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Human Organic Anion Transporters 1 (hOAT1/SLC22A6) and 3 (hOAT3/SLC22A8) Transport Edaravone (MCI-186; 3-methyl-1-phenyl-2-pyrazolin-5-one) and Its Sulfate Conjugate

Pharmacokinetics Laboratory, Mitsubishi Pharma Corporation, Chiba, Japan (N.M., T.T., Y.I., T.N.); and Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan (H.K., Y.S.)

(Received December 10, 2006; accepted May 10, 2007)

	Abstract

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3-Methyl-1-phenyl-2-pyrazolin-5-one (MCI-186; edaravone), a novel free radical scavenger, is used for the treatment of acute cerebral infarction. After marketing, a few cases of acute renal failure were reported in patients following treatment with this drug. Because edaravone is mainly excreted into the urine following conjugation to glucuronide or sulfate, the renal excretion mechanisms of edaravone should help provide important information when considering the clinical cases. We examined the transport of edaravone and its sulfate and glucuronide conjugates via human organic anion transporter 1 (hOAT1) and 3 (hOAT3), expressed on the basolateral membranes of proximal tubules. The hOAT1- and hOAT3-transfected human embryonic kidney (HEK)-293 cells exhibited a markedly higher uptake of edaravone sulfate and a slightly higher uptake of edaravone than vector-transfected cells. The K_m values of edaravone sulfate uptake by hOAT1 and hOAT3 were 11 and 15 µM, respectively. Estimation of the relative contribution of hOAT1 and hOAT3 using reference compounds suggested that hOAT1 and hOAT3 might contribute to the renal uptake of edaravone sulfate to the same extent. However, edaravone and its sulfate showed no cytotoxicity toward both hOAT1-HEK and control cells, suggesting that higher uptake in hOAT1-HEK did not associate with cytotoxicity of these compounds. In conclusion, our results suggest that both hOAT1 and hOAT3 are responsible for the basolateral uptake of edaravone sulfate in the kidney.

View larger version (7K): [in this window] [in a new window]   FIG. 1. Chemical structures of edaravone (A), edaravone sulfate (B), and edaravone glucuronide (C).

Organic anion transport systems, such as human organic anion transporter 1 (hOAT1/SLC22A6) and 3 (hOAT3/SLC22A8), are predominantly expressed in the kidney and are expressed on the basolateral membrane of the proximal tubules in humans (Kusuhara and Sugiyama, 2002; Russel et al., 2002; Lee and Kim, 2004). hOAT1 and hOAT3 play an important role in the transport of organic anions across the basolateral membrane of human proximal tubules. The substrates of hOAT1 include a variety of drugs, such as p-aminohippuric acid (PAH), ochratoxin A, nonsteroidal anti-inflammatory agents, ß-lactam antibiotics, diuretics, methotrexate and antiviral drugs, and endogenous compounds, such as cyclic nucleotides, prostaglandins, and uremic toxins (Hosoyamada et al., 1999; Ho et al., 2000; Khamdang et al., 2002; Enomoto et al., 2003; Deguchi et al., 2004), whereas the substrates of hOAT3 include drugs, such as ß-lactam antibiotics [benzylpenicillin (PCG)], HMG-CoA reductase inhibitors (pravastatin), and H₂ receptor antagonists (cimetidine), as well as endogenous compounds, such as uremic toxins and conjugated steroids (dehydroepiandrosterone sulfate, estradiol-17ß-glucuronide, and estrone-3-sulfate) (Cha et al., 2001; Hasegawa et al., 2002; Enomoto et al., 2003; Deguchi et al., 2004; Hasannejad et al., 2004; Khamdang et al., 2004). To date, only limited information is available concerning the interaction between organic anion transporter (OAT) and sulfate- and glucuronide-conjugated drug metabolites.

In the present study, we examined the transport of edaravone and its conjugated metabolites in hOAT1- and hOAT3-transfected human embryonic kidney (HEK)-293 cells to investigate the renal uptake mechanisms of edaravone and its conjugates. In addition, we examined the effect of hOAT1 expression on the cytotoxicity of these compounds.

