Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2016/08/08/dmd.116.069807.DC1.html 1521-009X/44/10/1562–1568$25.00 DRUG METABOLISM AND DISPOSITION Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/dmd.116.069807 Drug Metab Dispos 44:1562–1568, October 2016
Expression of Organic Anion Transporting Polypeptide 1A2 in Red Blood Cells and Its Potential Impact on Antimalarial Therapy s Andrea Hubeny, Markus Keiser, Stefan Oswald, Gabriele Jedlitschky, Heyo K. Kroemer,1 Werner Siegmund, and Markus Grube Department of Pharmacology (A.H., G.J., H.K.K., M.G.) and Department of Clinical Pharmacology (M.K., S.O., W.S.) at the Center of Drug Absorption and Transport (C_DAT), University Medicine, Greifswald, Germany Received February 2, 2016; accepted August 4, 2016
ABSTRACT chloroquine: 1.0 6 1.5 mM), but no or only moderate effects were observed for OATP2B1. Subsequently, quinine was identified as a substrate of OATP1A2 (Km 23.4 mM). The OATP1A2-mediated uptake was sensitive to the OATP1A2-specific inhibitor naringin. Both OATPs were expressed in human RBCs, and ex vivo transport studies demonstrated naringin-sensitive accumulation of quinine in these cells (60 pmol versus 38 pmol/5 3 105 RBCs). Additional transport studies using OCT1–3 and organic cation transporter novel type 1 (OCTN1) indicated only significant quinine uptake by OCT1, which was not detected in RBCs. In conclusion, our data demonstrate expression of OATP2B1 and OATP1A2 in RBCs as well as OATP1A2-mediated uptake of quinine. Therefore, modulation of OATP1A2 function may affect quinine uptake into erythrocytes.
Introduction
ABCC1) and multidrug-resistance protein 4 (MRP4, ABCC4) (Wu et al., 2005), and chloroquine and quinine have been identified as inhibitors and substrates of multidrug and toxin extrusion protein 1 (MATE1, SLC47A1) and organic cation transporters (OCTs) (Muller et al., 2011; Nies et al., 2012). In addition, there is evidence that quinine interacts with organic anion transporting polypeptides (OATPs), in particular OATP1A2, which has been shown to transport N-methyl-quinine (Kullak-Ublick et al., 2001; Shitara et al., 2002). Interestingly, the impact of OATPs has not been addressed in detail in this context. Among the 11 human OATP transporters, OATP1B1, OATP1B3, OATP2B1, and OATP1A2 are of the most pharmacologic relevance. OATP1B1 and OATP1B3 are predominantly, or even exclusively, expressed in the liver, but OATP1A2 and OATP2B1 show broad expression profiles. Thus, OATP1A2 and OATP2B1 are especially interesting candidates for local drug distribution (Konig et al., 2013). We therefore characterized the expression of OATP1A2 and OATP2B1 in red blood cells (RBCs) and studied the interaction of antimalarial drugs with these transporters in comparison with the organic cation transporters OCT1, OCT2, OCT3, and organic cation transporter novel type 1 (OCTN1). Thus, we were able to demonstrate expression of OATP1A2 and OATP2B1 in RBCs and identified OATP1A2 as an uptake transporter for quinine in these cells.
Malaria remains one of the world’s most important infectious diseases, with 214 million new cases and 438,000 deaths in 2015 (WHO, 2015). For treatment of malaria, only a limited number of effective drugs are available. To prevent replication of blood-stage malaria parasites, quinoline-based drugs, such as quinine and chloroquine, that inhibit heme detoxification in the food vacuole are widely used (Kumar et al., 2007). Thus, to reach their intracellular target structures, these compounds have to cross the erythrocyte membrane. For a wide variety of drugs, including several antimalarial compounds, drug transporters of the ATP-binding cassette (ABC), solute carrier (SLC), or solute carrier organic anion (SLCO) family are involved in transmembrane transfer (Konig et al., 2013). For example, mefloquine, quinine, and chloroquine are inhibitors or substrates of efflux pumps such as P-glycoprotein (P-gp/ABCB1) (Riffkin et al., 1996; Pham et al., 2000; Rijpma et al., 2014), multidrug-resistance protein 1 (MRP1,
This work was supported by the German Federal Ministry for Education and Research [grant 03IPT612X]. 1 Current affiliation: University Medicine Göttingen, Göttingen, Germany. dx.doi.org/10.1124/dmd.116.069807. s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: ABC, ATP-binding cassette; E1S, estrone-3-sulfate; MDCKII, Madin-Darby canine kidney cells; MPP+, 1-methyl-4-phenylpyridinium; OATP, organic anion transporting polypeptide; OCT, organic cation transporter; OCTN, organic cation transporter novel type; PBS, phosphate-buffered saline; P-gp, P-glycoprotein; RBC, red blood cell; SLC, solute carrier; TEA, tetraethylammonium. 1562
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 4, 2016
