Inhibitory effects of hinokiflavone on human liver cytochrome P450 enzymes

Hinokiflavone (HF) exhibits multiple pharmacological activities. This study aimed to evaluate the effect of HF on cytochrome P450 (CYP450s), which may provide a theoretical basis for its rational clinical application.

Human pooled liver microsomes (HLMs) were incubated with HF and CYP isoform-specific probe substrates, with the vehicle group (no HF and positive inhibitors) serving as a negative control group and positive inhibitors as a positive control group. The inhibitory effect of HF on CYP2C9, 2C19, and 3A4 was evaluated by using HF at concentrations of 0, 2, 5, 10, 20, 50, and 100 μM. The inhibitory model and parameters of HF on CYP2C9, 2C19, and 3A4 were assessed by the Lineweaver-Burk plots, secondary linear and nonlinear regression analysis.

Selective inhibition of HF was observed on CYP2C9, 2C19, and 3A4 among the eight main CYP enzymes. HF showed concentration-dependent inhibition on CYP2C9, 2C19, and 3A4 with the IC₅₀ values of 6.79 μM, 13.59 μM, 3.52 μM for 3A4-T (testosterone as the substrate), and 3.95 μM for 3A4-M (midazolam as the substrate), respectively. Through the inhibitory model evaluation, HF was revealed to be a competitive inhibitor of 2C9 (Kᵢ = 2.951 μM) and 2C19 (Kᵢ = 5.958 μM). The inhibitory model of HF on CYP3A4 was a non-competitive and time-dependent inhibition with the K_i of 1.226 μM, K_I of 1.410 μM, and K_inact of 0.095 min^-1.

HF competitively inhibited the activities of CYP2C9 and CYP2C19, whereas it exhibited non-competitive and TDI against CYP3A4. Concomitant use of HF with medications primarily metabolized by CYP2C9, CYP2C19, or CYP3A4 may pose a risk of clinically relevant drug-drug interactions (DDI).