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Role of Organic Anion Transporters in the Pharmacokinetics of Zonampanel, an -Amino-3-hydroxy-5-methylisoxazole-4-propionate Receptor Antagonist, in Rats

Drug Metabolism Research Laboratories, Astellas Pharma Inc., Tokyo, Japan (T.M., T.H., T.U., H.K.); and Astellas Research Technologies Co., Ltd. (T.A.), Tokyo, Japan

(Received December 5, 2007; Accepted April 25, 2008)

	Abstract

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Zonampanel monohydrate ([2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl] acetic acid monohydrate, YM872) is a novel -amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist. In humans, almost all administered zonampanel is excreted in the urine unchanged. Furthermore, zonampanel is transported by human organic anion transporter (OAT) 1, and OAT3 but not by OAT2, suggesting the contribution of OATs to renal excretion. In rats also, zonampanel is predominantly eliminated via urine but partly also via bile as the unchanged form. In this study, the molecular mechanism of the excretion of zonampanel was elucidated using cells expressing rat Oat1, Oat2, and Oat3. Furthermore, zonampanel (15 mg/kg) was given i.v. to rats with or without probenecid (50 mg/kg) or cimetidine (40 mg/kg), and pharmacokinetic parameters were compared. Zonampanel inhibited the uptake of typical substrates by Oat1, Oat2, and Oat3 with inhibition constant (K_i) values of 7.02 to 10.4 µM. A time- and saturable concentration-dependent increase in [¹⁴C]zonampanel uptake was observed in these cells [Michaelis-Menten constant (K_m) values: 13.4 to 53.6 µM]. Probenecid and cimetidine inhibited [¹⁴C]zonampanel uptake by Oats. In in vivo experiments, probenecid and cimetidine decreased intrinsic clearance for both the renal secretion and biliary excretion of zonampanel. Considering the tissue distribution and localization of each transporter, these results suggest that in rats zonampanel is taken up from the blood into proximal tubular cells via Oat1 and Oat3 and, unlike the case in humans, also into hepatocytes via Oat2 and Oat3. The interspecies differences in the excretion of zonampanel between rats and humans may thus be explained by those in the substrate selectivity and tissue distribution of OATs.

View larger version (8K): [in this window] [in a new window]   FIG. 1. Chemical structure of zonampanel monohydrate. * position of the U-ring-14C radiolabel.

Although renal excretion is also the major elimination route of zonampanel in animal species, interspecies differences in the renal/nonrenal excretion ratio have been observed. After i.v. administration of [¹⁴C]zonampanel to humans, urinary excretion of radioactivity and unchanged zonampanel accounted for 94.9 and 90.6% of the dose, respectively, whereas fecal excretion of radioactivity was only 0.5% (Minematsu et al., 2005). After i.v. administration of [¹⁴C]zonampanel to rats, in contrast, approximately 30% of administered radioactivity was excreted in the feces, and no metabolite was found in the bile (unpublished data), indicating that approximately 30% of administered zonampanel is excreted unchanged via bile into the feces in rats. In addition, urinary excretions of radioactivity and unchanged zonampanel were 75.6 and 71.5% of the dose, respectively (Sohda et al., 2004). The molecular mechanisms of the excretion of zonampanel in rats as well as the reasons for the interspecies differences remain unknown.

Here, we investigated the interaction of zonampanel with rat Oats using cell lines stably expressing these transporters. Transport of zonampanel was also investigated using rat organic anion-transporting polypeptide (oatp) 1-, oatp2-, and oatp4-expressing Xenopus laevis oocytes and multidrug resistance associated protein 2 (Mrp2)-expressing membrane vesicles. We also evaluated the effects of organic anion transporter inhibitors on the pharmacokinetics of zonampanel in rats using the inhibitors probenecid and cimetidine (Burckhardt and Burckhardt, 2003). Whereas cimetidine is a well known inhibitor of organic cation transporters, it is also known as an Oat3 inhibitor (Burckhardt and Burckhardt, 2003).

	Materials and Methods

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Materials. Zonampanel monohydrate (molecular weight 349.26) and its internal standard, YM-53362, were synthesized at Astellas Pharma Inc. [¹⁴C]Zonampanel (specific radioactivity 3.50 MBq/mg; radiochemical purity 99.2%) was synthesized by GE Healthcare (Buckinghamshire, UK). [³H]p-Aminohippuric acid, [¹⁴C]salicylic acid, [³H]estrone-3-sulfate, [³H]estradiol-17β-D-glucuronide, and [³H]taurocholic acid (138.8, 1.74, 2120, 1735, and 185 GBq/mmol, respectively) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). All other chemicals and reagents used were commercially available and of guaranteed purity. Dose levels and concentrations of zonampanel were expressed as zonampanel monohydrate. Water-injected and rat oatp1-, oatp2-, and oatp4-expressing Xenopus laevis oocytes were purchased from BD Gentest (Woburn, MA). Membrane vesicles prepared from rat Mrp2-expressing and mock Sf9 insect cells were purchased from Genomembrane (Kanagawa, Japan).