Important antimalarial drugs, including quinolines, act against blood schizonts by interfering with hemoglobin metabolism. To reach their site of action, these compounds have to cross the plasma membrane of red blood cells (RBCs). Organic cation transporters (OCTs) and organic anion transporting polypeptides (OATPs) are important uptake transporters and interesting candidates for local drug transport. We therefore studied their interaction with antimalarial compounds (quinine, chloroquine, mefloquine, pyrimethamine, artemisinin, and artesunate) and characterized the expression of OATP1A2 and OATP2B1 in RBCs. Competition assays using transporter-overexpressing MadinDarby canine kidney (MDCKII) cells and the model substrate estrone-3-sulfate identified quinine and chloroquine as potent inhibitors of OATP1A2 function (IC50 quinine: 0.7 6 1.2 mM;
1563
OATP1A2 and Antimalarial Compounds Materials and Methods
the test compound. After 5 minutes, substrate uptake was stopped by aspiration of incubation buffer and washing 3 times with ice-cold PBS. Cells were lysed with 800 ml 0.2% SDS and 5 mM EDTA, and an aliquot of 200 ml was dissolved in 2 ml of scintillation mixture (Rotiszint Eco Plus, Roth, Karlsruhe, Germany). Radioactivity was measured in a scintillation b-counter (type 1409; LKB-Wallac/PerkinElmer, Freiburg, Germany). For normalization, the protein concentration of the whole-cell lysate was determined using the BCA method (Thermo Scientific, Waltham, MA). Quinine uptake experiments were performed in transporter-overexpressing and control MDCKII cells for the indicated time points using tritium-labeled quinine and the OATP1A2 inhibitor naringin (100 mM) (Bailey, 2010). Quinine uptake into RBCs was studied by a rapid filtration method using polyvinylidene difluoride (0.2 mm pore size) filter membranes. We suspended 5 105 RBCs in prewarmed transport buffer, then incubated them with quinine in the presence or absence of naringin (100 mM) for 30 minutes at 37C and 4C, respectively. The reaction was stopped by adding 1 ml of ice-cold PBS containing naringin (100 mM). RBC samples were filtered immediately through a polyvinylidene difluoride filter (presoaked in PBS containing 100 mM naringin) and rinsed 3 times with 3 ml of ice-cold PBS. For measurement of radioactivity, the filter was dissolved in 5 ml of scintillation mixture (Rotiszint; Roth) and measured as described earlier. All transport studies were performed with incubation/transport buffer containing 140 mM NaCl2, 5 mM KCl, 1 mM KH2PO4, 1.5 mM CaCl2, 5 mM glucose, and 12.5 mM HEPES (pH 7.3). For pH 6.0 studies, the pH of the incubation buffer was adjusted using HCl. Statistical Methods. For calculations, graphs, and statistical analyses, we used Excel (Microsoft Corp., Redmond, WA) and GraphPad Prism 5.01 (GraphPad, San Diego, CA). Competition experiments were calculated as the percentage of transporter-overexpressing MDCKII cells treated with vehicle. The IC50 values were determined by nonlinear regression from sigmoidal dose–response curves using the following constraints: top = 0% inhibition and bottom #100% inhibition. To determine statistical significance, one-way or two-way analysis of variance followed by Dunnett’s or Tukey’s multiple comparison test were used for analyzing multiple groups. P , 0.05 was considered statistically significant.
Results Using transporter-overexpressing MDCKII cells and the probe substrates E1S (1 mM) for OATP1A2 and OATP2B1, MPP+ (500 nM) for the OCTs, and TEA (0.3 mM) for OCTN1, the interactions between the antimalarial drugs quinine, chloroquine, mefloquine, pyrimethamine, artemisinin, and artesunate and the individual transporters were studied. Transporter overexpression was confirmed by immunofluorescence staining, demonstrating a basolateral localization for OATP2B1 and the OCT(N)s, while OATP1A2 was localized to the apical membrane (Fig. 1; Supplemental Fig. 1). Functional studies in transporteroverexpressing and vector-transfected MDCKII cells revealed transporterspecific uptake of the respective probe substrates (Fig. 1). In competition studies, all the compounds with the exception of artesunate (100 mM each) showed inhibition of OATP1A2-, OCT1-, and OCT2-mediated transport. OATP2B1 function was only slightly affected by mefloquine, but OCT3 and OCTN1 were not inhibited by any of the antimalarial drugs (Fig. 1). The most potent interactions were further characterized by determination of the respective half-maximal inhibitory concentrations (IC50 values), identifying quinine and chloroquine as higher affinity inhibitors of OATP1A2 (IC50 quinine: 0.7 6 1.2 mM; chloroquine: 1.0 6 1.5 mM) compared with mefloquine (IC50 6.0 6 3.4 mM) (Fig. 2). The respective IC50 values for OCT1 and/or OCT2 were higher than those for OATP1A2, but also in low micromolar range (Table 1). Subsequently, we examined the direct uptake of quinine, the most potent inhibitor. Although no OATP2B1-mediated transport was observed, OATP1A2-overexpressing cells displayed time-dependent accumulation of quinine, compared with the vector-transfected MDCKII cells (Fig. 3A). The total quinine uptake was pH-sensitive, with higher uptake rates at pH 7.3 compared with pH 6.0, whereas the OATP1A2-
