Advertisement
Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Characterization of Methotrexate Transport and Its Drug Interactions with Human Organic Anion Transporters

Michio Takeda, Suparat Khamdang, Shinichi Narikawa, Hiroaki Kimura, Makoto Hosoyamada, Seok Ho Cha, Takashi Sekine and Hitoshi Endou
Journal of Pharmacology and Experimental Therapeutics August 2002, 302 (2) 666-671; DOI: https://doi.org/10.1124/jpet.102.034330

Abstract

Life-threatening drug interactions are known to occur between methotrexate and nonsteroidal anti-inflammatory drugs (NSAIDs), probenecid, and penicillin G. The purpose of this study was to characterize methotrexate transport, as well as to determine the site and the mechanism of drug interactions in the proximal tubule. Mouse proximal tubule cells stably expressing basolateral human organic anion transporters (hOAT1 and hOAT3) and apical hOAT (hOAT4) were established. The Km values for hOAT1-, hOAT3-, and hOAT4-mediated methotrexate uptake were 553.8 μM, 21.1 μM, and 17.8 μM, respectively. NSAIDs (salicylate, ibuprofen, ketoprofen, phenylbutazone, piroxicam, and indomethacin), probenecid, and penicillin G dose dependently inhibited methotrexate uptake mediated by hOAT1, hOAT3, and hOAT4. Kinetic analysis of inhibitory effects of these drugs on hOAT3-mediated methotrexate uptake revealed that these inhibitions were competitive. TheKi values for the effects of salicylate, phenylbutazone, indomethacin, and probenecid on hOAT3-mediated methotrexate uptake were comparable with therapeutically relevant plasma concentrations of unbound drugs. In addition, in the presence of human serum albumin, the Ki values were comparable with therapeutically relevant total plasma concentrations of drugs. In conclusion, these results suggest that methotrexate is taken up via hOAT3 and hOAT1 at the basolateral side of the proximal tubule and effluxed or taken up at the apical side via hOAT4. In addition, hOAT1, hOAT3, and hOAT4 are the sites of drug interactions between methotrexate and NSAIDs, probenecid, and penicillin G. Furthermore, it was predicted that hOAT3 is the site of drug interactions between methotrexate and salicylate, phenylbutazone, indomethacin, and probenecid in vivo.

Methotrexate is widely used at high dosages in the treatment of malignancies, whereas it is used at low dosages in rheumatoid arthritis. Methotrexate is eliminated almost entirely in an unchanged form in urine, which involves glomerular filtration and active tubular secretion (Shen and Azarnoff, 1978). Therefore, renal insufficiency or drug interactions, which reduce the clearance of methotrexate, are potentially toxic events.

Interactions between methotrexate and drugs including nonsteroidal anti-inflammatory drugs (NSAIDs), probenecid, and penicillin G have been reported by several groups of investigators (Ellison and Servi, 1985; Thyss et al., 1986; Basin et al., 1991; Frenia and Long, 1992;Tracy et al., 1992; Kremer and Hamilton, 1995). Severe and even life-threatening interactions have been observed, including bone marrow suppression and acute renal failure (Ellison and Servi, 1985; Thyss et al., 1986; Basin et al., 1991; Frenia and Long, 1992). The interactions may have been caused by protein binding displacement, inhibitory effects on the renal secretion of methotrexate, and a decline in glomerular filtration as a result of inhibition of prostaglandin synthesis (Tracy et al., 1992; Kremer and Hamilton, 1995). Among these possible causes, the renal tubular secretion of methotrexate has been thought to be a major site for drug interaction (Frenia and Long, 1992).

Two different types of human multispecific OATs (hOAT1 and hOAT3) were recently isolated (Reid et al., 1998; Hosoyamada et al., 1999; Cha et al., 2001). In the kidney, hOAT1 and hOAT3 are localized at the basolateral membrane of the proximal tubule (Hosoyamada et al., 1999;Cha et al., 2001). Since hOAT1 and hOAT3 mediate the transport of various drugs, endogenous substances, and xenobiotics, these transporters are considered to be responsible for the basolateral uptake of organic anions in renal epithelial cells. On the other hand, OAT-K1 (Saito et al., 1996; Masuda et al., 1999b), OAT-K2 (Masuda et al., 1999a), hOAT4 (Cha et al., 2000), organic anion-transporting peptide 1 (oatp1) (Jacquemin et al., 1994), oatp3 (Abe et al., 1998), multidrug-resistance protein 2 (MRP2) (Leier et al., 2000), and human type I sodium-dependent inorganic phosphate transporter (NPT1) (Uchino et al., 2000) were isolated and identified as transporters of anionic drugs and substances at the apical membrane of the proximal tubule.

