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Methotrexate-Loxoprofen Interaction: Involvement of Human Organic Anion Transporters hOAT1 and hOAT3
Drug Metab. Pharmacokin. 19 (5): 369–374 (2004).
Regular Article Methotrexate-Loxoprofen Interaction: Involvement of Human Organic Anion Transporters hOAT1 and hOAT3 Yuichi UWAI, Risa TANIGUCHI, Hideyuki MOTOHASHI, Hideyuki SAITO, Masahiro OKUDA and Ken-ichi INUI Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan Full text of this paper is available at http://www.jssx.org
Summary: Human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8) are responsible for renal tubular secretion of an antifolic acid methotrexate, and are considered to be involved in drug interaction of methotrexate with nonsteroidal anti-in‰ammatory drugs (NSAIDs). In our hospital, a delay of methotrexate elimination was experienced in a patient with Hodgkin's disease, who took loxoprofen, a commonly used NSAID in Japan, which suggested a cause. In this study, we examined the drug interaction via hOAT1 and hOAT3, using Xenopus laevis oocytes. hOAT1 and hOAT3 mediated the methotrexate transport with low a‹nity (Km of 724.0 mM) and high a‹nity (Km of 17.2 mM), respectively. Loxoprofen and its trans-OH metabolite, an active major metabolite, markedly inhibited the methotrexate transport by both transporters. Their inhibition concentrations (IC50 ) were in the range of the therapeutic levels. These ˆndings suggest that loxoprofen retards the elimination of methotrexate, at least in part, by inhibiting hOAT1 and hOAT3.
Key words: organic anion transporter; renal tubular secretion; drug interaction; methotrexate; loxoprofen the OATs, mRNA expression levels of hOAT1 and hOAT3, both localized at the basolateral membrane of renal proximal epithelial cells, were reported to be markedly higher than other organic ion transporters in the human kidney cortex.8) Furthermore, OAT1 and OAT3 were shown to be responsible for tubular uptake of several anionic drugs from functional aspects.9,10) Since methotrexate is a substrate of OAT1 and OAT3, these transporters were suggested to contribute to the renal excretion of methotrexate.7,9,11) Moreover, Uwai et al.12) and Takeda et al.13) represented that various NSAIDs have inhibitory eŠects on methotrexate transport by the renal organic anion transporters, suggesting that the organic anion transporters are involved in the drug interaction between methotrexate and NSAIDs. NSAIDs alleviate pain by inhibiting prostaglandins synthesis mediated by cyclooxygenases. NSAIDs often induce severe toxicity including gastric mucosal injury. A propionic acid derivative loxoprofen has a strong inhibitory potency against cyclooxygenases by converting into trans-OH metabolite, a major metabolite (Fig. 1; ref, 14). The side eŠect of loxoprofen on gastric mucosa was shown to be weaker than indomethacin,
Introduction Methotrexate is used for the treatment of malignancies and rheumatoid arthritis.1–3) Administered methotrexate is mostly excreted into urine as the unchanged form, and organic anion transporters, expressed in the renal proximal tubules, are responsible for its tubular secretion.4) It was reported that methotrexate often induces severe adverse eŠects such as bone marrow depression, hepatitis or acute renal failure through an increase in blood methotrexate levels when nonsteroidal anti-in‰ammatory drugs (NSAIDs) are simultaneously administered.5,6) The mechanisms of the drug interaction have been suggested to decrease renal clearance of methotrexate via reduction of renal blood ‰ow and via inhibition of its tubular secretion by NSAIDs in addition to competing at protein binding.3) For a safe and eŠective drug administration regimen, it is important to specify the molecule(s) responsible for drug elimination and interaction. Since Sekine et al.7) reported the cDNA isolation of organic anion transporter 1 (OAT1) from a rat kidney cDNA library, several additional isoforms of OAT1 were cloned. Among
Received; May 28, 2004, Accepted; August 26, 2004 To whom correspondence should be addressed : Prof. Ken-ichi INUI, Ph.D., Department of Pharmacy, Kyoto University Hospital, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel. ＋81-75-751-3577, Fax. ＋81-75-751-4207, E-mail: inui＠kuhp.kyoto-u.ac.jp
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Fig. 1. The chemical structures of loxoprofen and trans-OH metabolite.
because the conversion occurs after absorption.15) Therefore, loxoprofen is one of the most used NSAIDs in Japan. In our hospital, one patient with Hodgkin's disease who was treated with methotrexate at high dose showed a delay of the elimination of plasma methotrexate. Values of serum creatinine and serum blood urea nitrogen indicated that renal function of the patient was not deteriorated. When the methotrexate elimination was delayed, the patient had taken loxoprofen for fever. Then, the drug interaction between methotrexate and loxoprofen was suspected. Although, at present, the drug interaction between loxoprofen and methotrexate is described in the package insert of Loxonin} (loxoprofen), the mechanisms of the drug interaction with organic anion transporters have not been demonstrated. From these backgrounds, we performed methotrexate uptake experiments using the Xenopus laevis oocyte expression system to assess the contribution of hOAT1 and hOAT3 to the drug interaction between methotrexate and loxoprofen. Materials and Methods Materials: Loxoprofen sodium and the trans-OH metabolite were kindly gifted by Sankyo Co. (Tokyo, Japan). [3?,5?,7-3H(N)]Methotrexate (851–1073 GBq W mmol) was purchased from Moravek Biochemicals (Brea, CA, USA). Phenylbutazone, salicylate and unlabeled methotrexate (L-(＋)-amethopterin) were obtained from Nacalai Tesque (Kyoto, Japan). Flufenamate, ibuprofen and indomethacin were from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals used were of the highest purity available. Isolation of hOAT1 cDNA: The speciˆc primer-set for hOAT1-1 (sense strand with a Sal I site, 5?-CCG-
