Advertisement
Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Involvement of Rat and Human Organic Anion Transporter 3 in the Renal Tubular Secretion of Topotecan [(S)-9-Dimethylaminomethyl-10-hydroxy-camptothecin hydrochloride]

Shin-ichi Matsumoto, Kenji Yoshida, Naoki Ishiguro, Tomoji Maeda and Ikumi Tamai
Journal of Pharmacology and Experimental Therapeutics September 2007, 322 (3) 1246-1252; DOI: https://doi.org/10.1124/jpet.107.123323

Abstract

Topotecan [(S)-9-dimethylaminomethyl-10-hydroxy-camptothecin hydrochloride] is primarily excreted into urine in humans, with approximately 49% of the dose recovered as total topotecan (topotecan lactone plus topotecan hydroxyl acid form). The renal elimination of topotecan involves tubular secretion in addition to glomerular filtration, but little is known about the molecular mechanism of the renal tubular secretion. In the present study, we investigated the transport characteristics of topotecan hydroxyl acid across the renal basolateral membrane using rat kidney slices and rat or human transporter-expressing Xenopus laevis oocytes. Pravastatin and probenecid significantly inhibited the uptake of topotecan hydroxyl acid by rat kidney slices with Ki values of 10.6 and 8.1 μM, respectively, and p-aminohippurate was weakly inhibitory at high concentrations, whereas excess tetraethylammonium had no effect. The uptake of topotecan hydroxyl acid by oocytes injected with complementary RNA of either rat or human organic anion transporter 3 (rOAT3 or hOAT3) was greater than that of water-injected oocytes. Kinetic analysis showed that the Km values for rOAT3 and hOAT3 were 21.9 and 56.5 μM, respectively. Neither rOAT1 nor hOAT1 stimulated topotecan hydroxyl acid transport. These results suggest that the urinary excretion of topotecan hydroxyl acid is accounted for by transport via OAT3, as well as glomerular filtration, in both rats and humans; therefore, drug-drug interactions involving OAT3 may cause a change in clearance of topotecan.

Topotecan, a water-soluble analog of camptothecin, inhibits DNA replication and RNA transcription by stabilizing the cleavable complexes formed between topoisomerase I and DNA (Hsiang et al., 1989). It has antitumor activity in several tumor types, including ovarian cancer, small cell and nonsmall cell lung cancer, lymphoma, leukemia, and pediatric tumors (Burris, 1999; Kollmannsberger et al., 1999; Arun and Frenkel, 2001). As illustrated in Fig. 1, topotecan has a lactone moiety and is reversibly hydrolyzed from the active, lactone form to a less potent, hydroxyl acid form in a pH-dependent reaction; at physiologic pH, the equilibrium favors the hydroxyl acid form (Underberg et al., 1990).

Topotecan undergoes both renal and hepatic elimination (Stewart et al., 1994; Furman et al., 1996). It is primarily excreted in urine with both topotecan lactone and the hydroxyl acid form, accounting for approximately 49% of the administered i.v. dose in cancer patients (Herben et al., 2002). In a study of adults with normal (creatinine clearance, 64–171 ml/min) and impaired renal function (creatinine clearance, 18–59 ml/min), renal clearance of topotecan was decreased in patients with renal dysfunction (O'Reilly et al., 1996a). On the other hand, adults with liver dysfunction (serum total bilirubin, 1.2–14.9 mg/dl) showed only a little change of topotecan disposition (O'Reilly et al., 1996b). These data suggest that renal clearance is a primary elimination pathway for topotecan.

A population pharmacokinetic analysis based on the pooled data from clinical trials showed that the estimated renal clearance of topotecan was larger than the glomerular filtration rate, indicating that topotecan undergoes tubular secretion (Mould et al., 2002). In mice, probenecid, a typical inhibitor of the organic anion transporter family (Tahara et al., 2005), inhibited the tubular secretion of topotecan hydroxyl acid and, apparently, increased the systemic exposure to topotecan lactone (Zamboni et al., 1998). Therefore, organic anion transporter (OAT) is probably responsible for the tubular secretion of topotecan hydroxyl acid. However, the exact mechanism of renal elimination of topotecan has not been identified. Because patients with high topotecan systemic exposure have an increased probability of severe neutropenia (Mould et al., 2002), which is the dose-limiting toxicity of topotecan (Dennis et al., 1997), it is important to elucidate the mechanisms of renal clearance of topotecan. Accordingly, we investigated transporter(s) involved in the renal uptake of topotecan hydroxyl acid.

Fig. 1.
Fig. 1.

Chemical structures of topotecan lactone form and hydroxyl acid form.

To date, rOAT1, rOAT2, and rOAT3 have been identified in the basolateral membranes of rat kidney (Sekine et al., 1997, 1998; Kusuhara et al., 1999). These studies demonstrated that rOAT1 and rOAT3 are mainly expressed in the kidney (Sekine et al., 1997; Kusuhara et al., 1999), whereas rOAT2 is predominantly expressed in the liver and only weakly in the kidney (Sekine et al., 1998). Functional characterization revealed that rOAT1 and rOAT3 selectively transport p-aminohippurate (PAH) and pravastatin, an HMG-CoA reductase inhibitor, respectively (Hasegawa et al., 2002). Therefore, these substrates can be useful as relatively specific inhibitors of rOAT1 and rOAT3, respectively. Human (h) OAT1 and hOAT3 and their orthologs in the rat (rOAT1 and rOAT3) are predominantly expressed in the kidney and localized at the basolateral membranes of renal proximal tubules (Hosoyamada et al., 1999; Cha et al., 2001). Moreover, it has been reported that the mRNA levels of hOAT1 and hOAT3 were much higher than those of other organic ion transporters in the human kidney cortex (Motohashi et al., 2002). Thus, hOAT1 and hOAT3 are considered to play important roles in the tubular secretion of organic anions, including various clinically used drugs, as well as endogenous compounds, from the circulation. In the present study, to elucidate the mechanisms of renal clearance of topotecan, we attempted to characterize the transport of topotecan hydroxyl acid, using rat kidney slices and rat and human transporter-expressing Xenopus laevis oocytes.