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 4, 2016
Materials. Radiolabeled tracer compounds [3H]estrone-3-sulfate ([3H]E1S, 50 Ci/mmol), [3H]1-methyl-4-phenylpyridinium ([3H]MPP+, 80 Ci/mmol), [14C]tetraethylammonium ([14C]TEA, 55 mCi/mmol), and [3H]quinine (20 Ci/mmol) were obtained from Hartmann Analytic (Braunschweig, Germany). Antimalarial compounds were purchased from Sigma-Aldrich (Steinheim, Germany). Cell Culture. Madin-Darby canine kidney (MDCKII) cells overexpressing human OATP2B1 or OCT were described previously elsewhere (Grube et al., 2006, 2011). OATP1A2-overexpressing MDCKII cells were generated as described elsewhere for transporter-overexpressing human embryonic kidney 293 (HEK293) cells (Leonhardt et al., 2010). The cells were grown in 75 cm2 cell culture flasks in Dulbecco’s modified Eagle’s medium supplemented with 2 mM L-glutamine, 1% minimal essential medium nonessential amino acids, 10% fetal calf serum, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37C in a humidified atmosphere containing 5% CO2. Except for the experiments, transfected cell lines were cultured in selection medium containing neomycin 0.5 mg/ml (OATP1A2), hygromycin B 350 mg/ml (OATP2B1, OCT3), or blasticidin 10 mg/ml (OCT1–2). Preparation of Red Blood Cells. After approval by the local ethics committee of the University Medicine Greifswald, blood samples were taken from male and female healthy volunteers. RBCs were prepared from fresh, EDTA-treated venous blood by layering on Mono-Poly Resolving Medium (MP Biomedicals, Eschwege, Germany) with subsequent centrifugation at 300g for 30 minutes. The resulting pellet of RBCs was washed with isotonic phosphate-buffered saline (PBS) solution and immediately used for transport studies or stored at 280C for immunoblotting. Immunofluorescence Staining. Protein localization in transfected MDCKII cells was studied as described elsewhere for transporter-overexpressing human embryonic kidney 293 (HEK293) cells (Grube et al., 2006; Leonhardt et al., 2010; Mandery et al., 2010). For detection of OCT, the cells were fixed with ethanol (70% or 99%) for 10 minutes at room temperature, permeabilized with 0.4% Tween-20 in PBS, and blocked using 5% fetal calf serum in PBS. OCT1 was detected using the antibody ab123128 (rabbit, 1/25; Abcam, Cambridge, United Kingdom). For OCT2 detection the antibody HPA008567 (rabbit, 1/25 dilution; Sigma-Aldrich) was used; OCT3 was detected by C-14 (goat, 1/50 dilution; Santa Cruz Biotechnology, Heidelberg, Germany), and OCTN1 by A01 (mouse, 1/25 dilution; Abnova, Taipei City, Taiwan). Localization of OATP1A2 and OATP2B1 in human RBCs was also characterized by immunofluorescence staining. EDTA-treated whole blood smears were fixed with methanol (OATP1A2, room temperature) or acetone (OATP2B1, 220C) for 10 minutes, permeabilized with 0.4% Tween-20 in PBS for 10 minutes, and blocked using 5% fetal calf serum in PBS. OATP1A2 was detected using the polyclonal antibody C18884 (rabbit, 1/20 dilution; Assay Biotechnology, Sunnyvale, CA). OATP2B1 detection was performed using transporter-specific rabbit antiserum (1/200 dilution; Grube et al., 2005). RBCs were additionally stained with phycoerythrin-labeled CD235a (1/200 dilution; BD Pharmingen, Heidelberg, Germany). All immunofluorescence stainings were analyzed using the LSM780 confocal laser scanning system (Carl Zeiss MicroImaging, Jena, Germany). Only signals above the background fluorescence determined by control stainings using preimmune serum (rabbit) were shown. Immunoblot Analysis. Membrane fractions of MDCKII cells and RBCs were separated by a 7.5% sodium dodecyl sulfate (SDS) polyacrylamide gel after denaturation at 95C for 5 minutes. Immunoblotting was performed using a tank blotting system (Bio-Rad Laboratories, Munich, Germany). Primary antibodies were diluted in Tris-buffered saline containing 0.05% Tween-20 and 0.1% bovine serum albumin to the following final concentration: OATP1A2 antibody (C18884, 1/2000; Assay Biotechnology) and OATP2B1 antibody (1/1000); secondary horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Bio-Rad Laboratories) was used at a 1/2000 dilution. Detection was performed using the enhanced chemiluminescence detection system (Thermo Scientific, Schwerte, Germany). Transport Assays. For functional experiments in the transporter overexpressing MDCKII cells, radiolabeled transporter substrates (E1S: OAT1A2 and OATP2B1; MPP+: OCT1, OCT2, and OCT3; TEA: OCTN1) were used. Cells (70,000 cells/well) were cultured in 24-well plates for 4 days. One day before the experiment, the medium was changed, and the cells were incubated with 2.5 mM sodium butyrate to stimulate transporter expression. For inhibition studies, the cells were washed once with prewarmed PBS and incubated at 37C with transport buffer containing the radiolabeled substrate and
1564
Hubeny et al.