TCGACGCCTGGCCCAATGGCCTTTAA-3? corresponding to position 258 to 278; antisense strand with a Not I site, 5?-CCGCGGCCGCAAGGCCCCTTCTCAGTCCTCA-3? corresponding to position 1957 to 1977; accession number AB009697 in GenBank database; ref, 16) was used for reverse transcribed-polymerase chain reaction (PCR) with human kidney poly(A)＋ RNA (Clontech, Palo Alto, CA, USA). The obtained PCR product was ligated into pSPORT1 and recognized as hOAT1-2 from the sequence analysis (accession number AB009698; ref, 16). Then, Human Kidney lTriplExTM Library (Clontech) was screened by hybridization with the PCR product. An isolated positive clone with a 2.1-kb insert was subcloned into pSPORT1 cut with Xba I and Eco RI. The complete sequence of the clone was determined, and the deduced amino acid sequence was identical to hOAT1-2.16) Isolation of hOAT3 cDNA: Human Kidney RapidScreenTM cDNA Library Panels (OriGene Technologies, Rockville, MD, USA) were screened by PCR method with the hOAT3-speciˆc primers (Sense strand, 5?-AATGCCAGCCTGCCCAATGA-3? corresponding to position 365 to 384; antisense strand, 5?-GTCTGCAAGTCCAAGGGCAC-3? corresponding to position 1331 to 1350; accession number AB042505; ref, 11). A positive clone with a 2.2-kb insert was isolated and subcloned into pSPORT1 cut with Xba I and Eco RI. The complete sequence of the clone was determined and the deduced amino acid sequence was identical to that of hOAT3 cloned by Cha et al.11) except amino acid 271, that is lysine in hOAT3 isolated in this study and glutamic acid in their hOAT3. Methotrexate uptake experiment using Xenopus laevis oocytes expressing hOAT1 and hOAT3: An uptake experiment using Xenopus laevis oocytes was performed as previously described.12) The capped RNA encoding hOAT1 or hOAT3 was transcribed from XbaI-linearized pSPORT1 containing hOAT1 or hOAT3, respectively, with T7 RNA polymerase. After 50 nL of water or the cRNA (25 ng) were injected into defolliculated oocytes, the oocytes were maintained in modiˆed Barth's medium (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3 )2, 0.4 mM CaCl2, 0.8 mM MgSO4, 2.4 mM NaHCO3 and 5 mM HEPES) containing 50 mg W mL gentamicin at 189 C. Three days after the injection, the uptake reaction was initiated by incubating the oocytes in 500 mL of uptake buŠer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2 and 5 mM HEPES; pH 7.4) with [3H]methotrexate at 259 C in the presence or absence of an inhibitor for the indicated periods. The uptake reaction was terminated by adding 2 mL of ice-cold uptake buŠer to each well, and the oocytes were washed four times with 2 mL of the ice-cold buŠer. After washing, each oocyte was transferred to a scintillation counting vial and solubilized in 300 mL of 10z
Methotrexate-loxoprofen Interaction with hOAT1 and hOAT3
Fig. 2. Time-dependence of methotrexate uptake by hOAT1-(A) or hOAT3-(B) expressing oocytes. Water-injected (open circle) and hOAT-expressing (closed circle) oocytes were incubated with 500 nM [3H]methotrexate for the indicated periods. The uptake amounts of [3H]methotrexate in each oocyte were determined. Each point represents the mean±S.E. of [3H]methotrexate uptake in 6 to 10 oocytes.
sodium lauryl sulfate. Three milliliters of ACSII (Amersham International, Buckinghamshire, UK) were added to each solubilized oocyte, and the radioactivity was determined in a liquid scintillation counter. Statistical analysis: Multiple comparisons were performed with ScheŠ áe's test following analysis of variance. DiŠerences were considered signiˆcant at Pº0.05. Results Transport characteristics of methotrexate by hOAT1 and hOAT3: To assess the involvement of hOAT1 and hOAT3 in the drug interaction between methotrexate and loxoprofen, we performed the transport experiments using Xenopus oocytes. At the beginning, transport characteristics of methotrexate by hOAT1 and hOAT3 were investigated. Methotrexate uptake by hOAT1- and hOAT3-expressing oocytes was increased
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Fig. 3. Dose-dependence of methotrexate uptake by hOAT1-(A) or hOAT3-(B) expressing oocytes. Water-injected (open circle) and hOAT-expressing (closed circle) oocytes were incubated with [3H]methotrexate at various concentrations for 1 h. The uptake amounts of [3H]methotrexate in each oocyte were determined. Each point represents the mean±S.E. of [3H]methotrexate uptake in 8 to 10 oocytes.
linearly up to 1 h, compared with water-injected oocytes (Fig. 2). Accordingly, further experiments were performed with 1-h incubation. To compare the kinetic parameters of methotrexate transport by hOAT1 and hOAT3, concentration dependence of methotrexate uptake was examined. With the use of nonlinear least squares regression analysis, kinetic parameters were estimated according to the Michaelis-Menten equation. A typical result of three separate experiments is represented in Fig. 3. The apparent Michaelis-Menten constant (Km) and maximal velocity (Vmax ) values were 724.0±74.9 mM and 217.1±44.7 pmol W oocyte W h for hOAT1, and 17.2±3.6 mM and 6.5±1.4 pmol W oocyte W h for hOAT3, respectively (mean±S.E., n＝3). EŠect of loxoprofen and trans-OH metabolite on methotrexate uptake by hOAT1- or hOAT3-expressing oocytes: Next, the inhibitory eŠects of loxoprofen and the trans-OH metabolite on the methotrexate transport by hOAT1 and hOAT3 were assessed. As shown in
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Table 1. EŠects of various NSAIDs on methotrexate uptake by hOAT1- or hOAT3-expressing oocytes. Water-injected and hOAT-expressing oocytes were incubated with 500 nM [3H]methotrexate in the absence (control) or presence of each NSAID at 0.1 mM for 1 h. The uptake amounts of [3H]methotrexate in each oocyte were determined. Each value represents the mean± S.E. of [3H]methotrexate uptake as a percentage of control in 6 to 10 oocytes. Inhibitors Water-injected Control Loxoprofen trans-OH metabolite Ibuprofen Indomethacin Flufenamate Phenylbutazone Salicylate
Methotrexate uptake (z of control) hOAT1
hOAT3
41.0±3.8 100.0±8.7 33.2±2.6* 39.8±4.3* 43.7±4.4* 30.7±2.2* 44.1±2.4* 56.1±5.0* 106.7±5.3
23.4±1.0 100.0±9.5 31.5±1.3* 34.3±2.2* 45.4±2.8* 55.0±2.9* 38.8±3.3* 47.6±5.8* 61.5±5.3*
*Pº0.05, signiˆcantly diŠerent from control values.