Establishment of Transfectants and Cell Culture. Construction of stable transfectants expressing rat Oat1, Oat2, and Oat3 was performed as follows. The full coding region of rat Oat1, Oat2, and Oat3 was amplified from rat liver or kidney cDNA by reverse transcription-polymerase chain reaction following the reported sequences given by accession numbers NM_017224 [GenBank] , AB017446 [GenBank] , and NM_053537 [GenBank] , respectively. Full-length rat Oat1, Oat2, and Oat3 were subcloned into mammalian expression vector pcDNA3.1/zeo(+) (Invitrogen, Carlsbad, CA). The vector constructs of rat Oat1 and Oat3, as well as the empty vector, were introduced into parental HEK293 cells (rat Oat1-HEK293, rat Oat3-HEK293, and mock-HEK293, respectively) with Lipofectamine 2000 (Invitrogen) transfection reagent according to the manufacturer's protocol. Stably transfected cells were selected by adding Zeocin (Invitrogen) to the culture medium. Rat Oat1-HEK293, rat Oat3-HEK293, and mock-HEK293 were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin, 50 µg/ml streptomycin, and 100 µg/ml Zeocin at 37°C with 5% CO₂ under humidified conditions on the bottom of a dish. When the vector construct of rat Oat2 was introduced into parental HEK293 cells, no rat Oat2 function (the uptake of [¹⁴C]salicylic acid) was observed. Therefore, the vector construct of rat Oat2 and the empty vector were subsequently introduced into parental LLC-PK1 cells (rat Oat2-LLC-PK1 and mock-LLC-PK1, respectively) with Lipofectamine 2000 transfection reagent. Stably transfected cells were selected by adding Zeocin to the culture medium. Rat Oat2-LLC-PK1 and mock-LLC-PK1 were grown in medium 199 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 100 µg/ml Zeocin at 37°C with 5% CO₂ under humidified conditions on the bottom of a dish. The cells were seeded in poly-D-lysine-coated 12-well plates (BD Biosciences, Franklin Lakes, NJ) at a density of 2.0 x 10⁵ cells/well. Cell culture medium was replaced with culture medium supplemented with approximately 5 mM sodium butyrate 1 day before transport studies to induce the expression of proteins.

Transport Studies Using Rat Oat-Expressing Cells. Uptake was initiated by adding Dulbecco's phosphate-buffered saline (DPBS) (Sigma-Aldrich, St. Louis, MO) containing a radiolabeled ligand at a designated concentration after cells had been washed twice and preincubated with DPBS. The DPBS consisted of 137 mM NaCl, 2.68 mM KCl, 1.47 mM KH₂PO₄, 8.10 mM Na₂HPO₄, 0.904 mM CaCl₂, and 0.492 mM MgCl₂ (pH 7.4). Uptake was terminated at the designated time by the addition of ice-cold DPBS after removal of the incubation buffer. Cells were then washed with 1 ml of ice-cold DPBS. To determine the uptake of radiolabeled ligands, cells were dissolved in 500 µl of 1 M NaOH and, after cell lysis, were neutralized with 250 µl of 2 M HCl. Aliquots (600 µl) were transferred to vials, and the scintillation cocktail Hionic-Fluor (PerkinElmer Life and Analytical Sciences) was added. Radioactivity was measured using TriCarb 2900TR and 3100TR liquid scintillation counters (PerkinElmer Life and Analytical Sciences). Aliquots (40 µl) were used to determine protein concentrations by the method of Lowry et al. (1951) using a DC protein assay kit (Bio-Rad, Hercules, CA) with bovine serum albumin as a standard.

Transport Studies Using Rat Oatp1-, Oatp2-, and Oatp4-Expressing Oocytes. Upon receipt, purchased oocytes were kept at 16°C until use in the transport studies on the same day. The uptake experiment was performed at room temperature and initiated by pipetting oocytes into 2 ml of Na⁺ buffer containing a radiolabeled ligand at a designated concentration after oocytes had been washed with the buffer. The Na⁺ buffer consisted of 100 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, and 10 mM HEPES (pH 7.4). Uptake was terminated at the designated time by pipetting the oocytes into ice-cold Na⁺ buffer (2 ml). Oocytes were then washed by transferring them into 2 ml of fresh ice-cold Na⁺ buffer (repeated three times). To determine the uptake of radiolabeled ligands, each oocyte was transferred to a scintillation vial and dissolved using 10% (w/v) sodium dodecyl sulfate solution. For water-injected and transporter-expressing oocytes, 10 and 15 oocytes, respectively, were used. For the uptake of [¹⁴C]zonampanel, 5 oocytes were pooled to obtain a radioactivity level above the lower limit of detection (consequently, two and three samples of pooled oocytes for water-injected and transporter-expressing oocytes, respectively, were prepared). Subsequently, Hionic-Fluor scintillation cocktail was added to vials, and the radioactivity was measured using a TriCarb 3100TR liquid scintillation counter.

