Elsevier

Biochemical Pharmacology

Volume 68, Issue 12, 15 December 2004, Pages 2409-2416
Biochemical Pharmacology

In vitro and ex vivo evidence for modulation of P-glycoprotein activity by progestins

https://doi.org/10.1016/j.bcp.2004.08.026Get rights and content

Abstract

The well known gender-related differences in drug action may partly be explained by changes in activity and expression of drug metabolising enzymes, but also by modulation of active drug transport systems (e.g. P-glycoprotein, Pgp) by sexual steroids, which is yet not well investigated. Because many women are using hormones (e.g. as oral contraceptives) we investigated the influence of different synthetic progestins on Pgp activity. Pgp inhibition of progesterone, medroxyprogesterone, chlormadinone, cyproterone, levonorgestrel, norethisterone, desogestrel, and norgestimate was measured in vitro in two Pgp over-expressing cell lines (L-MDR1, P388/dx cells) and the corresponding parental cell lines by means of calcein assay, and ex vivo in human peripheral blood mononuclear cells (PBMCs) by rhodamine123 efflux. For most progestins tested, concentrations needed to double baseline fluorescence (f2) in L-MDR1 cells were similar to that of the potent Pgp inhibitor quinidine, whereas levonorgestrel and norethisterone did not reach f2. The results in P388/dx cells essentially confirmed our findings in L-MDR1 cells. Additionally, Pgp inhibitory activity of all progestins tested was also shown ex vivo in PBMCs. The potent Pgp inhibition by several synthetic progestins in vitro and ex vivo suggests that such an interaction might be clinically relevant despite generally low plasma concentrations of progestins. The results may be of particular importance for Pgp substrates, such as protease inhibitors and chemotherapeutic agents, for which intracellular concentrations are critical.

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Materials and drugs

Culture media, FCS, medium supplements, antibiotics, and HBSS were purchased from Invitrogen, collagen-R was from Serva, DMSO and Triton X-100 were from AppliChem, doxorubicin hydrochloride from Sigma–Aldrich, calcein-AM from MoBiTec, rhodamine123 from Calbiochem, vincristine from Merck Biosciences, 96-well microtiter plates and culturing bottles were from Nunc. The PE labelled antibodies against human CD8+ and mouse IgG2a as well as the vacutainer®CPT™ were purchased from BD Biosciences.

Evaluation of the Pgp inhibitory potency of progestins with the calcein assay

Due to the low solubility of the progestins and the very high expression level of Pgp in L-MDR1 cells, none of the progestins revealed plateau effects in L-MDR1 cells and levonorgestrel and norethisterone even did not reach f2 values (Table 1; Fig. 1A–C). We therefore also tested all compounds in P388/dx cells, a murine cell line over-expressing Pgp as well, but to a lower extent than L-MDR1 cells. In this cell line some progestins showed plateau effects and only levonorgestrel did not reach f2

Discussion

The results of the present in vitro/ex vivo study revealed a potent Pgp inhibitor effect of several progestins, which are widely used for OC and HRT. The extent of Pgp inhibition in L-MDR1 cells by most progestins was comparable to that of the potent Pgp inhibitor quinidine and slightly weaker than that of verapamil. The rank order of inhibition was essentially confirmed in P388/dx cells with one notable exception. In contrast to other progestins whose f2 values were roughly one order of

Acknowledgements

The authors thank Mrs. Stephanie Fuchs for her excellent technical assistance and Mrs. Jennifer Lohr for conducting some of the calcein assays. Furthermore, we thank Janssen-Cilag GmbH, Eli Lilly and Co., and Grünenthal GmbH for kindly providing the test compounds, Dr. Dario Ballinari (Pharmacia and Upjohn) for providing the cell lines P388 and P388/dx, and Dr. Alfred H. Schinkel for providing the cell line L-MDR1. Moreover, we would like to thank Dr. Nina Wettschurek for her help with the flow

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