The purpose of this study was to characterize methotrexate transport in the proximal tubule, as well as to determine the site and mechanism of interactions between methotrexate and NSAIDs, probenecid, and penicillin G using the second portion of the proximal tubule (S2) cells stably expressing hOAT1, hOAT3, and hOAT4 (S2 hOAT1, S2 hOAT3, and S2 hOAT4, respectively).

Experimental Procedures

Materials.

[3H]Methotrexate (547.6 GBq/mmol) was purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). Methotrexate, various NSAIDs, probenecid, penicillin G, transferrin, and human serum albumin were obtained from Sigma Chemical Co. (St. Louis, MO). Other materials used included fetal bovine serum, trypsin and geneticin from Invitrogen (Carlsbad, CA), recombinant epidermal growth factor from Wakunaga (Hiroshima, Japan), insulin from Shimizu (Shizuoka, Japan), RITC 80–7 culture medium from Iwaki Co. (Tokyo, Japan), and TfX-50 from Promega (Madison, WI).

Cell Culture and Establishment of S2 hOAT1, S2 hOAT3, and S2 hOAT4.

S2cells, derived from transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene, were established as described previously by us (Takeda et al., 1999). S2 is the segment of the proximal tubule in which hOAT1, hOAT3, and hOAT4 were localized (Hosoyamada et al., 1999; Cha et al., 2001; Babu et al., 2002). The full-length cDNAs of hOAT1, hOAT3, and hOAT4 were subcloned into pcDNA 3.1 (Invitrogen, Carlsbad, CA), a mammalian expression vector. S2 hOAT1, S2 hOAT3, and S2 hOAT4 were obtained by transfecting S2 cells with pcDNA3.1-hOAT1, pcDNA3.1-hOAT3, and pcDNA3.1-hOAT4 coupled with pSV2neo, a neomycin-resistant gene, using TfX-50 according to the manufacturer's instructions. S2 cells transfected with pcDNA3.1 lacking an insert, and pSV2neo were designated as S2 pcDNA 3.1 and used as a control (mock). These cells were grown in a humidified incubator at 33°C and under 5% CO2 using RITC 80–7 medium containing 5% fetal bovine serum, 10 μg/ml transferrin, 0.08 U/ml insulin, 10 ng/ml recombinant epidermal growth factor, and 400 μg/ml Geneticin. The cells were subcultured in a medium containing 0.05% trypsin-EDTA solution (containing 137 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 4 mM NaHCO3, 0.5 mM EDTA, and 5 mM Hepes, pH 7.2) and used for 10 to ∼35 passages. Clonal cells were isolated using a cloning cylinder and screened by determining the uptake of the optimal substrate for each transporter, i.e.,para-[14C]aminohippuric acid for hOAT1 (Hosoyamada et al., 1999) and [3H]estrone sulfate for hOAT3 (Cha et al., 2001) and hOAT4 (Cha et al., 2000). The S2 monolayer was determined to be leaky, based on the results of a study in which we estimated paracellular secretion from cells cultured on a permeable support, usingd-[3H]mannitol as an indicator. In addition, vertical sections of S2 hOAT1, S2 hOAT3, and S2 hOAT4 stained with polyclonal antibodies against hOAT1, hOAT3, and hOAT4, respectively, showed that the subcellular localization of hOAT1, hOAT3, and hOAT4 proteins was mainly on the cell membrane. Both the basolateral and apical portions of the membrane showed positive staining. Therefore, the cells were cultured on a solid support for these experiments.

Uptake Experiments.

Uptake experiments were performed as previously described (Takeda et al., 1999). As described above, the cells were cultured at 33°C, which is suitable for cell growth for cells encoding temperature-sensitive simian virus 40 large T-antigen gene (Takeda et al., 1999). In contrast, uptake experiments were performed at 37°C, which is commonly used for evaluating transporter activities. There was no difference in the amount of uptake between 33°C and 37°C (data not shown). Based on these results, the cells were cultured at 33°C, whereas uptake experiments were performed at 37°C. The S2 cells were seeded in 24-well tissue culture plates at a cell density of 1 × 105cells/well. After the cells were cultured for 2 days, the cells were washed three times with Dulbecco's modified phosphate-buffered saline (D-PBS) solution (containing 137 mM NaCl, 3 mM KCl, 8 mM NaHPO4, 1 mM KH2PO4, 1 mM CaCl2, and 0.5 mM MgCl2, pH 7.4), and then preincubated in the same solution for 10 min in a water bath at 37°C. The cells were then incubated in D-PBS containing [3H]methotrexate at various concentrations as indicated in each experiment. The uptake was stopped by the addition of ice-cold D-PBS, and the cells were washed three times with the same solution. The cells in each well were lysed with 0.5 ml of 0.1 N sodium hydroxide and 2.5 ml of Aquasol-2, and radioactivity was determined using a β-scintillation counter (Aloka, Tokyo, Japan; LSC-3100).

