Bidirectional membrane transport: Simulations of transport inhibition in uptake studies explain data obtained with flavonoids

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Abstract

The purpose of the simulations was to obtain an estimate of concentration-dependent uptake curves when two counteracting transporters are present. On the basis of this experimental data obtained with a pair of ovarian carcinoma cell lines, one of which was not expressing the exsorptive transporter P-glycoprotein and one of which was an MDR1-transfected, P-glycoprotein expressing variant, the kinetics of cellular uptake of the radiolabel 3H-talinolol were calculated and the inhibitory constants at P-gp were determined for different flavonoids. With respect to the inhibition of P-gp function, among others, naringenin and isoquercitrin were identified as inhibitors, yet estimation of the inhibitory constant was only possible for uptake values corrected for non-P-glycoprotein-mediated processes. It was assumed that an additional inside-directed transporting protein (Carrier B), which is inhibited by the presence of test compounds, uptake of radiolabel was simulated as a function of the concentration of test-compound, with exemplary parameters for the rate constant (kB) of the additional Carrier B and the inhibition constants (KI-values) for both transporting proteins. The obtained uncorrected experimental data, which showed either inhibition or enhancement of radiolabel uptake as a function of the inhibitor concentration, were appropriately explained by the respective model. The respective model included an exsorptive transporter as well as carrier-mediating facilitated diffusion.

It is concluded that flavonoids, such as naringenin and isoquercitrin, inhibit an inside-directed process in addition to their inhibition of P-glycoprotein-mediated exsorption.

Introduction

Not only regarding anticancer chemotherapy in tumors, also for other drugs with an intracellular site of action membrane passaging is crucial. This process may – usually – occur in both directions, i.e., into the cell and out of the cell, where in both directions active and/or passive processes may be involved. This is of particular relevance in such tumors, which are exhibiting or developing the multi-drug resistance (MDR) phenotype, in which the inside/outside balance is or becomes unfavourable for the therapeutic agent. This MDR phenotype is most often characterized by a reduced intracellular drug level and overexpression of the ATP-binding cassette transporter P-glycoprotein (P-gp), which is capable of transporting a wide range of xenobiotic compounds out of tumor cells. The transport activity of P-gp can be blocked and multidrug resistance reversed by the addition of substrate analogs or so-called chemosensitizers or modulators (Krishna and Mayer, 2001)

Recently, a great effort was made to detect and quantify the expression of transporters in various tissues (e.g., Anderle et al., 2005). This gives experimental evidence for the occurrence of many pharmacokinetically –potentially– relevant transporters in normal tissues. Such transporters are not always of high specificity, but transport many different substrates with a wide structural variety (e.g., Seelig and Landwojtowicz, 2000). Moreover, when studying a particular transporter, also related transporters out of the same family with considerable structural overlap may be present in a cell membrane and also transport the respective compound, but at different rates.

Transporter expression may vary dependent upon the circumstances. For example, Ginsburg and Stein (2005) described modulations of functional transport pathways in the membranes of Plasmodium falciparum host erythrocytes. Diseases, nutrition, drug therapy, many factors may have an influence and increase or decrease the expression or function of particular transporters and, hence, affect the ratio between inside- and outside-directed processes.

The functional characterization of P-gp as well as other ABC transporters involved in multi-drug resistance, the family of multi-drug resistance-associated proteins (MRP1-7) and the ABC half-transporter MXR (=BCRP) includes the conceptual problem of identifying substrates and inhibitors of the different MDR-relevant proteins.

Transporter affinities may be characterized via transport inhibition of model substrates, and numerous experimental models, for P-gp mainly P-gp-overexpressing cell lines, have been established. However, significant variabilities in transport characteristics were detected, not only for different cellular systems and different model substrates, yet even for well defined and established test systems, when data were obtained from, e.g., different laboratories. Studies performed in our laboratories showed a deviation of the expression of various transporters depending upon the passage number and the number of days in culture and of the resulting membrane permeabilities of drugs (Dressler, 2002, Hilgendorf et al., 2005). And it is, hence, to be expected that also the in/out transport ratio is affected.

Affinity screening with studies on the influence of a test compound on the transport of a model substrate into cells are frequently used to characterize the nature of a compound as transport inhibitor and define its affinity to a particular transporter. However, IC50 values (the concentrations that give half-maximal inhibition) for a particular compound may differ considerably between models, even though the same model substrate is used. For verapamil, the reversal of daunorubicin extrusion by P-gp ranges from 0.4 μM (SW2780AD cells, Javaheri et al., 1983) to 10 μM (CEM/vinblastine1000 cells, Hu et al., 1990). Comparable results were also obtained with cyclosporine A and other P-gp inhibitors. Litman et al. (2003) mention higher IC50 values with increasing resistance of carcinoma cell lines and the potential relevance of an increasing number of pump molecules.

