Rapid detection of ABC transporter interaction: Potential utility in pharmacology

Abstract

Introduction: The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp/ABCB1), multidrug resistance-associated protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2) are known to transport a wide range of structurally diverse compounds. Their high level of expression at the blood–brain, maternal–fetal, and blood–testis barriers as well as their purported roles in oral absorption suggests that ABC transporters play important pharmacologic roles. Methods: We have developed a method to characterize the function and inhibition of ABC transporters using an automated cell counter with fluorescence detection capability. The assay was performed using stably-transfected HEK293 cells expressing P-gp, MRP1, or ABCG2 and examining transport of fluorescent substrates in the presence or absence of known inhibitors and compared to results obtained with a flow cytometer. Fold increase in intracellular fluorescence was then calculated for cells incubated with fluorescent substrate in the absence of inhibitor versus in the presence of inhibitor. Results: Fold increase values obtained either with the cell counter or flow cytometer were comparable for cells expressing either MRP1 or ABCG2; slightly higher fold increase values were observed when cells expressing P-gp were read on a flow cytometer compared to the cell counter. Discussion: The assay described provides an inexpensive detection method to aid in the development of novel ABC transporter inhibitors or to characterize potential drug–drug interactions.

Introduction

The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp, encoded by the MDR-1 or ABCB1 gene), multidrug resistance-associated protein 1 (MRP1, encoded by the MRP1 or ABCC1 gene), and breast cancer resistance protein (BCRP/ABCG2, encoded by the ABCG2 gene) have been shown to mediate energy-dependent transport of a variety of structurally dissimilar compounds out of cells, against a concentration gradient (Gottesman, Fojo, & Bates, 2002). P-gp, the first ABC transporter discovered and by far the best characterized, has been shown to transport a wide variety of substrates including anthracyclines, vinca alkaloids, taxanes and tyrosine kinase inhibitors; HIV protease inhibitors such as nalfinavir, ritonavir and amprenavir; as well as steroids and HMG-CoA inhibitors (Gottesman et al., 2002, Cascorbi, 2006). It has been shown to participate in oral drug absorption and is also a component of the blood–brain barrier, suggesting that P-gp plays a role in normal tissue protection (Gottesman et al., 2002, Deeken and Loscher, 2007). The MRP1 transporter has been shown to confer resistance to a narrower range of antineoplastics, including the anthracyclines, vinca alkaloids, etoposide and teniposide, and it has also been shown to transport glucuronide and glutathione conjugates (e.g. leukotriene C4) (Cascorbi, 2006, Bakos and Homolya, 2007). Expression of MRP1 at the blood–brain barrier and choroid plexus suggests that it, too, serves a protective role by preventing accumulation of drugs in these sanctuary sites (Bakos and Homolya, 2007, Rao et al., 1999). ABCG2 has also been shown to transport a growing list of substrates including mitoxantrone, the camptothecin analogs topotecan and irinotecan, and the tyrosine kinase inhibitors gefitinib and imatinib as well as antibiotics, HMG-CoA inhibitors and HIV protease inhibitors (Xu, Peng, & Zhang, 2007). ABCG2 is believed to form part of the blood–brain barrier, blood–testis barrier, and maternal–fetal barrier and has also been shown to modulate oral drug absorption (Robey, Polgar, Deeken, To, & Bates, 2007). Therefore, ABC transporters play significant roles in the pharmacology of substrate compounds.

The purported roles for ABC transporters in drug disposition have led to increased interest in describing the interactions between ABC transporters and novel drug therapies. Recognizing the significant pharmacologic role of ABC transporters, the FDA has published several guidance documents to address the role of ABC transporters in drug development and to determine potential drug–drug interactions (Giacomini et al., 2010). Additionally, the purported role of ABC transporters in clinical drug resistance has sparked the development of inhibitors that can block efflux of substrate compounds (Gottesman et al., 2002). Such inhibitors may also be used to improve drug penetration into sanctuary sites such as the brain or to increase oral drug bioavailability. Several animal studies have demonstrated increased brain penetration of the tyrosine kinase inhibitors gefitinib and imatinib when the drugs were coadministered with the dual P-gp and ABCG2 inhibitor elacridar (GF120918) (Chen et al., 2009, Kawamura et al., 2009, Lagas et al., 2009). Additionally, human clinical trials have shown that oral coadministration of elacridar with topotecan leads to significantly increased serum levels of topotecan compared to oral administration of topotecan alone (Kruijtzer et al., 2002, Kuppens et al., 2007).

Flow cytometry-based functional assays are often used to characterize interactions between drugs and ABC transporters and usually involve the use of fluorescent transporter substrates such as rhodamine 123 and calcein AM for P-gp (Feller et al., 1995); calcein AM for MRP1 (Feller et al., 1995, Dogan et al., 2004); and BODIPY-prazosin and pheophorbide a for ABCG2 (Robey et al., 2001, Robey et al., 2004). Flow cytometers have the sensitivity to provide accurate and reliable results, but they are often costly and require extensive calibration and user training. Therefore, the development of alternative and cost-effective methods would be advantageous to researchers in this field.

Herein we report a convenient method for analyzing the function of ABC transporters and characterizing drug–transporter interactions using an automated cell counter with fluorescence detection, the Cellometer® Vision (Nexcelom Bioscience, Lawrence, MA). The Vision allows for rapid detection of intracellular fluorescence of ABC transporter substrates using an LED excitation light source, optical filtering, and cooled CCD camera technology for fluorescence detection. The instrument can automatically analyze acquired cell images and measure cell concentration, viability, and cell size. A proprietary image process algorithm is utilized to analyze cell images. Furthermore, the analyzed results (i.e. cell image, size, and fluorescence distribution histogram) may be saved for research records.

In this work, we have developed a robust detection method using a cell counter with fluorescence detection for measuring intracellular fluorescence of P-gp, MRP1, and ABCG2 substrates. The assay and results are validated by comparing overlays generated with the cell counter to overlays generated when the samples were analyzed on a flow cytometer. The methods presented here have the potential to identify compounds that could mediate drug–drug interactions through ABC transporter inhibition. Additionally, these methods could identify novel inhibitors of ABC transporters that might be used to increase drug uptake in the CNS, modulate drug oral bioavailability, or alter drug uptake in tumors.