Inhibition Study.

To evaluate the inhibitory effects of various NSAIDs, probenecid, and penicillin G on methotrexate uptake mediated by hOAT1, hOAT3, and hOAT4, S2 hOAT1, S2 hOAT3, and S2 hOAT4 were incubated in a solution containing [3H]methotrexate in the absence or presence of various drugs at 37°C. Considering theKm values for hOAT1-, hOAT3-, and hOAT4-mediated methotrexate uptake, hOAT1-, hOAT3-, and hOAT4-mediated methotrexate uptake was estimated using a methotrexate concentration of 1 μM (hOAT1) or 100 nM (hOAT3 and hOAT4). In addition, based on the results of time course experiments (Fig.1), hOAT3-mediated methotrexate uptake was evaluated during the 2-min incubation. On the other hand, since the specific methotrexate uptake activity, represented as the amount of hOAT1- and hOAT4-mediated methotrexate uptake subtracted by that by mock, was about two times larger during the 15-min incubation than that during the 2-min incubation, we chose 15 min as the incubation time for the inhibition experiments. Salicylate, probenecid, and penicillin G were dissolved in distilled water. Ibuprofen, ketoprofen, phenylbutazone, piroxicam, and indomethacin were dissolved in dimethyl sulfoxide and diluted with the incubation medium. The final concentration of dimethyl sulfoxide in the incubation medium was adjusted to less than 1%.

Figure 1
Figure 1

Time course of methotrexate uptake in S2hOAT1 (A), S2 hOAT3 (B), and S2 hOAT4 (C). These cells were incubated in a solution containing 1 μM [3H]methotrexate (hOAT1) or 100 nM [3H]methotrexate (hOAT3 and hOAT4) for 1 to 30 min at 37°C. The values for the specific uptake in S2 hOAT1, S2 hOAT3, and S2 hOAT4 (open squares) were obtained by subtracting that in mock (closed circles) from those in S2 hOAT1, S2 hOAT3, and S2 hOAT4 (open circles). Each value represents the mean ± S.E. of four determinations from one typical experiment of the two separate experiments.

Kinetic Analysis.

After preincubation as described above, S2 hOAT3 was incubated in D-PBS containing [3H]methotrexate at different concentrations in the absence or presence of various drugs for 2 min. For the kinetic analysis of inhibitory effects, double reciprocal plot analyses were performed based on methotrexate under each condition (Apiwattanakul et al., 1999). When the inhibition was competitive, theKi values were calculated based on the following equation,Ki=concentration of inhibitor/ [(Km of methotrexate with inhibitor/ Km of methotrexate without inhibitor)−1]

Statistical Analysis.

Data are expressed as means ± S.E. Statistical differences were determined using one-way ANOVA with Dunnett's post hoc test. Differences were considered significant atP < 0.05.

Results

Methotrexate Uptake.

To evaluate the time-dependent uptake of methotrexate in S2 hOAT1, S2 hOAT3 and S2 hOAT4, S2 cell monolayers were incubated in a solution containing 1 μM methotrexate (hOAT1) or 100 nM methotrexate (hOAT3 and hOAT4) for various periods at 37°C. As shown in Fig. 1, when the amount of methotrexate uptake in mock was subtracted from those in S2 hOAT1, S2 hOAT3, and S2 hOAT4, the specific uptake in S2 hOAT1 (Fig. 1A) and S2 hOAT4 (Fig. 1C) increased in a time-dependent manner and reached steady state, whereas that in S2 hOAT3 (Fig. 1B) decreased after reaching peak. The latter may be due to the observation that nonspecific methotrexate efflux overwhelmed hOAT3-mediated methotrexate uptake depending on the nature of monoclonal cells into which hOAT3 cDNA was transfected.

