Advertisement
Research ArticleArticle

Coproporphyrin-I: A Fluorescent, Endogenous Optimal Probe Substrate for ABCC2 (MRP2) Suitable for Vesicle-Based MRP2 Inhibition Assay

Ravindranath Reddy Gilibili, Sagnik Chatterjee, Pravin Bagul, Kathleen W. Mosure, Bokka Venkata Murali, T. Thanga Mariappan, Sandhya Mandlekar and Yurong Lai
Drug Metabolism and Disposition June 2017, 45 (6) 604-611; DOI: https://doi.org/10.1124/dmd.116.074740

Abstract

Inside-out–oriented membrane vesicles are useful tools to investigate whether a compound can be an inhibitor of efflux transporters such as multidrug resistance–associated protein 2 (MRP2). However, because of technical limitations of substrate diffusion and low dynamic uptake windows for interacting drugs used in the clinic, estradiol-17β-glucuronide (E17βG) remains the probe substrate that is frequently used in MRP2 inhibition assays. Here we recapitulated the sigmoidal kinetics of MRP2-mediated transport of E17βG, with apparent Michaelis-Menten constant (Km) and Vmax values of 170 ±17 µM and 1447 ± 137 pmol/mg protein/min, respectively. The Hill coefficient (2.05 ± 0.1) suggests multiple substrate binding sites for E17βG transport with cooperative interactions. Using E17βG as a probe substrate, 51 of 97 compounds tested (53%) showed up to 6-fold stimulatory effects. Here, we demonstrate for the first time that coproporphyrin-I (CP-I) is a MRP2 substrate in membrane vesicles. The uptake of CP-I followed a hyperbolic relationship, adequately described by the standard Michaelis-Menten equation (apparent Km and Vmax values were 7.7 ± 0.7 µM and 48 ± 11 pmol/mg protein/min, respectively), suggesting the involvement of a single binding site. Of the 47 compounds tested, 30 compounds were inhibitors of human MRP2 and 8 compounds (17%) stimulated MRP2-mediated CP-I transport. The stimulators were found to share the basic backbone structure of the physiologic steroids, which suggests a potential in vivo relevance of in vitro stimulation of MRP2 transport. We concluded that CP-I could be an alternative in vitro probe substrate replacing E17βG for appreciating MRP2 interactions while minimizing potential false-negative results for MRP2 inhibition due to stimulatory effects.

Introduction

Multidrug resistance–associated protein 2 [MRP2 (also called ABCC2)], a member of the ATP-binding cassette (ABC) transporters, is expressed exclusively on the apical membrane of hepatocytes, enterocytes, and kidney proximal tubule cells. MRP2 functional deficiency caused by the ABCC2 gene mutation in humans is the molecular basis of conjugated hyperbilirubinemia, known as Dubin-Johnson syndrome. MRP2 also transports many structurally diverse drugs and their metabolites and plays a key role in drug disposition and detoxification processes (Nies and Keppler, 2007). For example, impaired function of MRP2 could increase the systemic exposure of pravastatin due to the increased absorption and the reduced biliary and/or urinary excretion (Niemi et al., 2006). As a result, inhibition of MRP2 can cause accumulation of compounds and/or their metabolites in the liver to reach a toxic level (Isley, 2003; Tang, 2007). Although drug-drug interactions caused by MRP2 inhibition are still not well-defined in the clinic, given the importance of MRP2 in drug disposition and elimination, it has become one of the emerging transporters of clinical importance as recognized by the International Transporter Consortium (ITC) (Zamek-Gliszczynski et al., 2012; Hillgren et al., 2013). ITC has also rationalized the importance of MRP2 in hepatotoxicity due to its role in regulating drug concentration in the liver (Yoshida et al., 2013).

As aforementioned, understanding the interaction of a new chemical entity with MRP2 becomes important from drug-drug interaction and toxicity perspective. Currently, two endogenous compounds, namely estradiol-17β-glucuronide (E17βG) and leukotriene C4 (LTC4), are commonly used in membrane vesicle uptake assays to understand whether a compound is an inhibitor of MRP2 (Brouwer et al., 2013). LTC4 displays a very short duration of uptake linearity (only up to 2 minutes) in membrane vesicles (Heredi-Szabo et al., 2008), leading to a chance of variability during experiments. These limitations hinder the use of LTC4 in characterizing MRP2 inhibition. As a result, E17βG is the endogenous in vitro probe substrate of choice for MRP2 interaction. However, E17βG uptake in membrane vesicles suffers from large interlaboratory variability in reported Michaelis-Menten constant (Km) and Vmax values (Supplemental Table 1) (Borst et al., 2006). E17βG has displayed homotropic cooperativity with MRP2-mediated uptake, yielding sigmoidal kinetics in membrane vesicles (Supplemental Table 1). To explain cooperative interactions, a two–binding site theory has been proposed for MRP2: E17βG binds to the substrate binding site S that mediates the transport of it; and as E17βG concentration increases it binds to the modulator site M, modulating the affinity of the transport site (Fig. 1). In addition, complex modulations (stimulation and/or inhibition) of MRP2-mediated E17βG transport have been well described in the literature (Bakos et al., 2000; Evers et al., 2000; Zelcer et al., 2003a). The complex modulation suggests that MRP2-interacting compounds could bind to S sites to inhibit, to M sites to stimulate, or to both sites to stimulate and inhibit MRP2-mediated transport (also known as “bell-shape” kinetics) (Zelcer et al., 2003a). This stimulatory effect is considered to be substrate dependent. For example, probenecid is reported to stimulate E17βG uptake but inhibits methotrexate uptake in MRP2 membrane vesicles (Zelcer et al., 2003b). In another study (Herédi-Szabó et al., 2009), benzbromarone, sulfasalazine, probenecid, and indomethacin are reported to stimulate both human and rat MRP2-mediated E17βG transport, albeit to different extents. As MRP2-mediated E17βG transport displays sigmoidal kinetics, the self-cooperative effects form a unique interaction for each E17βG modulator pair within the complex binding sites of MRP2 protein. Therefore, the stimulatory effects could potentially mask the MRP2 inhibition of many modulating compounds and yield false-negative MRP2 inhibition. Collectively, an alternative in vitro probe substrate that displays classic Michaelis-Menten kinetics is needed for better understanding of MRP2 interactions with new chemical entities.