	Materials and Methods

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Materials. [³H]PAH (156 GBq/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). [³H]PCG (814 GBq/mmol) was purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). Edaravone, edaravone sulfate, edaravone glucuronide, [¹⁴C]edaravone (516 MBq/mmol), and adefovir were synthesized by Mitsubishi Pharma Corporation (Osaka, Japan). [³⁵S]Edaravone sulfate and [¹⁴C]edaravone glucuronide were biosynthesized by incubating edaravone in rat liver cytosol and microsomes, respectively. In the case of [³⁵S]edaravone sulfate, the reaction buffer contained 5 mM HEPES, 1 mM MgCl₂, 2 mM edaravone, 2 mg/ml rat liver cytosol, and 1.85 MBq [³⁵S]3'-phosphoadenosine 5'-phosphosulfate (63 GBq/mmol) (PerkinElmer Life Sciences) in a final volume of 1.0 ml. For [¹⁴C]edaravone glucuronide, the reaction buffer contained 5 mM HEPES, 1 mM MgCl₂,1mM dithiothreitol, 0.1 mM D-saccharic acid-1,4-lactone, 0.01% (v/v) Triton X-100, 2 mM edaravone, 2 mg/ml rat liver microsomes, and 185 kBq [¹⁴C]UDP-glucuronic acid (11.58 GBq/mmol) (MP Biomedicals, Irvine, CA) in a final volume of 1.0 ml. After incubation for 2 h at 37°C, both reactions were terminated by adding 4 ml of ice-cold methanol. After centrifugation, the supernatant was evaporated under N₂ and injected into a high-performance liquid chromatography system. The chromatographic conditions were as follows: column, CAPCELL PAK C18MG column (5 µm, 4.6 x 250 mm, SHISEIDO, Tokyo, Japan); mobile phase, methanol/10 mM ammonium acetate (pH 5.5) = 35:65 (v/v); flow rate, 0.8 ml/min; UV 254 nm. Purification was conducted by comparison with the retention time of each standard compound. The radiochemical purity of [³⁵S]edaravone sulfate and [¹⁴C]edaravone glucuronide prepared by this method was found to be 99.1 and 92.7%, respectively. Unlabeled PAH and PCG were purchased from Sigma-Aldrich (St. Louis, MO). All the other chemicals were of analytical grade and commercially available.

Cell Culture. The hOAT1- and hOAT3-transfected HEK-293 cells (hOAT1- and hOAT3-HEK) were established as described previously (Deguchi et al., 2004). hOAT1- and hOAT3-HEK were grown in Dulbecco's modified Eagle's medium (Invitrogen, Scotland, UK) supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 U/ml), streptomycin (100 µg/ml), and G418 sulfate (400 µg/ml) at 37°C with 5% CO₂ and 95% humidity. Cells were seeded in polylysine-coated 24-well plates (BD Biosciences, Bedford, MA) at a density of 1.2 x 10⁵ cells/well. The cell culture medium was replaced with culture medium supplemented with 5 mM sodium butyrate 24 h before the transport studies to induce the expression of hOAT1 and hOAT3.

Transport Studies. Transport studies were carried out as described previously (Hasegawa et al., 2002). The composition of the transport buffer was as follows: 118 mM NaCl, 23.8 mM NaHCO₃, 4.83 mM KCl, 0.96 mM KH₂PO₄, 1.20 mM MgSO₄, 12.5 mM HEPES, 5 mM glucose, and 1.53 mM CaCl₂,pH 7.4. The cells were preincubated with 0.5 ml of incubation medium for 15 min at 37°C. After preincubation, the medium was replaced with 0.25 ml of incubation medium containing radiolabeled ligands. At the end of the incubation period, the medium was aspirated, and cells were washed twice with 1 ml of ice-cold incubation medium. The cells were lysed in 250 µl of 1N NaOH. Aliquots (400 µl) were transferred to scintillation vials after adding 250 µlof 1N HCl. The radioactivity associated with the cells and medium was determined by liquid scintillation counting after adding 8 ml of ACSII (Amersham Biosciences). The protein content of the solubilized cells was determined using a BCA Protein Assay Kit (Pierce, Rockford, IL).