Materials and Methods

Materials. Topotecan was provided by GlaxoSmithKline (Collegeville, PA). [14C]Inulin (2.5 mCi/g) and 1-methyl-4-phenylpyridinium (80 Ci/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO). PAH and probenecid were purchased from Sigma-Aldrich (St. Louis, MO). Tetraethylammonium (TEA) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Pravastatin was kindly donated by Sankyo Co. (Tokyo, Japan). All other chemicals used in the present study were of the highest purity available.

cDNA Cloning of Transporters. The rOAT1 gene was PCR-amplified using a Marathon-ready rat kidney cDNA library (Clontech Laboratory Inc., San Francisco, CA) as a template, with upstream primer 5′-ACTGGAGGTCCTCAGTCATTGACC-3′ and downstream primer 5′-GAAGCCCTTCCGTTCTCAAAGTCC-3′ (both synthesized by Hokkaido System Science, Sapporo, Japan), based on the reported rOAT1 gene sequence (Sekine et al., 1997) (accession no. AB004559). A major 1.7-kb polymerase chain reaction product was ligated into the pCR Blunt II TOPO cloning vector (Invitrogen, Carlsbad, CA), and then rOAT1-cDNA was digested with HindIII and XbaI and ligated into the gene expression vector pcDNA3.1 (Invitrogen). The rOAT3 gene was PCR-amplified using the Marathon-ready rat kidney cDNA library as a template, with upstream primer 5′-GGTTCATCTTGCCTGGTGCCATGAC-3′ and downstream primer 5′-GGATCAGTCTCTTGTGGCCAGGAAAGAG-3′, based on the reported rOAT3 gene sequence (Kusuhara et al., 1999) (accession no. AB017446). A major 1.7-kb polymerase chain reaction product was ligated into pCR Blunt II TOPO cloning vector (Invitrogen), and then rOAT3-cDNA was digested with BamHI and XbaI and ligated into expression plasmid vector pGEMHE, which was kindly provided by Dr. Liman (Liman et al., 1992). The hOAT1 gene was PCR-amplified using the Marathon-ready human kidney cDNA library (Clontech Laboratory Inc.) as a template, with upstream primer 5′-CAATGGCCTTTAATGACCTC-3′ and downstream primer 5′-GTCCTCAGAGTCCATTC-3′, based on the reported hOAT1 gene sequence (Hosoyamada et al., 1999) (accession no. NM00479). A major 1.8-kb polymerase chain reaction product was ligated into pCR 2.1 TOPO cloning vector (Invitrogen), and the hOAT1-cDNA was digested with EcoRI and ligated into pcDNA3.1. The hOAT3 gene was PCR-amplified using the Cap Site cDNA dT human kidney (Nippon Gene, Tokyo, Japan) as a template, with upstream primer 5′-CACCAGCCCCATCGGATCCA-3′ and downstream primer 5′-TCACCAAGCTCTCAGAAGGCTTCA-3′, based on the reported hOAT3 gene sequence (Cha et al., 2001) (accession no. AB042505). A major 1.8-kb polymerase chain reaction product was ligated into the TA cloning vector pGEM-T Easy (Promega, Madison, WI), and then hOAT3-cDNA was digested with EcoRI and ligated into pGEMHE.

Uptake of Topotecan Hydroxyl Acid by Rat Kidney Slices. All animal care and experimentation were conducted according to the guidelines of the Tokyo University of Science. Male Wistar rats (7 or 8 weeks; Sankyo Co.) were housed three per cage with free access to commercial chow and tap water and maintained on a 12-h dark/light cycle (8:00 AM–8:00 PM light) in an air-controlled room (temperature, 24.5 ± 1°C; humidity, 55 ± 5%). Uptake studies using kidney slices were carried out as described in a previous report (Ishiguro et al., 2005). Kidney slices (0.3 mm in thickness) from rats were put in ice-cold oxygenated incubation buffer. The incubation buffer consists of 120 mM NaCl, 16.2 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, and 10 mM NaH2PO4/Na2HPO4, adjusted to pH 7.5. Three slices, weighing 6 to 20 mg, were randomly selected and then preincubated in a 12-well plate with 1 ml of oxygenated incubation buffer in each well at 37°C for 5 min. [14C]Inulin was used to estimate the amount of water adhering to the kidney slices in each experiment. Topotecan was dissolved in 50 mM phosphate buffer, pH 9.0, and left overnight at 100 μM in a refrigerator to prepare topotecan hydroxyl acid solution. The conversion ratio of topotecan to its hydroxyl acid form was approximately 97% at pH 9.0. We also confirmed that the hydroxyl acid form was stable in the buffer, pH 9.0, for 17 h when stored at 4°C. To minimize the conversion of topotecan hydroxyl acid to its lactone form, the stock solutions were diluted with uptake medium, pH 7.5, to the appropriate concentration just before uptake studies. The extent of the lactone form to total topotecan (the lactone plus the hydroxyl acid form) was 3.3% for the initial, 11% after a 30-min incubation, and 25% after a 60-min incubation in uptake medium, pH 7.5, at 37°C, suggesting that the initial uptake rates (15 min in kidney slice experiments and 30 min in X. laevis oocyte experiments) in the present study mainly reflect the transport of topotecan hydroxyl acid form. The uptake studies were performed at 37°C for an appropriate time, and then each slice was rapidly removed from the incubation buffer, washed in ice-cold saline, blotted on filter paper, and weighed. To measure the uptake of topotecan hydroxyl acid, the slices were lysed in 0.25 ml of 0.5 N NaOH solution with sonication and neutralized by adding the same volume of 0.5 N HCl. Then, 50 μl of methanol/distilled water/perchloric acid (5:4:1) was added to a 50-μl aliquot of the sample and mixed for 10 s. The samples were transferred to a MultiScreen Solvinert (pore size, 0.45 μm; PTFE filter; Millipore Corp., Bedford, MA) equipped with a 96-well collection plate. Then, the samples were centrifuged (900g, 5 min, 4°C) to obtain the filtrates in the 96-well collection plate. Each filtrate was analyzed by HPLC. When a radiolabel was involved, the slices were dissolved in 100 μl of 1 N NaOH, and radioactivity was determined in a liquid scintillation counter (LSC-5100; Aloka, Tokyo, Japan) after addition of a liquid scintillation cocktail, Clearsol-1 (Nacalai Tesque, Kyoto, Japan). Uptake was expressed as the tissue/medium ratio (microliters per gram of kidney) obtained by dividing the uptake amount by the initial concentration of substrate in the incubation buffer. The apparent inhibition constant, Ki value, was calculated from inhibition plots based on the following equation: Math where S/M represents the slice/medium ratio, the subscript (+I) represents the value in the presence of inhibitor, and I is the concentration of inhibitor (micromolar). The Ki value was estimated by the nonlinear least-squares method using KaleidaGraph (Synergy Software, Reading, PA).

