Abstract

Ethinylestradiol (EE) is one of the key constituents of oral contraceptives. Major metabolites of EE in humans are the glucuronide and sulfate conjugates, EE-3-O-glucuronide (EE-G) and EE-3-O-sulfate (EE-S). In the present study, transport of EE-G and EE-S by the human multidrug resistance proteins MRP1, MRP2, and MRP3 was investigated using inside-out membrane vesicles, isolated from Sf9 cells expressing human MRP1, MRP2, or MRP3. Vesicular uptake studies showed that EE-G was not a substrate for MRP1, whereas an ATP-dependent and saturable transport of [³H]EE-G was observed in MRP2 (K_m of 35.1 ± 3.5 μM) and MRP3 (K_m of 9.2 ± 2.3 μM) containing vesicles. EE-S was not transported by either MRP1, MRP2, or MRP3. However, low concentrations of EE-S stimulated MRP2-mediated uptake of ethacrynic acid glutathione. EE-S also stimulated MRP2 and MRP3-mediated uptake of 17β-estradiol-17β-d-glucuronide. Interestingly, EE-S stimulated strongly MRP2- and MRP3-mediated uptake of EE-G by increasing its apparent transport affinity, whereas no reciprocal stimulation of EE-S uptake by EE-G was observed. These data indicate that EE-S allosterically stimulates MRP2- and MRP3-mediated transport of EE-G and is not cotransported with EE-G. Our studies demonstrate specific active transport of a pharmacologically relevant drug conjugate by human MRP2 and MRP3, involving complex interactions with other organic anions. We also suggest that caution needs to be taken when using only competition studies as screening tools to identify substrates or inhibitors of MRP-mediated transport.