Vesicular Transport Assays Using Rat Mrp2-Expressing Membrane Vesicles. Aliquots (65 µl) of reaction mixture (50 mM MOPS, 21 mM Tris, 70 mM KCl, 12.1 mM MgCl₂, 2.3 mM glutathione, and either 4.62 mM ATP or AMP, pH 7.0) containing radiolabeled compound was prewarmed for 5 min at 37°C. Uptake was initiated by adding 10 µl of membrane vesicle solution (5 mg of protein/ml in 50 mM Tris-HCl, 50 mM mannitol, 2 mM EGTA, 8 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 mM dithiothreitol) to the reaction mixture. After incubation at 37°C for the designated time, the uptake was terminated by diluting the reaction mixture with 1 ml of ice-cold washing buffer (40 mM MOPS, 17 mM Tris, and 70 mM KCl, pH 7.0). Diluted samples were rapidly filtered through 0.7-µm-pore glass microfiber filter [GF/F (Whatman, Maidstone, UK), presoaked in saline containing 10% (w/v) bovine serum albumin], followed by two washings with 5 ml of ice-cold washing buffer. Subsequently, the radioactivity on the filter was measured by liquid scintillation counting using the TriCarb 3100TR, after addition of the liquid scintillation cocktail Filter-Count (PerkinElmer Life and Analytical Sciences) to the filter. The "sidedness" (inside-out/right side-out ratio) of the membrane vesicles was not determined (ATP-dependent uptake can only occur in inside-out vesicles).

Kinetic Analysis. Model fitting was performed by WinNonlin (Pharsight, Mountain View, CA) with use of the Gauss-Newton algorithms with Levenberg and Hartley modification. Kinetic parameters were obtained by simultaneous fitting of the data in transporter-expressing cells and mock cells with the weighting factor 1/y² to the following equations: V = V_max · [S]/(K_m + [S]) + P_dif · [S] for transporter-expressing cells and V = P_dif · [S] for mock cells, where V is uptake velocity of the substrate (picomoles per minute per milligram of protein), [S] is substrate concentration in the medium (micromolar concentration), K_m is the Michaelis-Menten constant (micromolar concentration), V_max is maximum uptake velocity (picomoles per minute per milligram of protein), and P_dif is uptake clearance of the nonsaturable component (microliters per minute per milligram of protein). Inhibition constant (K_i) values were calculated by simultaneous fitting of the data in transporter-expressing cells and mock cells without a weighting factor to the following equations, assuming competitive inhibition: CL_{uptake, +inh} = (CL_uptake - P_dif)/(1 + [I]/K_i) + P_dif for transporter-expressing cells and CL_{uptake, +inh} (= CL_uptake) = P_dif for mock cells, where CL_uptake is uptake clearance in the absence of inhibitor and CL_{uptake, +inh} is that in the presence of inhibitor and [I] is the inhibitor concentration. The substrate concentration was low compared with the K_m value in the inhibition study.

View larger version (10K): [in this window] [in a new window]   FIG. 2. Inhibitory effects of zonampanel on the rat Oat1-, Oat2-, and Oat3-mediated uptake of [3H]p-aminohippuric acid, [14C]salicylic acid, and [3H]estrone-3-sulfate, respectively. Rat Oat1-, Oat3-, and mock-HEK293 cells and rat Oat2- and mock-LLC-PK1 cells were incubated in DPBS containing designated concentrations of zonampanel in the presence of [3H]p-aminohippuric acid (1 µM) for Oat1, [14C]salicylic acid (5 µM) for Oat2, and [3H]estrone-3-sulfate (1 µM) for Oat3 at 37°C for 1, 2, and 1 min, respectively. Data are expressed as the mean ± S.D. of three samples. A broken line represents the best-fit line drawn using the equations described in the text.