Fig. 1.
Fig. 1.

Scheme of the two–binding site model for MRP2. Substrates or modulating compounds bind to the substrate binding site S that mediates the transport of the substrate and can also bind to the modulator site M that modulates the affinity of the transport site. The proposed mechanism of E17βG is that it first binds to the S site followed by the M site at a higher concentration, resulting in sigmoidal kinetics. conc., concentration.

Coproporphyrin I (CP-I) is one of coproporphyrin byproducts of heme biosynthesis. In the liver, CP-I is taken up into hepatocytes by organic anion–transporting polypeptide transporters and effluxed into bile likely by MRP2 (Benz-de Bretagne et al., 2011, 2014; Lai et al., 2016; Shen et al., 2016). As such, a higher proportion of CP-I is secreted into the urine of subjects with Dubin-Johnson syndrome compared with normal subjects. This information indicates a central role of MRP2 in CP-I disposition. However, no in vitro corroboration is provided to substantiate these findings to date. In the present study, we aim to do the following: 1) to provide a definitive in vitro proof of involvement of human MRP2 in transport of CP-I; 2) to characterize in depth the kinetics of CP-I transport in human MRP2 vesicles; and 3) to compare known modulators of human MRP2 in the E17βG assay, with a special emphasis on reported stimulators.

Materials and Methods

Chemicals and Reagents.

Metformin HCl was purchased from RT Corporation (Laramie, WY), rosuvastatin calcium was purchased from Angene International Limited (Hong Kong). Human MRP2-expressing inside-out membrane vesicles (protein concentration, 5 mg/ml) derived from Sf9 insect cells were purchased from GenoMembrane, Co., Ltd (Yokohama, Japan). Assay incubation plates (96 well, ultralow attachment, polystyrene, flat bottom, clear; Costar) were purchased from Corning (Corning, NY). Assay plates (96 well, black polystyrene) were used for fluorescence measurement. All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).

Vesicular Transport Assay.

MRP2-mediated transport assay was performed using inside-out membrane vesicles, according to the manufacturer protocol. Briefly, membrane vesicles were diluted to an appropriate concentration with incubation buffer containing 50 mM 4-morpholinepropanesulfonic acid-Tris (pH 7.0), 70 mM KCl, and 7.5 mM MgCl2. Diluted membrane vesicles (20µl) were transferred into individual wells of 96-well plates and coincubated with 0.5 µl of test substrates (CP-I or E17βG) at 37°C for 3 minutes. Then, the reaction was initiated by adding prewarmed incubation buffer (29.5 µl) containing 4 mM ATP or 4 mM AMP and 2 mM glutathione. After incubation for a designated time at 37°C on rotary shaker (Innova 40; New Brunswick Scientific Co., Edison, NJ) at 100 rpm, the reaction was stopped by the addition of 150 µl of cold wash buffer containing 40 mM 4-morpholinepropanesulfonic acid-Tris (pH 7.0) and 70 mM KCl. The reaction mixture was then transferred into a prewet, 96-well filter plate, which was placed onto a filtration device (FiltrEX 96 Well Filter Plates; Corning Technologies India Pvt Ltd, Mumbai, India) and filtered by a rapid filtration technique (MultiScreenHTS Vacuum Manifold; EMD Millipore, Billerica, MA). To remove excess reaction mixture, wells were washed five times, each time with 200 μl of ice-cold wash buffer. After the final washing step, the filter plate was dried at room temperature for 1 hour. Next, as an extraction solvent, 100 µl of 0.5% SDS dissolved in Milli-Q water (EMD Millipore) was added to the filter plate wells, and the plate was kept on microplate shaker (VWR International, Radnor, PA) for 15 minutes at 230 rpm. Then the filter plate was centrifuged (Eppendorf Inc., Westbury, NY) for 2 minutes at 778g along with a receiver plate attached to its bottom to receive the filtrate. In case of E17βG, 100 µl of acetonitrile was used as an extraction solvent. The filtrate was then used to quantify the CP-I or E17βG levels using either a fluorimeter or LC-MS/MS, respectively.

Inhibition of MRP2-Mediated CP-I or E17βG Uptake in Membrane Vesicles.

