In vitro evaluation of norwogonin as a potential inhibitor of cytochrome P450 forms 3A4, 2D6, and 2C9

Norwogonin, a bioactive flavonoid with anti-inflammatory and anticancer potential. Its interactions with cytochrome P450 enzymes remain uncharacterised. This study aimed to investigate the effect of norwogonin on the activity of P450s.The inhibitory effects of norwogonin on P450 forms (1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4) were evaluated using pooled human liver microsomes (HLMs). Dose-dependent inhibition assays (0–100 μM norwogonin) and time-dependent studies (0–30 min incubation) were conducted with form-specific probe substrates. Metabolites were quantified via High Performance Liquid Chromatography (HPLC).Norwogonin selectively inhibited 2C9, 2D6, and 3A4 (IC50: 13.05 ± 4.62, 11.33 ± 2.21, and 7.52 ± 0.61 μM, respectively), with 3A4 exhibiting the highest sensitivity. Kinetic analysis revealed distinct inhibitory mechanisms of norwogonin: non-competitive for 3A4 (Ki = 3.755 μM) and competitive for 2C9 (Ki = 6.879 μM) and 2D6 (Ki = 6.028 μM). Time-dependent inhibition (TDI) of norwogonin was observed exclusively for 3A4, characterised by increasing suppression with prolonged incubation (KI = 2.323 μM−1, Kinact = 0.035 min−1), while 2C9/2D6 inhibition remained static over time.Norwogonin selectively inhibited 2C9, 2D6, and 3A4 via distinct mechanisms, with time-dependent 3A4 suppression suggesting the potential interactions, necessitating caution in co-administered therapies.