Analytical Methods. Plasma zonampanel concentrations were determined using a validated high-performance liquid chromatography (HPLC) assay with UV detection (Noguchi et al., 2008). Briefly, 0.1-ml portions of internal standard solution (10 µg/ml YM-53362 solution in 0.01 M HCl) were added to 1 ml of plasma, followed by the addition of 0.1 M Tris-HCl buffer (pH 7). The mixture was vortexed for approximately 5 s and centrifuged at 830g for 5 min at 4°C, and the supernatant was applied to a Sep-Pak cartridge (Waters, Milford, MA) preconditioned using methanol and water. After the cartridge was washed twice using 2.5 ml each of 0.1 M Tris-HCl buffer (pH 7), zonampanel and internal standard were eluted using 75% methanol. After addition of 0.1 ml of 0.5 M HCl, the eluate was vortexed for approximately 5 s. Then, after addition of 6 ml of tri-n-butyl phosphate with 5% water, the sample was shaken for 10 min (200 strokes/min) and centrifuged at 830g for 5 min at 4°C, and the organic phase was aspirated off (performed twice). An aliquot (0.1 ml) of aqueous phase was injected into the HPLC system. Chromatographic separation was done with a TSKgel ODS-80Ts column (4.6 mm i.d. x 250 mm; Tosoh Corporation, Tokyo, Japan) and a TSKguardgel ODS-80Ts guard column (3.2 mm i.d. x 15 mm; Tosoh Corporation) at 35°C. The mobile phase consisted of 0.5 M phosphoric acid, acetonitrile, and water (100:20:880, v/v/v), pumped at a rate of 1.0 ml/min. The detection absorbance was set at 333 nm, and calibration ranged from 10 to 5000 ng/ml. Accuracy and precision at concentrations including the lower limit of quantification (LLOQ) were 0.78 to 5.36% and 0.70 to 4.51%, respectively. When the concentration of a sample was expected to exceed the upper limit of quantification, the sample was diluted using blank plasma. For the calculation of means ± S.D., measured concentrations below the LLOQ (10 ng/ml) were regarded as 0.00 ng/ml.

Urinary zonampanel concentrations were determined using a validated HPLC-UV method, which was modified from the method for plasma. Briefly, 0.1-ml portions of internal standard solution (10 µg/ml YM-53362 solution in 0.01 M HCl) were added to 0.2 ml of the urine sample diluted 100-fold with water. After mixing, 0.1 ml of aqueous phase was injected into the HPLC system under the same conditions as above. Calibration ranged from 10 to 5000 µg/ml. Accuracy and precision at concentrations including the LLOQ were –2.37 to –1.00% and 0.62 to 2.36%, respectively. The cumulative urinary excretion ratio of zonampanel (A_e, percentage of dose) was calculated using dose, urine volume, and urinary concentration.

Pharmacokinetic Analysis. Time profiles of the mean plasma concentration of zonampanel were applied to a noncompartmental model (linear trapezoidal method) using the WinNonlin program and the following parameters were calculated: estimated plasma concentration of zonampanel at time 0 (C₀), area under the time-concentration curve based on plasma concentration (AUC), half-life at the terminal phase (t_1/2), distribution volume at the steady state (V_ss), and total body clearance based on plasma concentration (CL_{tot, p}). Using calculated values of these parameters and the hypotheses and eqs. 3, 4, 5, 6, 7, 8 below, the following pharmacokinetic parameters were calculated: total body clearance based on blood concentration (CL_{tot, b}), renal clearance based on blood concentration (CL_{renal, b}), nonrenal clearance based on blood concentration (CL_{nonrenal, b}), renal secretion clearance based on blood concentration (CL_{renal, sr, b}), intrinsic renal secretion clearance (CL_{renal, sr, nt}), and overall intrinsic clearance of biliary excretion (CL_{bile, int, all}).

The hypothesis for renal clearance is as follows:

1. It was hypothesized that resorption is negligible because CL_{renal, b} is much larger than (f_p/R_b) · GFR.
   
2. It has been reported that the parallel tube model is more suitable for the description of renal tubular secretion than the well stirred model (Jankù, 1993; Jankù and Zvara, 1993). It was thus hypothesized that CL_{renal, sr, b} can be described according to the parallel tube model using the following equation:
   

(1)

where R_b is blood/plasma concentration ratio and Q_renal is renal blood flow.

The hypothesis for biliary excretion clearance is as follows:

1. Nonrenal clearance represents biliary clearance (CL_{bile, b}), because zonampanel is hardly metabolized (Sohda et al., 2004).
   
2. Nonrenal clearance can be described by the following equation (Shitara et al., 2005):
   

(2)

where Q_hepatic is hepatic blood flow.

Equations for parameters are as follows:

(3)

(4)

(5)

(6)

Equation 1 gives

(7)

Equation 2 gives

(8)

where A_{e 06 h} is urinary excretion of zonampanel from 0 to 6 h after dosing.