Table 1, methotrexate uptake by both transporters was signiˆcantly inhibited by both loxoprofen and the trans-OH metabolite similar to other NSAIDs such as ibuprofen, indomethacin, ‰ufenamate and phenylbutazone. Salicylate at 100 mM showed an inhibitory eŠect on hOAT3, but not on hOAT1 (Table 1). To determine the inhibition concentration (IC50 ) values of loxoprofen and the trans-OH metabolite, we investigated the dose-dependence of their inhibitory eŠects on the methotrexate transport mediated by hOAT1 and hOAT3. From three independent experiments, a representative result is shown in Fig. 4. Loxoprofen and the trans-OH metabolite inhibited methotrexate transport by hOAT1 and hOAT3 dose-dependently, and the estimated IC50 were 27.1±8.7 mM for loxoprofen and 12.2±3.4 mM for the trans-OH metabolite in hOAT1, and 8.7±3.6 mM for loxoprofen and 4.5±1.7 mM for the trans-OH metabolite in hOAT3 (mean±S.E., n＝3). Discussion Several reports represent that coadministration of methotrexate and NSAIDs often induces a marked elevation of blood methotrexate levels and resultant severe toxicity.5,6) The possible mechanisms for this interaction have been suggested to be displacement of protein binding, decreased renal blood ‰ow via inhibition of prostaglandin synthesis by NSAIDs, and competition at renal organic anion transporters.3,12,13) In our hospital, interaction between methotrexate and loxoprofen was suspected. The purpose of the present study is to estimate the involvement of hOAT1 and hOAT3 to the interaction between methotrexate and loxoprofen. To our knowledge, this is the ˆrst report
Fig. 4. Dose-dependence of inhibitory eŠect of loxoprofen and trans-OH metabolite on methotrexate uptake by hOAT1-(A) or hOAT3-(B) expressing oocytes. hOAT-expressing oocytes were incubated with 500 nM [3H]methotrexate in the absence (control) or presence of loxoprofen (open circle) or trans-OH metabolite (closed circle) at various concentrations for 1 h. The uptake amounts of [3H]methotrexate in each oocyte were determined. Each point represents the mean±S.E. of [3H]methotrexate uptake in 5 to 10 oocytes.
representing the inhibitory eŠects of loxoprofen and the trans-OH metabolite on hOAT1 and hOAT3. OAT1 and OAT3 were suggested to play important roles in the renal uptake of anionic drugs in terms of functional and expressional ˆndings.8–10) Although various compounds were reported to be substrates of OAT1 and OAT3, it is important to determine their responsibility for the renal uptake of each substrate. In the case of methotrexate, the studies by Nierenberg17) and Besseghir et al.18) suggested that the renal organic anion transporter mediating basolateral uptake of p-aminohippuric acid and methotrexate is identical in rabbits. Accordingly, OAT1, the predominant transporter of p-aminohippuric acid, is considered to be responsible for renal uptake of methotrexate in rabbits. In rats, it was reported that indomethacin, which is a potent inhibitor for rOAT1 but not for rOAT3, inhibited the tubular clearance of methotrexate to
Methotrexate-loxoprofen Interaction with hOAT1 and hOAT3
one eighth.19) In addition to the evidence that methotrexate is a substrate of rOAT1,7,9) it was thought that rOAT1 works for the basolateral transport of methotrexate in rats. However, there is no information suggesting which transporter is important for renal uptake of methotrexate in humans. In the present study, we compared the transport characteristics of methotrexate by hOAT1 with those by hOAT3. The estimated Km values for hOAT1 and hOAT3 were 724.0 mM and 17.2 mM, respectively, indicating that hOAT1 is a low-a‹nity type transporter of methotrexate, and that hOAT3 is a high-a‹nity type. When high-dose therapy of methotrexate is performed for treatment of malignancy, the blood levels of methotrexate reach 1 mM. Accordingly, it is speculated that hOAT1 and hOAT3 would work at high concentrations of serum methotrexate and at low concentrations, respectively. The contribution of hOAT1 and hOAT3 to tubular secretion of methotrexate has remained to be elucidated. This study shows that loxoprofen and the trans-OH metabolite have strong inhibitory eŠects on hOAT1 and hOAT3 like other NSAIDs (Table 1). The inhibition degrees by the NSAIDs shown in Table 1 are comparable with previous reports.12,13,20,21) Because Sosogi et al. reported that 1-h incubation with several NSAIDs at 100 mM did not aŠect ATP content of IEC-6 cells, it is likely that the decrease of methotrexate uptake is independent of uncoupling eŠect of NSAIDs.22) The estimated IC50 values of loxoprofen and the trans-OH metabolite for hOAT1 and hOAT3 were similar to those of other NSAIDs, such as ibuprofen, indomethacin, ketoprofen, diclofenac, etodolac, naproxen, piroxicam for hOAT1 and hOAT3.20,21) Usually, loxoprofen is administered orally at 180 mg per day divided into three doses, or is administered in potions of 60 to 120 mg in Japan. The administered loxoprofen immediately disappears from blood (its elimination half life is about 1.2 h), and is mainly excreted into urine as glucuronide conjugates of loxoprofen and the trans-OH metabolite. When one tablet of loxoprofen (60 mg as loxoprofen anhydrous) is administered, maximal plasma concentrations of loxoprofen and the trans-OH metabolite reach about 20 mM and 3 mM, respectively, suggesting that their IC50 values for hOAT1 and hOAT3 are comparable with their therapeutic range. However, because protein bindings of loxoprofen and the trans-OH metabolite are greater than 90z, unbound concentrations of them are lower than the IC50 values. In the present clinical case, the albumin concentration of the dL, following to liver patient had decreased to 2.6 g W insu‹ciency, suggesting that the free fraction ratio could be increased up to two-fold. Also, the decrease in glucuronidation activity against loxoprofen and the trans-OH metabolite in addition to the increase of
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unbound fractions of the two compounds might have in‰uenced the renal uptake of methotrexate via hOAT1 and hOAT3. Furthermore, eŠect of the trans-OH metabolite on renal blood ‰ow should be considered. There are two reports examining whether NSAIDs are substrates of hOATs.20,21) The results for some NSAIDs are controversial. Mulato et al. showed that ibuprofen and ketoprofen were not transported by hOAT1.20) However, Khamdang et al. reported that hOAT1 and hOAT3 mediated transport of these NSAIDs.21) In addition, Khamdang et al. represented that the hOATs transported indomethacin and salicylate.21) The mechanisms of interaction between NSAIDs and OATs should be elucidated in the future. According to the reports by Dannhardt and Kiefer,23) Mulato et al.20) and Khamdang et al,21) several NSAIDs have similar inhibition constants against cyclooxygenases, hOAT1 and hOAT3. There are many reports representing that NSAIDs interact with various drugs via receptors, serum proteins and via metabolic enzymes, in addition to via renal organic anion transporters.24) Because NSAIDs are very commonly used, new NSAIDs that do not interact with proteins except cyclooxygenases are desirable. In future studies, analysis on the molecular structures of cyclooxygenases and other proteins would help the development of NSAIDs that avoid the drug interactions. In summary, the drug interaction between methotrexate and loxoprofen was suspected in a patient with Hodgkin's disease in our hospital. The experimental data shows that loxoprofen and the trans-OH metabolite have inhibitory eŠects on the methotrexate transport by hOAT1 and hOAT3 like other NSAIDs. It is suggested that loxoprofen induces the reduction of the elimination rate of methotrexate by inhibiting hOAT1 and hOAT3. Acknowledgements: This work was supported by a Grant-in-aid for Research on Human Genome, Tissue Engineering, and Food Biotechnology from the Ministry of Health, Labor, and Welfare of Japan (H12Genome-019), a Grant-in-Aid for Scientiˆc Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Japan Research Foundation for Clinical Pharmacology. References 1)
2)
3)
Frei, E., JaŠe, N., Tattersall, M. H., Pitman, S. and Parker, L.: New approaches to cancer chemotherapy with methotrexate. N. Engl. J. Med., 292: 846–851 (1975). Jolivet, J., Cowan, K. H., Curt, G. A., Clendeninn, N. J. and Chabner, B. A.: The pharmacology and clinical use of methotrexate. N. Engl. J. Med., 309: 1094–1104 (1983). Bannwarth, B., P áehourcq, F., Schaeverbeke, T. and
374
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
Yuichi UWAI, et al.
