PXR-ABC drug transporters/CYP-mediated ursolic acid transport and metabolism in vitro and vivo.

The transporting kinetics and metabolic kinetics of ursolic acid were studied in transgenic cell models. Then, the pharmacokinetics features of ursolic acid and the expression of ATP-binding cassette transporters (ABC transporter) and cytochrome P450 (CYP) enzymes in tissues after pregnane X receptor (PXR) activation by 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN) were investigated in rats. After silencing of PXR in Caco2-siRNA-PXR cells, there was a decrease in the protein abundance of P-glycoprotein, breast cancer-resistant protein, multidrug resistance-associated protein 2 (MRP2), and CYP2C9. The apparent permeability (PDR) values of 10, 20, and 50 µM ursolic acid in Caco2 cells were 2.19 ± 0.44, 1.40 ± 0.17, and 1.25 ± 0.07, respectively, whereas in Caco2-siRNA-PXR cells, they were 1.85 ± 0.36, 1.24 ± 0.11, and 1.19 ± 0.04, respectively. PXR-RXRα would significantly activate ABC transporter expression in Caco2 cells. Compared with Caco2 cells, when the concentrations of ursolic acid were 10, 20, and 50 µM, the PDR values increased in Caco2-PXR-RXRα cells after PXR activation: 1.60 ± 0.31 versus 1.97 ± 0.21, 1.46 ± 0.08 versus 2.01 ± 0.19, and 1.32 ± 0.26 versus 2.09 ± 0.22, respectively. Simultaneously, PXR-RXRα would activate the expression of CYP2C9; metabolic kinetics of ursolic acid in CYP metabolizing enzyme lysate of Caco2 cells and Caco2-PXR-RXR cells was studied and it was found that the Km values were 81.99 ± 44.32 and 60.05 ± 29.62 µg/ml, and Vmax values were 3.77 ± 0.86 and 3.41 ± 0.96 µg · ml-1  · min-1 , respectively. However, in human CYP metabolizing recombinase, we found that both CYP2C9 and CYP34A were involved in the metabolism of ursolic acid. Vm and Km values for CYP3A4 and CYP2C9 were 3.57 ± 1.12 µg · ml-1  · min-1 and 81.71 ± 18.38 µg/ml, 3.85 ± 1.46 µg · ml-1  · min-1 and 62.18 ± 14.56 µg/ml, respectively. As a strong agonist for mouse pxr, PCN could significantly affect pharmacokinetics of ursolic acid in rats, and it showed discrepant effects on messenger RNA expression of cyp and transporters in tissues.