Research Articles
Interactions of azole antifungal agents with the human breast cancer resistance protein (BCRP)

https://doi.org/10.1002/jps.20963Get rights and content

ABSTRACT

Breast cancer resistance protein (BCRP) is an efflux transporter that plays an important role in drug disposition. The goal of this study was to investigate the interactions of azole antifungal agents, ketoconazole, itraconazole, fluconazole, and voriconazole, with BCRP. First, the effect of the azoles on BCRP efflux activity in BCRP‐overexpressing HEK cells was determined by measuring intracellular pheophorbide A (PhA) fluorescence using flow cytometry. We found that keotoconazole and itraconazole significantly inhibited BCRP‐mediated efflux of PhA at low µM concentrations. However, fluconazole only mildly inhibited and voriconazole did not inhibit BCRP efflux activity at concentrations up to 100 µM. The IC50 value of ketoconazole for inhibition of BCRP‐mediated PhA efflux was 15.3 ± 6.5 µM. Ketoconazole and itraconazole also effectively reversed BCRP‐mediated resistance of HEK cells to topotecan. When direct efflux of [3H]ketoconazole was measured in BCRP‐overexpressing HEK cells, we found that [3H]ketoconazole was not transported by BCRP. Consistent with this finding, BCRP did not confer resistance to ketoconazole and itraconazole in HEK cells. Taken together, ketoconazole and itraconazole are BCRP inhibitors, but fluconazole and voriconazole are not. These results suggest that BCRP could play a significant role in the pharmacokinetic interactions of ketoconazole or itraconazole with BCRP substrate drugs. © 2007 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 96: 3226–3235, 2007

Section snippets

INTRODUCTION

The human breast cancer resistance protein (BCRP, gene symbol ABCG2) belongs to the subfamily G of the large ATP‐binding cassette (ABC) transporter superfamily.1., 2., 3. Recent studies have illustrated that BCRP is capable of transporting a broad range of substrates, from highly lipophilic anticancer agents (e.g., mitoxantrone and the anthracyclines) to hydrophilic organic anions (e.g., estrone‐3‐sulfate and 17β‐estradiol 17‐(β‐D‐glucuronide)).4., 5., 6. The molecular mechanism by which BCRP

Materials

Ketoconazole and fluconazole were purchased from Sigma (St. Louis, MO). Itraconazole was from Research Diagnostics (Flanders, NJ). Voriconazole was a gift from Dr. Kenneth E. Thummel (Department of Pharmaceutics, University of Washington, Seattle, WA). [3H]ketoconazole (10 Ci/mmol) was from American Radiolabeled Chemicals (St. Louis, MO). Pheophorbide A (PhA) was from Frontier Scientific (Logan, UT). Topotecan was a gift from GlaxoSmithKline (Research Triangle Park, NC). Fumitremorgin C (FTC)

Inhibition of BCRP‐Mediated PhA Efflux by Azole Antifungals

Since PhA is a fluorescent compound and is known to be a specific substrate for BCRP, but not for P‐gp and MRP1,27 it was used as a model BCRP substrate in determining the effects of azole antifungals on BCRP efflux activity. We used BCRP‐overexpressing HEK cells for PhA efflux experiments. We have shown that the HEK cells express little endogenous P‐gp, MRP1, or MRP2.22 In the present study, we further determined the endogenous expression of MRP4 in HEK cells by immunoblotting. No MRP4 was

DISCUSSION

In the present study, we have for the first time investigated the interactions of BCRP with four azole antifungals with different chemical structures (Fig. 1). We found that ketoconazole can inhibit BCRP‐mediated PhA efflux with an IC50 value of approximately 15 µM (Fig. 3). This inhibitory potency is lower than that of FTC which has an IC50 value of approximately 1 µM for BCRP.23 Itraconazole also significantly inhibited BCRP at concentrations below 1 µM (Fig. 2). However, we could not measure

Acknowledgements

We gratefully acknowledge financial support from NIH grant HD044404 (to QM and JDU). The authors thank Dr. Robert W. Robey and Dr. Susan E. Bates (National Cancer Institute, Bethesda, MD) for providing the HEK cell lines. We also acknowledge GlaxoSmithKline (Research Triangle Park, NC) and Dr. Kenneth E. Thummel (Department of Pharmaceutics, University of Washington, Seattle, WA) for providing topotecan and voriconazole, respectively.

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