The kinetics of methotrexate uptake was examined to evaluate the pharmacological characteristics of hOAT1, hOAT3, and hOAT4 on methotrexate transport. The concentration-dependent uptake of methotrexate was observed in S2 hOAT1, S2hOAT3, and S2 hOAT4 after subtraction of uptake by mock (data not shown). As shown in Fig. 2, Eadie-Hofstee plot analysis of hOAT1-, hOAT3-, and hOAT4-mediated methotrexate uptake gave a single straight line. The estimatedKm values of methotrexate uptake by hOAT1, hOAT3, and hOAT4 were 553.8 ± 43.2 μM, 21.1 ± 2.8 μM, and 17.8 ± 1.6 μM, respectively (from three determinations in one typical experiment of two separate experiments). These results suggest that hOAT1, hOAT3, and hOAT4 mediate transport of methotrexate.

Figure 2
Figure 2

Eadie-Hofstee plot analysis of methotrexate uptake in S2 hOAT1 (A), S2 hOAT3 (B), and S2hOAT4 (C). S2 hOAT1, S2 hOAT3, S2hOAT4, and mock were incubated with various concentrations of [3H]methotrexate for 2 min at 37°C. The values for the uptake in mock were subtracted from those in S2 hOAT1, S2 hOAT3, and S2 hOAT4. Each value represents mean ± S.E. of three determinations from one typical experiment of the two separate experiments.

Inhibitory Effects on Methotrexate Uptake.

We examined the effects of various drugs on methotrexate uptake mediated by hOAT1, hOAT3, and hOAT4. The drugs used were NSAIDs (salicylate, ibuprofen, ketoprofen, phenylbutazone, piroxicam, and indomethacin), probenecid, and penicillin G. These drugs inhibited methotrexate uptake mediated by hOAT1, hOAT3, and hOAT4 in a dose-dependent manner, whereas penicillin G did not inhibit hOAT1-mediated methotrexate uptake and salicylate did not inhibit hOAT4-mediated methotrexate uptake (data not shown). The effects of these NSAIDs on methotrexate uptake by hOAT1, hOAT3, and hOAT4 are listed in Table 1.

View this table:
Table 1

The effects of various NSAIDs on methotrexate uptake mediated by hOAT1, hOAT3, and hOAT4

Kinetic Analysis of Inhibition.

To further elucidate the inhibitory effects of NSAIDs, probenecid, and penicillin G on hOAT3-mediated methotrexate uptake, we have examined the inhibitory effects of these drugs at different concentrations of methotrexate. Typical results are shown in Fig. 3. Analysis of a Lineweaver-Burk plot of the effects of salicylate (Fig.3A), phenylbutazone (Fig. 3B), indomethacin (Fig. 3C), and probenecid (Fig. 3D) on the hOAT3-mediated methotrexate uptake demonstrated that these drugs inhibited the methotrexate uptake in a competitive manner. The inhibitory effects of ibuprofen, ketoprofen, piroxicam, and penicillin G were also competitive (data not shown). TheKi values for each drug tested are shown in Table 2. In addition, we performed a kinetic analysis of the inhibitory effects of salicylate, phenylbutazone, indomethacin, and probenecid on hOAT3-mediated methotrexate uptake in the presence of 5% human serum albumin. The inhibitory effects of these drugs on hOAT3-mediated methotrexate uptake in the presence of 5% human serum albumin were also competitive, and the Ki values are listed in Table 2.

Figure 3
Figure 3

Kinetic analysis of inhibition by various drugs on methotrexate uptake in S2 hOAT3. Methotrexate uptake was measured at various concentrations of [3H]methotrexate in the presence or absence of various drugs (A, salicylate; B, phenylbutazone; C, indomethacin; and D, probenecid) for 2 min at 37°C, and Lineweaver-Burk plot analyses were performed. The values for the uptake in mock were subtracted from those in S2hOAT3. Each value represents the mean ± S.E. of four determinations from one typical experiment.

View this table:
Table 2

The Ki values of various drugs that inhibit methotrexate uptake in S2 hOAT3

Discussion

hOAT1 and hOAT3 were recently cloned and characterized as multispecific OATs, and were found to mediate active transport of organic anions from the interstitium to the cells in the basolateral membrane of the proximal tubule (Hosoyamada et al., 1999; Cha et al., 2001). Some differences in characteristics exist between hOAT1 and hOAT3, such as substrate specificity and localization: hOAT1 at the basolateral side of the S2 segment of the proximal tubule (Hosoyamada et al., 1999) versus hOAT3 at the first, second, and third segments (S1, S2, and S3) of the proximal tubule (Cha et al., 2001). In addition, hOAT1, but not hOAT3, exhibits transport properties as an exchanger (Hosoyamada et al., 1999;Cha et al., 2001). On the other hand, hOAT4 is localized to the apical membrane of the proximal tubule (Babu et al., 2002), and hOAT4 exhibits a relatively narrow substrate specificity compared with hOAT1 and hOAT3 (Cha et al., 2000).