Various compounds were selected from the literature as modulators to evaluate their inhibitory and/or stimulatory effects on MRP2-mediated E17βG or CP-I transport in membrane vesicles. All modulators were tested at different concentrations. The final assay concentrations of CP-I and E17βG were 5 and 50 µM, respectively. For E17βG as a substrate, modulators were tested at 20 and 200 µM to determine their inhibitory or stimulatory effects on MRP2-mediated transport. With CP-I as a substrate, modulators were typically tested at 0.1, 1, 10, 50, 100, 250, 500, and 1000 µM to determine IC50 values. All compound stock solutions were prepared in dimethylsulfoxide (DMSO), were spiked directly into assay mixture containing MRP2 vesicle protein (for CP-I and E17βG are 50 and 25 µg, respectively), and were preincubated for 3 minutes at 37°C. The solubility of inhibitors and the stability of incubation at high concentrations were monitored during the experiments. Then the reaction was initiated with the addition of substrate containing ATP or AMP. The rest of the assay was conducted as described in the above section Vesicular Transport Assay. Controls included within each experiment were with substrate alone (either CP-I or E17βG) in the presence of ATP or AMP. ATP-dependent transport of CP-I or E17βG was measured in the presence of modulators and compared with control data. The effect of DMSO on the CP-I transport was also evaluated. It was observed that the final DMSO concentrations up to 2% have negligible effect on CP-I transport (data not shown). In this study, the DMSO content used was not more than 1.15%.

Fluorimetry Analysis of CP-I.

Fluorescence measurements were conducted with a microplate reader (SpectraMax M2e; Molecular Devices, Sunnyvale, CA), using 401 and 595 nm, respectively, as excitation and emission wavelengths for CP-1 ATP-dependent net transport were calculated by subtracting the values obtained in the presence of AMP from those obtained in the presence of ATP. All the experiments related to CP-I were conducted in dim light to minimize fluorescent bleaching.

LC-MS/MS Analysis of E17βG.

LC-MS/MS analysis of E17βG was performed using a Waters Corporation (Milford, MA) UPLC system coupled with a Triple Quad 5500 System (AB Sciex, Framingham, MA), fitted with Electrospray ionization source. Naphthyl glucuronide was used as internal standard (IS). The analyte and IS were separated on a BEH C18-A 50 × 2.1 mm column (Waters Corporation). An elution gradient with two solvents was used: 1) water with 0.1% formic acid; and 2) acetonitrile with 0.1% formic acid. A linear gradient was performed as follows: 0.2 minute at 5% solvent B; in 0.5 minute, solvent B was increased from 5% to 95% and remained constant at 95% solvent B for 1.20 minutes. Then in 1.5 minutes solvent B was decreased from 95% to 5%, and it remained constant for 0.5 minute. The flow rate was set at 0.6 ml/min. The electrospray ionization source conditions were as follows: capillary voltage, ×4500 V; drying gas temperature, 450°C; and nebulizer gas pressure, 50 psi (both nebulizer and drying gas were high-purity nitrogen). E17βG and IS were monitored in negative ion mode with the transition of mass/charge ratios 447 → 112.7 and 547 → 112.7, respectively. Analyst version 1.6.2 was used for system control and data processing.

Data Analysis.

All data were presented as the mean ± SD. Fluorescence intensities from three wells were used to generate the mean and SD. To determine the kinetic parameters (Km and Vmax) and IC50 values, data were analyzed by nonlinear regression using GraphPad Prism software version 5.02 (GraphPad Software, San Diego, CA) with the following appropriate equations.

For CP-I, Km and Vmax were generated from the direct uptake transport measurements using the Michaelis-Menten equation:Embedded ImageFor E17βG, the active transport followed sigmoidal fit. Therefore, to obtain best fit, the Michaelis-Menten equation was modified as given below:Embedded Imagewhere V is the velocity (pmol of substrate per milligram of protein per minute), [S] is the substrate concentration in μM, and h is the Hill coefficient characterizing the degree of cooperativity.

Results

MRP2-Mediated CP-I and E17βG Transport in Membrane Vesicles.

The transport kinetics of E17βG or CP-I were first characterized through concentration-dependent transport of E17βG or CP-I in membrane vesicles overexpressing human MRP2 protein. Prior to the kinetics characterization, ATP-dependent activities and time linearity of MRP2-mediated CP-I uptake were determined at a concentration of 5 µM for different time points, including 5, 10, 15, 30, 45, and 60 minutes, at 37°C in the presence of ATP or AMP. MRP2-mediated CP-I transport in membrane vesicles was linear with time up to 45 minutes (Supplemental Fig. 1). CP-I transport was negligible in the presence of AMP (control) and 35-fold more in the presence of ATP than control. As such, all inhibition studies were optimized to a 30-minute time point of incubation. For E17βG, as described previously (Zhang et al., 2016), a 15-minute incubation was chosen as the optimal time point of incubations.

As depicted in Fig. 2A, the concentration-dependent MRP2-mediated E17βG uptake in membrane vesicles appears to follow sigmoidal kinetics with cooperative interaction, which suggests the existence of multiple binding sites. The apparent Km and Vmax values (mean ± S.D.) for E17βG were 170 ± 17 µM and 1447 ± 137 pmol/mg protein/min, respectively. The Hill coefficient for E17βG was 2.05 ± 0.1. In contrast, MRP2-mediated CP-I uptake in membrane vesicles followed a hyperbolic relationship, as the rate of uptake increased in a linear fashion at low concentrations and was saturated at high concentrations. The kinetics curve could be adequately modeled by the standard Michaelis-Menten equation (Fig. 2B), which suggests the involvement of single binding site in the transport of CP-I. The apparent Km and Vmax values (mean ± S.D.) for CP-I were 7.7 ± 0.7 µM and 48 ± 11 pmol/mg protein/min, respectively.