Values of R_b, f_p, GFR, Q_renal, and Q_hepatic were fixed at 0.64 for R_b (unpublished data), 0.125 for f_p (unpublished data), 11.4 ml/min/kg for GFR (Fischer et al., 2000), 40.71 ml/min/kg for Q_renal (Delp et al., 1991), and 55.2 ml/min/kg for Q_hepatic (Davies and Morris, 1993) for the calculation of parameters CL_{tot, b}, CL_{renal, b}, CL_{nonrenal, b}, CL_{renal, sr, b}, CL_{renal, sr, int}, and CL_{bile, int, all}. Two studies reported that probenecid does not affect GFR in rats (Darling and Morris, 1991; Foote and Halstenson, 1998), whereas, given that it has no effect on blood flow in the renal vein in dogs (Barza et al., 1975), probenecid is unlikely to change renal blood flow in rats. Cimetidine did not affect GFR or renal blood flow rate in rats (Rothwell et al., 1984; Foote and Halstenson, 1998). The change in the CL_{bile, int, all} should be carefully considered (the decrease in CL_{bile, int, all} may be underestimated), given that probenecid increases blood flow in the portal vein of dogs (Barza et al., 1975).

	Results

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Functional Verification of Cells Stably Expressing Rat Oat1, Oat2, and Oat3 Using Their Prototypical Substrates and Inhibitors. Time- and concentration-dependent uptakes of [³H]p-aminohippuric acid, [¹⁴C]salicylic acid, and [³H]estrone-3-sulfate were observed for rat Oat1, Oat2, and Oat3, respectively (data not shown). Respective K_m, V_max, and P_dif values were 32.5 ± 3.0 µM, 4320 ± 310 pmol/min/mg of protein, and 4.21 ± 0.88 µl/min/mg of protein for rat Oat1-mediated uptake of [³H]p-aminohippuric acid (1 min); 122 ± 23 µM, 2120 ± 370 pmol/min/mg of protein, and 3.86 ± 0.46 µl/min/mg of protein for rat Oat2-mediated uptake of [¹⁴C]salicylic acid (2 min); and 7.13 ± 0.92 µM, 642 ± 67 pmol/min/mg of protein, and 5.80 ± 0.96 µl/min/mg of protein for rat Oat3-mediated uptake of [³H]estrone-3-sulfate (1 min) (estimate ± S.E.). The K_i values of probenecid on rat Oat1-mediated uptake of [³H]p-aminohippuric acid (1 µM, 1 min), rat Oat2-mediated uptake of [¹⁴C]salicylic acid (5 µM, 2 min), and rat Oat3-mediated uptake of [³H]estrone-3-sulfate (1 µM, 1 min) were 6.16 ± 0.96, 372 ± 69, and 0.920 ± 0.053 µM, respectively, and those of cimetidine were >1000, >1000, and 33.7 ± 12.8 µM, respectively (estimate ± S.E.). In this study, approximately 25% of the rat Oat2-mediated uptake activity for [¹⁴C]salicylic acid remained in the presence of 1 mM probenecid, which was comparable with the results of the previous study (Morita et al., 2001). Values for the kinetic parameters K_m and K_i were comparable with those in the literature (Burckhardt and Burckhardt, 2003). Therefore, functional verification of cells stably expressing rat Oat1, Oat2, and Oat3 using their prototypical substrates and inhibitors was successfully performed.

Inhibitory Effects of Zonampanel on Rat Oat1, Oat2, and Oat3-Mediated Uptake of Their Prototypical Substrates. Zonampanel inhibited rat Oat1, Oat2 and Oat3-mediated uptake of [³H]p-aminohippuric acid, [¹⁴C]salicylic acid, and [³H]estrone-3-sulfate, respectively, in a concentration-dependent manner (Fig. 2; Table 1). Estimated K_i values were similar among rat Oats, ranging from 7.02 to 10.4 µM.

Zonampanel Uptake Mediated by Rat Oat1, Oat2, and Oat3. Uptake of [¹⁴C]zonampanel by the three tested transporters, rat Oat1, Oat2, and Oat3, was time- and concentration-dependent (Fig. 3). The Eadie-Hofstee plots indicated that there was one saturable component. Kinetic parameters are listed in Table 2. Estimated K_m values were similar among rat Oats, ranging from 13.4 to 53.6 µM, and relatively comparable with the K_i values (Tables 1 and 2).

View larger version (23K): [in this window] [in a new window]   FIG. 3. Time profiles and concentration dependence (Eadie-Hofstee plots) for the rat Oat1-, Oat2-, and Oat3-mediated uptake of [14C]zonampanel. Rat Oat1-, Oat3-, and mock-HEK293 cells and rat Oat2- and mock-LLC-PK1 cells were incubated in DPBS containing [14C]zonampanel (3 µM for time profiles; various concentrations for concentration dependence) at 37°C for the designated incubation time (time profiles) and for 5 min (concentration dependence). Data are expressed as the mean ± S.D. of three samples. A broken line represents the best-fit line drawn using the parameters in Table 2.

