Abstract

Buprenorphine (BUP) is a synthetic derivative of the morphine alkaloid thebaine. BUP is metabolized by N-dealkylation to form the active metabolite nor-buprenorphine (Nor-BUP), and both undergo subsequent glucuronidation. Although BUP has been used clinically for years, its metabolism has still not been fully elucidated. The aim of this study was to clarify the identity of the human hepatic cytochromes P450 (P450s) involved in BUP metabolism and to investigate other potential metabolites. The metabolism of BUP was examined using human liver microsomes (HLM) and Ad293 P450-transfected cell lines, as well as CYP 3A4 and 2C8 recombinant isoforms. The kinetic parameters of metabolite formation were calculated for HLM and competent isoforms. Individual contribution of P450 isoforms in BUP metabolism as well as Nor-BUP production was evaluated using chemical inhibition experiments, as well as the relative activity factor approach. The analytical method used was based on liquid chromatography-mass spectrometry. Among the 13 P450 isoforms tested, CYP 3A4, 2C8, 3A5, and 3A7 produced Nor-BUP. Based on the results of chemical inhibition, CYP 3A4 accounts for about 65% of Nor-BUP production and CYP 2C8 for about 30%. BUP utilization by either HLM or P450-transfected cells revealed that another oxidative metabolic pathway exists, which was found to involve CYP 2C9, 2C18, 2C19, and mainly CYP 3A. Incubation of BUP or Nor-BUP with HLM led to the formation of new metabolites, identified by tandem mass spectrometry as being hydroxy-BUP and hydroxy-Nor-BUP. Hydroxy-BUP was produced by the CYP 3A, but not the 2C isoforms.