Dehais, J.: Clinical pharmacokinetics of low-dose pulse methotrexate in rheumatoid arthritis. Clin. Pharmacokinet., 30: 194–210 (1996). Liegler, D. G., Henderson, E. S., Hahn, M. A. and Oliverio, V. T.: The eŠect of organic acids on renal clearance of methotrexate in man. Clin. Pharmacol. Ther., 10: 849–857 (1969). Maiche, A. G.: Acute renal failure due to concomitant action of methotrexate and indomethacin. Lancet, 1: 1390 (1986). Thyss, A., Milano, G., Kubar, J., Namer, M. and Schneider, M.: Clinical and pharmacokinetic evidence of a life-threatening interaction between methotrexate and ketoprofen. Lancet, 1: 256–258 (1986). Sekine, T., Watanabe, N., Hosoyamada, M., Kanai, Y. and Endou, H.: Expression cloning and characterization of a novel multispeciˆc organic anion transporter. J. Biol. Chem., 272: 18526–18529 (1997). Motohashi, H., Sakurai, Y., Saito, H., Masuda, S., Urakami, Y., Goto, M., Fukatsu, A., Ogawa, O. and Inui, K.: Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J. Am. Soc. Nephrol., 13: 866–874 (2002). Uwai, Y., Okuda, M., Takami, K., Hashimoto, Y. and Inui, K.: Functional characterization of the rat multispeciˆc organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney. FEBS Lett., 438: 321–324 (1998). Hasegawa, M., Kusuhara, H., Sugiyama, D., Ito, K., Ueda, S., Endou, H. and Sugiyama, Y.: Functional involvement of rat organic anion transporter 3 (rOat3; Slc22a8 ) in the renal uptake of organic anions. J. Pharmacol. Exp. Ther., 300: 746–753 (2002). Cha, S. H., Sekine, T., Fukushima, J., Kanai, Y., Kobayashi, Y., Goya, T. and Endou, H.: Identiˆcation and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol. Pharmacol., 59: 1277–1286 (2001). Uwai, Y., Saito, H. and Inui, K.: Interaction between methotrexate and nonsteroidal anti-in‰ammatory drugs in organic anion transporter. Eur. J. Pharmacol., 409: 31–36 (2000). Takeda, M., Khamdang, S., Narikawa, S., Kimura, H., Hosoyamada, M., Cha, S. H., Sekine, T. and Endou, H.: Characterization of methotrexate transport and its drug interactions with human organic anion transporters. J. Pharmacol. Exp. Ther., 302: 666–671 (2002). Matsuda, K., Tanaka, Y., Ushiyama, S., Ohnishi, K. and Yamazaki, M.: Inhibition of prostaglandin synthe-
15)
16)
17)
18)
19)
20)
21)
22)
23)
24)
sis by sodium 2-[4-(2-oxocyclopentylmethyl)phenyl] propionate dihydrate (CS-600), a new anti-in‰ammatory drug, and its active metabolite in vitro and in vivo. Biochem. Pharmacol., 33: 2473–2478 (1984). Kawano, S., Tsuji, S., Hayashi, N., Takei, Y., Nagano, K., Fusamoto, H. and Kamada, T.: EŠects of loxoprofen sodium, a newly synthesized non-steroidal anti-in‰ammatory drug, and indomethacin on gastric mucosal haemodynamics in the human. J. Gastroenterol. Hepatol., 10: 81–85 (1995). Hosoyamada, M., Sekine, T., Kanai, Y. and Endou, H.: Molecular cloning and functional expression of a multispeciˆc organic anion transporter from human kidney. Am. J. Physiol., 276: F122–F128 (1999). Nierenberg, D. W.: Drug inhibition of penicillin tubular secretion: concordance between in vitro and clinical ˆndings. J. Pharmacol. Exp. Ther., 240: 712–716 (1987). Besseghir, K., Mosig, D. and Roch-Ramel, F.: Transport of methotrexate by the in vitro isolated rabbit proximal tubule. J. Pharmacol. Exp. Ther., 250: 688–695 (1989). Statkevich, P., Fournier, D. J. and Sweeney, K. R.: Characterization of methotrexate elimination and interaction with indomethacin and ‰urbiprofen in the isolated perfused rat kidney. J. Pharmacol. Exp. Ther., 265: 1118–1124 (1993). Mulato, A. S., Ho, E. S. and Cihlar, T.: Nonsteroidal anti-in‰ammatory drugs e‹ciently reduce the transport and cytotoxicity of adefovir mediated by the human renal organic anion transporter 1. J. Pharmacol. Exp. Ther., 295: 10–15 (2000). Khamdang, S., Takeda, M., Noshiro, R., Narikawa, S., Enomoto, A., Anzai, N., Piyachaturawat, P. and Endou, H.: Interactions of human organic anion transporters and human organic cation transporters with nonsteroidal anti-in‰ammatory drugs. J. Pharmacol. Exp. Ther., 303: 534–539 (2002). Sosogi, A., Gao, F., Tomimatsu, T., Hirata, K. and Horie, T.: Non-steroidal anti-in‰ammatory drugs aŠect the methotrexate transport in IEC-6 cells. Life Sci., 73: 437–446 (2003). Dannhardt, G. and Kiefer, W.: Cyclooxygenase inhibitors-current status and future prospects. Eur. J. Med. Chem., 36: 109–126 (2001). Johnson, A. G., Seidemann, P. and Day, R. O.: NSAID-related adverse drug interactions with clinical relevance. An update. Int. J. Clin. Pharmacol. Ther., 32: 509–532 (1994).