Carrier-mediated transport of methotrexate in the basolateral side of the proximal tubule was suggested based on the results of the experiments using renal slices (Nierenberg, 1983; Williams et al., 1984) or isolated proximal tubule (Besseghir et al., 1989). The current results suggest that hOAT3 and hOAT1 mediate the transport of methotrexate in the basolateral side of the proximal tubule. Human OAT cloned by Lu was designated as human kidney p-aminohippurate transporter (hPAHT) (Lu et al., 1999), and there was 98.2% amino acid sequence homology between hPAHT and hOAT1 (data not shown). In contrast to hOAT1, hPAHT exhibited no significant methotrexate uptake activity. The discrepancy in methotrexate uptake activity between hOAT1 and hPAHT may be due to this small difference in amino acid sequence or the experimental conditions. To clarify this, a site-directed mutagenesis study should be performed. The current results were also consistent with the previous results that rat OAT1 mediates methotrexate uptake (Uwai et al., 2000). In addition, hOAT2, which was recently identified to be localized to the basolateral side of the proximal tubule, showed no significant uptake activity of methotrexate (M. Takeda, unpublished observation). This is in contrast to the recent report that cells expressing hOAT2 showed uptake of methotrexate (Sun et al., 2001). The reason for the discrepancy remains unknown, and further studies should be performed. It is also possible that unidentified OATs other than hOAT3 and hOAT1 are also involved in methotrexate transport in the basolateral membrane of the proximal tubule.

In addition to hOAT4, human transporters mediating the transport of organic anions in the apical membrane of the proximal tubule have been cloned, including MRP2 (Leier et al., 2000) and NPT1 (Uchino et al., 2000). Moreover, human MRP2 was shown to mediate the efflux of methotrexate using stable cells (Hooijberg et al., 1999). Thus, the relative contribution of hOAT4 and MRP2 to apical methotrexate transport should be clarified. In addition to human transporters, rodent transporters mediating the apical transport of organic anions have been cloned, including OAT-K1 (Saito et al., 1996; Masuda et al., 1999b), OAT-K2 (Masuda et al., 1999a), oatp1 (Jacquemin et al., 1994), and oatp3 (Abe et al., 1998). Among them, OAT-K1 and OAT-K2 were reported to mediate bidirectional methotrexate transport (Saito et al., 1996; Masuda et al., 1999a,b), and theKm value for OAT-K1-mediated methotrexate transport was determined to be 1.0 μM (Masuda et al., 1997). Thus, the cloning of human homologs of these rodent transporters and the functional analysis for the role of these transporters in apical methotrexate transport should be performed

Since various drugs tested in this study dose dependently inhibited hOAT1-, hOAT3-, and hOAT4-mediated methotrexate uptake, it was suggested that hOAT1, hOAT3, and hOAT4 are sites of drug interactions between methotrexate and these drugs. Among these interactions, it appears that those between methotrexate and salicylate, phenylbutazone, indomethacin, or probenecid at hOAT3 may have clinical significance, as follows. The total and unbound plasma concentrations of various drugs are as listed in the Table 2. Therapeutically relevant concentrations of unbound drugs in the plasma are thought to be within 5-fold of the maximum steady-state concentrations of unbound drugs in the plasma (Zhang et al., 2000). Comparing the Kivalues with therapeutically relevant concentrations of unbound drugs in the plasma, those for salicylate, phenylbutazone, indomethacin, and probenecid were comparable. In addition, theKi values obtained in the presence of 5% human serum albumin were comparable with total plasma concentrations of salicylate, phenylbutazone, indomethacin, or probenecid. Based on these, it was predicted that these drugs could inhibit the hOAT3-mediated methotrexate uptake in vivo, leading to the increased plasma concentration of methotrexate.

