Effect of pregnane X receptor ligands on transport mediated by human OATP1B1 and OATP1B3
Introduction
Nuclear receptors are ligand-activated transcription factors that play an important role in xenobiotic disposition and human diseases like diabetes, obesity and cancer (Giguere, 1999, Wang and LeCluyse, 2003). Numerous clinically prescribed drugs including dexamethasone and rifampicin are ligands of the human pregnane X receptor, which is highly expressed in hepatocytes. By binding to their receptors, these drugs regulate the expression of many drug-metabolizing enzymes (Xie et al., 2004) and transporters (Guo et al., 2002). Therefore they can affect the metabolism of the majority of drugs. With the exception of rifampicin (Vavricka et al., 2002), paclitaxel (Smith et al., 2005) and lithocholate (Yamaguchi et al., 2006), it is largely unknown how pregnane X receptor ligands enter hepatocytes to bind to their receptor, but in general it is assumed to be by simple diffusion.
Organic anion transporting polypeptides (rodents: Oatps; human: OATPs) belong to a growing superfamily of transport proteins that mediate uptake of structurally diverse amphiphilic organic solutes (Hagenbuch and Meier, 2004). Among the 11 human OATPs, OATP1B1 and OATP1B3 are liver specific transporters that are multispecific and mediate the uptake of numerous drugs into hepatocytes (Hagenbuch and Meier, 2003). Previous work has shown that the glucocorticoid dexamethasone and the antibiotic rifampicin directly interact with OATPs. We demonstrated that dexamethasone inhibits OATP1A2-mediated dehydroepiandrosterone sulfate transport (Kullak-Ublick et al., 1998) in addition to being a substrate of rat Oatp1a1 (Bossuyt et al., 1996). Furthermore, we reported that rifampicin inhibits transport mediated by Oatp1a1 and Oatp1a4 (Fattinger et al., 2000). A more recent study showed that rifampicin not only inhibits human OATP1B1 and OATP1B3 but is directly transported by both OATPs (Vavricka et al., 2002).
Because drug–drug interactions can occur due to direct inhibition of uptake into liver in which OATP1B1 and OATP1B3 are expressed, we hypothesized that additional, readily available pregnane X receptor ligands including carbamazepine, clotrimazole, estradiol, lithocholate, metyrapone, mevinolin, mifepristone, paclitaxel, phenytoin, and troglitazone (Goodwin et al., 1999, Honkakoski et al., 2003) would interact with OATP1B1- and OATP1B3-mediated transport. To test this hypothesis, we developed Chinese Hamster Ovary (CHO) cells stably expressing either OATP1B1 or OATP1B3 and determined substrate uptake into these cells in the presence as well as absence of pregnane X receptor ligands.
Section snippets
Materials
Radiolabeled [3H]estradiol-17β-glucuronide (39.8 Ci/mmol) and [3H]estrone-3-sulfate (57.3 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA), and [3H]clotrimazole (7.5 Ci/mmol) from Moravek Biochemicals Inc. (Brea, CA). The cell-impermeant Fluo-3, pentapotassium salt was obtained from Invitrogen (Carlsbad, CA). Cell culture reagents were from Invitrogen (Carlsbad, CA) and fetal bovine serum from Hyclone (Logan, UT). All pregnane X receptor ligands were purchased from Sigma (St.
Characterization of OATP1B1 and OATP1B3 cell lines
We constructed CHO cell lines that stably express the most frequent variants of OATP1B1 (OATP1B1⁎1b or N130D) (Tirona et al., 2001) and OATP1B3 (haplotype 1 or S112A and M233I) (Smith et al., 2007). To demonstrate expression of OATP1B1 and 1B3 in the generated CHO cell lines, we used confocal microscopy. The results shown in Fig. 1(A and B) demonstrate that the respective cell lines expressed OATP1B1 or 1B3 at the plasma membrane. To functionally characterize these OATP1B1- and 1B3-expressing
Discussion
The experiments presented here show that several pregnane X receptor ligands interact with uptake mediated by human OATP1B1 and 1B3 at pharmacological concentrations. Therefore, these pregnane X receptor ligands have the potential to cause adverse interactions with other drugs that are transported by the two liver specific OATPs. In agreement with previously reported studies for several drugs (Campbell et al., 2004, Hirano et al., 2006, Shitara et al., 2003, Vavricka et al., 2002) most tested
Acknowledgments
Confocal images were acquired at KUMC core facility (http://www.kumc.edu/cic), supported by NIH Shared Resource Grant (NCRR RR14637-01) and the Kansas Biomedical Research Infrastructure network. This work was supported by National Institute of Health grants RR021940, and GM077336 to BH and in part by A-Cute-Tox (EU LSHD-CT-2004-512051) to BS.
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