Method. Due to limited solubility and ionization of bilirubin, various probe substrates were utilized to examine the activities of UGT1A1, MRP2 and OATP1B1. Specifically, the inhibition of UGT1A1 activity was examined in human liver microsomes and recUGT1A1 by monitoring the formation of estradiol 3-glucuronide (E3G) in the absence or presence of various concentrations of inhibitors. The inhibition of MRP2 activity was examined by monitoring the accumulation of carboxy dichlorofluorescein (CDCF) in MRP2 membrane vesicles in the absence or presence of various concentrations of inhibitors. The inhibition of OATP1B1 activity was examined by monitoring the flux of pitavastatin in OATP1B1 expressing HEK cells in the absence or presence of various concentrations of inhibitors.
Results. Ritonavir was the most potent inhibitor of E3G formation with IC50 value of 0.8 µM. This was followed by troglitazone, bromfenac and labetalol at IC50 value of 7.7, 10 and 55 µM, respectively. Troglitazone was the more potent inhibitor of MRP2 with IC50 value of 16 µM. Labetalol and ritonavir did not inhibit MRP2 while bromfenac had only a modest affect on MRP2 activity. Ritonavir and troglitazone were potent inhibitors of OATP1B1 with IC50 value of 0.4 and 0.5 µM, respectively.
Conclusions. UGT1A1 inhibition alone is not sufficient to explain hepatotoxicity characterized by Hy’s Law because the more potent inhibitor of UGT1A1 activity was ritonavir which does not induce elevated bilirubin levels. Troglitazone exhibits significant hepatotoxicity and can be described by Hy’s Law. It was shown to block the activity of UGT1A1, MRP2 and OATP1B1. However, although bromfenac and labetalol can also be described by Hy’s Law, they were poor inhibitors of OATP1B1 and MRP2. These results suggest that classification under Hy’s Law cannot be predicted by UGT1A1, MRP2 nor OATP1B1, and that other factors such as BSEP may be important.