Inhibitory Effects of Probenecid and Cimetidine on Rat Oat1, Oat2, and Oat3-Mediated Uptake of Zonampanel. Probenecid inhibited rat Oat1- and Oat3-mediated uptake of [¹⁴C]zonampanel but had only a weak inhibitory effect on rat Oat2-mediated uptake (Fig. 4; Table 3). In contrast with the results for rat Oat2-mediated uptake of [¹⁴C]salicylic acid, more than 50% of the activity remained for Oat2-mediated transport of [¹⁴C]zonampanel in the presence of 1 mM probenecid (Fig. 4) (the concentration of zonampanel was 2 µM, which was less than its K_m value of 13.4 µM). This difference in the inhibitory effects of probenecid indicates that there may be substrate differences. Cimetidine inhibited rat Oat3-mediated uptake of [¹⁴C]zonampanel with a K_i value of 8.74 µM, but had only weak inhibitory effects on rat Oat1- and Oat2-mediated uptake (Fig. 4; Table 3)

View larger version (22K): [in this window] [in a new window]   FIG. 4. Inhibitory effects of probenecid and cimetidine on the rat Oat1-, Oat2-, and Oat3-mediated uptake of [14C]zonampanel. Rat Oat1-, Oat3-, and mock-HEK293 cells and rat Oat2- and mock-LLC-PK1 cells were incubated in DPBS containing [14C]zonampanel (1 µM for Oat1, 2 µM for Oat2, and 5 µM for Oat3) in the presence or absence of designated concentrations of probenecid or cimetidine at 37°C for 5 min. Data are expressed as the mean ± S.D. of three samples. A broken line represents the best-fit line drawn using the equations described in the text.

Zonampanel Uptake Mediated by Rat Oatp1, Oatp2, Oatp4, and Mrp2. Marked uptake of [¹⁴C]zonampanel (10 µM) by rat oatp1, oatp2, oatp4, and Mrp2 (Fig. 5, B and C) was not observed, but marked transporter-mediated transport was observed for the prototypical substrates of each transporter ([³H]estrone-3-sulfate for oatp1 and oatp4, [³H]taurocholic acid for oatp2, and [³H]estradiol-17β-D-glucuronide for Mrp2) (Fig. 5, A and D). In addition, zonampanel (1 mM) did not affect the Mrp2-mediated transport of [³H]estradiol-17β-D-glucuronide. The uptake of [¹⁴C]zonampanel into oatp4-expressing oocytes was slightly higher (approximately 1.8-fold) than that into water-injected oocytes.

View larger version (22K): [in this window] [in a new window]   FIG. 5. A, uptake of prototypical substrates (2 µM; [3H]estrone-3-sulfate for rat oatp1 and oatp4 and [3H]taurocholic acid for rat oatp2) into water-injected oocytes and rat oatp1-, oatp2-, and oatp4-expressing oocytes for 60 min. Data are expressed as the mean ± S.D. of 10 or 15 oocytes. B, uptake of [14C]zonampanel (10 µM) into water-injected oocytes and rat oatp1, oatp2, and oatp4-expressing oocytes for 60 and 120 min. Data are expressed as the mean (a) or the mean ± S.D. of two or three samples of pooled oocytes, respectively. C, time courses of the uptake of [14C]zonampanel (10 µM) into membrane vesicles prepared from mock- and rat Mrp2-expressing Sf-9 insect cells in the presence and absence of ATP. Data are expressed as the mean of two samples. D, Effects of zonampanel (1 mM) on the 5-min uptake of [3H]estradiol-17β-D-glucuronide (2.2 µM) into membrane vesicles prepared from rat Mrp2-expressing Sf-9 insect cells in the presence and absence of ATP. Data are expressed as the mean of two samples.

View larger version (22K): [in this window] [in a new window]   FIG. 6. Time profiles of plasma zonampanel concentrations after i.v. bolus administration of zonampanel (15 mg/kg) with or without probenecid (50 mg/kg) or cimetidine (40 mg/kg) to rats. Data are expressed as the mean ± S.D. of four rats.

	Discussion

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Although zonampanel is metabolized only slightly in both rats and humans (Sohda et al., 2004; Minematsu et al., 2005), fecal excretion of [¹⁴C]zonampanel is much greater in rats (30% of dose) (unpublished data) than in humans (0.5% of dose) (Minematsu et al., 2005). Here, we investigated the molecular mechanisms of zonampanel excretion in rats and the reason for the interspecies differences in excretion between rats and humans using cell lines stably expressing rat Oat1, Oat2, and Oat3. In addition, we also tested the effects of the organic anion transporter inhibitors probenecid and cimetidine on the pharmacokinetics of zonampanel in rats. Species differences in the substrate selectivity and distribution/localization of OATs well explained the difference in the excretion.

