Development of a fluorescence-based assay for drug interactions with human Multidrug Resistance Related Protein (MRP2; ABCC2) in MDCKII-MRP2 membrane vesicles

doi:10.1016/j.ejpb.2010.03.008

European Journal of Pharmaceutics and Biopharmaceutics

Volume 75, Issue 2, June 2010, Pages 284-290

https://doi.org/10.1016/j.ejpb.2010.03.008 Get rights and content

Abstract

Purpose

To establish a fluorescence-based assay for drug interactions with the ABC-export-protein MRP2 (ABCC2).

Methods

Apical membrane vesicles were isolated by differential centrifugation from polarized MDCKII cells and MDCKII cells transfected with human MRP2. Vesicle fractions were characterized by electron microscopy, determination of the marker enzyme alkaline phosphatase and Western blot analysis of MRP2. Vesicle orientation was determined by measurement of 5′-nucleotidase activity in the absence and in the presence of detergents. To assess MRP2 activity, the uptake of the fluorescent MRP2-substrate 5-(6)-carboxy-2′,7′-dichlorofluorescein (CDF) was determined in the absence and in the presence of other compounds potentially interacting with MRP2.

Results

Apical membrane vesicles could be isolated from cells in considerable purity as indicated by electron microscopy, enrichment of alkaline phosphatase and high enrichment of MRP2 in vesicles of MDCKII-MRP2 cells. About half of the vesicles showed “inside-out” orientation. CDF was taken up into the membrane vesicles in a time- and concentration-dependent manner following a Michaelis–Menten type of kinetics with a K_M of 39 μM and a V_max of 465.3 fmol/(mg protein × min). Thereby, uptake into vesicles from transfected cells was significantly higher than uptake into vesicles from control cells. Presence of known MRP2-substrates/inhibitors in the incubation medium decreased CDF uptake into the vesicles in a concentration-dependent manner, whereas nonsubstrates/inhibitors had no effect.

Conclusions

This CDF-based uptake assay can be used as a rapid and sensitive screening system to assess drug interactions with human MRP2 and therefore represents a useful tool in compound profiling.

Section snippets

Materials

Murine antibody against MRP2 (clone M2III-6) and MK571 were obtained from Alexis Biochemicals (Lörrach, FRG). Alkaline phosphatase antibody (clone 3A8) was from Abnova (Heidelberg, FRG). Secondary anti-mouse-horseradish peroxidase-conjugated antibody was from KPL (Wedel, FRG). Cyclosporine A was purchased from Novartis (Basle, CH). Sf9-MRP2-VT vesicles were from Solvo Biotechnology (Budapest, HU). All other materials were obtained from the usual commercial sources at the highest purity

Results and discussion

MRP2 (ABCC2) is an export protein with an apparent molecular weight of approximately 190 KDa, which is expressed in most barrier tissues throughout the body [15]. Besides p-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), it significantly contributes to the active extrusion of many drugs and drug metabolites [16], [17]. Therefore, we aimed to develop an assay, which can easily be used to study a potential impact of MRP2 on bioavailability and disposition of novel drug

Acknowledgements

The authors thank the group of Prof. D. Robinson, University of Heidelberg, for the help with electron microscopy.

