Effects of bergapten on the pharmacokinetics of macitentan in rats both in vitro and in vivo

Abstract

Macitentan was approved by the United States Food and Drug Administration (FDA) in 2013 for the treatment of pulmonary arterial hypertension (PAH). Bergapten is a furanocoumarin that is abundant in Umbelliferae and Rutaceae plants and is widely used in many Chinese medicine prescriptions. Considering the possible combination of these two compounds, this study is aimed to investigate the effects of bergapten on the pharmacokinetics of macitentan both in vitro and in vivo. Rat liver microsomes (RLMs), human liver microsomes (HLMs), and recombinant human CYP3A4 (rCYP3A4) were used to investigate the inhibitory effects and mechanisms of bergapten on macitentan in vitro. In addition, pharmacokinetic parameters were also studied in vivo. Rats were randomly divided into two groups (six rats per group), with or without bergapten (10 mg/kg), and pretreated for 7 days. An oral dose of 20 mg/kg macitentan was administered to each group 30 min after bergapten or 0.5% CMC-Na administration on day 7. Blood was collected from the tail veins, and the plasma concentrations of macitentan and its metabolites were assessed by ultra-performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS). Finally, we analyzed the binding force of the enzyme and two small ligands by in silico molecular docking to verify the inhibitory effects of bergapten on macitentan. The in vitro results revealed that the IC₅₀ values for RLMs, HLMs, and rCYP3A4 were 3.84, 17.82 and 12.81 μM, respectively. In vivo pharmacokinetic experiments showed that the AUC_(0-t), AUC_(0-∞), and C_max of macitentan in the experimental group (20,263.67 μg/L*h, 20,378.31 μg/L*h and 2,999.69 μg/L, respectively) increased significantly compared with the control group (7,873.97 μg/L*h, 7,897.83 μg/L*h and 1,339.44 μg/L, respectively), while the CL_z/F (1.07 L/h/kg) of macitentan and the metabolite-parent ratio (MR) displayed a significant decrease. Bergapten competitively inhibited macitentan metabolism in vitro and altered its pharmacokinetic characteristics in vivo. Further molecular docking analysis was also consistent with the experimental results. This study provides a reference for the combined use of bergapten and macitentan in clinical practice.

Keywords: bergapten; drug-drug interaction; macitentan; pharmacokinetics; pulmonary arterial hypertension.

FIGURE 1

Macitentan metabolism to ACT-132577 and ACT-373898. Notes: * Main metabolic pathway.

FIGURE 2

SRM chromatograms of macitentan, ACT-132577 and IS.

FIGURE 3

Michaelis-Menten kinetics and IC₅₀ plots of effect of bergapten on macitentan in RLMs (A, B), HLMs (C, D) and human recombinant (E, F) (n = 3, mean ± SD).

FIGURE 4

Michaelis-Menten model (A), Lineweaver–Burk plots (B), and slope of primary plot (C) for bergapten inhibition of macitentan in RLMs (n = 3, mean ± SD).

FIGURE 5

Michaelis-Menten model (A), Lineweaver–Burk plots (B), and slope of primary plot (C) for bergapten inhibition of macitentan in HLMs (n = 3, mean ± SD).

FIGURE 6

Michaelis-Menten model (A), Lineweaver–Burk plots (B), and slope of primary plot (C) for bergapten inhibition of macitentan in rCYP3A4 (n = 3, mean ± SD).

FIGURE 7

Mean plasma concentration-time profiles of macitentan (A) and ACT-132577 (B) (n = 6, mean ± SD).

FIGURE 8

Overall view of the crystal structure of CYP3A4 and small ligands (A). Detailed view of the crystal structure of CYP3A4 docked with bergapten (Blue) and macitentan (Pink) (B).