Almost all commonly used substrates were shown to exhibit affinity towards several transporting proteins. For example, vinblastine is substrate for P-gp, MRP1, and MRP2 (Evers et al., 1998), fexofenadine was identified to be transported by P-gp and members of the OATP family (Cvetkovic et al., 1999), Verapamil showed – beside its interaction with P-glycoprotein – inhibition of MRP1 (Loe et al., 2000), OCT1 (Zhang et al., 1998b), and OCT2 (Koepsell et al., 2003). When compounds with affinity to different transporters are included into screening studies as model substrates, the overall transport rates mostly depend on more than one transporter. Even in transfected cell lines, which were selected because of overexpression of one particular transporter, other transporters are – and need to be – present as well, since for example uptake of nutrients into cells is necessary. Hence, in general transport may happen and – in fact – happens in both directions, into and out of the cells. Since various inside- and outside-directed transport processes occur simultaneously in cells and transport inhibition is not necessarily specific, it may be difficult to interpret uptake data under initial screening conditions, where the number of inhibitor concentrations is usually limited.

This hypothesis was incorporated into the modelling of P-gp blockade quantified by studying the ability of inhibition of daunorubicin and calcein-AM efflux from cells with different levels of drug resistance, where a pump/leak model was used (Litman et al., 2003) with simultaneous bidirectional passive membrane transport plus exsorptive transport. However, this concept is not appropriate for modelling the outcome of inhibition studies performed with different flavonoids as well as some other compounds (Ofer et al., 2006).

Experimental data obtained under different conditions – in cells with low and cells with high exsorptive transport – have raised evidence that the outcome of such studies may yield in either overall increased or overall reduced uptake, dependent on the concentration of the inhibitor, when both inside- and outside-directed active or carrier-mediated transport occurs (in addition to passive diffusion) and the transport characteristics are different (Ofer, 2004).

In order to visualize the effects of the simultaneous/sequential inhibition of more than one transporter, the uptake of model substrates was simulated dependent on the addition of transport inhibitors. The simulations are based on experimentally determined parameters in closely related cell lines, which express P-gp to a different extent and which cannot be explained by the presence of one exsorptive transporter only, as assumed in models such as the pump/leak model.

Section snippets

Transport model

With respect to the transport model, major assumptions included the presence of one secretory and one –potentially– absorptive transport protein within the cell membrane (Fig. 1).

The secretory transporter (here ‘Transporter A’) was assumed to mediate active export of its substrates handling substrate present in the cell membrane as well as substrate present in the intracellular space (as, e.g., described for P-gp by Stein (1997)). Absorptive transport was assumed to follow the characteristics

Results

Previous studies had shown an apparently unexplainable, yet reproducible deviation in studies on radiolabel uptake inhibition, which was detected for a number of compounds, not only for flavonoids (e.g., Fig. 2). The purpose of the simulations was to obtain an estimate of concentration-dependent uptake curves when two counteracting transporters (exsorptive Transporter A (P-gp), Carrier B) are present and the influence of transport inhibitors with different A/B selectivities. Assuming the

Discussion

In the simulations introduced here, two transport systems were included into the model in addition to passive diffusion, in order to simulate the inhibitor concentration dependence of radioligand uptake into a particular cell system, which was shown to express P-glycoprotein. In addition to initial functional (=inhibition) studies, the clear demonstration of P-gp expression in A2780/M250 cells and its lack in A2780 cells (Western blot and vinblastine-resistance assay) had confirmed the

Nomenclature

    Ce

    concentration in the extracellular space

    Ci

    concentration in the intracellular space

    Cm–i

    concentration in the membrane in proximity of the intracellular compartment

    Cm–e

    concentration in the membrane in proximity of the extracellular compartment

    e

    extracellular space

    i

    intracellular space

    kB

    rate constant for Carrier B-mediated facilitated diffusion

    ke

    rate constant for active export of substrate present in the extracellular compartment of the membrane

    ki

    rate constant for active export of substrate present

Acknowledgements

This study was in part supported by a grant from the Dr. Robert-Pfleger-Stiftung to HSL and a grant from the Deutsche Forschungsgemeinschaft to PL (LA 563/3-2, LA 563/3-3).

References (19)

  • H. Ginsburg et al.

    How many functional transport pathways does Plasmodium falciparum induce in the membrane of its host erythrocyte

    Trends Parasitol.

    (2005)
  • A. Seelig et al.

    Structure-activity relationship of P-glycoprotein substrates and modifiers

    Eur. J. Pharm. Sci.

    (2000)
  • P. Anderle et al.

    Changes in the transcriptional profile of transporters in the intestine along the anterior–posterior and crypt-villus axes

    BMC Genomics

    (2005)
  • M. Cvetkovic et al.

    Oatp and P-glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine

    Drug Metab. Dispos.

    (1999)
  • C Dressler

    Interactions of Losartan and its Major Metabolite EXP3174 with Membrane Transporters in vitro, in situ, and in vivo

    (2002)
  • R. Evers et al.

    Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA

    J. Clin. Invest.

    (1998)
  • J. Gabrielsson et al.

    Pharmacokinetic and Pharmacodynamic Data Analysis: Concepts and Application

    (1997)
  • C. Hilgendorf et al.

    Selective downregulation of the MDR1 gene product in Caco-2 cells by stable transfection to prove its relevance in secretory drug transport

    Mol. Pharm.

    (2005)
  • X.F. Hu et al.

    Effect of cyclosporine and verapamil on the cellular kinetics of daunorubicin

    Curr. Drug Targets

    (1990)
There are more references available in the full text version of this article.

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