Zonampanel is a substrate of rat Oat1, Oat2, and Oat3 (Fig. 3) and human OAT1 and OAT3 but is not transported by human OAT2 (Hashimoto et al., 2004). Expression levels of OAT1 mRNA in the liver are much lower than those in the kidney in both rats and humans (Race et al., 1999; Buist et al., 2002). In both species, OAT2 is expressed on the basolateral membranes of the hepatocytes (Shitara et al., 2005). In contrast, although the mRNA of OAT3 is expressed in rat liver (Kusuhara et al., 1999; Buist et al., 2002), it is not detected in human liver (Race et al., 1999). In rats, OAT2 and OAT3 may accelerate the uptake of zonampanel from the blood into the liver, resulting in greater fecal excretion via bile than in humans. Regarding renal excretion, a recent review article described the expression of OAT1 and OAT3 on the basolateral membranes of rat and human proximal renal tubules (Shitara et al., 2005). It is therefore likely that zonampanel is rapidly secreted from blood into the urine via OAT1 and OAT3 in both rats and humans. OAT2 is localized in different regions of the kidney in rats and humans. For rats, it is found on the apical membrane of the tubules in the medullary thick ascending limb of Henle's loop and collecting ducts, whereas for humans, it is found on the basolateral membrane of the tubule (Kojima et al., 2002; Shitara et al., 2005). Therefore, rat Oat2 may mediate zonampanel reabsorption rather than its secretion into the urine.

In this study, after i.v. administration of zonampanel (15 mg/kg) without probenecid or cimetidine to rats, the plasma concentration of zonampanel was approximately 30 µg/ml (86 µM) at the first sampling time (5 min), which rapidly decreased to around 0.1 µg/ml within 120 min. Because the unbound fraction of zonampanel in rat plasma was 0.125, when the total plasma concentration of zonampanel is 30 µg/ml, the unbound plasma concentration is 11 µM, which is lower than the K_m values of zonampanel for rat Oat1, Oat2, and Oat3. Similar plasma concentrations were obtained in a preliminary study (unpublished results). In a clinical study, using intravenous constant infusion, the plasma concentration of zonampanel reached 2.93 µg/ml (8.4 µM with 0.84 µM as unbound) as we reported previously (Minematsu et al., 2005). Intravenous bolus administration of 15 mg/kg zonampanel to rats can result in this concentration. Therefore, the dose of zonampanel was chosen as 15 mg/kg. Whereas C₀ ranged from 57.8 to 102 µg/ml (165–291 µM) (Table 4), the unbound concentrations were approximately 21 to 36 µM, which are still around the K_m values, suggesting only slight saturation for kinetics just after administration. In this study, zonampanel was excreted unchanged in the urine, accounting for 76.2 to 79.3% of the dose within 6 h in rats (Table 4). In addition, the renal clearance of zonampanel was higher than the product of f_p and GFR, indicating that tubular secretion plays an important role in the renal excretion of this drug.

In combination with probenecid, renal clearance and intrinsic renal secretion clearance decreased to 33.8 and 17.4%, respectively, compared with values for the control group (Table 4), suggesting a transporter inhibition-induced decrease in tubular secretion. Nonrenal and intrinsic nonrenal clearance of zonampanel in the probenecid-coadministered group also decreased to 36.9 and 32.9%, respectively, of that in the control (Table 4). Probenecid has been reported to inhibit the transporter-mediated biliary excretion of 6-nitro-7-sulfamoyl-benzo(f)quinoxaline-2,3-dione, which has a chemical structure similar to that of zonampanel, in rats (Hansen, 1995). On this basis, probenecid was considered to inhibit transporter-mediated biliary excretion (in this case the uptake into the hepatocytes) of zonampanel. In a rat study, radioactivity after i.v. administration of [¹⁴C]zonampanel was distributed mainly in the kidney and liver (unpublished data). The reduced V_ss (Table 4) agrees with decreased cellular uptake due to the inhibition of transporters by probenecid in the basolateral membrane of the proximal renal tubule and of hepatocytes. When probenecid was given to rats at 50 mg/kg, unbound plasma concentrations were approximately 100 µg/ml (350 µM) at 2.5 min, >10 µg/ml (35 µM) at 1 h, and >2 µg/ml (7 µM) at 2 h after dosing (Emanuelsson and Paalzow, 1988), which were higher than the K_i values for probenecid on [¹⁴C]zonampanel uptake by rat Oat1 and Oat3 (1.44 and 1.13 µM, respectively) (Table 3). Considering the K_i value for the effect of probenecid on the rat Oat2-mediated zonampanel transport (>1000 µM), it is unlikely that probenecid inhibited Oat2 in the in vivo experiments. The lack of complete inhibition of the renal and nonrenal clearance of zonampanel could also be due to lack of Oat2 inhibition, slight saturation for kinetics just after administration as described above, and/or involvement of the other transporters.

