Abstract

Buprenorphine (BN) is a thebaine derivative with analgesic properties. To identify and characterize the cytochrome P450 (CYP) enzyme(s) involved in BN N-dealkylation, in vitro studies using human liver microsomes and recombinant human CYP enzymes were performed. Norbuprenorphine formation from BN was measured by a simple HPLC-UV assay method, without extraction. The BNN-dealkylation activities in 10 human liver microsomal preparations were strongly correlated with microsomal CYP3A-specific metabolic reactions, i.e. triazolam 1′-hydroxylation (r = 0.954), midazolam 1′-hydroxylation (r = 0.928), and testosterone 6β-hydroxylation (r = 0.897). Among the eight recombinant CYP enzymes studied (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), only CYP3A4 could catalyze BN N-dealkylation. The apparent K_M value for recombinant CYP3A4 was similar to that for human liver microsomes (23.7 vs.39.3 ± 9.2 μM). The demonstration of BNN-dealkylation by recombinant CYP3A4 and the agreement in the affinities (apparent K_M values) of human liver microsomes and recombinant CYP3A4 provide the most supportive evidence for BN N-dealkylation being catalyzed by CYP3A4.