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 4, 2016 Fig. 1. Interaction between OATP and OCT(N) transporters and antimalarial compounds. E1S (1 mM, 5 minutes, OATP1A2, OATP2B1), MPP+ (0.5 mM, 1.5 minutes, OCT1–3) and TEA (0.3 mM, 5 minutes, OCTN1) uptake was studied in transporter-overexpressing MDCKII cells in the presence and absence of the respective compound (each 100 mM) as well as in vector-transfected control cells (MDCKII). Data are represented in relation to transporter-overexpressing cells (100%, dotted line), mean+ S.D., n = 2–4, one-way analysis of variance and Dunnett’s multiple comparison test versus control-treated MDCKII-OATP1A2 cells; *P , 0.05, **P , 0.01, ***P , 0.001. Inserts: Immunofluorescence staining of respective transporter (bars: 20 mm).
1565
OATP1A2 and Antimalarial Compounds
Transporter expression was analyzed in RBCs by use of immunofluorescence microscopy. OATP1A2 and OATP2B1 were expressed in the membrane of human RBCs and colocalized with the erythrocyte surface marker CD235a (Fig. 4A). Concerning the organic cation transporters, no signal was detected for OCT1–3 (data not shown) whereas OCTN1 was localized to the plasma membrane (Supplemental Fig. 3A). For the OATPs as well as OCTN1 these findings were verified by Western blot analysis, demonstrating transporter-specific signals at the expected molecular masses (OATP2B1 ;84 kDa; OATP1A2;74 kDa; OCTN1 ;62 kDa, Fig. 4B; Supplemental Fig. 3B). Finally, quinine uptake into RBCs was studied ex vivo using erythrocytes from healthy volunteers. Significant temperature-dependent transport was observed (60 6 10 pmol/5 105 RBCs at 37C versus 34 6 7 pmol/5 105 RBCs at 4C), which was inhibited by the OATP1A2-inhibitor naringin (100 mM) at 37C, but not at 4C (Fig. 4C).
Fig. 2. Determination of half-maximal inhibitory concentrations (IC50 values) of quinine, chloroquine, and mefloquine for OATP1A2-mediated E1S transport (1 mM, 5 minutes). Data represent the background corrected mean values (mean 6 S.D., n = 3–4).
specific uptake rate was comparable at both pH values (Fig. 3B). In addition, OATP1A2-mediated quinine uptake was sensitive to the OATP1A2specific inhibitor naringin at both pH 7.3 and pH 6.0 (Fig. 3C). The kinetic parameters of the OATP1A2-mediated quinine transport were 23.4 6 6.1 mM for Km, and 241 6 24 pmol/mg/min for Vmax (Fig. 3D). Quinine uptake by OCT1 and OCT2 was also studied. Slight, but significant, quinine uptake was observed only for OCT1 (Supplemental Fig. 2A). However, OCT1 function was not affected by naringin, as shown by using the standard OCT1 substrate MPP+ (Supplemental Fig. 2B).
In the present study, we were able to show interactions between antimalarial drugs and OATPs as well as identified quinine as a substrate of OATP1A2. In addition, we demonstrated expression of OATP2B1 and OATP1A2 in erythrocytes. To reach their target sites, antimalarial drugs have to cross the erythrocyte plasma membrane. OATPs, especially OATP1A2 and OATP2B1, are interesting candidates for local drug uptake into RBCs (Konig et al., 2013). We therefore characterized the interaction of both transporters with antimalarial drugs acting against blood schizonts (quinine, chloroquine, mefloquine, pyrimethamine, artemisinin, and artesunate) using transporter-overexpressing MDCKII cells in addition to studying transporter expression in RBCs. Transporter expression and function were shown for each transporter-overexpressing cell line. Of note, in contrast to other OATPs, OATP1A2 was localized in the apical membrane of MDCKII cells. This finding was in line with a recent study using a similar model (Liu et al., 2015) and corresponds to the physiologic localization of this transporter in tissues such as the blood– brain barrier (Bronger et al., 2005) or the kidney (Lee et al., 2005). Using these systems, interaction studies between antimalarial drugs and OATP1A2 and OATP2B1 revealed no significant influence on OATP2B1 function. OATP1A2 was inhibited by almost all tested compounds (except artesunate). Among them, chloroquine and quinine were identified as the most potent inhibitors, although the maximal inhibitory effect for chloroquine was much lower compared with quinine. Half-maximal inhibitory concentrations of 1.0 and 0.7 mM were calculated for chloroquine and quinine, respectively. These findings are in line with the results of a very recent study on the interaction between chloroquine and OATP1A2 (Xu et al., 2016). Compared with therapeutic concentrations, this value is only slightly above the plasma concentration of chloroquine (300–600 nM) (Muller et al., 2011) and even an order of magnitude below the levels reached in quinine therapy (6–25 mM) (Powell and McNamara, 1972; Hall et al., 1973). Hence, coapplication of chloroquine or quinine with other OATP1A2 substrates might lead to drug–drug interactions at the transporter level. The impact of OATP1A2 in drug absorption and systemic bioavailability is still a controversial issue (Glaeser et al., 2007; Groer et al., 2013), but its luminal localization in the blood–brain barrier points to an important function for distribution processes into the brain (Cheng et al., 2012). Therefore, antimalarial therapy using chloroquine or quinine may interfere with OATP1A2 substrates such as neurosteroids (Gao et al., 2015) and opioid peptides (Gao et al., 2000), and drugs such as triptans (Cheng et al., 2012). Besides its inhibitory function, quinine was shown to be a substrate of OATP1A2 with an affinity in the low micromolar range. This finding