Uwai et al. (2000) have already demonstrated that the IC50 values of salicylate, ketoprofen, and indomethacin for rat OAT1-mediated methotrexate uptake were 1410, 0.5, and 2.7 μM, respectively. However, we found that the IC50 values of salicylate, ketoprofen, and indomethacin for hOAT1-mediated methotrexate uptake were >2000, 1200, and >2000 μM, respectively (data not shown). This discrepancy in findings may be due to the species difference, i.e., between rats and humans. Compared with rat OAT1, the amino acid sequence of hOAT1 exhibited 86.0% homology (Sekine et al., 1997). On the other hand, the IC50 values of ibuprofen, ketoprofen, piroxicam, indomethacin, and probenecid for adefovir uptake in Chinese hamster ovary cells stably expressing hOAT1 were 8.0, 1.3, 20.5, 3.0, and 7.4 μM, respectively (Mulato et al., 2000). The results were similar to those for rat OAT1-mediated methotrexate uptake (Uwai et al., 2000), but much lower than those for hOAT1-mediated methotrexate uptake. The discrepancy may be due to the fact that theKm value for hOAT1-mediated adefovir uptake was 23.8 μM (Ho et al., 2000), whereas that for hOAT1-mediated methotrexate uptake was 553.8 μM.

At present, the mechanism for the interaction between methotrexate and ibuprofen, ketoprofen, or piroxicam, or penicillin G remains unclear. It is possible that other transporters mediate methotrexate transport and are responsible for drug interactions. Since protein-binding displacement of methotrexate by NSAIDs except salicylate is generally thought to account for more than a small transient increase in free methotrexate concentration, protein binding changes would probably not be of major clinical significance (Taylor and Halprin, 1977). In addition, since prostaglandin synthesis is stimulated to maintain the glomerular filtration rate in the setting of prerenal volume contraction (Ciabattoni et al., 1984; Dunn, 1984), NSAIDs may decrease glomerular filtration of methotrexate as a result of inhibition of prostaglandin synthesis (Kremer and Hamilton, 1995). However, patients who do not have a condition that predisposes them to activation of renal prostaglandins would not have a decrease in renal function following treatment with NSAIDs (Kremer and Hamilton, 1995).

In conclusion, these results suggest that methotrexate is taken up in the basolateral membrane by hOAT3 and hOAT1, and taken up or effluxed in the apical membrane of the proximal tubule via hOAT4. In addition, hOAT1, hOAT3, and hOAT4 were sites of drug interactions between methotrexate and NSAIDs, probenecid, and penicillin G. Furthermore, it was predicted that hOAT3 is a site of drug interactions between methotrexate and salicylate, phenylbutazone, indomethacin, or probenecid in vivo. Stable cells established in this study provide a good opportunity for evaluating drug interaction of methotrexate and newly developed drugs, particularly NSAIDs, in the preclinical stage.

Footnotes

  • This study was supported in part by grants-in-aid from the Ministry of Education, Sports, Science and Technology (11671048, 11694310, and 13671128), the Science Research Promotion Fund of the Japan Private School Promotion Foundation, and Research on Health Sciences focusing on Drug Innovation from Japan Health Sciences Foundation.

  • DOI: 10.1124/jpet.102.034330

  • Abbreviations:
    NSAID
    nonsteroidal anti-inflammatory drug
    OAT
    organic anion transporter
    hOAT
    human organic anion transporter
    OAT-K1
    OAT-K2, renal organic anion transporter 1 and 2
    oatp1
    oatp2, organic anion-transporting polypeptide 1 and 2
    MRP2
    multidrug resistance protein 2
    NPT1
    human type I sodium-dependent inorganic phosphate transporter
    S1
    S2, S3, the first, second, and third segment of proximal tubule
    D-PBS
    Dulbecco's modified phosphate-buffered saline
    hPAHT
    human kidneyp-aminohippurate transporter
    • Received February 20, 2002.
    • Accepted April 8, 2002.

