Abstract
Purpose. Although sister-P-glycoprotein (SPGP, BSEP) is closely related to P-glycoprotein, it is much more selective in distribution and substrate recognition. Moreover, because inhibition or lack of BSEP function has severe consequences including cholestasis, hepatotoxicity, exposure to toxic xenobiotics, and drug interactions, in vitro methods are necessary for quantifying and characterizing specific inhibition of BSEP. Therefore, the objective is to discern a method and quantitatively characterize several example BSEP inhibitors.
Methods. With fluorescent markers having been used successfully to evaluate and quantify inhibition of P-gp-mediated transport, this study evaluates several compounds for specific cell retention caused by BSEP inhibitors. In addition to the several compounds asserted to be BSEP inhibitors, the compounds suggested to be BSEP substrates might also inhibit BSEP competitively. Retained fluorescence of possible BSEP substrates was measured by a flow cell cytometer using transfected cells presenting the BSEP transporter specifically and abundantly.
Results. Several compounds were shown to inhibit BSEP active transport of the fluorescent substrates dihydrofluorescein and bodipy. The inhibition potency was quantified (i.e., cyclosporin A IC50 ∼ 7 μM), revealing incongruent relative sensitivities among the substrate markers, with H2FDA generally the most sensitive of the series of substrate markers evaluated.
Conclusions. The inconsistent sensitivities of the transport markers (H2FDA and bodipy) were reminiscent of the apparent multiple binding site behaviors observed for P-gp and could indicate opposing and unequal yet interacting binding sites akin to those of P-gp. Nonetheless, notable differences between P-gp and BSEP in marker substrate recognition/transport were apparent despite the observed overlap in xenobiotic recognition and transport. Thus far the most potent inhibitors seem to be cyclosporin, tamoxifen, and valinomycin. There are likely to be much more potent inhibitors, and other substrates also may be more sensitive to inhibition of transport.
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REFERENCES
S. V. Ambudkar, S. Dey, C. A. Hrycyna. M. Ramachandra, I. Pastan, and M. M. Gottesman. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu. Rev. Pharmacol. Toxicol. 39:361-398 (1999).
F. J. Sharom, R. Liu, Q. Qu, and Y. Romsicki. Exploring the structure and function of the P-glycoprotein multidrug transporter using fluorescence spectroscopic tools. Semin. Cell Dev. Biol. 12:257-265 (2001).
S. Childs, R. L. Yeh, E. Georges, and V. Ling. Identification of a sister gene to P-glycoprotein. Cancer Res. 55:2029-2034 (1995).
R. Thompson and S. Strautnieks. BSEP: function and role in progressive familial intrahepatic cholestasis. Semin. Liver Dis. 21:545-550 (2001).
P. J. Meier and B. Stieger. Bile salt transporters. Annu. Rev. Physiol. 64:635-661 (2002).
S. S. Strautnieks, L. N. Bull, A. S. Knisely, S. A. Kocoshis, N. Dahl, H. Arnell, E. Sokal, K. Dahan, S. Childs, V. Ling, M. S. Tanner, A. F. Kagalwalla, A. Nemeth, J. Pawlowska, A. Baker, G. Mieli-Vergani, N. B. Freimer, R. M. Gardiner, and R. J. Thompson. A gene encoding a liver-specific ABC transporter is mutated in progressive familial intrahepatic cholestasis. Nat. Genet. 20:233-238 (1998).
R. Wang, M. Salem, I. M. Yousef, B. Tuchweber, P. Lam, S. J. Childs, C. D. Helgason, C. Ackerley, M. J. Phillips, and V. Ling. Targeted inactivation of sister of P-glycoprotein gene (BSEP) in mice results in non-progressive but persistent intrahepatic cholestasis. Proc. Natl. Acad. Sci. USA 98:2011-2016 (2001).
B. Stieger, K. Fattinger, J. Madon, G. A. Kullak-Ublick, and P. J. Meier. Drug-and estrogen-induced cholestasis through inhibition of the hepatocellular bile salt export pump (Bsep) of rat liver. Gastroenterology 118:422-430 (2000).
S. Childs, R. L. Yeh, D. Hui, and V. Ling. Taxol resistance mediated by transfection of the liver-specific sister gene of P-glycoprotein. Cancer Res. 58:4160-4167 (1998).