Uptake of Topotecan Hydroxyl Acid byX. laevisOocytes. Uptake studies using X. laevis oocytes were performed as described previously (Tamai et al., 2000; Ishiguro et al., 2005; Nozawa et al., 2005). cRNAs of OATs and organic cation transporters (OCTs) were prepared by in vitro transcription with T7 RNA polymerase in the presence of ribonuclease inhibitor and an RNA cap analog using a mMessage mMACHINE kit (Ambion, Austin, TX). For transport experiments, defolliculated oocytes were injected with 25 ng of each transporter's cRNA or the same volume of water and incubated in modified Barth's solution [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, and 10 mM HEPES, pH 7.4] containing 50 μg/ml gentamicin at 18°C for 3 days. The oocytes were transferred to a 24-well plate and preincubated in ND96 buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.4) at room temperature for at least 15 min. After preincubation, the buffer was replaced with ND96 buffer containing topotecan hydroxyl acid to initiate the uptake reaction at room temperature for the designated time. Uptake was terminated by washing three times with ice-cold ND96 buffer. To determine the uptake of topotecan hydroxyl acid, the cells were sonicated in 0.2 ml of 0.1 M HCl/methanol (1:1) and then centrifuged at 12,000g for 5 min. The supernatant was diluted 2-fold with 50 mM phosphoric acid and subjected to HPLC. For radioactive compounds, the oocytes were solubilized in 5% sodium dodecyl sulfate solution, and then the radioactivity in aliquots was determined by liquid scintillation counting. Uptake was represented as the oocyte/medium ratio (microliters per oocyte) obtained by dividing the uptake amount by the initial concentration of substrate in the incubation buffer. To estimate kinetic parameters for saturable uptake, the initial uptake rate was fitted to the following equation by nonlinear least-squares regression analysis using KaleidaGraph: Math where v and s are the initial uptake rate and concentration of substrate, respectively, and Km and Vmax represent the Michaelis-Menten constant (micromolar) and the maximal uptake rate (picomoles per 30 min per oocyte), respectively.

HPLC Analysis. Sample preparation was performed under acidic conditions as described above for the determination of topotecan lactone form. The chromatographic system consisted of a Waters 2795 Alliance separation module (Waters, Milford, MA) equipped with a Waters 2475 Multi λ fluorescence detector. Topotecan was separated on a Capcell pak C18 MGII analytical column (4.6 × 100 mm, 5 μm; Shiseido, Tokyo, Japan). The mobile phase was 75 mM phosphate buffer, pH 6.0/methanol (7/3, v/v), and was used at the flow rate of 1.0 ml/min. The column was kept at 50°C, and the detection was performed at excitation and emission wavelengths of 382 and 524 nm. Topotecan was quantitated using absolute calibration method. The standard curves for substrate concentrations in rat kidney slices and X. laevis oocytes were linear over the range of 0.04 to 40 nM and 0.05 to 5 nM, respectively. The interassay precision and accuracy of these methods for topotecan using rat kidney slices were less than 5% and from –4.5 to 2.5%, respectively, and those using oocytes were within 3% and from –1.9 to 1.3%, respectively.

Statistical Analysis. The significance of differences was determined by one-way analysis of variance followed by nonpaired Student's t test. Differences were considered to be significant at p < 0.05.

Results

Uptake of Topotecan Hydroxyl Acid by Rat Kidney Slices.Figure 2 shows the time course of the uptake of topotecan hydroxyl acid by rat kidney slices. The uptake of topotecan hydroxyl acid increased linearly over 15 min. The initial uptake rate was assessed as the uptake at 15 min in the following inhibition study.

