New insights into the carrier-mediated transport of estrone-3-sulfate in the Caco-2 cell model.

The current studies were undertaken to gain new insights into the interplay and mechanism of membrane transporters involved in the permeability of estrone-3-sulfate (E1S) in Caco-2 cells cultured either on the bottom of multiwell plastic dishes or on filter support. We demonstrate that Caco-2 cells from the "Deutsche sammlung von mikroorganismen und zellkulturen" (DSMZ) exhibit extensive and consistent carrier-mediated uptake of [(3)H]-E1S after a culture period of 11-13 days. The kinetic characterization, the inhibitory profile and the pH dependence for the initial linear uptake permeability (PUP) of [(3)H]-E1S suggest that the organic anion transporting polypeptide (OATP) 2B1 is the main transporter involved in the apical E1S PUP in Caco-2 cells from the DSMZ. Furthermore, our results indicate that the efflux transporter breast cancer resistance protein (BCRP) affects E1S PUP, even when uptake is measured at the initial linear uptake phase. Although almost identical results were obtained for cells cultured on plastic dishes and on filter supports, the OATP2B1 stimulator dexamethasone did not affect the PUP for cells grown on dishes but increased [(3)H]-E1S PUP by more than 2-fold for filter grown cells. The basolateral PUP of [(3)H]-E1S of filter grown cells was inhibited by several inhibitors of the bidirectional transporter organic solute transporter α/ß (OSTα/ß). Efflux studies were performed by loading the cells with either [(3)H]-E1S or [(3)H]-taurocholic acid (TCA) and subsequently measuring the efflux of radio labeled substance in the absence or presence of BCRP or OST α/ß inhibitors. Similar effluxes of [(3)H]-E1S was observed across the apical and basolateral membrane, and the apical efflux was greatly decreased in the presence of the BCRP inhibitor fumitremorgin C. In contrast, efflux of [(3)H]-TCA to the basolateral compartment was clearly larger than to the apical compartment. Trans-stimulation of basolateral [(3)H]-E1S efflux was observed in the presence of taurolithocholic acid (TLC), although none of the applied OSTα/ß inhibitors were able to confirm the existence of carrier-mediated efflux at the basolateral membrane, neither for [(3)H]-E1S nor for [(3)H]-TCA. These results highlight the importance of transporter interplay for E1S and drug compounds in Caco-2 cells and emphasize the importance of identifying the basolateral transporters in these cells.

Intestinal transporters for endogenic and pharmaceutical organic anions: the challenges of deriving in-vitro kinetic parameters for the prediction of clinically relevant drug-drug interactions.

OBJECTIVES: This review provides an overview of intestinal human transporters for organic anions and stresses the need for standardization of the various in-vitro methods presently employed in drug-drug interaction (DDI) investigations. KEY FINDINGS: Current knowledge on the intestinal expression of the apical sodium-dependent bile acid transporter (ASBT), the breast cancer resistance protein (BCRP), the monocarboxylate transporters (MCT) 1, MCT3-5, the multidrug resistance associated proteins (MRP) 1-6, the organic anion transporting polypetides (OATP) 2B1, 1A2, 3A1 and 4A1, and the organic solute transporter α/ß (OSTα/ß) has been covered along with an overview of their substrates and inhibitors. Furthermore, the many challenges in predicting clinically relevant DDIs from in-vitro studies have been discussed with focus on intestinal transporters and the various methods for deducting in-vitro parameters for transporters (K(m) /K(i) /IC50, efflux ratio). The applicability of using a cut-off value (estimated based on the intestinal drug concentration divided by the K(i) or IC50) has also been considered. SUMMARY: A re-evaluation of the current approaches for the prediction of DDIs is necessary when considering the involvement of other transporters than P-glycoprotein. Moreover, the interplay between various processes that a drug is subject to in-vivo such as translocation by several transporters and dissolution should be considered.

Interactions between organic anions on multiple transporters in Caco-2 cells.

In drug development, Caco-2 cells are often employed to study the influence of membrane transporters on drug permeability. The aim of the current study was to characterize permeability and kinetic parameters of selected organic anionic compounds in Caco-2 cells, and to investigate whether the Caco-2 cell line may be used as an overall model to predict interactions on multiple membrane transporters in the intestine. Taurocholic acid (TCA) and estrone-3-sulfate (E(1) S) were used as model substrates. Possible inhibitors studied were TCA, E(1) S, taurolithocholic acid, fluvastatin, and glipizide. The effects of these compounds on initial uptake, apparent permeability, and intracellular end-point accumulations of the probe substrates were studied. Both interactions on apical and basolateral influx transporters were observed. These interactions were proposed to be mediated mainly by the apical sodium-dependent bile acid transporter and the organic solute transporter α/ß, and to less extent by the organic anion transporting polypeptide 2B1. However, interactions on efflux transporters were not detected, although they were expected from the literature on the investigated compounds. Biosimulation methods may be the key to further distinguish between interactions on multiple transporters, including the involvement of efflux transporters, by taking into account the changes in dynamics that drug interactions may cause.