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 4, 2016
Discussion
1566
Hubeny et al. TABLE 1 Half-maximal inhibitory concentrations (IC50 values) and maximal inhibitory effects of selected antimalarial compounds for OATP1A2, OCT1, and OCT2 Data represent background corrected IC50 values (mean 6 S.D.) from three to four experiments. OATP1A2
OCT1
OCT2
Compound IC50
Quinine Chloroquine Mefloquine Pyrimethamine Artemisinin
Maximum Inhibitory Effect
mM
%
0.7 6 1.2 1.0 6 1.5 6.0 6 3.4 — —
94 60 93 — —
IC50 mM
3.5 12.8 6.6 8.5 4.2
6 7.6 691 6 6.5 6.1.9 6 5.5
IC50
%
mM
85 80 70 88 88
5.9 6 8.9 6 14.6 6 3.4 6 —
Maximum Inhibitory Effect %
3.2 5.4 5.8 1.4
92 87 87 100 —
parasite. Quinine rapidly enters the erythrocyte; however, intracellular concentrations are below the corresponding plasma levels, indicating transporter proteins are involved in quinine distribution (Salako and Sowunmi, 1992). It is well established that quinine is transported by P-gp, thereby limiting the neurotoxic side effects of the drug (Kerb et al., 2009). However, P-gp expression in erythrocytes is very low (Abraham et al., 2001), so the presence of a saturable quinine uptake transporter in RBCs may also help to explain this observation. We were able to show the expression of OATP2B1 and OATP1A2 in the plasma membrane of erythrocytes, pointing to a possible role for OATP1A2 in quinine uptake from the blood plasma to the target site. This hypothesis was confirmed by our in vitro results demonstrating temperature-dependent and naringin-sensitive uptake of quinine into human erythrocytes. As a consequence of this finding, the concomitant
Fig. 3. Quinine uptake into transporter-overexpressing MDCKII cells. (A) Time-dependent quinine uptake (1 mM) at pH 7.3 into OATP1A2- and OATP2B1-overexpressing MDCKII cells (mean 6 S.D., n = 3–4). (B) OATP1A2-mediated quinine uptake (1 mM) at pH 7.3 and pH 6.0 (hatched columns) (mean 6 S.D., n = 3, two-way analysis of variance and Tukey’s multiple comparison test, *P , 0.05, **P , 0.01, ***P , 0.001). (C) Sensitivity of OATP1A2-mediated quinine uptake (1 mM, 30 minutes) to the OATP1A2 inhibitor naringin (100 mM) at pH 7.3 and pH 6.0 (mean 6 S.D., n = 3, one-way analysis of variance and Tukey’s multiple comparison test, *P , 0.05, **P , 0.01). (D) Concentration-dependent uptake of quinine (0.78–100 mM, 5 minutes) at pH 7.3 by OATP1A2. OATP1A2-specific transport was used to calculate kinetic parameters (mean 6 S.D., n = 6).
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 4, 2016
was in line with a previous report demonstrating OATP1A2-mediated uptake of the closely related N-methyl-quinine (Kullak-Ublick et al., 2001). The transport of other OATP1A2 substrates such as E1S or methotrexate is enhanced at lower pH (Badagnani et al., 2006; Leuthold et al., 2009), but the total uptake of quinine was reduced at pH 6.0, most likely due to reduced passive diffusion caused by an increase in the fraction of ionized molecule (pKa 8.15–8.58). In addition, quinine is another example of an OATP1A2 substrate with cationic character, such as trospium chloride (Bexten et al., 2015) or rocuronium (van Montfoort et al., 2001), indicating a more amphiphilic substrate spectrum of OATP1A2 compared with other OATPs. Malaria is the most important therapeutic indication for quinine. Here, it acts against blood schizonts by inhibition of heme detoxification, resulting in free cytotoxic heme, which accumulates and kills the
Maximum Inhibitory Effect
OATP1A2 and Antimalarial Compounds
1567
Acknowledgments The authors thank Tina Sonnenberger (Department of Pharmacology, University Medicine, Greifswald, Germany) for the excellent technical assistance.
Fig. 4. (A) Immunofluorescence staining of OATP1A2 and OATP2B1 in RBC. Wholeblood smears were probed against OATP1A2 and OATP2B1 (both green). Costaining was performed with the erythrocyte membrane marker CD235a (red). Control stainings with preimmune serum exhibited no specific signals (inserts). Scale bar: 5 mm. (B) Immunoblot of crude membrane fractions of RBC and transporter-overexpressing MDCKII cells (control) using OATP-specific antibodies. (C) In vitro uptake of quinine (100 nM) into RBCs measured at 37C and 4C in the presence and absence of the OATP1A2 inhibitor naringin (100 mM) (mean 6 S.D., n = 4 RBC preparations, oneway analysis of variance and Tukey’s multiple comparison test, *P , 0.05, **P , 0.01).
