Category: Preclinical Development
Purpose: Hepatic uptake transporter Organic Anion Transporting Polypeptides (OATPs) are involved in the uptake of glucuronides. The purpose of the current study is to investigate the involvement of the three most important hepatic uptake transporters OATP 1B1, 1B3 and 2B1 in the uptake of flavonoid glucuronides. Based on the affinity of three model compounds i.e. wogonoside (wogonin 7-glucuronide), luteolin 3’-glucuronide and apigenin 7-glucuronide, we further aimed at developing a screening method to evaluate the substrate affinity of the transporters more efficiently.
Methods: OATP 1B1, 1B3 and 2B1 overexpressed HEK-293 cell lines were used in the current study. Concentration dependent cell uptake study (1, 5, 10, 25, 50 µM) were performed to acquire the Km and Vmax of the model compounds. Cross-inhibition studies were performed to screen the substrate in OATP 1B1 overexpressed cells. Wogonoside, luteolin 3’-glucuronide and estradiol 17-glucuronide, a widely used in vitro substrate for OATP 1B1, were used as substrates. A series of compounds (including a well-recognized OATP 1B1 inhibitor rifampicin) were incubated with the substrates as inhibitors to determine their inhibition ability. All experiments were performed in triplicates. The intracellular concentrations were analyzed by LC-MS.
Results: Despite the fact that the model compounds have similar chemical structures, the Km and Vmax for wogonoside, luteolin 3’-glucuronide and apigenin 7-glucuronide varied dramatically. The Km of OATP 1B1 and 2B1 for luteolin 3’-glucuronide was 4.46 and 0.89 µM, with Vmax of 22.8 and 4.1 pmol/min, respectively. The results suggested that OATP 1B1 and 2B1 competed for luteolin 3’-glucuronide, and OATP1B1 acted as a relatively low affinity, high capacity enzyme while OATP 2B1 acted as a high affinity, low capacity enzyme. For wogonoside, the Km of OATP 1B1 was determined to be 27.68 µM with a Vmax of 55.4 pmol/min. Apigenin 7-glucuronide had a Km of 14.8 µM for OATP 2B1 and a Vmax of 5.8 pmol/min. Other substrate-transporter pairs did not display a saturable kinetics up to 50 µM which is beyond relevant biological concentration in human (in nM range), suggesting they were not significantly involved in the uptake process. In the cross-inhibition study in cells overexpressing OATP 1B1, all three compounds were significantly inhibited by the OATP 1B1 inhibitor rifampicin ( >97%). The results also showed no significant inhibition by luteolin 7-glucuronide, which was determined not a substrate of the transporter. For wogonoside, inhibition effects from other compounds were determined as rifampicin (98%) > luteolin (93%) > estradiol (90%) > estradiol 17-glucuronide (84%) > luteolin 3’-glucuronide (72%) > wogonin (61%) > luteolin 7-glucuronide (5.9%). For E2G, the inhibition effects were rifampicin (99%) > estradiol (90%) > luteolin (89%) = luteolin 3’-glucuronide (89%) > wogonin (79%) > wogonoside (67%) > luteolin 7-glucuronide (32%). For luteolin 3’-glucuronide, the inhibition effects were rifampicin (98%) > luteolin (97%) > estradiol (92%) = estradiol 17-glucuronide (92%) > wogonin (87%) > wogonoside (61%) > luteolin 7-glucuronide (11%). The compounds used as inhibitors showed similar inhibition ability toward the three substrates. Comparing with the compound affinity of the transporter, compounds with higher affinity of the transporter tended to have better inhibition ability over the transporting process. In addition, parent compounds generally had better or similar inhibition effects on the transporter than their glucuronides.
Conclusion: In spite of the structural similarity, wogonoside, luteolin 3’-glucuronide and apigenin 7-glucuronide have different affinity towards OATP 1B1, 1B3 and 2B1. A cross-inhibition method is developed for screening the affinity of a compound toward the transporter efficiently.
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, Oklahoma City, Oklahoma
Maria Karlgren– Researcher, Uppsala University, Uppsala, Uppsala Lan, Sweden
Ming Hu– Professor of Pharmaceutics, University of Houston, Houston, Texas