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Commercial vesicles expressed the Mrp2 and MRP2 proteins, with notably lower expression of mMrp2 compared with hMRP2 protein (Figure 1A). The probe Mrp2/MRP2 substrate, CDCF, was used for inhibition assays as previously described.24,25 Cisplatin inhibited the adenosine triphosphate–dependent transport of CDCF in plasma membrane vesicles (Figure 1B).
The chemotherapeutic drug cisplatin is actively transported into proximal tubules, leading to acute renal injury. Previous studies suggest that the multidrug resistance–associated protein 2 (Mrp2) transporter may efflux cisplatin conjugates from cells. We sought to determine whether the absence of Mrp2 alters the accumulation and toxicity of platinum in the kidneys of mice and whether transgenic expression of the human MRP2 gene could protect against cisplatin injury in vivo. Plasma, kidneys, and livers from vehicle- and cisplatin-treated wild-type and Mrp2-null mice were collected for quantification of platinum and toxicity. By 24 hours, twofold higher concentrations of platinum were detected in the kidneys and livers of Mrp2-null mice compared with wild types. Enhanced platinum concentrations in Mrp2-null mice were observed in DNA and cytosolic fractions of the kidneys. Four days after cisplatin treatment, more extensive proximal tubule injury was observed in Mrp2-null mice compared with wild-type mice. Kidneys from naive Mrp2-null mice had elevated glutathione S-transferase mRNA levels, which could increase the formation of cisplatin-glutathione conjugates that may be metabolized to toxic thiol intermediates. Transgenic expression of the human MRP2 gene in Mrp2-null mice reduced the accumulation and nephrotoxicity of cisplatin to levels observed in wild-type mice. These data suggest that deficiency in Mrp2 lowers platinum excretion and increases susceptibility to kidney injury, which can be rescued by the human MRP2 ortholog.
Interactions between human multidrug resistance related protein (MRP2; ABCC2) and excipients commonly used in self-emulsifying drug delivery systems (SEDDS)
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It is performed using purified vesicles from Spodoptera frugiperda (Sf9) insect cells expressing high level of MRP2 proteins. Previous studies have relied on an indirect determination by measuring the inhibition of transport of fluorescent or radioisotope labeled marker 17β-estradiol-17-β-d-glucuronide ([3H] E2-17βG) as substrate (Wortelboer et al., 2005; Lechner et al., 2010). However, to simplify the method, other substrates of MRP2 have also been used in the transport assay in conjunction with liquid chromatography–tandem mass spectrometry (LC–MS) for quantification of substrates.
The aim of this study was to investigate the multidrug resistance related protein-2 (MRP2) inhibition effect of excipients commonly employed in the formulation of self-emulsifying drug delivery systems (SEDDS). Cytotoxicity and safe dosages of fifteen excipients, including five surfactants, three oils, three co-surfactants and four solid carriers, were compared using MTT assay. Caco-2 cell permeability and MRP2 vesicles transport assays were used in the inhibition analysis. Scutellarin was shown to be a substrate of MRP2 and used as a probe in the inhibition assay. Twelve excipients decreased efflux ratio of scutellarin in Caco-2 model, among them six excipients showed inhibition effect on MRP2 in MRP2 transport model. In Caco-2 model, Cremophor^® EL, Maisine^® 35-1, PEG 2000 and β-cyclodextrin reduced efflux ratio of scutellarin the most in the four different excipients groups. The efflux reduction mechanism was further validated by MRP2 transport assay in inhibiting MRP2 activity in Cremophor^® EL and PEG 2000. It is suggested that scutellarin quantified by LC–MS can be used as a model drug for MRP2 inhibition analysis using Caco-2 permeability and vesicles transport analyses. Together they constitute a sensitive and rapid screening method for assessing interactions between excipients and MRP2 in SEDDS formulation.
Characterization of rhodamine-123, calcein and 5(6)-carboxy-2′, 7′-dichlorofluorescein (CDCF) export via MRP2 (ABCC2) in MES-SA and A549 cells
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On the other hand, MRP2 is known to transport calcein and according to Lai et al. (2007), maximal inhibition is observed with 25 μM MK571, exactly as in our study. Our results with cyclosporin A, MK571 and benzbromarone inhibition of CDCF transport via MRP2 are in agreement with those reported by Lechner et al. (2010), who found that benzbromarone and MK571 achieve much higher maximal inhibition than cyclosporin A. In another study, cyclosporin A was found to inhibit CDCF transport in membrane vesicles overexpressing MRP2 over the same concentration range as in our study (Heredi-Szabo et al., 2008).
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Research paperDevelopment of a fluorescence-based assay for drug interactions with human Multidrug Resistance Related Protein (MRP2; ABCC2) in MDCKII-MRP2 membrane vesicles

Abstract

Purpose

Methods

Results

Conclusions

Section snippets

Materials

Results and discussion

Acknowledgements

Toxicol. Lett.

Cancer Treat. Rev.

Adv. Drug Deliv. Rev.

Protein Expr. Purif.

Anal. Biochem.

Gastroenterology

Biochim. Biophys. Acta

J. Biol. Chem.

Kidney Int.

J. Biol. Chem.

Biochem. Pharmacol.

Kidney Int.

J. Biomol. Screen.

Eur. J. Pharm. Biopharm.

Transporters and drug therapy: implications for drug disposition and disease

Clin. Pharmacol. Ther.

Intestinal efflux transporters and drug absorption

Expert Opin. Drug Metab. Toxicol.

The role of ATP binding cassette transporters in tissue defense and organ regeneration

J. Pharmacol. Exp. Ther.

Research paper
Development of a fluorescence-based assay for drug interactions with human Multidrug Resistance Related Protein (MRP2; ABCC2) in MDCKII-MRP2 membrane vesicles