therapy of quinine and other OATP1A2 substrates and/or inhibitors may interfere with quinine distribution and action. Indeed, there is evidence for an interaction between quinine and the OATP1A2 inhibitor ritonavir, which has been shown to significantly increase quinine Cmax and the area under the curve (Cvetkovic et al., 1999; Soyinka et al., 2010). Although those investigators attributed this observation to the inhibition of quinine metabolism via CYP3A4 by ritonavir, it is likewise possible that transporters are involved. Ritonavir is also known as a potent inhibitor of P-gp (Lee et al., 1998). However, because quinine is well absorbed in the gut with a bioavailability
Authorship Contributions Participated in research design: Hubeny, Grube. Conducted experiments: Hubeny, Grube. Contributed new reagents or analytic tools: Hubeny, Keiser, Oswald. Performed data analysis: Hubeny, Grube. Wrote or contributed to the writing of the manuscript: Hubeny, Grube, Jedlitschky, Siegmund, Kroemer. References Abraham EH, Shrivastav B, Salikhova AY, Sterling KM, Johnston N, Guidotti G, Scala S, Litman T, Chan KC, Arceci RJ, et al. (2001) Cellular and biophysical evidence for interactions between adenosine triphosphate and P-glycoprotein substrates: functional implications for adenosine triphosphate/drug cotransport in P-glycoprotein overexpressing tumor cells and in P-glycoprotein low-level expressing erythrocytes. Blood Cells Mol Dis 27:181–200. Arndt P, Volk C, Gorboulev V, Budiman T, Popp C, Ulzheimer-Teuber I, Akhoundova A, Koppatz S, Bamberg E, Nagel G, et al. (2001) Interaction of cations, anions, and weak base quinine with rat renal cation transporter rOCT2 compared with rOCT1. Am J Physiol Renal Physiol 281: F454–F468. Badagnani I, Castro RA, Taylor TR, Brett CM, Huang CC, Stryke D, Kawamoto M, Johns SJ, Ferrin TE, Carlson EJ, et al. (2006) Interaction of methotrexate with organic-anion transporting polypeptide 1A2 and its genetic variants. J Pharmacol Exp Ther 318:521–529. Bailey DG (2010) Fruit juice inhibition of uptake transport: a new type of food-drug interaction. Br J Clin Pharmacol 70:645–655. Bexten M, Oswald S, Grube M, Jia J, Graf T, Zimmermann U, Rodewald K, Zolk O, Schwantes U, Siegmund W, et al. (2015) Expression of drug transporters and drug metabolizing enzymes in the
Downloaded from dmd.aspetjournals.org at ASPET Journals on September 4, 2016
above 80%, inhibition of intestinal P-gp is unlikely to be the reason for the observed increase in the plasma levels (Paintaud et al., 1993; Soyinka et al., 2010). Inhibition of P-gp in RBCs would result in the opposite effect, but inhibition of OATP1A2 uptake into the erythrocytes could also contribute to increased drug levels in the plasma. Moreover, not only may drug–drug interactions influence quinine uptake into RBCs, but also genetic polymorphisms or environmental factors may have an effect. For example, genetic variants in the SLCO1A2 gene encoding OATP1A2, as well as several food components, including fruit juices, green tea, and the flavonoid quercetin, have been shown to affect OATP1A2 function (Bailey, 2010; Mandery et al., 2010; Yamakawa et al., 2011; Misaka et al., 2014). These results indicate an involvement of OATP1A2 for quinine uptake into RBCs, but the possible impact of other transporters must also be taken in account. According to its cationic character, members of the OCT family are especially interesting candidates. Indeed, we were able to show inhibition of OCT1- and OCT2-mediated MPP+ uptake by quinine, and direct transport of quinine by OCT1, which was in line with previous reports (Arndt et al., 2001; Koepsell et al., 2007). Although quinine transport by OCT1 might be relevant for the hepatic uptake and in turn the metabolism of quinine (Kerb et al., 2009), we were not able to detect OCT1 in erythrocytes, and OCT1 function was not affected by naringin. Another possible candidate for quinine uptake into RBCs is OCTN1. This transporter is expressed in erythrocytes (Kobayashi et al., 2004), is inhibited by quinine, and has been shown to transport the quinine diastereomer quinidine (Yabuuchi et al., 1999). However, in contrast to these previous reports, we could not detect an interaction between quinine and OCTN1, which is most likely a result of the lower concentrations used in our experiments. In summary, we characterized OATP2B1 and OATP1A2 expression in RBCs and were able to demonstrate OATP1A2-dependent transport of the antimalarial drug quinine. Therefore, intracellular drug concentration within RBCs is determined not only by passive uptake and active efflux processes but also by uptake transporters such as OATP1A2. As a consequence, environmental, pharmacologic, and/or genetic modification of OATP1A2 function may affect intracellular concentration and activity of quinine in antimalarial therapy.