Inhibitory Effect of Various Compounds on the Uptake of Topotecan Hydroxyl Acid by Rat Kidney Slices. To confirm the involvement of transporter(s) in the renal uptake of topotecan hydroxyl acid at the tissue level, we examined the inhibitory effects of several typical inhibitors, including PAH and pravastatin, which are selective substrates of rOAT1 (Hasegawa et al., 2002) and rOAT3 (Hasegawa et al., 2002), respectively, probenecid, a general inhibitor of organic anion transporter family (Tahara et al., 2005), and TEA, a substrate of OCTs (Arndt et al., 2001; Ishiguro et al., 2005) (Fig. 3). With the exception of TEA, all of the compounds examined had a significant effect on the uptake of topotecan hydroxyl acid. Pravastatin and probenecid significantly inhibited the uptake of topotecan hydroxyl acid by rat kidney slices in a concentration-dependent manner, with Ki values of 10.6 and 8.1 μM, respectively. On the other hand, PAH was weakly inhibitory at high concentrations. These data suggested that topotecan hydroxyl acid is transported by rOATs but not by rOCTs.

Fig. 2.
Fig. 2.

Time course of topotecan hydroxyl acid uptake by rat kidney slices. Rat kidney slices were incubated at 37°C with 50 nM topotecan hydroxyl acid, pH 7.5, over 60 min. The apparent uptake of [14C]inulin was evaluated to estimate the volume of water adhering to the kidney slices during incubation. The mean value of representative adherent water volume was used to correct the apparent uptake of topotecan hydroxyl acid (0.121 ± 0.004 ml/15 min/g kidney). Each point represents the mean ± S.E. for three kidney slices.

Fig. 3.
Fig. 3.

Inhibitory effect of various compounds on topotecan hydroxyl acid uptake by rat kidney slices. Rat kidney slices were incubated for 15 min at 37°C with 50 nM topotecan hydroxyl acid, pH 7.5, in the presence of pravastatin (A), probenecid (B), PAH (C), or TEA (D). The solid lines represent the fitted line obtained by nonlinear regression analysis. Each point represents the mean ± S.E. for three kidney slices.

Uptake of Topotecan Hydroxyl Acid by rOAT1- and rOAT3-ExpressingX. laevisOocytes. The time course of topotecan hydroxyl acid uptake by X. laevis oocytes injected with rOAT3 cRNA or water is shown in Fig. 4A. The uptake of topotecan hydroxyl acid was markedly stimulated in rOAT3-expressing oocytes. In contrast, the uptake of topotecan hydroxyl acid at 60 min by rOAT1-expressing oocytes was comparable with that by water-injected oocytes (Fig. 4B). In addition, the uptake of topotecan hydroxyl acid by both rOCT1- and rOCT2-expressing oocytes was comparable with that by water-injected oocytes. It was confirmed that rOCT1 and rOCT2 significantly transported 1-methyl-4-phenylpyridinium as a positive control for OCT-mediated transport (data not shown). As shown in Fig. 5, the rOAT3-mediated uptake was saturable, and kinetic analysis revealed that the Km and Vmax values of topotecan hydroxyl acid transport by rOAT3 were 21.9 ± 4.08 μM and 0.666 ± 0.044 pmol/30 min/oocyte, respectively.

Fig. 4.
Fig. 4.

Uptake of topotecan hydroxyl acid by X. laevis oocytes expressing rOAT1 or rOAT3. A, oocytes injected with rOAT3 (closed circle) or water (open circle) were incubated for the specified periods at room temperature with 5 μM topotecan hydroxyl acid. Each point represents the mean ± S.E. from 11 to 14 oocytes. *, significant difference from the uptake of water-injected oocytes (p < 0.05). B, oocytes injected with rOAT1 (shaded column) or water (open column) were incubated for 60 min at room temperature with 5 μM topotecan hydroxyl acid. Each column represents the mean ± S.E. from 11 to 12 oocytes.

Fig. 5.
Fig. 5.

Concentration dependence of topotecan hydroxyl acid uptake by X. laevis oocytes expressing rOAT3. X. laevis oocytes injected with rOAT3 cRNA (closed circle) or water (open circle) were incubated at room temperature for 30 min with topotecan hydroxyl acid. Broken and dotted lines represent the uptake obtained from the rOAT3 cRNA-injected oocytes and the water-injected oocytes, respectively. Solid line (R2 = 0.998) represents the rOAT3-mediated uptake after subtraction of the uptake obtained from the water-injected oocytes from the rOAT3 cRNA-injected oocytes by nonlinear least-squares regression analysis. Each point represents the mean ± S.E. from 10 to 14 oocytes. Inset, Eadie-Hofstee plots of topotecan hydroxyl acid uptake after correction for uptake by water-injected oocytes. v, uptake rate (picomoles per 30 min per oocyte); s, substrate concentration (micromolar).

Uptake of Topotecan Hydroxyl Acid byX. laevisOocytes Expressing hOAT1 and hOAT3. The time course of topotecan hydroxyl acid uptake by X. laevis oocytes injected with hOAT3 cRNA or water was examined (Fig. 6A). The uptake of topotecan hydroxyl acid by X. laevis oocytes injected with hOAT3 cRNA was significantly greater than that by water-injected oocytes and increased linearly with time up to 60 min. In contrast, X. laevis oocytes injected with hOAT1 cRNA did not stimulate the transport of topotecan hydroxyl acid compared with water-injected oocytes, indicating that hOAT1 does not transport topotecan hydroxyl acid (Fig. 6B). The uptake of topotecan hydroxyl acid by hOCT2-expressing oocytes was comparable with that by water-injected oocytes (data not shown). To characterize the transport of topotecan hydroxyl acid by hOAT3, the concentration dependence of its uptake by X. laevis oocytes injected with hOAT3 cRNA, after subtraction of the uptake by water-injected oocytes, was studied in the concentration range from 1 to 200 μM (Fig. 7). The Km and Vmax values of topotecan hydroxyl acid transport by hOAT3 were 56.5 ± 6.98 μM and 0.604 ± 0.027 pmol/30 min/oocyte, respectively. These results demonstrate that human, as well as rat, OAT3 recognizes topotecan hydroxyl acid.

Fig. 6.
Fig. 6.