Fig. 2.
Fig. 2.

Concentration-dependent uptake transport of E17βG (A) and CP-I (B) in MRP2-expressing Sf9 membrane vesicles. MRP2 membrane vesicles were incubated at 37°C for 15 minutes (E17βG) or 30 minutes (CP-I). ATP-dependent uptake is measured by subtracting uptake in the presence of AMP from that in the presence of ATP. For MRP2-mediated E17βG uptake, Km and Vmax values were 170 ± 17 µM and 1447 ± 137 pmol/mg protein/min, respectively. The Hill plot was shown as insert graph, and the Hill coefficient is 2.046 ± 0.1. For MRP2-mediated CP-I uptake, Km and Vmax values were 7.7 ± 0.7 µM and 48 ± 11 pmol/mg protein/min, respectively. The Hill coefficient is 1 for the best fit. The data are presented as the mean and SD of a single experiment performed in triplicate.

Effects of Modulators on MRP2-Mediated E17βG Transport in Membrane Vesicles.

To investigate the compound-dependent stimulation, we tested total of 97 compounds selected from the literature to evaluate their effect on E17βG transport by MRP2 in membrane vesicles. All compounds for E17βG transport were tested at 20 and 200 µM. Of those compounds tested, 51 compounds displayed stimulatory effects (>10% of control) with at least one concentration (Fig. 3; Supplemental Table 2). Stimulatory effects appeared to be related to the concentration of modulators. Mixed effects were also observed, as a subset of compounds showed stimulatory effect at lower concentration and inhibitory effect at higher concentration or vice versa for other subset molecules (Fig. 3).

Fig. 3.
Fig. 3.

Graphical representation of effect of various modulators on MRP2-mediated E17βG transport. MRP2-mediated E17βG transport was measured in inside-out membrane vesicles, derived from MRP2-expressing Sf9 insect cells, after incubation with E17βG at 50 µM with and without modulators at 20 and 200 µM. A total of 96 modulators were tested and, among those, 51 modulators (53%) stimulated MRP2-mediated E17βG transport by ≥10%. Sixteen modulators (16%) significantly inhibited E17βG transport at least either one or both of the concentrations tested. The data were presented as the mean from a single experiment (CV <20%).

Effects of Modulators on MRP2-Mediated CP-I Uptake in Membrane Vesicles.

We further tested 47 compounds that show either stimulatory effects in the inhibition of MRP2-mediated E17βG uptake or bile acids and statins that share the cholestane structure and are potential stimulators to assess their effects on ATP-dependent CP-I transport by MRP2. The compounds selected were reported in the literature for their inhibitory and/or stimulatory effects on E17βG transport by MRP2. For example, stimulatory effects of 32 of 47 compounds selected on MRP2-meidated E17βG transport were reported in literature (Pedersen et al., 2008; Morgan et al., 2013). In addition, a few representative statins, bile acids, and steroidal compounds were also included to test their effects on CP-I uptake transport by MRP2.

Of the compounds assessed, 30 compounds were determined to be inhibitors for MRP2-mediated CP-I transport (Table 1). Of 30 inhibitors, 7 compounds were obtained with an IC50 of <100 µM, ranging from 11 to 84 µM as benzbromarone > bromosulfophthalein > MK-571 > troglitazone > rifampicin > atorvastatin > losartan potassium. Ten compounds, including nodolol, alpha-bilirubin, metformin, acetaminophen, taurocholic acid, and cholic acid, displayed no effect on ATP-dependent CP-I uptake transport by MRP2. Eight compounds (17%) were identified as stimulators, compared with 32 of 47 compounds when E17βG was used as the probe substrate. The net changes in stimulation with these compounds varied from 34% to 181% (Table 1). A bell-shaped inhibition curve of MRP2-mediated CP-I transport was observed with E17βG, with stimulatory effects appearing at low concentrations (0.1–100 µM) and inhibitory effects appearing at high concentrations >100 µM, with an IC50 value of 187 µM.

View this table:
TABLE 1

List of compounds tested for their modulatory effect against CP-I as substrate, and their classification into type of modulator: compounds with no modulation are listed first, followed by inhibitors in order of increasing IC50, followed by stimulators

Structure Similarity of Stimulators of MRP2-Mediated CP-I Uptake in Membrane Vesicles.

The chemical structures of the stimulators of MRP2-mediated CP-I uptake in membrane vesicles are depicted in Fig. 5. These stimulators, except for mitoxantrone and pyrimethamine, share the general pattern of the 17–carbon ring backbone, which is the same structure used by other biologically important steroid molecules including steroid hormones, bile acids, and vitamins (Fig. 5). Mitoxantrone has two identical side chains containing both amino and hydroxyl moieties that were rich in oxygen- and nitrogen-containing planar tricyclic anthraquinone rings (9.08 pKa). Pyrimethamine (7.34 pKa) belongs to phenylpyrimidines that contain a benzene ring linked to a pyrimidine ring through a C-C or C-N bond (Fig. 5).