Similar to the case with probenecid, the renal and intrinsic renal secretion clearances of zonampanel in the cimetidine-coadministered group were decreased to 64.9 and 49.6%, respectively, of those in the control group (Table 4), suggesting inhibition of Oat3. The nonrenal and intrinsic nonrenal clearances of zonampanel in the cimetidine-coadministered group were also decreased to 57.2 and 53.5%, respectively, of those in the control group (Table 4), also suggesting the inhibition of Oat3. Furthermore, the V_ss of zonampanel also decreased in the cimetidine-coadministered group (Table 4). When cimetidine was given to rats at 40 mg/kg, the plasma cimetidine concentration was approximately 20 µg/ml (80 µM) at approximately 5–10 min, 10 µg/ml (40 µM) at 0.5 h, >3 µg/ml (12 µM) at 1 h, and >1 µg/ml (4.0 µM) at 2 h after administration, and f_p was 0.78 to 0.83 (5–50 µg/ml) in rats (Adedoyin et al., 1987), suggesting an unbound plasma cimetidine concentration of approximately 64 µM at approximately 5–10 min, 32 µM at 0.5 h, >9.5 µM at 1 h, and >3.2 µM at 2 h (K_i values for cimetidine on [¹⁴C]zonampanel uptake by rat Oat3, 8.74 µM) (Table 3). With consideration of the K_i values for cimetidine on the rat Oat1- and Oat2-mediated zonampanel transport (>1000 µM), it was unlikely that cimetidine inhibited Oat1 and Oat2 in the in vivo experiments. Although cimetidine did not completely inhibit renal and nonrenal clearance of zonampanel, this may be due to lack of Oat1/Oat2 inhibition, slight saturation for kinetics just after administration as described above, and/or involvement of the other transporters.

In rats, organic anion transporters other than Oats, such as oatp1, oatp2, and oatp4 are localized on the basolateral membrane of hepatocytes: oatp1 is on the apical membrane of the renal tubules, and Mrp2 is on the apical membranes of the hepatocytes and renal tubules (Shitara et al., 2005). The importance of these transporters in the pharmacokinetics of zonampanel may be low, but oatp4 might be partly involved in the uptake of zonampanel into hepatocytes (Fig. 5) (any statistical significance in the difference between the uptake into water-injected and oatp4-expressing oocytes could not be tested). A future detailed study may reveal the involvement of oatp4 in a more quantitative manner. Because zonampanel is not a good substrate of rat Mrp2, we could not find the transporters involved in the secretion across the apical membrane of hepatocytes and renal tubules. For the accumulation of zonampanel in the liver and kidney, the tissue/plasma radioactivity concentration ratio was approximately 1.6 for the liver and 7 for the kidney after the i.v. administration of [¹⁴C]zonampanel to rats (unpublished data) (radioactivity represents the unchanged zonampanel, because zonampanel is metabolized only slightly as described above). Therefore, the uptake process on the basolateral membrane is probably dominant to the efflux process on the apical membrane.

In conclusion, zonampanel was transported by rat Oat1, Oat2, and Oat3 in a concentration-dependent manner with K_m values of 13.4, 13.4, and 53.6 µM, respectively. The Oats inhibitor probenecid and Oat3 inhibitor cimetidine decreased the intrinsic clearance for renal secretion and biliary excretion of zonampanel in rats, as calculated using a physiological model. Considering the tissue distribution and localization of each transporter, these results suggest that, in rats, zonampanel is taken up via Oat1 and Oat3 from the blood into proximal tubular cells and, unlike in humans, via Oat2 and Oat3 from the blood into hepatocytes. The interspecies differences in the excretion of zonampanel between rats and humans may be explained by these differences in the substrate selectivity and tissue distribution/localization of OATs. The approaches considering interspecies-differences in transporters using in vitro transporter-expressing systems and in vivo chemical inhibition experiments would be useful in drug development, especially in cases of extrapolation from animals to humans. It should be kept in mind that localization of the transporters requires careful attention when interspecies differences are discussed.

	Acknowledgments

 
We thank Dr. Ken-ichi Umehara for his technical instruction during the molecular biology experiments.

	Footnotes

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

doi:10.1124/dmd.107.019828.

ABBREVIATIONS: YM872, zonampanel monohydrate, [2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl] acetic acid monohydrate; GFR, glomerular filtration rate; OAT/Oat, organic anion transporter; oatp, organic anion-transporting polypeptide; Mrp, multidrug resistance-associated protein; YM-53362, 3-[7-(1H-imidazol-1-yl)-6-nitro-2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-1-yl] propionic acid monohydrochloride 0.7 hydrate; DPBS, Dulbecco's phosphate-buffered saline; MOPS, 3-[N-morpholino]propanesulfonic acid; HPLC, high-performance liquid chromatography; LLOQ, lower limit of quantification.

Address correspondence to: Dr. Tsuyoshi Minematsu, Drug Metabolism Research Laboratories, Astellas Pharma Inc., 1-8, Azusawa 1-chome, Itabashiku, Tokyo 174-8511, Japan. E-mail: tsuyoshi.minematsu{at}jp.astellas.com

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