Uptake of topotecan hydroxyl acid by X. laevis oocytes expressing hOAT1 or hOAT3. A, oocytes injected with hOAT3 (closed circle) or water (open circle) were incubated for the specified periods at room temperature with 5 μM topotecan hydroxyl acid. Each point represents the mean ± S.E. from 13 to 15 oocytes. *, significant difference from the uptake of water-injected oocytes (p < 0.05). B, oocytes injected with hOAT1 (shaded column) or water (open column) were incubated for 60 min at room temperature with 5 μM topotecan hydroxyl acid. Each column represents the mean ± S.E. for 12 oocytes.

Discussion

Many investigators have reported on the systemic and renal disposition of topotecan in various animals and humans (Herben et al., 1996, 2002; Zamboni et al., 1998; Hartmann and Lipp, 2006; Mustafa et al., 2006). Topotecan is primarily excreted into urine as both lactone and hydroxyl acid forms, accounting for approximately 49% of the dose administered i.v. in cancer patients (Herben et al., 2002). Although topotecan undergoes both renal and hepatobiliary excretion (Stewart et al., 1994; Furman et al., 1996), it has been shown to undergo only limited metabolism (Herben et al., 1997, 2002; Rosing et al., 1997). A population pharmacokinetic analysis indicated that tubular secretion in addition to glomerular filtration is involved in topotecan renal clearance in humans (Mould et al., 2002). Prior studies in murine species demonstrated that the renal clearance of topotecan was significantly reduced by probenecid, a typical inhibitor of organic anion transporters, suggesting that topotecan undergoes renal tubular secretion via organic anion transporter(s) (Zamboni et al., 1998; Mustafa et al., 2006). However, the molecular mechanism of renal tubular secretion of topotecan has not been elucidated. In kidney, OAT1 and OAT3 are expressed at the basolateral membranes and play important roles in the first step of the renal secretion of organic anions. Thus, we hypothesized that OATs mediate the basolateral uptake of topotecan hydroxyl acid in renal epithelial cells. Therefore, in the present study, we examined the transport characteristics of topotecan hydroxyl acid from blood to kidney proximal tubular cells by using rat kidney slices and X. laevis oocytes expressing rat and human OAT1 and OAT3. We present here the first evidence that OAT3, but not OAT1, plays a primary role in the basolateral uptake of topotecan hydroxyl acid into epithelial cells from the blood in both rat and human kidney.

Fig. 7.
Fig. 7.

Concentration dependence of topotecan hydroxyl acid uptake by X. laevis oocytes expressing hOAT3. Oocytes injected with hOAT3 cRNA (closed circle) or water (open circle) were incubated at room temperature for 30 min with topotecan hydroxyl acid. Broken and dotted lines represent the uptake obtained from the hOAT3 cRNA-injected oocytes and the water-injected oocytes, respectively. Solid line (R2 = 0.992) represents the hOAT3-mediated uptake after subtraction of the uptake obtained from the water-injected oocytes from the hOAT3 cRNA-injected oocytes by nonlinear least-squares regression analysis. Each point represents the mean ± S.E. for from 12 to 14 oocytes. Inset, Eadie-Hofstee plots of topotecan hydroxyl acid uptake after subtraction of the uptake by water-injected oocytes. v, uptake rate (picomoles per 30 min per oocyte); s, substrate concentration (micromolar).

The uptake of topotecan hydroxyl acid into rat kidney slices was potently inhibited by pravastatin and probenecid with Ki values of 10.6 and 8.1 μM, respectively, whereas PAH showed only weak inhibition at high concentration (Fig. 3). TEA did not block the uptake up to 10 mM. Hasegawa et al. (2002) previously demonstrated different inhibitory potencies of PAH and pravastatin for rOAT1- and rOAT3-mediated uptake; the Ki value of PAH for the uptake of pravastatin by rOAT3-expressing LLC-PK1 cells was 1.4 mM, which was 23-fold greater than the Km value for the rOAT1-mediated uptake of PAH (Km = 60 μM), and the Ki value of pravastatin for the uptake of PAH by rOAT1-expressing cells was 1.2 mM, which is 86-fold greater than the Km value for the rOAT3-mediated uptake of pravastatin (Km = 13 μM). The Ki values of probenecid for cimetidine and famotidine uptake by rOAT3-HEK cells were found to be 5.77 and 2.55 μM, respectively (Tahara et al., 2005). The Ki values of pravastatin and probenecid for topotecan hydroxyl acid uptake in the present study are close to the reported Km or Ki values. In addition, topotecan hydroxyl acid was shown to be a substrate for rOAT3, with a Km value of 21.9 μM in rOAT3-expressing oocytes (Fig. 5), whereas the uptake of topotecan hydroxyl acid by rOAT1-expressing oocytes was comparable with that by water-injected oocytes (Fig. 4). These results support the view that topotecan hydroxyl acid is taken up by rat kidney via rOAT3 but not rOAT1. Although contribution of rOAT3 but not rOAT1 was suggested, apparent uptake of topotecan hydroxyl acid was inhibited by OAT3 substrate by 40% (Fig. 3). The remaining uptake in the presence of OAT3 inhibitors may suggest a contribution of other transporters than rOAT3. However, contribution of rOAT2, which is also weakly expressed in the basolateral membrane of rat kidney (Sekine et al., 1998), may be excluded as the following reason. Khamdang et al. (2004) previously demonstrated the IC50 values of pravastatin and probenecid for the uptake of PGF2 by rOAT2-expressing S2 cells were 449 and 977 μM, respectively. The Ki values of pravastatin (10.6 μM) and probenecid (8.1 μM) for topotecan hydroxyl acid uptake observed in the present study are fairly low compared with those reported IC50 values. These results support that topotecan hydroxyl acid is taken up by rat kidney via mainly rOAT3, and the extent of contribution of rOAT3 is estimated as at least 60% in the apparent uptake into slices. Two possible explanations for the remaining 40% are considered: 1) the part of uptake is simple diffusion of topotecan hydroxyl acid and the converted lactone form, and/or 2) the unknown transporters that are probenecid and pravastatin-insensitive may be involved. The adsorption amount of topotecan hydroxyl acid is also possible, although the amount may not be extensive, because the uptake of topotecan hydroxyl acid at 0 min is extrapolated as near origin as shown in Fig. 2.