Discussion

ABC transporter MRP2 transports its substrates from the inside to the outside of cells. As a result, identifying a substrate in an intact cell system overexpressing MRP2 protein is difficult to accomplish through the monitoring of the direct transport of the substrate. Alternatively, substrate transport can be determined using inside-out–oriented membrane vesicles demonstrating ATP-dependent transport of a substrate into the vesicles. For inhibition studies, probe substrates should ideally be potential victim drugs used in the clinic. However, technical difficulties, such as extensive substrate diffusion into membrane vesicles and the lack of an optimal dynamic window of ATP-dependent uptake, make most xenobiotic substrates unsuitable in the vesicle assay. Currently, LTC4 and E17βG are recommended by the ITC as probe substrates for assessing MRP2 inhibition (Brouwer et al., 2013).

In the present study, we first tested 97 compounds in MRP2-mediated E17βG transport at two concentrations, 20 and 200 µM (Figs. 3 and 4). Of these compounds, 51 (53%) were found to be the stimulators of MRP2-mediated E17βG transport in at least one of the two concentrations tested. Our data are in good agreement with results reported previously, where in most instances, marketed drugs have been shown to stimulate the MRP2-mediated E17βG transport (Pedersen et al., 2008; Morgan et al., 2013). For some compounds (dacarbazine and sulfasalazine), a strong stimulation was observed at a lower concentration (20 µM), whereas E17βG transport was inhibited at a high concentration (200 µM), demonstrating a bell-shaped curve of modulation. The stimulatory effects of compounds on MRP2-mediated E17βG transport in membrane vesicles limits the use of E17βG, as concerns of false-negative inhibition remain for many modulating compounds. The concern for false-negative inhibition becomes important in explaining hyperbilirubinemia caused by MRP2 inhibition. A compound can cause hyperbilirubinemia by causing direct hepatotoxicity or by inhibiting MRP2-mediated bilirubin transport. Hyperbilirubinemia mediated by MRP2 inhibition, may not be fatal for the progression of the compound down clinical development; however, hepatotoxicity signal can prevent a compound from further progress. Therefore, providing a clinically relevant MRP2 probe that is devoid of the risk of identifying a compound as having false-negative inhibitor, becomes very important for further use a compound. In this relation, CP-I as a probe substrate identified 64% of the 47 compounds as inhibitors (Fig. 4). This will help to differentiate hyperbilirubinemia as a consequence of MRP2 inhibition from hyperbilirubinemia via hepatotoxicity. In contrast, using E17βG as a probe substrate, it is difficult to classify compounds as inhibitors, because compounds vary significantly in their behavior depending on concentration (bell-shaped curves). Therefore, in our study, we separated the compounds into stimulators and inhibitors and/no effects based on their behavior using E17βG as the probe substrate (Fig. 4).

Fig. 4.
Fig. 4.

Graphical representation of modulators of E17βG and CP-I transport. Pie chart of the segregation of compounds based on their transport properties with either 50 µM E17βG (A) or 5 µM CP-I (B) in MRP2-transfected Sf9 membrane vesicles. For E17βG transport, compounds were classified into two classes: stimulators (showing >10% stimulation of E17βG transport), and no effect/inhibitors. For CP-I transport, compounds were classified into three classes: inhibitors, stimulators, and compounds with no effect.

Although the urinary concentration ratio of CP-I to that of sum of CP-I and CP-III has been used as a surrogate marker to assess MRP2 activity clinically (Benz-de Bretagne et al., 2011), in vitro evidence of the transport of CP-I by MRP2 was lacking. Herein, we have shown for the first time that CP-I is taken up into membrane vesicles overexpressing MRP2 and that the ATP-dependent uptake was about 30-fold higher than the control, which provided an excellent dynamic window to assess MRP2 inhibitory effects. In addition, the transport of CP-I by MRP2 was linear up to 45 minutes (Supplemental Fig. 1), which provides a time range long enough to decrease variations, compared with LTC4 (Heredi-Szabo et al., 2008). Low Km values of LTC4 (695 nM) results in rapid deviation from linearity of uptake, therefore, making the experiments variation prone. In addition, because CP-I is fluorescent, it is easily measurable compared with LTC4, which requires radioactivity analysis. In addition to LTC4, carboxy-dichlorofluorescein (CDF) is also recommended as a probe substrate for MRP2 by the ITC (Brouwer et al., 2013). CDF follows the Michaelis-Menten kinetics and is a fluorescent substrate; however, it is not endogenous. CDF is administered in cell system as di-acetate prodrug, because CDF is a poor permeable molecule. CDF transport is also reported to be stimulated by different compounds such as verapamil, budesonide, and thioridazine (Munić et al., 2011; Kidron et al., 2012). However, it is challenging to further understand the significance of the stimulation of MRP2-mediated CDF transport in vivo, because of the permeability and prodrug factor.

Unlike E17βG, CP-I uptake in MRP2-expressed membrane vesicles followed Michaelis-Menten kinetics (Fig. 2B), which indicates that CP-I does not have affinity for the modulator site for its own transport. Most importantly, although 32 of the 47 compounds chosen to assess CP-I transport are identified as stimulators in E17βG transport (Fig. 3), either in the literature (Pedersen et al., 2008; Morgan et al., 2013) or in our data; only 8 compounds were found to stimulate CP-I transport. Therefore, with CP-I as the probe substrate the percentage of compounds showing in vitro stimulation was largely reduced. Although stimulators of E17βG transport varied widely in their structural features, stimulators of CP-I transport displayed common structural feature of 17–carbon ring steroid moiety (Fig. 5). The other two compounds, mitoxantrone and pyrimethamine, are strongly basic. Mitoxantrone forms a head-to-tail dimer and binds at two opposite grooves of the G-quadruplex. These structural features are being investigated with in vivo studies to understand the physiologic relevance of the stimulators with structure similarities.