References

  1. ↵
    1. Abe T,
    2. Kakyo M,
    3. Sakagami B,
    4. Tokui T,
    5. Nishio T,
    6. Tanemoto M,
    7. Nomura H,
    8. Hebert SC,
    9. Matsuno S,
    10. Kondo H,
    11. et al.
    (1998) Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2. J Biol Chem 273:22395–22401.
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Apiwattanakul N,
    2. Sekine T,
    3. Chairoungdua A,
    4. Kanai Y,
    5. Nakajima N,
    6. Sophasan S,
    7. Endou H
    (1999) Transport properties of nonsteroidal anti-inflammatory drugs by organic anion transporter 1 expressed in Xenopus laevis oocytes. Mol Pharmacol 55:847–854.
    OpenUrlAbstract/FREE Full Text
  3. ↵
  4. ↵
    1. Basin KS,
    2. Escalante A,
    3. Beardmore TD
    (1991) Severe pancytopenia in a patient taking low dose methotrexate and probenecid. J Rheumatol 18:609–610.
    OpenUrlPubMed
  5. ↵
    1. Hardman JG,
    2. Limbird LE
    1. Benet LZ,
    2. Oie S,
    3. Schwartz JB
    (1996) Design and optimization of dosage regimens; pharmacokinetic data. in Goodman & Guilman's The Pharmacological Basis of Therapeutics, eds Hardman JG, Limbird LE (McGraw-Hill, New York), 9th ed, pp 1707–1792.
  6. ↵
    1. Besseghir K,
    2. Mosig D,
    3. Roch-Ramel F
    (1989) Transport of methotrexate by the in vitro isolated rabbit proximal tubule. J Pharmacol Exp Ther 250:688–695.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Cha SH,
    2. Sekine T,
    3. Fukushima JI,
    4. Kanai Y,
    5. Kobayashi Y,
    6. Goya T,
    7. 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
  8. ↵
    1. Cha SH,
    2. Sekine T,
    3. Kusuhara H,
    4. Yu E,
    5. Kim YJ,
    6. Kim DK,
    7. Sugiyama Y,
    8. Kanai Y,
    9. Endou H
    (2000) Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta. J Biol Chem 275:4507–4512.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Ciabattoni G,
    2. Cinotti GA,
    3. Pierucci A,
    4. Simonetti BM,
    5. Manzi M,
    6. Pugliese F,
    7. Barsotti P,
    8. Pecci G,
    9. Taggi F,
    10. Patrono C
    (1984) Effects of sulindac and ibuprofen in patients with chronic glomerular disease. Evidence for the dependence of renal function on prostaglandin. N Engl J Med 310:279–283.
    OpenUrlPubMed
  10. ↵
    1. Dunn MJ
    (1984) Nonsteroidal antiinflammtory drugs and renal function. Annu Rev Med 35:411–428.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Ellison NM,
    2. Servi RJ
    (1985) Acute renal failure and sudden death following sequential intermediate-dose methotrexate and 5-FU: a possible adverse effect due to concomitant indomethacin administration. Cancer Treat Rev 69:342–343.
    OpenUrl
  12. ↵
    1. Frenia ML,
    2. Long KS
    (1992) Methotrexate and nonsteroidal antiinflammatory drug interactions. Ann Phramacother 26:234–237.
  13. ↵
    1. Ho ES,
    2. Lin DC,
    3. Mendel DB,
    4. Cihlar T
    (2000) Cytotoxicity of antiviral nucleotides adefovir and cidofovir is induced by the expression of human renal organic anion transporter 1. J Am Soc Nephrol 11:383–393.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Holland SM,
    2. Livesey EF,
    3. Owen J,
    4. Pennington GW,
    5. Roberts JB,
    6. Somers GF
    (1960) Blood and urine levels following intramuscular administration of potassium penicillin V. Antibiot Agents Chemother 10:25–29.
  15. ↵
    1. Hooijberg JH,
    2. Broxterman HJ,
    3. Kool M,
    4. Assaraf YG,
    5. Peters GJ,
    6. Noordhuis P,
    7. Scheper RJ,
    8. Borst P,
    9. Pinedo HM,
    10. Jansen G
    (1999) Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2. Cancer Res 59:2532–2535.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Hosoyamada M,
    2. Sekine T,
    3. Kanai Y,
    4. Endou H
    (1999) Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney. Am J Physiol 276:F122–F128.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Jacquemin E,
    2. Hagenbuch B,
    3. Stieger B,
    4. Wolkoff AW,
    5. Meier PJ
    (1994) Expression cloning of a rat liver Na+-independent organic anion transporter. Proc Natl Acad Sci USA 91:133–137.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Kremer JM,
    2. Hamilton RA
    (1995) The effects of nonsteroidal antiinflammatory drugs on methotrexate (MTX) pharmacokinetics: impairment of renal clearance of MTX at weekly maintenance doses but not at 7.5 mg. J Rheumatol 22:2072–2077.
    OpenUrlPubMed
  19. ↵
    1. Leier I,
    2. Hummel-Eisenbeiss J,
    3. Cui Y,
    4. Keppler D
    (2000) ATP-dependent para-aminohippurate transport by apical multidrug resistance protein MRP2. Kidney Int 57:1636–1642.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Lu R,
