human urine from subjects taking buprenorphine showed the presence of M1 and M3; most of M1 was conjugated, whereas 60 to 70% of M3 was unconjugated.
Footnotes
-
This study was supported by National Institute on Drug Abuse Grants R01 DA10100 (D.E.M.), RO1 DA 13004 (E.M.K.), and KO2 DA00478 (E.M.K.), and by the General Clinical Research Center at Virginia Commonwealth University (M01RR00065, National Center for Research Resources/National Institutes of Health).
-
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
-
doi:10.1124/dmd.105.006148.
-
ABBREVIATIONS: HLM, human liver microsome; P450, cytochrome P450; MAb, monoclonal antibody; NADPH GS, NADPH generating system; LC-ESI-MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; MS, mass spectrometer; SRM, selected reaction monitoring; SIM, selected ion monitoring; CID, collision-induced dissociation; RAF, relative activity factor.
- Received June 17, 2005.
- Accepted December 19, 2005.
- The American Society for Pharmacology and Experimental Therapeutics