Regular Article Methotrexate-Loxoprofen Interaction: Involvement of Human Organic Anion Transporters hOAT1 and hOAT3 Yuichi UWAI, Risa TANIGUCHI, Hideyuki MOTOHASHI, Hideyuki SAITO, Masahiro OKUDA and Ken-ichi INUI Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan Full text of this paper is available at http://www.jssx.org
Summary: Human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8) are responsible for renal tubular secretion of an antifolic acid methotrexate, and are considered to be involved in drug interaction of methotrexate with nonsteroidal anti-in‰ammatory drugs (NSAIDs). In our hospital, a delay of methotrexate elimination was experienced in a patient with Hodgkin's disease, who took loxoprofen, a commonly used NSAID in Japan, which suggested a cause. In this study, we examined the drug interaction via hOAT1 and hOAT3, using Xenopus laevis oocytes. hOAT1 and hOAT3 mediated the methotrexate transport with low a‹nity (Km of 724.0 mM) and high a‹nity (Km of 17.2 mM), respectively. Loxoprofen and its trans-OH metabolite, an active major metabolite, markedly inhibited the methotrexate transport by both transporters. Their inhibition concentrations (IC50 ) were in the range of the therapeutic levels. These ˆndings suggest that loxoprofen retards the elimination of methotrexate, at least in part, by inhibiting hOAT1 and hOAT3.
Key words: organic anion transporter; renal tubular secretion; drug interaction; methotrexate; loxoprofen the OATs, mRNA expression levels of hOAT1 and hOAT3, both localized at the basolateral membrane of renal proximal epithelial cells, were reported to be markedly higher than other organic ion transporters in the human kidney cortex.8) Furthermore, OAT1 and OAT3 were shown to be responsible for tubular uptake of several anionic drugs from functional aspects.9,10) Since methotrexate is a substrate of OAT1 and OAT3, these transporters were suggested to contribute to the renal excretion of methotrexate.7,9,11) Moreover, Uwai et al.12) and Takeda et al.13) represented that various NSAIDs have inhibitory eŠects on methotrexate transport by the renal organic anion transporters, suggesting that the organic anion transporters are involved in the drug interaction between methotrexate and NSAIDs. NSAIDs alleviate pain by inhibiting prostaglandins synthesis mediated by cyclooxygenases. NSAIDs often induce severe toxicity including gastric mucosal injury. A propionic acid derivative loxoprofen has a strong inhibitory potency against cyclooxygenases by converting into trans-OH metabolite, a major metabolite (Fig. 1; ref, 14). The side eŠect of loxoprofen on gastric mucosa was shown to be weaker than indomethacin,
Introduction Methotrexate is used for the treatment of malignancies and rheumatoid arthritis.1–3) Administered methotrexate is mostly excreted into urine as the unchanged form, and organic anion transporters, expressed in the renal proximal tubules, are responsible for its tubular secretion.4) It was reported that methotrexate often induces severe adverse eŠects such as bone marrow depression, hepatitis or acute renal failure through an increase in blood methotrexate levels when nonsteroidal anti-in‰ammatory drugs (NSAIDs) are simultaneously administered.5,6) The mechanisms of the drug interaction have been suggested to decrease renal clearance of methotrexate via reduction of renal blood ‰ow and via inhibition of its tubular secretion by NSAIDs in addition to competing at protein binding.3) For a safe and eŠective drug administration regimen, it is important to specify the molecule(s) responsible for drug elimination and interaction. Since Sekine et al.7) reported the cDNA isolation of organic anion transporter 1 (OAT1) from a rat kidney cDNA library, several additional isoforms of OAT1 were cloned. Among
Received; May 28, 2004, Accepted; August 26, 2004 To whom correspondence should be addressed : Prof. Ken-ichi INUI, Ph.D., Department of Pharmacy, Kyoto University Hospital, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel. ＋81-75-751-3577, Fax. ＋81-75-751-4207, E-mail: inui＠kuhp.kyoto-u.ac.jp
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Fig. 1. The chemical structures of loxoprofen and trans-OH metabolite.
because the conversion occurs after absorption.15) Therefore, loxoprofen is one of the most used NSAIDs in Japan. In our hospital, one patient with Hodgkin's disease who was treated with methotrexate at high dose showed a delay of the elimination of plasma methotrexate. Values of serum creatinine and serum blood urea nitrogen indicated that renal function of the patient was not deteriorated. When the methotrexate elimination was delayed, the patient had taken loxoprofen for fever. Then, the drug interaction between methotrexate and loxoprofen was suspected. Although, at present, the drug interaction between loxoprofen and methotrexate is described in the package insert of Loxonin} (loxoprofen), the mechanisms of the drug interaction with organic anion transporters have not been demonstrated. From these backgrounds, we performed methotrexate uptake experiments using the Xenopus laevis oocyte expression system to assess the contribution of hOAT1 and hOAT3 to the drug interaction between methotrexate and loxoprofen. Materials and Methods Materials: Loxoprofen sodium and the trans-OH metabolite were kindly gifted by Sankyo Co. (Tokyo, Japan). [3?,5?,7-3H(N)]Methotrexate (851–1073 GBq W mmol) was purchased from Moravek Biochemicals (Brea, CA, USA). Phenylbutazone, salicylate and unlabeled methotrexate (L-(＋)-amethopterin) were obtained from Nacalai Tesque (Kyoto, Japan). Flufenamate, ibuprofen and indomethacin were from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals used were of the highest purity available. Isolation of hOAT1 cDNA: The speciˆc primer-set for hOAT1-1 (sense strand with a Sal I site, 5?-CCG-