Ligand uptake is given as the cell-to-medium concentration ratio determined as the amount of ligand associated with the cells divided by the medium concentration. Specific uptake was obtained by subtracting the uptake into vector-transfected cells from that into cDNA-transfected cells. Kinetic parameters were obtained using the following equation:

where v is the uptake velocity of the substrate (pmol/min/mg), S is the substrate concentration in the medium (µM), K_m is the Michaelis-Menten constant (µM), and V_max is the maximum uptake rate (pmol/min/mg). Fitting was performed by the nonlinear least-squares method using WinNonlin version 4.1 (Pharsight Corporation, Mountain View, CA).

Cytotoxicity Test. Vector- and hOAT1-transfected HEK-293 cells were seeded in parallel into a 96-well clear-bottom black microplate at 2000 cells/well (n = 3) in Dulbecco's modified Eagle's medium (without phenol red) supplemented with 10% fetal bovine serum. The next day, the cells were treated with edaravone or edaravone sulfate at 0.3 to 1000 µM for 3 days. Dimethyl sulfoxide was used as a negative control at a final concentration of 0.2% (v/v). Adefovir was used as a positive control and diluted with the medium to give a concentration of 0.3 to 1000 µM. Then, the incubation medium was removed from the wells, and the plate was stored at –80°C until analysis. The cellular nucleic acid content was measured according to the protocol of the CyQUANT cell proliferation assay kit (Invitrogen-Molecular Probes, Eugene, OR). The fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm using a fluorescence microplate reader (FLEXstation, software SOFTmax PRO 4.3.1, Molecular Devices, Sunnyvale, CA). The mean fluorescence of the solvent control was defined as representing 100% growth, and the results were expressed as a percentage of the controls. The concentration producing 50% inhibition (IC₅₀) was calculated from a 4-parameter curve fit using SOFTmax PRO 4.3.1. Three independent experiments were carried out.

Estimation of Uptake Clearance in Kidney Slices from cDNA-Transfected Cells. Using the relative activity factor (RAF) concept (Hasegawa et al., 2003), we estimated the contribution of hOAT1 and hOAT3 to the total uptake of test compound by human kidney slices. Because PAH and PCG are relatively selective substrates of hOAT1 and hOAT3, respectively, they were used as reference compounds for hOAT1- or hOAT3-mediated uptake (Deguchi et al., 2004; Nozaki et al., 2007). Briefly, the ratio of the uptake clearance of reference compounds in human kidney slices to that in the expression system was calculated and defined as R_hOAT1 and R_hOAT3. The uptake clearance of the test compound by hOAT1 and hOAT3 in kidney slices was separately calculated by multiplying the uptake clearance of the test compound in transporter-expressing cells (CL_test,hOAT1 and CL_test,hOAT3) by R_hOAT1 and R_hOAT3, respectively, as described in the following equations:

View larger version (18K): [in this window] [in a new window]   FIG. 2. Concentration dependence of the uptake of [3H]PAH by hOAT1 and that of [3H]PCG by hOAT3. The concentration dependence of the uptake of [3H]PAH by hOAT1-HEK (A) and that of [3H]PCG by hOAT3-HEK (B) are shown as Michaelis-Menten plots. The uptake of [3H]PAH by hOAT1-HEK was measured for 1 min at 0.24 to 160 µM. The uptake of [3H]PCG by hOAT3-HEK was measured for 1 min at 0.045 to 320 µM. The hOAT1- and hOAT3-mediated transports were obtained by subtracting the transport velocity in vector-transfected cells from that in hOAT1-HEK and hOAT3-HEK. Each point represents the mean ± S.E. (n = 3).

	Results

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Uptake of Edaravone and Its Metabolites by hOAT1- and hOAT3-Transfected HEK-293 Cells. Figure 1 shows the chemical structures of edaravone, edaravone sulfate, and edaravone glucuronide. Figure 2A shows the concentration dependence of the uptake of [³H]PAH, a typical substrate of OAT1, in hOAT1-HEK. Figure 2B shows the concentration dependence of the uptake of [³H]PCG, a typical substrate of OAT3, in hOAT3-HEK. The estimated K_m values for the uptake of [³H]PAH by hOAT1-HEK and [³H]PCG by hOAT3-HEK were 27.4 ± 1.2 and 66.0 ± 3.0 µM, respectively, and these were consistent with previous reported values (PAH, 15.4–28.0 µM; PCG, 52.1–54.0 µM) (Ho et al., 2000; Deguchi et al., 2004; Tahara et al., 2005; Ueo et al., 2005). The V_max values for hOAT1-HEK and hOAT3-HEK were 503 ± 14 and 869 ± 26 pmol/min/mg, respectively.

