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Front Pharmacol. 2019 May 7;10:496. doi: 10.3389/fphar.2019.00496. eCollection 2019.

The Efflux Mechanism of Fraxetin-O-Glucuronides in UGT1A9-Transfected HeLa Cells: Identification of Multidrug Resistance-Associated Proteins 3 and 4 (MRP3/4) as the Important Contributors.

Qin Z1,2, Zhang B1,2, Yang J1,2, Li S3, Xu J3, Yao Z3,4, Zhang X1,2, Gonzalez FJ5, Yao X3,4.

Author information

1
Department of Pharmacy, First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
2
Henan Key Laboratory of Precision Clinical Pharmacy, Zhengzhou University, Zhengzhou, China.
3
College of Pharmacy, Jinan University, Guangzhou, China.
4
Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou, China.
5
Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States.

Abstract

Fraxetin, a natural compound present in many dietary supplements and herbs, is useful in the treatment of acute bacillary dysentery and type 2 diabetes. Previously, several metabolic studies have revealed extensive first-pass metabolism causing formation of fraxetin-O-glucuronides (G1 and G2), resulting in poor bioavailability of fraxetin. Active transport processes play an important role in the excretion of fraxetin-O-glucuronides. Nevertheless, the transporters involved are yet to be elucidated. In this study, we aimed to determine the active efflux transporters, including breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs), involved in the excretion of fraxetin-O-glucuronides. A chemical inhibitor, MK571 (5 and 20 μM), a pan-MRP inhibitor, led to a significant decrease in excreted G1 (maximal 59.1%) and G2 levels (maximal 42.4%), whereas Ko143 (5 and 20 μM), a selective BCRP inhibitor, caused moderate downregulation of excreted G1 (maximal 29.4%) and G2 (maximal 28.5%). Furthermore, MRP3 silencing resulted in a marked decrease of excretion rates (by 29.1% for G1 and by 21.1% for G2) and of fraction metabolized (f met; by 24.1% for G1 and by 18.6% for G2). Similar results, i.e., a significant reduction in excretion rates (by 34.8% for G1 and by 32.3% for G2) and in f met (by 22.7% for G1 and by 23.1% for G2) were obtained when MRP4 was partially silenced. No obvious modifications in the excretion rates, intracellular levels, and f met values of glucuronides were observed after short hairpin RNA (shRNA)-mediated silencing of transporters BCRP and MRP1. Taken together, our results indicate that MRP3 and MRP4 contribute more to the excretion of fraxetin-O-glucuronides than the other transporters do.

KEYWORDS:

HeLa1A9 cells; MRPs; UGT1A9; efflux transporters; fraxetin; glucuronidation

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