It has been shown that hOAT3 can transport topotecan hydroxyl acid with a Km value of 56.5 μM in hOAT3-expressing X. laevis oocytes (Fig. 7). The maximal plasma concentration of total topotecan reached approximately 100 nM after a 30-min i.v. infusion at the clinical dose of 1.5 mg/m2 in humans (Herben et al., 2002), and the plasma protein binding of topotecan appears to be low (7–35%; Herben et al., 1996). Accordingly, it was suggested that topotecan hydroxyl acid can be transported efficiently by hOAT3. On the other hand, topotecan hydroxyl acid is not likely to be a substrate of hOAT1 in oocytes expressing hOAT1 (Fig. 6). It was reported that the mRNA level of hOAT3 was approximately 3 times that of hOAT1 in human kidney (Motohashi et al., 2002). Accordingly, transport via hOAT3 should be more important than that via hOAT1 in the renal secretion of topotecan hydroxyl acid in humans. This finding is consistent with the characteristics of hOAT3, which prefers organic anions with bulky side groups as substrates compared with hOAT1 (Cha et al., 2001).

It has been suggested that there is some overlap in substrates between OAT3 and the organic anion-transporting polypeptide (OATP) family (Müller and Jansen, 1997; Suzuki and Sugiyama, 2000). Among the OATP family members, OATP1B1, OATP1B3, and OATP2B1 are expressed at the sinusoidal membrane of hepatocytes and are involved in hepatic uptake of organic anions (König et al., 2000; Tamai et al., 2000; Abe et al., 2001). In humans, the urinary recovery of topotecan is approximately 50% (Herben et al., 2002), and the other 50% of administered topotecan undergoes elimination via other routes, including hepatobiliary excretion. In fact, a previous study demonstrated considerable concentrations of topotecan in the unchanged form in bile (Saltz et al., 1993). Recently, we have showed that SN-38 hydroxyl acid, a camptothecin derivative like topotecan, is transported by OATP1B1, using cDNA-transfected HEK293 cells and cRNA-injected oocytes (Nozawa et al., 2005). Therefore, it is possible that OATPs are involved in the hepatic uptake of topotecan hydroxyl acid, although further studies are required to test this hypothesis.

Tahara et al. (2005) demonstrated that probenecid is a potent inhibitor of r/hOAT3 by using cDNA-transfected cells. They found that the Ki values of probenecid for cimetidine and famotidine uptake by hOAT3-HEK cells were 3.37 and 4.17 μM, respectively. The unbound plasma concentration of probenecid (18–80 μM) at clinical doses (0.5–2.0 g; Selen et al., 1982) was greater than its Ki value for hOAT3. Furthermore, some cephalosporin antibiotics such as cefazolin and ceftriaxone exhibit higher plasma unbound concentrations than their Ki values for hOAT3 (Shitara et al., 2005). The present study showed that hOAT3 mediates the transport of topotecan hydroxyl acid. Thus, combined administration of probenecid and some cephalosporin antibiotics may result in a delay in the clearance of topotecan by inhibiting the renal clearance of topotecan hydroxyl acid. As illustrated in Fig. 1, a pH-dependent reversible conversion occurs between the topotecan lactone and hydroxyl acid forms after administration of topotecan. An increase in systemic exposure to the hydroxyl acid because of inhibition of the tubular secretion of the hydroxyl acid form, therefore, may shift the hydrolysis equilibrium toward the lactone form, leading to an increase in systemic exposure to the lactone form. Indeed, compared with topotecan alone, coadministration of topotecan with probenecid decreased the systemic clearance of topotecan lactone in mice (Zamboni et al., 1998). Given the narrow therapeutic index of topotecan, it is possible that concomitant administration of topotecan and probenecid or some cephalosporin antibiotics may lead to clinically relevant drug-drug interactions, although there is no clinical evidence for this at present. Our findings provide useful information for considering potential dose modifications when topotecan is given with certain concomitant medications.

In conclusion, OAT3 plays an important role in the renal secretion of topotecan hydroxyl acid in humans and rats. hOAT3 is expressed in the skeletal muscle and brain as well as kidney (Cha et al., 2001; Takeda et al., 2004). hOAT3 could also play a predominant role in the tissue distribution of topotecan hydroxyl acid. Drug-drug interactions at hOAT3 and interindividual variability of hOAT3 expression could affect the disposition of not only topotecan hydroxyl acid but also topotecan lactone, at least indirectly, by shifting the hydrolysis equilibrium toward systemic formation of the lactone form. Therefore, hOAT3 is a potential molecular target for the prevention of adverse effects arising from topotecan treatment.

Footnotes

  • This study was supported in part by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology Japan.

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

  • doi:10.1124/jpet.107.123323.

  • ABBREVIATIONS: Topotecan, (S)-9-dimethylaminomethyl-10-hydroxy-camptothecin hydrochloride; OAT, organic anion transporter; r, rat; PAH, p-aminohippurate; h, human; TEA, tetraethylammonium; PCR, polymerase chain reaction; OCT, organic cation transporter; HPLC, high-performance liquid chromatography; OATP, organic anion-transporting polypeptide; SN-38, 7-ethyl-10-hydroxycamptothecin.