Fig. 5.
Fig. 5.

Chemical structure of stimulators for MRP2-mediated CP-I transport.

E17βG kinetics is usually explained by distinct modulator and transport sites (Zelcer et al., 2003a; Borst et al., 2006). The substantial stimulatory effect of the binding of E17βG to the modulator site changes the transport kinetics to a sigmoidal nature. However, E17βG stimulated the transport of CP-I only to a low extent (<40% to 10 µM, whereas E17βG was an inhibitor of CP-I transport at higher concentrations). Therefore, an assumption can be made that the binding of E17βG to the modulator site has a minimum effect on transport of CP-I. This proves that the stimulation is a probe substrate–dependent phenomenon, further necessitating the importance of identifying a clinically relevant probe substrate.

Among the compounds assessed, bile acids inhibited MRP2-mediated CP-I transport. Previous studies indicated GCDCA, taurochenodeoxycholic acid, TDCA, and glycocholic acid as stimulators of MRP-2–mediated E17βG transport (Bodo et al., 2003). With CP-1 as substrate, chenodeoxycholic acid, deoxycholic acid, GCDCA, glycodeoxycholic acid, and TDCA were found to be inhibitors of MRP2. MRP2 is responsible for bile acid–independent bile flow in the case of cholestasis. As a result, accumulated intrahepatic bile acids due to cholestasis might inhibit MRP2 function and result in hyperbilirubinemia, providing another mechanism of bilirubin increase apart from direct hepatotoxicity.

Although diclofenac, indomethacin, glyburide, losartan, and troglitazone are stimulators of E17βG transport (Fig. 3) (Morgan et al., 2013), these compounds appeared to be inhibitors in MRP2-mediated CP-I transport (Table 1). Troglitazone was withdrawn from the market for the cases of liver failure. Cholestasis mediated by the bile salt export pump (ABCB11) inhibition and reactive metabolite formation from troglitazone sulfate have been shown to be two major mechanisms of toxicity. Although it has been reported to downregulate MRP2 expression (Foster et al., 2012), MRP2 functional inhibition by troglitazone can potentially contribute to the observed cholestatic injury with troglitazone. In addition, troglitazone sulfate has been reported (Funk et al., 2001) to be a more potent inhibitor of canalicular transporter bile salt export pump and hence its hepatic accumulation increases toxicity. Troglitazone sulfate is also reported to be a substrate of MRP2 (Kostrubsky et al., 2001). Troglitazone, by inhibiting MRP2, can contribute to the higher accumulation of troglitazone sulfate and hence to toxicity. Higher intrahepatic levels of troglitazone sulfate corroborate our findings (Chojkier, 2005). Therefore, the interaction of troglitazone sulfate with MRP2 and its contribution to the accumulation of troglitazone and its sulfate conjugates remains to be further determined. Similarly, losartan and glyburide are drugs associated with clinical liability of liver toxicity. They are reported to be MRP2 inhibitors in our study for the first time. Although the relationship of MRP2 inhibition and liver toxicity remains to be further explored, our data could shed new light on the mechanism of hepatotoxicity caused by troglitazone, losartan, and glyburide.

To summarize, to better understand MRP2 inhibition and the in vivo consequence, an alternative in vitro probe substrate is needed to minimize the stimulatory effects and consequent false-negative occurrences in membrane vesicle assays using E17βG as a substrate. Herein, we have for the first time shown CP-I to be an MRP2 in vitro substrate for appreciating MRP2 inhibition. The transport kinetics were obtained in membrane vesicles transfected with MRP2. Only eight compounds with structural similarity stimulated CP-I transport among the 47 compounds evaluated. With CP-1, previously classified stimulators including diclofenac, indomethacin, glyburide, losartan and troglitazone in MRP2-mediated E17βG vesicle uptake were redetermined to be potent MRP2 inhibitors. In addition, bile acids and atorvastatin, simvastatin, lovastatin, and rosuvastatin were also found to be inhibitors of MRP2-mediated CP-I transport. We conclude that CP-I is a better probe substrate in membrane vesicle uptake assays for characterizing MRP2 inhibition effects.

Acknowledgments

The authors thank Punit Marathe, William Humphreys, Devang Shah, Murali Subramanian, and Mike Sinz for help during various aspects of this work, such as data analysis, experimental setup, and reviewing of the manuscript.

Authorship Contributions

Participated in research design: Lai, Chatterjee and Gilibili.

Conducted experiments: Gilibili, Chatterjee, Bagul, Mosure, Murali.

Contributed new reagents or analytic tools: Gilibili, Chatterjee, Bagul, Mosure and Lai.

Performed data analysis: Lai, Chatterjee, Gilibili, Mosure, Bagul and Mariappan.

Wrote or contributed to the writing of the manuscript: Chatterjee, Lai, Gilibili, Mosure, Bagul, Mandlekar and Mariappan.

