Substrate-Dependent Inhibition of P-glycoprotein Mediated Efflux Transport of Digoxin

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Purpose: P-glycoprotein (P-gp, MDR1, ABCB1) is an efflux transporter expressed in most of the major barriers of the human body, including the jejunum, colon, liver, kidney, placenta, testis and the blood-brain barrier. Expression of P-gp in cells of these barriers affects the absorption, distribution and excretion of a wide range of structurally diverse drug compounds and P-gp therefore constitutes a potential site for drug-drug interactions. The U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) recommend that all new investigational drugs be screened for interactions with P-gp among other transporters in vitro. This includes an evaluation of new drug compounds as inhibitors of P-gp, preferably in competition with a clinically relevant probe substrate such as digoxin. However, increasing evidence indicates that the binding pocket of P-gp contains several overlapping binding sites for substrates. This suggests that drug-drug interactions may be pairwise specific and observations of interaction from a single drug-drug combination may therefore not apply to a second drug combination. At present, it is not known which drug combinations should be considered to cover the range of different binding sites of P-gp. Recently, we developed a porcine epithelial cell line (iP-gp), which forms tight monolayers and displays a high expression of human P-gp together with a low expression of endogenous ABC transporters, making this cell line a useful in vitro tool for studying interactions with human P-gp. The purpose of the present work was to screen different known P-gp substrates and inhibitors for interactions with P-gp mediated efflux transport of digoxin using the iP-gp cell line. Potentially, to provide knowledge of substrates and inhibitors that share binding sites with digoxin or occupy different binding sites.

Methods: For transport experiments, iP-gp cells were cultured on permeable supports for 16-17 days in conventional growth media. Basolateral to apical transport of 3H-digoxin was measured in absence and presence of increasing concentrations of known P-gp substrates and inhibitors. Observed inhibition was characterized by estimation of the corresponding half-maximal inhibitory concentration (IC50) value.

Results: Concentration-dependent inhibition of P-gp mediated efflux transport of 3H-digoxin was observed in the presence of increasing concentrations of the well-described P-gp inhibitor zosuquidar (LY335979). The IC50-value for the zosuquidar-mediated inhibition of digoxin transport was estimated to be 0.05 ± 0.01 µM. Verapamil (P-gp substrate and inhibitor) and quinidine (P-gp substrate) were also shown to inhibit efflux transport of 3H-digoxin in a concentration-dependent manner, while the well-known P-gp substrate Rhodamine 123 did not affect digoxin efflux transport, not even at concentrations up to 200 µM. We have previously shown that atenolol and citalopram are substrates of P-gp using the iP-gp cell line. However, in the present work only citalopram was observed to affect P-gp mediated efflux transport of digoxin.

Conclusion: The results of the present work corroborate previous reports of substrate-dependent inhibition of P-gp mediated efflux transport. The observed inhibition of P-gp mediated 3H-digoxin efflux transport by zosuquidar, verapamil, quinidine and citalopram suggests that these compounds modulate or occupy the same or overlapping binding sites as digoxin. In contrast, atenolol and Rhodamine 123 did not affect the transport of 3H-digoxin, indicating that these substrates occupy binding sites different from digoxin. The fact that Rhodamine 123 did not affect P-gp mediated transport of digoxin indicates that digoxin does not occupy the hypothesized Rhodamine or R-binding sites of P-gp. Following the R, H, and P binding site hypothesis, the only remaining proposed active transport binding site is the Hoechst or H-site. Ongoing work is therefore aimed at investigating whether digoxin may occupy the hypothesized H-binding site of P-gp.
Original languageEnglish
Publication date14 Nov 2017
Publication statusPublished - 14 Nov 2017
Event2017 AAPS ANNUAL MEETING AND EXPOSITION - San Diego, United States
Duration: 12 Nov 201715 Nov 2017


CountryUnited States
CitySan Diego

ID: 195587877