  • Received March 24, 2007.
  • Accepted June 5, 2007.

References

  1. ↵
    Abe T, Unno M, Onogawa T, Tokui T, Kondo TN, Nakagomi R, Adachi H, Fujiwara K, Okabe M, Suzuki T, et al. (2001) LST-2, a human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers. Gastroenterology 120: 1689–1699.
    OpenUrlCrossRefPubMed
  2. ↵
    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.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    Arun B and Frenkel EP (2001) Topoisomerase I inhibition with topotecan: pharmacologic and clinical issues. Expert Opin Pharmacother 2: 491–505.
    OpenUrlCrossRefPubMed
  4. ↵
    Burris HA 3rd (1999) The evolving role of oral topotecan. Semin Hematol 36: 26–32.
    OpenUrlPubMed
  5. ↵
    Cha SH, Sekine T, Fukushima JI, Kanai Y, Kobayashi Y, Goya T, and Endou H (2001) Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol Pharmacol 59: 1277–1286.
    OpenUrlAbstract/FREE Full Text
  6. ↵
    Dennis MJ, Beijnen JH, Grochow LB and van Warmerdam J (1997) An overview of the clinical pharmacology of topotecan. Semin Oncol 24 (Suppl 5): S5–S18.
    OpenUrlPubMed
  7. ↵
    Furman WL, Baker SD, Pratt CB, Rivera GK, Evans WE, and Stewart CF (1996) Escalating systemic exposure of continuous infusion topotecan in children with recurrent acute leukemia. J Clin Oncol 14: 1504–1511.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    Hartmann JT and Lipp HP (2006) Camptothecin and podophyllotoxin derivatives: inhibitors of topoisomerase I and II: mechanisms of action, pharmacokinetics and toxicity profile. Drug Saf 29: 209–230.
    OpenUrlCrossRefPubMed
  9. ↵
    Hasegawa M, Kusuhara H, Sugiyama D, Ito K, Ueda S, Endou H, and Sugiyama Y (2002) Functional involvement of rat organic anion transporter 3 (rOat3; Slc22a8) in the renal uptake of organic anions. J Pharmacol Exp Ther 300: 746–753.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    Herben VM, ten Bokkel Huinink WW, and Beijnen JH (1996) Clinical pharmacokinetics of topotecan. Clin Pharmacokinet 31: 85–102.
    OpenUrlPubMed
  11. ↵
    Herben VM, ten Bokkel Huinink WW, Dubbelman AC, Mandjes IA, Groot Y, van Gortel-van Zomeren DM, and Beijnen JH (1997) Phase I and pharmacological study of sequential intravenous topotecan and oral etoposide. Br J Cancer 76: 1500–1508.
    OpenUrlPubMed
  12. ↵
    Herben VM, Schoemaker E, Rosing H, van Zomeren DM, ten Bokkel Huinink WW, Dubbelman R, Hearn S, Schellens JH, and Beijnen JH (2002) Urinary and fecal excretion of topotecan in patients with malignant solid tumours. Cancer Chemother Pharmacol 50: 59–64.
    OpenUrlCrossRefPubMed
  13. ↵
    Hosoyamada M, Sekine T, Kanai Y, and Endou H (1999) Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney. Am J Physiol 276: F122–F128.
    OpenUrlPubMed
  14. ↵
    Hsiang YH, Liu LF, Wall ME, Wani MC, Nicholas AW, Manikumar G, Kirschenbaum S, Silber R, and Potmesil M (1989) DNA topoisomerase I-mediated DNA cleavage and cytotoxicity of camptothecin analogues. Cancer Res 49: 4385–4389.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    Ishiguro N, Saito A, Yokoyama K, Morikawa M, Igarashi T, and Tamai I (2005) Transport of the dopamine D2 agonist pramipexole by rat organic cation transporters OCT1 and OCT2 in kidney. Drug Metab Dispos 33: 495–499.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    Khamdang S, Takeda M, Shimoda M, Noshiro R, Narikawa S, Huang XL, Enomoto A, Piyachaturawat P, and Endou H (2004) Interactions of human- and rat-organic anion transporters with pravastatin and cimetidine. J Pharmacol Sci 94: 197–202.
    OpenUrlCrossRefPubMed
  17. ↵
    Kollmannsberger C, Mross K, Jakob A, Kanz L, and Bokemeyer C (1999) Topotecan: a novel topoisomerase I inhibitor: pharmacology and clinical experience. Oncology 56: 1–12.
    OpenUrlCrossRefPubMed
  18. ↵
    König J, Cui Y, Nies AT, and Keppler D (2000) Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J Biol Chem 275: 23161–23168.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    Kusuhara H, Sekine T, Utsunomiya-Tate N, Tsuda M, Kojima R, Cha SH, Sugiyama Y, Kanai Y, and Endou H (1999) Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. J Biol Chem 274: 13675–13680.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    Liman ER, Tytgat J, and Hess P (1992) Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. Neuron 9: 861–871.
    OpenUrlCrossRefPubMed
  21. ↵
    Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, and Inui K (2002) Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J Am Soc Nephrol 13: 866–874.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    Mould DR, Holford NH, Schellens JH, Beijnen JH, Hutson PR, Rosing H, ten Bokkel Huinink WW, Rowinsky EK, Schiller JH, Russo M, et al. (2002) Population pharmacokinetic and adverse event analysis of topotecan in patients with solid tumors. Clin Pharmacol Ther 71: 334–348.
    OpenUrlCrossRefPubMed
  23. ↵
    Müller M and Jansen PL (1997) Molecular aspects of hepatobiliary transport. Am J Physiol 272: G1285–G1303.
    OpenUrlPubMed
  24. ↵