Footnotes

Abbreviations

ABC
ATP binding cassette
CDF
carboxy-dichlorofluorescein
CP-I
coproporphyrin I
DMSO
dimethylsulfoxide
E17βG
estradiol-17β-glucuronide
GCDCA
glycochenodeoxycholic acid
ITC
International Transporter Consortium
Km
Michaelis-Menten constant
LC-MS/MS
liquid chromatography-tandem mass spectrometry
LTC4
leukotriene C4
MRP2
multidrug-resistance associated protein 2
TDCA
taurodeoxycholic acid

References

  1. ↵
    1. Bakos E,
    2. Evers R,
    3. Sinkó E,
    4. Váradi A,
    5. Borst P, and
    6. Sarkadi B
    (2000) Interactions of the human multidrug resistance proteins MRP1 and MRP2 with organic anions. Mol Pharmacol 57:760–768.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Benz-de Bretagne I,
    2. Respaud R,
    3. Vourc’h P,
    4. Halimi JM,
    5. Caille A,
    6. Hulot JS,
    7. Andres CR, and
    8. Le Guellec C
    (2011) Urinary elimination of coproporphyrins is dependent on ABCC2 polymorphisms and represents a potential biomarker of MRP2 activity in humans. J Biomed Biotechnol 2011:498757.
    OpenUrlPubMed
  3. ↵
    1. Benz-de Bretagne I,
    2. Zahr N,
    3. Le Gouge A,
    4. Hulot JS,
    5. Houillier C,
    6. Hoang-Xuan K,
    7. Gyan E,
    8. Lissandre S,
    9. Choquet S, and
    10. Le Guellec C
    (2014) Urinary coproporphyrin I/(I + III) ratio as a surrogate for MRP2 or other transporter activities involved in methotrexate clearance. Br J Clin Pharmacol 78:329–342.
    OpenUrl
  4. ↵
    1. Bodo A,
    2. Bakos E,
    3. Szeri F,
    4. Varadi A, and
    5. Sarkadi B
    (2003) Differential modulation of the human liver conjugate transporters MRP2 and MRP3 by bile acids and organic anions. J Biol Chem 278:23529–23537.
    OpenUrlAbstract/FREE Full Text
  5. ↵
    1. Borst P,
    2. Zelcer N, and
    3. van de Wetering K
    (2006) MRP2 and 3 in health and disease. Cancer Lett 234:51–61.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Brouwer KL,
    2. Keppler D,
    3. Hoffmaster KA,
    4. Bow DA,
    5. Cheng Y,
    6. Lai Y,
    7. Palm JE,
    8. Stieger B,
    9. Evers R, and International Transporter Consortium
    (2013) In vitro methods to support transporter evaluation in drug discovery and development. Clin Pharmacol Ther 94:95–112.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Chojkier M
    (2005) Troglitazone and liver injury: in search of answers. Hepatology 41:237–246.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Evers R,
    2. Kool M,
    3. Smith AJ,
    4. van Deemter L,
    5. de Haas M, and
    6. Borst P
    (2000) Inhibitory effect of the reversal agents V-104, GF120918 and Pluronic L61 on MDR1 Pgp-, MRP1- and MRP2-mediated transport. Br J Cancer 83:366–374.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Foster JR,
    2. Jacobsen M,
    3. Kenna G,
    4. Schulz-Utermoehl T,
    5. Morikawa Y,
    6. Salmu J, and
    7. Wilson ID
    (2012) Differential effect of troglitazone on the human bile acid transporters, MRP2 and BSEP, in the PXB hepatic chimeric mouse. Toxicol Pathol 40:1106–1116.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Funk C,
    2. Pantze M,
    3. Jehle L,
    4. Ponelle C,
    5. Scheuermann G,
    6. Lazendic M, and
    7. Gasser R
    (2001) Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate. Toxicology 167:83–98.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Herédi-Szabó K,
    2. Glavinas H,
    3. Kis E,
    4. Méhn D,
    5. Báthori G,
    6. Veres Z,
    7. Kóbori L,
    8. von Richter O,
    9. Jemnitz K, and
    10. Krajcsi P
    (2009) Multidrug resistance protein 2-mediated estradiol-17beta-D-glucuronide transport potentiation: in vitro-in vivo correlation and species specificity. Drug Metab Dispos 37:794–801.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Heredi-Szabo K,
    2. Kis E,
    3. Molnar E,
    4. Gyorfi A, and
    5. Krajcsi P
    (2008) Characterization of 5(6)-carboxy-2,‘7’-dichlorofluorescein transport by MRP2 and utilization of this substrate as a fluorescent surrogate for LTC4. J Biomol Screen 13:295–301.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Hillgren KM,
    2. Keppler D,
    3. Zur AA,
    4. Giacomini KM,
    5. Stieger B,
    6. Cass CE,
    7. Zhang L, and International Transporter Consortium
    (2013) Emerging transporters of clinical importance: an update from the International Transporter Consortium. Clin Pharmacol Ther 94:52–63.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Isley WL
    (2003) Hepatotoxicity of thiazolidinediones. Expert Opin Drug Saf 2:581–586.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kidron H,
    2. Wissel G,
    3. Manevski N,
    4. Häkli M,
    5. Ketola RA,
    6. Finel M,
    7. Yliperttula M,
    8. Xhaard H, and
    9. Urtti A
    (2012) Impact of probe compound in MRP2 vesicular transport assays. Eur J Pharm Sci 46:100–105.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kostrubsky VE,
    2. Vore M,
    3. Kindt E,
    4. Burliegh J,
    5. Rogers K,
