Angular-type
pyranocoumarins (APs) show attractive prospects in anti-
hypertension,
chemotherapy and anti-HIV treatment. Previous studies revealed extensive hepatic metabolisms of several APs following similar pathways. This study investigated the
enzyme kinetics and the main CYP450
isozyme(s) involved in metabolism of (+)-
praeruptorin A (dPA), an AP with significant cardio-protective activities, in human liver microsomes (HLMs) using ultra high-performance liquid chromatography coupled with a hybrid quadrupole-linear ion trap mass spectrometry (UHPLC-QT-MS/MS). dPA produced 6 metabolites via hydrolysis (M1-M3), oxidation (M4-M6), and hydrolysis followed by acyl migration (M2 or M3). Oxidation at the C-3' side chain instead of the
coumarin ring was consolidated with the aromatic
proton signal in NMR spectra. The major metabolite (-)-cis-
khellactone (M1) followed biphasic kinetics in HLMs with high affinity (Km1 0.02μM) and intrinsic clearance (CLint1, invitro1.29mL/min/mg
protein), whereas other metabolites (M2-M6) fitted typical Michaelis-Menten kinetics with lower affinity (Km 3.85-39.13μM). Recombinant human
CYP3A4 showed the highest activity toward M1 and M4 formation, while it was
CYP2C19 for M2/M3 and M5 and
CYP2B6 for M6. Principal component analysis of the metabolite formation profile of dPA also revealed the highest similarity between
CYP3A4 and HLMs. Both
quercetin (
CYP2C8 inhibitor) and
ketoconazole (
CYP3A4 inhibitor) showed 60-100% inhibition of M1-M4 and M6 formations in HLMs, while M5 formation was mainly inhibited by α-naphthoflavone (
CYP1A2 inhibitor, 70-80%) and
quercetin (90%). Moreover, formations of all metabolites were predominantly inhibited by
CYP3A4 antibody (37-68%). These findings shed a light on main involvement of
CYP3A4 in human hepatic elimination of APs, indicating potential drug interactions.