In addition, we measured the uptake of [¹⁴C]edaravone, [³⁵S]edaravone sulfate, and [¹⁴C]edaravone glucuronide by these transfectants. The results of these uptake experiments are shown in Fig. 3. The uptake of [³⁵S]edaravone sulfate by hOAT1- or hOAT3-HEK was approximately 20 times higher than that by vector-transfected cells. hOAT1- or hOAT3-HEK also exhibited a 2-fold higher uptake of [¹⁴C]edaravone than control cells, but this uptake was much lower than the hOAT1- or hOAT3-dependent uptake of [³⁵S]edaravone sulfate. [¹⁴C]Edaravone glucuronide showed little uptake by any cells.

View larger version (25K): [in this window] [in a new window]   FIG. 3. Uptake of edaravone, edaravone sulfate, and edaravone glucuronide by hOAT1- and hOAT3-HEK cells. Vector-, hOAT1-, and hOAT3-transfected HEK-293 cells were incubated in a solution containing [14C]edaravone (7.2 µM), [35S]edaravone sulfate (0.10 µM), or [14C]edaravone glucuronide (0.32 µM) at 37°C for 10 min. Each value represents the mean ± S.E. (n = 3). ND, not detected. Significant differences from the vector control using Dunnett's test (***, p < 0.001).

View larger version (11K): [in this window] [in a new window]   FIG. 4. Time profiles of the uptake of [35S]edaravone sulfate by hOAT1 and hOAT3. A, the uptake of [35S]edaravone sulfate (0.13 µM) by hOAT1-HEK was examined at 37°C. Closed symbols and open symbols represent the uptake by hOAT1-HEK and vector-HEK, respectively. B, the uptake of [35S]edaravone sulfate (0.16 µM) by hOAT3-HEK was examined at 37°C. Closed symbols and open symbols represent the uptake by hOAT3-HEK and vector-HEK, respectively. Each value represents the mean ± S.E. (n = 3).

View larger version (17K): [in this window] [in a new window]   FIG. 5. Concentration dependence of the uptake of [35S]edaravone sulfate by hOAT1 and hOAT3. The concentration dependence of the uptake of [35S]edaravone sulfate by hOAT1-HEK (A) and hOAT3-HEK (B) is shown as Michaelis-Menten plots. The uptake by hOAT1-HEK or hOAT3-HEK was measured for 1 min at 0.13 to 32 µM or 0.16 to 64 µM, respectively. The hOAT1- and hOAT3-mediated transports were obtained by subtracting the transport velocity in vector-transfected cells from that in hOAT1-HEK and hOAT3-HEK. Each point represents the mean ± S.E. (n = 3).

Estimation of Uptake Clearance in Kidney Slices from cDNA-Transfected Cells. We estimated the relative contribution of hOAT1 and hOAT3 to the net uptake of edaravone sulfate based on the RAF concept (Hasegawa et al., 2003). It was predicted that hOAT1- and hOAT3-mediated uptake make similar contribution to the net uptake of edaravone sulfate in human kidney slices (Table 1).

Cytotoxicity Assay in hOAT1-Transfected HEK-293 Cells. We investigated the cytotoxicity of edaravone and edaravone sulfate in hOAT1-HEK cells. Adefovir, a substrate of hOAT1, was used as a positive control because Ho et al. (2000) reported that the IC₅₀ of adefovir was approximately 0.2 to 4 µM in hOAT1-transfected Chinese hamster ovary and V79 cells and approximately 100 to 400 µM in control cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium cytotoxicity tests after a 5-day treatment. Our data also showed markedly increased cytotoxicity of adefovir in hOAT1-transfected HEK-293 cells compared with control cells (Fig. 6C). The mean IC₅₀ calculated from three independent experiments was 146 µMinthe control cells and 2.34 µM in the hOAT1-HEK cells, suggesting that our cytotoxicity data on adefovir were similar to those of Ho et al. (2000). In contrast to adefovir, edaravone and its sulfate did not exhibit any cytotoxicity in both hOAT1-HEK and control cells at concentrations as high as 1 mM (Fig. 6, A and B).

