Purpose: Previously it was believed that hepatocytes were not able to efficiently take up xenobiotic glucuronides, and most compounds that got excreted into bile were supposed to undergo metabolism in liver. However, our recent published data showed that through rat portal vein perfusion, the recovery of certain flavonoid glucuronides (which could not be further metabolized in liver) from bile could reach up to 90% and even higher than that if we perfused flavonoid aglycone. Such high recovery from bile was not commonly known. We hypothesized that some flavonoid glucuronides were actually able to be efficiently taken up by liver and excreted into bile, suggesting hepatic uptake transporters (e.g. OATP1B1) were responsible for the high recovery of the glucuronides. The aim of the current study was to determine whether hepatic uptake transporters were involved in the uptake of glucuronides using a cell model. To verify the assumption, wogonoside (wogonin glucuronide) and apigenin glucuronide were chosen based on high recovery in bile from our unpublished perfusion data.
Methods: OATP1B1 overexpressed HEK 293 cell line was used to determine the uptake. Estradiol 17-beta-D-glucuronide (E2G) was known as a substrate of OATP1B1 transporter therefore was used in this experiment as a control compound. The cells were seeded in 24-well plate. After attachment for 3 days, cells were treated with 1 µM substrate in the absence or presence of 10 or 25 µM rifampicin at 37 °C. Incubation time was 0, 2, 5, 10 and 20 min. Intracellular concentration of the compounds was analyzed by LC/MS. To determine the concentration dependent inhibition effects of rifampicin on wogonoside uptake, a series concentration of rifampicin (0.1 µM, 0.5 µM, 1 µM, 5 µM and 25 µM) was used.
Results: The intracellular concentration of E2G increased with time and reached steady state at a concentration of 62 nM. In the presence of 10 µM rifampicin, the concentrations of E2G at all time points were lower than 1.72 nM which was close to its LLOQ, suggesting that the transporter overexpressed cell system was functioning as expected.
Wogonoside showed a significant uptake at a maximum of 82 nM at steady state around 20 min. However, for apigenin glucuronide, the concentrations detected with LC/MS were <7 nM and close to the LLOQ, indicating that apigenin glucuronide was not a substrate of OATP1B1.
Wogonoside was subsequently incubated with different concentrations of rifampicin and results showed concentration dependent inhibition effects. A higher concentration of rifampicin leads to a lower intracellular wogonoside concentration. The IC50 of rifampicin was determined to be 0.84 µM at 10 min.
Conclusion: With the cell uptake study, it was confirmed that OATP1B1 was responsible for the uptake of wogonoside, while apigenin glucuronide was not a substrate of this transporter.
Yifan Tu– Ph.D. Candidate, University of Houston, Houston, Texas
Taijun Yin– Lab Manager, University of Houston, HOUSTON, Texas
Wei Yue– Department of Pharmaceutical Sciences, University of Oklahoma Health Sciences Center
Ming Hu– Professor of Pharmaceutics, University of Houston, Houston, Texas