    6. Peter G,
    7. Altrogge D, and
    8. Sinz MW
    (2001) The effect of troglitazone biliary excretion on metabolite distribution and cholestasis in transporter-deficient rats. Drug Metab Dispos 29:1561–1566.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Lai Y,
    2. Mandlekar S,
    3. Shen H,
    4. Holenarsipur VK,
    5. Langish R,
    6. Rajanna P,
    7. Murugesan S,
    8. Gaud N,
    9. Selvam S,
    10. Date O, et al.
    (2016) Coproporphyrins in plasma and urine can be appropriate clinical biomarkers to recapitulate drug-drug interactions mediated by organic anion transporting polypeptide inhibition. J Pharmacol Exp Ther 358:397–404.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Morgan RE,
    2. van Staden CJ,
    3. Chen Y,
    4. Kalyanaraman N,
    5. Kalanzi J,
    6. Dunn 2nd RT,
    7. Afshari CA, and
    8. Hamadeh HK
    (2013) A multifactorial approach to hepatobiliary transporter assessment enables improved therapeutic compound development. Toxicol Sci 136:216–241.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Munić V,
    2. Hlevnjak M, and
    3. Eraković Haber V
    (2011) Characterization of rhodamine-123, calcein and 5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF) export via MRP2 (ABCC2) in MES-SA and A549 cells. Eur J Pharm Sci 43:359–369.
    OpenUrlPubMed
  20. ↵
    1. Niemi M,
    2. Arnold KA,
    3. Backman JT,
    4. Pasanen MK,
    5. Gödtel-Armbrust U,
    6. Wojnowski L,
    7. Zanger UM,
    8. Neuvonen PJ,
    9. Eichelbaum M,
    10. Kivistö KT, et al.
    (2006) Association of genetic polymorphism in ABCC2 with hepatic multidrug resistance-associated protein 2 expression and pravastatin pharmacokinetics. Pharmacogenet Genomics 16:801–808.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Nies AT and
    2. Keppler D
    (2007) The apical conjugate efflux pump ABCC2 (MRP2). Pflugers Arch 453:643–659.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Pedersen JM,
    2. Matsson P,
    3. Bergström CA,
    4. Norinder U,
    5. Hoogstraate J, and
    6. Artursson P
    (2008) Prediction and identification of drug interactions with the human ATP-binding cassette transporter multidrug-resistance associated protein 2 (MRP2; ABCC2). J Med Chem 51:3275–3287.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Shen H,
    2. Dai J,
    3. Liu T,
    4. Cheng Y,
    5. Chen W,
    6. Freeden C,
    7. Zhang Y,
    8. Humphreys WG,
    9. Marathe P, and
    10. Lai Y
    (2016) Coproporphyrins I and III as functional markers of OATP1B activity: in vitro and in vivo evaluation in preclinical species. J Pharmacol Exp Ther 357:382–393.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Tang W
    (2007) Drug metabolite profiling and elucidation of drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol 3:407–420.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Yoshida K,
    2. Maeda K, and
    3. Sugiyama Y
    (2013) Hepatic and intestinal drug transporters: prediction of pharmacokinetic effects caused by drug-drug interactions and genetic polymorphisms. Annu Rev Pharmacol Toxicol 53:581–612.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Zamek-Gliszczynski MJ,
    2. Hoffmaster KA,
    3. Tweedie DJ,
    4. Giacomini KM, and
    5. Hillgren KM
    (2012) Highlights from the international transporter consortium second workshop. Clin Pharmacol Ther 92:553–556.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Zelcer N,
    2. Huisman MT,
    3. Reid G,
    4. Wielinga P,
    5. Breedveld P,
    6. Kuil A,
    7. Knipscheer P,
    8. Schellens JH,
    9. Schinkel AH, and
    10. Borst P
    (2003a) Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2). J Biol Chem 278:23538–23544.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Zelcer N,
    2. Saeki T,
    3. Bot I,
    4. Kuil A, and
    5. Borst P
    (2003b) Transport of bile acids in multidrug-resistance-protein 3-overexpressing cells co-transfected with the ileal Na+-dependent bile-acid transporter. Biochem J 369:23–30.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Zhang Y,
    2. Han YH,
    3. Putluru SP,
    4. Matta MK,
    5. Kole P,
    6. Mandlekar S,
    7. Furlong MT,
    8. Liu T,
    9. Iyer RA,
    10. Marathe P, et al.
    (2016) Diclofenac and its acyl glucuronide: determination of in vivo exposure in human subjects and characterization as human drug transporter substrates in vitro. Drug Metab Dispos 44:320–328.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

Coproporphyrin-I as Novel In Vitro Probe Substrate for MRP2

Ravindranath Reddy Gilibili, Sagnik Chatterjee, Pravin Bagul, Kathleen W. Mosure, Bokka Venkata Murali, T. Thanga Mariappan, Sandhya Mandlekar and Yurong Lai
Drug Metabolism and Disposition June 1, 2017, 45 (6) 604-611; DOI: https://doi.org/10.1124/dmd.116.074740
Twitter logo Facebook logo Mendeley logo

Jump to section

Related Articles

Cited By...

More in this TOC Section

Similar Articles

Advertisement