View larger version (42K): [in this window] [in a new window]   FIG. 6. Cytotoxicity test of edaravone (A), edaravone sulfate (B), and adefovir (C) in vector- and hOAT1-transfected HEK-293 cells. The cells were treated with the compounds for 3 days in a 96-well clear-bottom black microplate, and the cellular nucleic acid content was measured using the CyQUANT cell proliferation assay kit. Values represent the mean ± S.E. from three independent experiments. The IC50 of adefovir calculated from three experiments were 105, 154, and 180 µM (mean, 146 µM) in vector-transfected HEK-293 cells. In hOAT1-transfected HEK-293 cells, the IC50 of adefovir were 1.47, 2.94, and 2.62 µM (mean, 2.34 µM). Significant differences from the solvent control by Dunnett's test (*, p < 0.05, **, p < 0.01) and from the vector-transfected HEK-293 cells shown by the t test (#, p < 0.05, ##, p < 0.01).

	Discussion

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In the present study, we examined the transport of edaravone and its conjugated metabolites in hOAT1- and hOAT3-expressing HEK-293 cells to investigate the renal uptake mechanisms of edaravone and its conjugates. As a result, hOAT1- and hOAT3-HEK exhibited markedly higher uptake of edaravone sulfate than vector-transfected cells (Fig. 3). A slight but significant increase was also observed in the uptake of edaravone by hOAT1- and hOAT3-HEK, but no significant uptake of edaravone glucuronide was observed (Fig. 3).

To date, only limited information is available concerning the interaction between OAT and sulfate- and glucuronide-conjugated drug metabolites. As far as edaravone is concerned, the sulfate conjugate is a better substrate of hOAT1 and hOAT3 than the glucuronide conjugate. In particular, the uptake clearances (V_max/K_m) of edaravone sulfate by hOAT1 and hOAT3 were greater than those of their typical substrates (PAH and PCG) (Table 1), indicating that edaravone sulfate is a good substrate of hOAT1 or hOAT3. Cumulative studies have identified several sulfate conjugates as OAT substrates. Bulky sulfate conjugates, such as dehydroepiandrosterone sulfate and estrone sulfate, are specifically transported by hOAT3 (Tahara et al., 2005). Indoxyl sulfate is a common substrate of hOAT1 and hOAT3, although its transport activity by OAT1 is much higher than that of hOAT3, and the K_m value for OAT1 is almost 13-fold lower than that for hOAT3 (Deguchi et al., 2004). Thus, edaravone sulfate is a unique sulfate conjugate that exhibits similar kinetic properties as far as interaction with hOAT1 and hOAT3 is concerned. The estimated K_m values for hOAT1 and hOAT3 were much higher than the unbound plasma concentration of edaravone sulfate (0.1 µM) in humans (Yokota et al., 1997), indicating that hOAT1 and hOAT3 are not saturated in clinical situations. In addition, estimation using RAF concept (Hasegawa et al., 2003) suggests that hOAT1 and hOAT3 make similar contribution to the renal uptake of edaravone sulfate in human kidney slices (Table 1).