1568
Hubeny et al. Liu H, Yu N, Lu S, Ito S, Zhang X, Prasad B, He E, Lu X, Li Y, Wang F, et al. (2015) Solute carrier family of the organic anion-transporting polypeptides 1A2- Madin-Darby canine kidney II: a promising in vitro system to understand the role of organic anion-transporting polypeptide 1A2 in blood-brain barrier drug penetration. Drug Metab Dispos 43:1008–1018. Mandery K, Bujok K, Schmidt I, Keiser M, Siegmund W, Balk B, König J, Fromm MF, and Glaeser H (2010) Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1. Biochem Pharmacol 80: 1746–1753. Misaka S, Yatabe J, Müller F, Takano K, Kawabe K, Glaeser H, Yatabe MS, Onoue S, Werba JP, Watanabe H, et al. (2014) Green tea ingestion greatly reduces plasma concentrations of nadolol in healthy subjects. Clin Pharmacol Ther 95:432–438. Müller F, König J, Glaeser H, Schmidt I, Zolk O, Fromm MF, and Maas R (2011) Molecular mechanism of renal tubular secretion of the antimalarial drug chloroquine. Antimicrob Agents Chemother 55:3091–3098. Nies AT, Damme K, Schaeffeler E, and Schwab M (2012) Multidrug and toxin extrusion proteins as transporters of antimicrobial drugs. Expert Opin Drug Metab Toxicol 8:1565–1577. Paintaud G, Alván G, and Ericsson O (1993) The reproducibility of quinine bioavailability. Br J Clin Pharmacol 35:305–307. Pham YT, Régina A, Farinotti R, Couraud P, Wainer IW, Roux F, and Gimenez F (2000) Interactions of racemic mefloquine and its enantiomers with P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT. Biochim Biophys Acta 1524:212–219. Powell RD and McNamara JV (1972) Quinine: side-effects and plasma levels. Proc Helminthol Soc Wash 39:331–338 http://science.peru.edu/COPA/ProcHelmSocWash_V39_SI_Basic_Research_ in_Malaria_1972I.pdf. Riffkin CD, Chung R, Wall DM, Zalcberg JR, Cowman AF, Foley M, and Tilley L (1996) Modulation of the function of human MDR1 P-glycoprotein by the antimalarial drug mefloquine. Biochem Pharmacol 52:1545–1552. Rijpma SR, van den Heuvel JJMW, van der Velden M, Sauerwein RW, Russel FGM, and Koenderink JB (2014) Atovaquone and quinine anti-malarials inhibit ATP binding cassette transporter activity. Malar J 13:359–367. Salako LA and Sowunmi A (1992) Disposition of quinine in plasma, red blood cells and saliva after oral and intravenous administration to healthy adult Africans. Eur J Clin Pharmacol 42: 171–174. Shitara Y, Sugiyama D, Kusuhara H, Kato Y, Abe T, Meier PJ, Itoh T, and Sugiyama Y (2002) Comparative inhibitory effects of different compounds on rat oatpl (slc21a1)- and Oatp2 (Slc21a5)-mediated transport. Pharm Res 19:147–153. Soyinka JO, Onyeji CO, Omoruyi SI, Owolabi AR, Sarma PV, and Cook JM (2010) Pharmacokinetic interactions between ritonavir and quinine in healthy volunteers following concurrent administration. Br J Clin Pharmacol 69:262–270. van Montfoort JE, Müller M, Groothuis GM, Meijer DK, Koepsell H, and Meier PJ (2001) Comparison of “type I” and “type II” organic cation transport by organic cation transporters and organic anion-transporting polypeptides. J Pharmacol Exp Ther 298:110–115. World Health Organization (WHO) (2015) World Malaria Report 2015, WHO, Geneva. Wu CP, Klokouzas A, Hladky SB, Ambudkar SV, and Barrand MA (2005) Interactions of mefloquine with ABC proteins, MRP1 (ABCC1) and MRP4 (ABCC4) that are present in human red cell membranes. Biochem Pharmacol 70:500–510. Xu C, Zhu L, Chan T, Lu X, Shen W, Madigan MC, Gillies MC, and Zhou F (2016) Chloroquine and hydroxychloroquine are novel inhibitors of human organic anion transporting polypeptide 1A2. J Pharm Sci 105:884–890. Yabuuchi H, Tamai I, Nezu J, Sakamoto K, Oku A, Shimane M, Sai Y, and Tsuji A (1999) Novel membrane transporter OCTN1 mediates multispecific, bidirectional, and pH-dependent transport of organic cations. J Pharmacol Exp Ther 289:768–773. Yamakawa Y, Hamada A, Shuto T, Yuki M, Uchida T, Kai H, Kawaguchi T, and Saito H (2011) Pharmacokinetic impact of SLCO1A2 polymorphisms on imatinib disposition in patients with chronic myeloid leukemia. Clin Pharmacol Ther 90:157–163.