C. Funk, C. Ponelle, G. Scheuermann, and M. Pantze. Cholestatic potential of troglitazone as a possible factor contributing to troglitazone-induced hepatotoxicity: in vivo and in vitro interaction at the canalicular bile salt export pump (Bsep) in the rat. Mol. Pharmacol. 59:627-635 (2001).
V. E. Kostrubsky, M. Vore, E. Kindt, J. Burliegh, K. Rogers, G. Peter, D. Altrogge, and M. W. Sinz. The effect of troglitazone biliary excretion on metabolite distribution and cholestasis in transporter-deficient rats. Drug Metab. Dispos. 29:1561-1566 (2001).
K. Fattinger, C. Funk, M. Pantze, C. Weber, J. Reichen, B. Stieger, and P. J. Meier. The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: a potential mechanism for hepatic adverse reactions. Clin. Pharmacol. Ther. 69:223-231 (2001).
E. J. Wang, C. N. Casciano, R. P. Clement, and W. W. Johnson. In vitro flow cytometry method to quantitatively assess inhibitors of P-glycoprotein. Drug Metab. Dispos. 28:522-528 (2000).
E. J. Wang, C. N. Casciano, R. P. Clement, and W. W. Johnson. Active transport of fluorescent P-glycoprotein substrates: evaluation as markers and interaction with inhibitors. Biochem. Biophys. Res. Commun. 289:580-585 (2001).
E. J. Wang, C. N. Casciano, R. P. Clement, and W. W. Johnson. Cooperativity in the inhibition of P-glycoprotein-mediated daunorubicin transport: evidence for half-of-the-sites reactivity. Arch. Biochem. Biophys. 383:91-98 (2000).
V. Lecureur, D. Sun, P. Hargrove, E. G. Schuetz, R. B. Kim, L. B. Lan, and J. D. Schuetz. Cloning and expression of murine sister of P-glycoprotein reveals a more discriminating transporter than MDR1/P-glycoprotein. Mol. Pharmacol. 57:24-35 (2000).
S. Dey, M. Ramachandra, I. Pastan, and M. M. Gottesman. Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein. Proc. Natl. Acad. Sci. USA 94:10594-10599 (1997).
E. J. Wang, C. N. Casciano, R. P. Clement, and W. W. Johnson. Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependency. Biochim. Biophys. Acta 1481:63-74 (2000).
G. Chang and C. B. Roth. Structure of MsbA from E. coli: a homolog of the multidrug resistance ATP binding cassette (ABC) transporters. Science 293:1793-1800 (2001).
T. W. Loo and D. M. Clarke. Determining the dimensions of the drug-binding domain of human P-glycoprotein using thiol cross-linking compounds as molecular rulers. J. Biol. Chem. 276:36877-36880 (2001).
U. Bolder, N. V. Trang, L. R. Hagey, C. D. Schteingart, H. T. Ton-Nu, C. Cerre, R. P. Elferink, and A. F. Hofmann. Sulindac is excreted into bile by a canalicular bile salt pump and undergoes a cholehepatic circulation in rats. Gastroenterology 117:962-971 (1999).
M. Torok, H. Gutmann, G. Fricker. and J. Drewe. Sister of P-glycoprotein expression in different tissues. Biochem. Pharmacol. 57:833-835 (1999).
E. G. Schuetz, S. Strom, K. Yasuda, V. Lecureur, M. Assem, C. Brimer, J. Lamba, R. B. Kim, V. Ramachandran, B. J. Komoroski, R. Venkataramanan, H. Cai, C. J. Sinal, F. J. Gonzalez, and J. D. Schuetz. Disrupted bile acid homeostasis reveals an unexpected interaction among nuclear hormone receptors, transporters, and cytochrome P450. J. Biol. Chem. 276:39411-39418 (2001).
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Wang, Ej., Casciano, C.N., Clement, R.P. et al. Fluorescent Substrates of Sister-P-Glycoprotein (BSEP) Evaluated as Markers of Active Transport and Inhibition: Evidence for Contingent Unequal Binding Sites. Pharm Res 20, 537–544 (2003). https://doi.org/10.1023/A:1023278211849
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DOI: https://doi.org/10.1023/A:1023278211849