The OAT-mediated transport of some drugs appears to be involved in their nephrotoxic effects. Most cephalosporin antibiotics or antiviral drugs, such as adefovir and cidofovir, are excreted into urine in nonmetabolized forms, and renal tubular secretion appears to be an important pathway for their renal clearance. These drugs are suggested to be not only filtered through the glomerulus but also actively secreted by the proximal tubules (Brown, 1993; Cundy et al., 1995a,b). The OAT-mediated transport of these drugs has been reported, and it is suggested that the accumulation of these drugs via OAT in the proximal tubules may be the primary step in their nephrotoxicity (Cihlar et al., 1999; Jariyawat et al., 1999; Takeda et al., 1999; Ho et al., 2000). Indeed, increased cytotoxicity of cidofovir, adefovir, or cephaloridine in OAT1-transfected cells compared with control cells has been observed (Cihlar et al., 1999; Jariyawat et al., 1999; Ho et al., 2000). Therefore, we also investigated whether the enhanced intracellular uptake of edaravone and its sulfate in hOAT1-HEK cells would contribute to increased cytotoxicity of these compounds. As shown in Fig. 6C, cell growth, an endpoint of cytotoxicity, of hOAT1-HEK cells was significantly aggravated by adefovir when compared with control cells. In contrast to adefovir, edaravone and its sulfate did not exhibit any cytotoxicity for both hOAT1-HEK and control cells even at 1 mM, suggesting that hOAT1 expression did not enhance the cytotoxicity of these compounds, probably because of their low cytotoxicity. Also in the previous study, it was found that edaravone and its conjugates exhibited little cytotoxicity for human renal proximal tubule epithelial cells and normal human cortical epithelial cells (Iwase et al., 2004). The cell viability of renal proximal tubule epithelial cells after 48-h treatment with edaravone, edaravone sulfate, and edaravone glucuronide was 66.4, 82.7, and 96.0%, respectively, even at 1 mM. In the case of human cortical epithelial cells, the cell viability was 69.5, 69.7, and 97.1%, respectively. Because the unbound plasma concentrations of edaravone, edaravone sulfate, and edaravone glucuronide were less than 1 µM, edaravone itself seems not to be directly nephrotoxic. The mechanism of acute renal failure in patients receiving edaravone treatment remains to be elucidated.

The main metabolite of edaravone in human urine is its glucuronide: approximately 70% of the dose was excreted into urine as the glucuronide (Yokota et al., 1997; Shibata et al., 1998), whereas in human plasma, the concentration of sulfate was higher than that of glucuronide (Yokota et al., 1997; Shibata et al., 1998). In the case of rats or dogs, the main metabolite in both urine and plasma was the sulfate (Komatsu et al., 1996). An in vitro study using human kidney S9 has suggested that edaravone sulfate undergoes conversion to edaravone by sulfatase, followed by glucuronidation in the human kidney (Yokota et al., 1997). Because the conversion activity of edaravone sulfate by sulfatase in the human kidney S9 was higher than that in rats (data not shown), the conversion from sulfate to glucuronide in the human kidney likely accounts for the difference between the abundance ratio of conjugates in human plasma and urine. Considering both the fact that edaravone sulfate is a substrate of hOAT1 and hOAT3 and the metabolic pathway of edaravone sulfate in human kidney, it appears that edaravone is taken up by the kidney as the sulfate form and excreted into urine as the glucuronide form following the conversion from sulfate to glucuronide via edaravone.

To achieve the vectorial transport of organic anions, such as edaravone sulfate, it is likely that transporter(s) are involved in not only the uptake across the basolateral membrane but also the efflux across the brush-border membrane of the proximal tubules. It is possible that edaravone glucuronide also undergoes efflux via some transporter(s) on the brush-border membranes because it is suggested that edaravone glucuronide produced in the kidney is excreted into urine in humans. It has been shown that the renal brush-border membrane possesses an influx/efflux transport system for organic anions, such as MRP2(ABCC2), MRP4(ABCC4), NPT1(SLC17A1), hOAT4(SLC22A11), and URAT1(SLC22A12), in humans (Enomoto et al., 2002; Kusuhara and Sugiyama, 2002; Russel et al., 2002; van Aubel et al., 2002). BCR-P(ABCG2) is also expressed on the brush-border membranes of the proximal tubules in rodents (Merino et al., 2005), and Bcrp is involved in the urinary excretion of E3040 sulfate (Mizuno et al., 2004), although it has been reported that BCRP protein is below the limit of detection in the normal human kidney (Maliepaard et al., 2001). These are candidate transporters responsible for the luminal secretion of the conjugated metabolites of edaravone, and further studies are necessary to elucidate the efflux mechanisms of the conjugated metabolites.

In conclusion, our results suggest that both hOAT1 and hOAT3 are responsible for the basolateral uptake of edaravone sulfate in the kidney, but expression of hOAT1 does not enhance its cytotoxicity.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.106.013912.

ABBREVIATIONS: edaravone, MCI-186, 3-methyl-1-phenyl-2-pyrazolin-5-one; hOAT, human organic anion transporter; PAH, p-aminohippuric acid; PCG, benzylpenicillin; OAT, organic anion transporter(s); HEK, human embryonic kidney; RAF, relative activity factor.

Address correspondence to: Yuichi Sugiyama, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp

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