In vitro metabolism of brucine by human liver microsomes and its interactions with CYP substrates

• Brucine has the potential to interact with CYP3A4 substrates.
• Brucine can inhibit the activity of CYP3A4.
• The enzyme kinetics of brucine in HLMs and CYP3A4 follow Michaelis–Menten kinetics.

Brucine, one of the main active ingredients in semen Strychni, has been included in many oral prescriptions of traditional Chinese medicine. In this study, we investigated the in vitro metabolism of brucine by human liver microsomes (HLMs) and the metabolic interactions of brucine with the substrates of cytochrome P450 (CYP450). Brucine was incubated with HLMs or CYP3A4 and then analysed by Liquid chromatography/mass spectrometry. The Km and Vmax values for HLMs were 30.53 ± 3.14 μM and 0.08 ± 0.0029 nmol/mg protein/min, respectively, while the corresponding values for CYP3A4 were 20.12 ± 3.05 μM and 6.40 ± 0.21 nmol/nmol P450/min. CYP3A4 may be the major enzyme responsible for brucine metabolism in HLMs, other human isoforms of CYP showed minimal or no effect on brucine metabolism. The inhibitory action of brucine was observed in CYP3A4 for the 1′-hydroxylation of midazolam, with inhibitory concentration 50 (IC50) of 8.4-fold higher than specific inhibitors in HLMs. Furthermore, brucine significantly inhibited the CYP3A4-catalyzed midazolam 1′-hydroxylation (Ki = 2.14 μM) at a concentration lower than 10 μM, but no obvious inhibitory effects were observed on other CYP substrates (IC50 > 50 μM). These results suggest that brucine has the potential to interact with a wide range of xenobiotics and endogenous chemicals especially CYP3A4 substrates.