TCGACGCCTGGCCCAATGGCCTTTAA-3? corresponding to position 258 to 278; antisense strand with a Not I site, 5?-CCGCGGCCGCAAGGCCCCTTCTCAGTCCTCA-3? corresponding to position 1957 to 1977; accession number AB009697 in GenBank database; ref, 16) was used for reverse transcribed-polymerase chain reaction (PCR) with human kidney poly(A)＋ RNA (Clontech, Palo Alto, CA, USA). The obtained PCR product was ligated into pSPORT1 and recognized as hOAT1-2 from the sequence analysis (accession number AB009698; ref, 16). Then, Human Kidney lTriplExTM Library (Clontech) was screened by hybridization with the PCR product. An isolated positive clone with a 2.1-kb insert was subcloned into pSPORT1 cut with Xba I and Eco RI. The complete sequence of the clone was determined, and the deduced amino acid sequence was identical to hOAT1-2.16) Isolation of hOAT3 cDNA: Human Kidney RapidScreenTM cDNA Library Panels (OriGene Technologies, Rockville, MD, USA) were screened by PCR method with the hOAT3-speciˆc primers (Sense strand, 5?-AATGCCAGCCTGCCCAATGA-3? corresponding to position 365 to 384; antisense strand, 5?-GTCTGCAAGTCCAAGGGCAC-3? corresponding to position 1331 to 1350; accession number AB042505; ref, 11). A positive clone with a 2.2-kb insert was isolated and subcloned into pSPORT1 cut with Xba I and Eco RI. The complete sequence of the clone was determined and the deduced amino acid sequence was identical to that of hOAT3 cloned by Cha et al.11) except amino acid 271, that is lysine in hOAT3 isolated in this study and glutamic acid in their hOAT3. Methotrexate uptake experiment using Xenopus laevis oocytes expressing hOAT1 and hOAT3: An uptake experiment using Xenopus laevis oocytes was performed as previously described.12) The capped RNA encoding hOAT1 or hOAT3 was transcribed from XbaI-linearized pSPORT1 containing hOAT1 or hOAT3, respectively, with T7 RNA polymerase. After 50 nL of water or the cRNA (25 ng) were injected into defolliculated oocytes, the oocytes were maintained in modiˆed Barth's medium (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3 )2, 0.4 mM CaCl2, 0.8 mM MgSO4, 2.4 mM NaHCO3 and 5 mM HEPES) containing 50 mg W mL gentamicin at 189 C. Three days after the injection, the uptake reaction was initiated by incubating the oocytes in 500 mL of uptake buŠer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2 and 5 mM HEPES; pH 7.4) with [3H]methotrexate at 259 C in the presence or absence of an inhibitor for the indicated periods. The uptake reaction was terminated by adding 2 mL of ice-cold uptake buŠer to each well, and the oocytes were washed four times with 2 mL of the ice-cold buŠer. After washing, each oocyte was transferred to a scintillation counting vial and solubilized in 300 mL of 10z
Methotrexate-loxoprofen Interaction with hOAT1 and hOAT3
Fig. 2. Time-dependence of methotrexate uptake by hOAT1-(A) or hOAT3-(B) expressing oocytes. Water-injected (open circle) and hOAT-expressing (closed circle) oocytes were incubated with 500 nM [3H]methotrexate for the indicated periods. The uptake amounts of [3H]methotrexate in each oocyte were determined. Each point represents the mean±S.E. of [3H]methotrexate uptake in 6 to 10 oocytes.
sodium lauryl sulfate. Three milliliters of ACSII (Amersham International, Buckinghamshire, UK) were added to each solubilized oocyte, and the radioactivity was determined in a liquid scintillation counter. Statistical analysis: Multiple comparisons were performed with ScheŠ áe's test following analysis of variance. DiŠerences were considered signiˆcant at Pº0.05. Results Transport characteristics of methotrexate by hOAT1 and hOAT3: To assess the involvement of hOAT1 and hOAT3 in the drug interaction between methotrexate and loxoprofen, we performed the transport experiments using Xenopus oocytes. At the beginning, transport characteristics of methotrexate by hOAT1 and hOAT3 were investigated. Methotrexate uptake by hOAT1- and hOAT3-expressing oocytes was increased
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Fig. 3. Dose-dependence of methotrexate uptake by hOAT1-(A) or hOAT3-(B) expressing oocytes. Water-injected (open circle) and hOAT-expressing (closed circle) oocytes were incubated with [3H]methotrexate at various concentrations for 1 h. The uptake amounts of [3H]methotrexate in each oocyte were determined. Each point represents the mean±S.E. of [3H]methotrexate uptake in 8 to 10 oocytes.
linearly up to 1 h, compared with water-injected oocytes (Fig. 2). Accordingly, further experiments were performed with 1-h incubation. To compare the kinetic parameters of methotrexate transport by hOAT1 and hOAT3, concentration dependence of methotrexate uptake was examined. With the use of nonlinear least squares regression analysis, kinetic parameters were estimated according to the Michaelis-Menten equation. A typical result of three separate experiments is represented in Fig. 3. The apparent Michaelis-Menten constant (Km) and maximal velocity (Vmax ) values were 724.0±74.9 mM and 217.1±44.7 pmol W oocyte W h for hOAT1, and 17.2±3.6 mM and 6.5±1.4 pmol W oocyte W h for hOAT3, respectively (mean±S.E., n＝3). EŠect of loxoprofen and trans-OH metabolite on methotrexate uptake by hOAT1- or hOAT3-expressing oocytes: Next, the inhibitory eŠects of loxoprofen and the trans-OH metabolite on the methotrexate transport by hOAT1 and hOAT3 were assessed. As shown in
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Table 1. EŠects of various NSAIDs on methotrexate uptake by hOAT1- or hOAT3-expressing oocytes. Water-injected and hOAT-expressing oocytes were incubated with 500 nM [3H]methotrexate in the absence (control) or presence of each NSAID at 0.1 mM for 1 h. The uptake amounts of [3H]methotrexate in each oocyte were determined. Each value represents the mean± S.E. of [3H]methotrexate uptake as a percentage of control in 6 to 10 oocytes. Inhibitors Water-injected Control Loxoprofen trans-OH metabolite Ibuprofen Indomethacin Flufenamate Phenylbutazone Salicylate
Methotrexate uptake (z of control) hOAT1
hOAT3
41.0±3.8 100.0±8.7 33.2±2.6* 39.8±4.3* 43.7±4.4* 30.7±2.2* 44.1±2.4* 56.1±5.0* 106.7±5.3
23.4±1.0 100.0±9.5 31.5±1.3* 34.3±2.2* 45.4±2.8* 55.0±2.9* 38.8±3.3* 47.6±5.8* 61.5±5.3*
*Pº0.05, signiˆcantly diŠerent from control values.