Address correspondence to: Dr. Markus Grube, Department of Pharmacology, University Medicine Greifswald, Center of Drug Absorption and Transport (C_DAT), Felix-Hausdorff-Str. 3, D-17489, Greifswald, Germany. E-mail:
[email protected] Downloaded from dmd.aspetjournals.org at ASPET Journals on September 4, 2016
bladder urothelium in man and affinity of the bladder spasmolytic trospium chloride to transporters likely involved in its pharmacokinetics. Mol Pharm 12:171–178. Bronger H, König J, Kopplow K, Steiner HH, Ahmadi R, Herold-Mende C, Keppler D, and Nies AT (2005) ABCC drug efflux pumps and organic anion uptake transporters in human gliomas and the blood-tumor barrier. Cancer Res 65:11419–11428. Cheng Z, Liu H, Yu N, Wang F, An G, Xu Y, Liu Q, Guan CB, and Ayrton A (2012) Hydrophilic anti-migraine triptans are substrates for OATP1A2, a transporter expressed at human blood-brain barrier. Xenobiotica 42:880–890. Cvetkovic M, Leake B, Fromm MF, Wilkinson GR, and Kim RB (1999) OATP and P-glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug Metab Dispos 27: 866–871. Gao B, Hagenbuch B, Kullak-Ublick GA, Benke D, Aguzzi A, and Meier PJ (2000) Organic aniontransporting polypeptides mediate transport of opioid peptides across blood-brain barrier. J Pharmacol Exp Ther 294:73–79. Gao B, Vavricka SR, Meier PJ, and Stieger B (2015) Differential cellular expression of organic anion transporting peptides OATP1A2 and OATP2B1 in the human retina and brain: implications for carrier-mediated transport of neuropeptides and neurosteriods in the CNS. Pflugers Arch 467:1481–1493. Glaeser H, Bailey DG, Dresser GK, Gregor JC, Schwarz UI, McGrath JS, Jolicoeur E, Lee W, Leake BF, Tirona RG, et al. (2007) Intestinal drug transporter expression and the impact of grapefruit juice in humans. Clin Pharmacol Ther 81:362–370. Gröer C, Brück S, Lai Y, Paulick A, Busemann A, Heidecke CD, Siegmund W, and Oswald S (2013) LC-MS/MS-based quantification of clinically relevant intestinal uptake and efflux transporter proteins. J Pharm Biomed Anal 85:253–261. Grube M, Ameling S, Noutsias M, Köck K, Triebel I, Bonitz K, Meissner K, Jedlitschky G, Herda LR, Reinthaler M, et al. (2011) Selective regulation of cardiac organic cation transporter novel type 2 (OCTN2) in dilated cardiomyopathy. Am J Pathol 178:2547–2559. Grube M, Köck K, Oswald S, Draber K, Meissner K, Eckel L, Böhm M, Felix SB, Vogelgesang S, Jedlitschky G, et al. (2006) Organic anion transporting polypeptide 2B1 is a high-affinity transporter for atorvastatin and is expressed in the human heart. Clin Pharmacol Ther 80: 607–620. Grube M, Meyer Zu Schwabedissen H, Draber K, Präger D, Möritz KU, Linnemann K, Fusch C, Jedlitschky G, and Kroemer HK (2005) Expression, localization, and function of the carnitine transporter octn2 (slc22a5) in human placenta. Drug Metab Dispos 33:31–37. Hall AP, Czerwinski AW, Madonia EC, and Evensen KL (1973) Human plasma and urine quinine levels following tablets, capsules, and intravenous infusion. Clin Pharmacol Ther 14:580–585. Kerb R, Fux R, Mörike K, Kremsner PG, Gil JP, Gleiter CH, and Schwab M (2009) Pharmacogenetics of antimalarial drugs: effect on metabolism and transport. Lancet Infect Dis 9:760–774. Kobayashi D, Aizawa S, Maeda T, Tsuboi I, Yabuuchi H, Nezu J, Tsuji A, and Tamai I (2004) Expression of organic cation transporter OCTN1 in hematopoietic cells during erythroid differentiation. Exp Hematol 32:1156–1162. Koepsell H, Lips K, and Volk C (2007) Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications. Pharm Res 24:1227–1251. König J, Müller F, and Fromm MF (2013) Transporters and drug-drug interactions: important determinants of drug disposition and effects. Pharmacol Rev 65:944–966. Kullak-Ublick GA, Ismair MG, Stieger B, Landmann L, Huber R, Pizzagalli F, Fattinger K, Meier PJ, and Hagenbuch B (2001) Organic anion-transporting polypeptide B (OATP-B) and its functional comparison with three other OATPs of human liver. Gastroenterology 120:525–533. Kumar S, Guha M, Choubey V, Maity P, and Bandyopadhyay U (2007) Antimalarial drugs inhibiting hemozoin (beta-hematin) formation: a mechanistic update. Life Sci 80:813–828. Lee CG, Gottesman MM, Cardarelli CO, Ramachandra M, Jeang KT, Ambudkar SV, Pastan I, and Dey S (1998) HIV-1 protease inhibitors are substrates for the MDR1 multidrug transporter. Biochemistry 37:3594–3601. Lee W, Glaeser H, Smith LH, Roberts RL, Moeckel GW, Gervasini G, Leake BF, and Kim RB (2005) Polymorphisms in human organic anion-transporting polypeptide 1A2 (OATP1A2): implications for altered drug disposition and central nervous system drug entry. J Biol Chem 280: 9610–9617. Leonhardt M, Keiser M, Oswald S, Kühn J, Jia J, Grube M, Kroemer HK, Siegmund W, and Weitschies W (2010) Hepatic uptake of the magnetic resonance imaging contrast agent Gd-EOB-DTPA: role of human organic anion transporters. Drug Metab Dispos 38:1024–1028. Leuthold S, Hagenbuch B, Mohebbi N, Wagner CA, Meier PJ, and Stieger B (2009) Mechanisms of pH-gradient driven transport mediated by organic anion polypeptide transporters. Am J Physiol Cell Physiol 296:C570–C582.