Effects of Acacetin on the Pharmacokinetics of Diazepam in vivo and in vitro.

Background: Herb-drug interactions (HDIs) have garnered significant attention in recent years. Objectives: To investigate the effects of acacetin on the pharmacokinetics of diazepam both in vivo and in vitro. Methods: Rat liver microsomes (RLMs) were incubated with diazepam and acacetin to determine the half-maximal inhibitory concentration (IC50) and inhibition constant (Ki) values of acacetin, as well as to evaluate its inhibitory effect on diazepam metabolism in vitro. For the in vivo experiment, twelve male Sprague-Dawley rats were randomly allocated into two groups (n = 6) and received either 50 mg/kg acacetin or vehicle for two weeks. Subsequently, diazepam (10 mg/kg) was administered to each rat. Blood samples (300 μL) were collected from the tail vein at 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, and 8 hours post-administration. The plasma concentrations of diazepam and its metabolites were quantified using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Results: The IC50 values for temazepam and nordiazepam in RLMs were 2.065 μM and 5.2 μM, respectively. The Ki values for temazepam and nordazepam demonstrated that acacetin inhibits diazepam metabolism in vitro. In vivo, pretreatment with acacetin increased the area under the curve (AUC) and maximum plasma concentration (Cmax) of diazepam, while significantly decreasing its apparent clearance (CLz/F, P < 0.05). The AUC values for temazepam and nordiazepam decreased, whereas their CLz/F values increased significantly (P < 0.05). PyMOL simulations indicated that acacetin and diazepam share the same cytochrome P450 3A4 (CYP3A4) or cytochrome P450 2C19 (CYP2C19) binding pocket, suggesting that acacetin inhibits diazepam metabolism via competitive inhibition. Conclusions: Acacetin significantly altered the pharmacokinetics of diazepam both in vivo and in vitro, indicating a potential interaction between acacetin and diazepam. Therefore, the concomitant use of acacetin and diazepam in clinical practice should be approached with caution.