Table 1, methotrexate uptake by both transporters was signiˆcantly inhibited by both loxoprofen and the trans-OH metabolite similar to other NSAIDs such as ibuprofen, indomethacin, ‰ufenamate and phenylbutazone. Salicylate at 100 mM showed an inhibitory eŠect on hOAT3, but not on hOAT1 (Table 1). To determine the inhibition concentration (IC50 ) values of loxoprofen and the trans-OH metabolite, we investigated the dose-dependence of their inhibitory eŠects on the methotrexate transport mediated by hOAT1 and hOAT3. From three independent experiments, a representative result is shown in Fig. 4. Loxoprofen and the trans-OH metabolite inhibited methotrexate transport by hOAT1 and hOAT3 dose-dependently, and the estimated IC50 were 27.1±8.7 mM for loxoprofen and 12.2±3.4 mM for the trans-OH metabolite in hOAT1, and 8.7±3.6 mM for loxoprofen and 4.5±1.7 mM for the trans-OH metabolite in hOAT3 (mean±S.E., n＝3). Discussion Several reports represent that coadministration of methotrexate and NSAIDs often induces a marked elevation of blood methotrexate levels and resultant severe toxicity.5,6) The possible mechanisms for this interaction have been suggested to be displacement of protein binding, decreased renal blood ‰ow via inhibition of prostaglandin synthesis by NSAIDs, and competition at renal organic anion transporters.3,12,13) In our hospital, interaction between methotrexate and loxoprofen was suspected. The purpose of the present study is to estimate the involvement of hOAT1 and hOAT3 to the interaction between methotrexate and loxoprofen. To our knowledge, this is the ˆrst report
Fig. 4. Dose-dependence of inhibitory eŠect of loxoprofen and trans-OH metabolite on methotrexate uptake by hOAT1-(A) or hOAT3-(B) expressing oocytes. hOAT-expressing oocytes were incubated with 500 nM [3H]methotrexate in the absence (control) or presence of loxoprofen (open circle) or trans-OH metabolite (closed circle) at various concentrations for 1 h. The uptake amounts of [3H]methotrexate in each oocyte were determined. Each point represents the mean±S.E. of [3H]methotrexate uptake in 5 to 10 oocytes.
representing the inhibitory eŠects of loxoprofen and the trans-OH metabolite on hOAT1 and hOAT3. OAT1 and OAT3 were suggested to play important roles in the renal uptake of anionic drugs in terms of functional and expressional ˆndings.8–10) Although various compounds were reported to be substrates of OAT1 and OAT3, it is important to determine their responsibility for the renal uptake of each substrate. In the case of methotrexate, the studies by Nierenberg17) and Besseghir et al.18) suggested that the renal organic anion transporter mediating basolateral uptake of p-aminohippuric acid and methotrexate is identical in rabbits. Accordingly, OAT1, the predominant transporter of p-aminohippuric acid, is considered to be responsible for renal uptake of methotrexate in rabbits. In rats, it was reported that indomethacin, which is a potent inhibitor for rOAT1 but not for rOAT3, inhibited the tubular clearance of methotrexate to
Methotrexate-loxoprofen Interaction with hOAT1 and hOAT3
one eighth.19) In addition to the evidence that methotrexate is a substrate of rOAT1,7,9) it was thought that rOAT1 works for the basolateral transport of methotrexate in rats. However, there is no information suggesting which transporter is important for renal uptake of methotrexate in humans. In the present study, we compared the transport characteristics of methotrexate by hOAT1 with those by hOAT3. The estimated Km values for hOAT1 and hOAT3 were 724.0 mM and 17.2 mM, respectively, indicating that hOAT1 is a low-a‹nity type transporter of methotrexate, and that hOAT3 is a high-a‹nity type. When high-dose therapy of methotrexate is performed for treatment of malignancy, the blood levels of methotrexate reach 1 mM. Accordingly, it is speculated that hOAT1 and hOAT3 would work at high concentrations of serum methotrexate and at low concentrations, respectively. The contribution of hOAT1 and hOAT3 to tubular secretion of methotrexate has remained to be elucidated. This study shows that loxoprofen and the trans-OH metabolite have strong inhibitory eŠects on hOAT1 and hOAT3 like other NSAIDs (Table 1). The inhibition degrees by the NSAIDs shown in Table 1 are comparable with previous reports.12,13,20,21) Because Sosogi et al. reported that 1-h incubation with several NSAIDs at 100 mM did not aŠect ATP content of IEC-6 cells, it is likely that the decrease of methotrexate uptake is independent of uncoupling eŠect of NSAIDs.22) The estimated IC50 values of loxoprofen and the trans-OH metabolite for hOAT1 and hOAT3 were similar to those of other NSAIDs, such as ibuprofen, indomethacin, ketoprofen, diclofenac, etodolac, naproxen, piroxicam for hOAT1 and hOAT3.20,21) Usually, loxoprofen is administered orally at 180 mg per day divided into three doses, or is administered in potions of 60 to 120 mg in Japan. The administered loxoprofen immediately disappears from blood (its elimination half life is about 1.2 h), and is mainly excreted into urine as glucuronide conjugates of loxoprofen and the trans-OH metabolite. When one tablet of loxoprofen (60 mg as loxoprofen anhydrous) is administered, maximal plasma concentrations of loxoprofen and the trans-OH metabolite reach about 20 mM and 3 mM, respectively, suggesting that their IC50 values for hOAT1 and hOAT3 are comparable with their therapeutic range. However, because protein bindings of loxoprofen and the trans-OH metabolite are greater than 90z, unbound concentrations of them are lower than the IC50 values. In the present clinical case, the albumin concentration of the dL, following to liver patient had decreased to 2.6 g W insu‹ciency, suggesting that the free fraction ratio could be increased up to two-fold. Also, the decrease in glucuronidation activity against loxoprofen and the trans-OH metabolite in addition to the increase of
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unbound fractions of the two compounds might have in‰uenced the renal uptake of methotrexate via hOAT1 and hOAT3. Furthermore, eŠect of the trans-OH metabolite on renal blood ‰ow should be considered. There are two reports examining whether NSAIDs are substrates of hOATs.20,21) The results for some NSAIDs are controversial. Mulato et al. showed that ibuprofen and ketoprofen were not transported by hOAT1.20) However, Khamdang et al. reported that hOAT1 and hOAT3 mediated transport of these NSAIDs.21) In addition, Khamdang et al. represented that the hOATs transported indomethacin and salicylate.21) The mechanisms of interaction between NSAIDs and OATs should be elucidated in the future. According to the reports by Dannhardt and Kiefer,23) Mulato et al.20) and Khamdang et al,21) several NSAIDs have similar inhibition constants against cyclooxygenases, hOAT1 and hOAT3. There are many reports representing that NSAIDs interact with various drugs via receptors, serum proteins and via metabolic enzymes, in addition to via renal organic anion transporters.24) Because NSAIDs are very commonly used, new NSAIDs that do not interact with proteins except cyclooxygenases are desirable. In future studies, analysis on the molecular structures of cyclooxygenases and other proteins would help the development of NSAIDs that avoid the drug interactions. In summary, the drug interaction between methotrexate and loxoprofen was suspected in a patient with Hodgkin's disease in our hospital. The experimental data shows that loxoprofen and the trans-OH metabolite have inhibitory eŠects on the methotrexate transport by hOAT1 and hOAT3 like other NSAIDs. It is suggested that loxoprofen induces the reduction of the elimination rate of methotrexate by inhibiting hOAT1 and hOAT3. Acknowledgements: This work was supported by a Grant-in-aid for Research on Human Genome, Tissue Engineering, and Food Biotechnology from the Ministry of Health, Labor, and Welfare of Japan (H12Genome-019), a Grant-in-Aid for Scientiˆc Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Japan Research Foundation for Clinical Pharmacology. References 1)
2)
3)
Frei, E., JaŠe, N., Tattersall, M. H., Pitman, S. and Parker, L.: New approaches to cancer chemotherapy with methotrexate. N. Engl. J. Med., 292: 846–851 (1975). Jolivet, J., Cowan, K. H., Curt, G. A., Clendeninn, N. J. and Chabner, B. A.: The pharmacology and clinical use of methotrexate. N. Engl. J. Med., 309: 1094–1104 (1983). Bannwarth, B., P áehourcq, F., Schaeverbeke, T. and
374
4)
5)
6)
7)
8)
9)
10)
11)
12)
13)
14)
Yuichi UWAI, et al.