    Mustafa S, Venkatesh P, Pasha K, Mullangi R, and Srinivas NR (2006) Altered intravenous pharmacokinetics of topotecan in rats with acute renal failure (ARF) induced by uranyl nitrate: do adenosine A1 antagonists (selective/non-selective) normalize the altered topotecan kinetics in ARF? Xenobiotica 36: 1239–1258.
    OpenUrlCrossRefPubMed
  25. ↵
    Nozawa T, Minami H, Sugiura S, Tsuji A, and Tamai I (2005) Role of organic anion transporter OATP1B1 (OATP-C) in hepatic uptake of irinotecan and its active metabolite, 7-ethyl-10-hydroxycamptothecin: in vitro evidence and effect of single nucleotide polymorphisms. Drug Metab Dispos 33: 434–439.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    O'Reilly S, Rowinsky EK, Slichenmyer W, Donehower RC, Forastiere AA, Ettinger DS, Chen TL, Sartorius S, and Grochow LB (1996a) Phase I and pharmacologic study of topotecan in patients with impaired renal function. J Clin Oncol 14: 3062–3073.
    OpenUrlAbstract
  27. ↵
    O'Reilly S, Rowinsky E, Slichenmyer W, Donehower RC, Forastiere A, Ettinger D, Chen TL, Sartorius S, Bowling K, Smith J, et al. (1996b) Phase I and pharmacologic studies of topotecan in patients with impaired hepatic function. J Natl Cancer Inst 88: 817–824.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Rosing H, Herben VM, van Gortel-van Zomeren DM, Hop E, Kettenes-van den Bosch JJ, ten Bokkel Huinink WW, and Beijnen JH (1997) Isolation and structural confirmation of N-desmethyl topotecan, a metabolite of topotecan. Cancer Chemother Pharmacol 39: 498–504.
    OpenUrlCrossRefPubMed
  29. ↵
    Saltz L, Sirott M, Young C, Tong W, Niedzwiecki D, Tzy-Jyun Y, Tao Y, Trochanowski B, Wright P, Barbosa K, et al. (1993) Phase I clinical and pharmacology study of topotecan given daily for 5 consecutive days to patients with advanced solid tumors, with attempt at dose intensification using recombinant granulocyte colony-stimulating factor. J Natl Cancer Inst 85: 1499–1507.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y, and Endou H (1998) Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 429: 179–182.
    OpenUrlCrossRefPubMed
  31. ↵
    Sekine T, Watanabe N, Hosoyamada M, Kanai Y, and Endou H (1997) Expression cloning and characterization of a novel multispecific organic anion transporter. J Biol Chem 272: 18526–18529.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Selen A, Amidon GL, and Welling PG (1982) Pharmacokinetics of probenecid following oral doses to human volunteers. J Pharm Sci 71: 1238–1242.
    OpenUrlCrossRefPubMed
  33. ↵
    Shitara Y, Sato H, and Sugiyama Y (2005) Evaluation of drug-drug interaction in the hepatobiliary and renal transport of drugs. Annu Rev Pharmacol Toxicol 45: 689–723.
    OpenUrlCrossRefPubMed
  34. ↵
    Stewart CF, Baker SD, Heideman RL, Jones D, Crom WR, and Pratt CB (1994) Clinical pharmacodynamics of continuous infusion topotecan in children: systemic exposure predicts hematologic toxicity. J Clin Oncol 12: 1946–1954.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    Suzuki H, and Sugiyama Y (2000) Transport of drugs across the hepatic sinusoidal membrane: sinusoidal drug influx and efflux in the liver. Semin Liver Dis 20: 251–263.
    OpenUrlCrossRefPubMed
  36. ↵
    Tahara H, Kusuhara H, Endou H, Koepsell H, Imaoka T, Fuse E, and Sugiyama Y (2005) A species difference in the transport activities of H2 receptor antagonists by rat and human renal organic anion and cation transporters. J Pharmacol Exp Ther 315: 337–345.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    Takeda M, Noshiro R, Onozato ML, Tojo A, Hasannejad H, Huang XL, Narikawa S, and Endou H (2004) Evidence for a role of human organic anion transporters in the muscular side effects of HMG-CoA reductase inhibitors. Eur J Pharmacol 483: 133–139.
    OpenUrlCrossRefPubMed
  38. ↵
    Tamai I, Nezu J, Uchino H, Sai Y, Oku A, Shimane M, and Tsuji A (2000) Molecular identification and characterization of novel members of the human organic anion transporter (OATP) family. Biochem Biophys Res Commun 273: 251–260.
    OpenUrlCrossRefPubMed
  39. ↵
    Underberg WJ, Goossen RM, Smith BR, and Beijnen JH (1990) Equilibrium kinetics of the new experimental anti-tumour compound SK&F 104864-A in aqueous solution. J Pharm Biomed Anal 8: 681–683.
    OpenUrlCrossRefPubMed
  40. ↵
    Zamboni WC, Houghton PJ, Johnson RK, Hulstein JL, Crom WR, Cheshire PJ, Hanna SK, Richmond LB, Luo X, and Stewart CF (1998) Probenecid alters topotecan systemic and renal disposition by inhibiting renal tubular secretion. J Pharmacol Exp Ther 284: 89–94.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools
Research ArticleGASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Involvement of Rat and Human Organic Anion Transporter 3 in the Renal Tubular Secretion of Topotecan [(S)-9-Dimethylaminomethyl-10-hydroxy-camptothecin hydrochloride]

Shin-ichi Matsumoto, Kenji Yoshida, Naoki Ishiguro, Tomoji Maeda and Ikumi Tamai
Journal of Pharmacology and Experimental Therapeutics September 1, 2007, 322 (3) 1246-1252; DOI: https://doi.org/10.1124/jpet.107.123323
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section