Dehais, J.: Clinical pharmacokinetics of low-dose pulse methotrexate in rheumatoid arthritis. Clin. Pharmacokinet., 30: 194–210 (1996). Liegler, D. G., Henderson, E. S., Hahn, M. A. and Oliverio, V. T.: The eŠect of organic acids on renal clearance of methotrexate in man. Clin. Pharmacol. Ther., 10: 849–857 (1969). Maiche, A. G.: Acute renal failure due to concomitant action of methotrexate and indomethacin. Lancet, 1: 1390 (1986). Thyss, A., Milano, G., Kubar, J., Namer, M. and Schneider, M.: Clinical and pharmacokinetic evidence of a life-threatening interaction between methotrexate and ketoprofen. Lancet, 1: 256–258 (1986). Sekine, T., Watanabe, N., Hosoyamada, M., Kanai, Y. and Endou, H.: Expression cloning and characterization of a novel multispeciˆc organic anion transporter. J. Biol. Chem., 272: 18526–18529 (1997). Motohashi, H., Sakurai, Y., Saito, H., Masuda, S., Urakami, Y., Goto, M., Fukatsu, A., Ogawa, O. and Inui, K.: Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J. Am. Soc. Nephrol., 13: 866–874 (2002). Uwai, Y., Okuda, M., Takami, K., Hashimoto, Y. and Inui, K.: Functional characterization of the rat multispeciˆc organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney. FEBS Lett., 438: 321–324 (1998). Hasegawa, M., Kusuhara, H., Sugiyama, D., Ito, K., Ueda, S., Endou, H. and Sugiyama, Y.: Functional involvement of rat organic anion transporter 3 (rOat3; Slc22a8 ) in the renal uptake of organic anions. J. Pharmacol. Exp. Ther., 300: 746–753 (2002). Cha, S. H., Sekine, T., Fukushima, J., Kanai, Y., Kobayashi, Y., Goya, T. and Endou, H.: Identiˆcation and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol. Pharmacol., 59: 1277–1286 (2001). Uwai, Y., Saito, H. and Inui, K.: Interaction between methotrexate and nonsteroidal anti-in‰ammatory drugs in organic anion transporter. Eur. J. Pharmacol., 409: 31–36 (2000). Takeda, M., Khamdang, S., Narikawa, S., Kimura, H., Hosoyamada, M., Cha, S. H., Sekine, T. and Endou, H.: Characterization of methotrexate transport and its drug interactions with human organic anion transporters. J. Pharmacol. Exp. Ther., 302: 666–671 (2002). Matsuda, K., Tanaka, Y., Ushiyama, S., Ohnishi, K. and Yamazaki, M.: Inhibition of prostaglandin synthe-
15)
16)
17)
18)
19)
20)
21)
22)
23)
24)
sis by sodium 2-[4-(2-oxocyclopentylmethyl)phenyl] propionate dihydrate (CS-600), a new anti-in‰ammatory drug, and its active metabolite in vitro and in vivo. Biochem. Pharmacol., 33: 2473–2478 (1984). Kawano, S., Tsuji, S., Hayashi, N., Takei, Y., Nagano, K., Fusamoto, H. and Kamada, T.: EŠects of loxoprofen sodium, a newly synthesized non-steroidal anti-in‰ammatory drug, and indomethacin on gastric mucosal haemodynamics in the human. J. Gastroenterol. Hepatol., 10: 81–85 (1995). Hosoyamada, M., Sekine, T., Kanai, Y. and Endou, H.: Molecular cloning and functional expression of a multispeciˆc organic anion transporter from human kidney. Am. J. Physiol., 276: F122–F128 (1999). Nierenberg, D. W.: Drug inhibition of penicillin tubular secretion: concordance between in vitro and clinical ˆndings. J. Pharmacol. Exp. Ther., 240: 712–716 (1987). Besseghir, K., Mosig, D. and Roch-Ramel, F.: Transport of methotrexate by the in vitro isolated rabbit proximal tubule. J. Pharmacol. Exp. Ther., 250: 688–695 (1989). Statkevich, P., Fournier, D. J. and Sweeney, K. R.: Characterization of methotrexate elimination and interaction with indomethacin and ‰urbiprofen in the isolated perfused rat kidney. J. Pharmacol. Exp. Ther., 265: 1118–1124 (1993). Mulato, A. S., Ho, E. S. and Cihlar, T.: Nonsteroidal anti-in‰ammatory drugs e‹ciently reduce the transport and cytotoxicity of adefovir mediated by the human renal organic anion transporter 1. J. Pharmacol. Exp. Ther., 295: 10–15 (2000). Khamdang, S., Takeda, M., Noshiro, R., Narikawa, S., Enomoto, A., Anzai, N., Piyachaturawat, P. and Endou, H.: Interactions of human organic anion transporters and human organic cation transporters with nonsteroidal anti-in‰ammatory drugs. J. Pharmacol. Exp. Ther., 303: 534–539 (2002). Sosogi, A., Gao, F., Tomimatsu, T., Hirata, K. and Horie, T.: Non-steroidal anti-in‰ammatory drugs aŠect the methotrexate transport in IEC-6 cells. Life Sci., 73: 437–446 (2003). Dannhardt, G. and Kiefer, W.: Cyclooxygenase inhibitors-current status and future prospects. Eur. J. Med. Chem., 36: 109–126 (2001). Johnson, A. G., Seidemann, P. and Day, R. O.: NSAID-related adverse drug interactions with clinical relevance. An update. Int. J. Clin. Pharmacol. Ther., 32: 509–532 (1994).