    2. Chan BS,
    3. Schuster VL
    (1999) Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C. Am J Physiol 276:F295–F303.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Masuda S,
    2. Ibaramoto K,
    3. Takeuchi A,
    4. Saito H,
    5. Hashimoto Y,
    6. Inui KI
    (1999a) Cloning and functional characterization of a new multispecific organic anion transporter, OAT-K2, in rat kidney. Mol Pharmacol 55:743–752.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Masuda S,
    2. Saito H,
    3. Inui KI
    (1997) Interactions of nonsteroidal anti-inflammatory drugs with rat renal organic anion transporter, OAT-K1. J Pharmacol Exp Ther 283:1039–1042.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Masuda S,
    2. Takeuchi A,
    3. Saito H,
    4. Hashimoto Y,
    5. Inui KI
    (1999b) Functional analysis of rat renal organic anion transporter OAT-K1: bidirectional methotrexate transport in apical membrane. FEBS Lett 459:128–132.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Mulato AS,
    2. Ho ES,
    3. Cihlar T
    (2000) Nonsteroidal anti-inflammatory drugs efficiently reduce the transport and cytotoxicity of adefovir mediated by the human renal organic anion transporter 1. J Pharmacol Exp Ther 295:10–15.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Nierenberg DW
    (1983) Competitive inhibition of methotrexate accumulation in rabbit kidney slices by nonsteroidal anti-inflammatory drugs. J Pharmacol Exp Ther 226:1–6.
    OpenUrlFREE Full Text
  26. ↵
    1. Peterson LR,
    2. Gerding DN,
    3. Zinneman HH,
    4. Moore BM
    (1977) Evaluation of three newer methods for investigating protein interactions of penicillin G. Antimicrob Agents Chemother 11:993–998.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Reid G,
    2. Wolff NA,
    3. Dautzenberg FM,
    4. Burckhardt G
    (1998) Cloning of a human renal p-aminohippurate transporter, hROAT1. Kidney Blood Press Res 21:233–237.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Saito H,
    2. Masuda S,
    3. Inui KI
    (1996) Cloning and functional characterization of a novel rat organic anion transporter mediating basolateral uptake of methotrexate in the kidney. J Biol Chem 271:20719–20725.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Sekine T,
    2. Watanabe N,
    3. Hosoyamada M,
    4. Kanai Y,
    5. Endou H
    (1997) Expression cloning and characterization of a novel multispecific organic anion transporter. J Biol Chem 272:18526–18529.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Shen DD,
    2. Azarnoff DL
    (1978) Clinical pharmacokinetics of methotrexate. Clin Pharmacokinet 3:1–13.
    OpenUrlPubMed
  31. ↵
    1. Sun W,
    2. Wu RR,
    3. van Poelje PD,
    4. Erison MD
    (2001) Isolation of a family of organic anion transporters from human liver and kidney. Biochem Biophys Res Commun 283:417–422.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Takeda M,
    2. Tojo A,
    3. Sekine T,
    4. Hosoyamada M,
    5. Kanei Y,
    6. Endou H
    (1999) Role of organic anion transporter 1 (OAT1) in cephaloridine-induced nephrotoxicity. Kidney Int 56:2128–2136.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Taylor JR,
    2. Halprin KM
    (1977) Effect of sodium salicylate and indomethacin on methotrexate-serum albumin binding. Arch Dermatol 113:588–591.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Thyss A,
    2. Milano G,
    3. Kubar J,
    4. Namer M,
    5. Schneider M
    (1986) Clinical and pharmacokinetic evidence of a life-threatening interaction between methotrexate and ketoprofen. Lancet 1:256–258.
    OpenUrlPubMed
  35. ↵
    1. Tracy TS,
    2. Krohn K,
    3. Jones DR,
    4. Bradley JD,
    5. Hall SD,
    6. Brater DC
    (1992) The effects of a salicylate, ibuprofen and naproxen on the disposition of methotrexate in patients with rheumatoid arthritis. Eur J Clin Pharmacol 42:121–125.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Uchino H,
    2. Tamai I,
    3. Yamashita K,
    4. Minemoto Y,
    5. Sai Y,
    6. Yabuuchi H,
    7. Miyamoto K,
    8. Takeda E,
    9. Tsuji A
    (2000) p-Aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1. Biochem Biophys Res Commun 270:254–259.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Uwai Y,
    2. Saito H,
    3. Inui KI
    (2000) Interaction between methotrexate and nonsteroidal anti-inflammatory drugs in organic anion transporter. Eur J Pharmacol 409:31–36.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Vietri M,
    2. DeSanti C,
    3. Pietrabissa A,
    4. Mosca F,
    5. Pacifici GM
    (2000) Inhibition of human liver phenol sulfotransferase by nonsteroidal anti-inflammatory drugs. Eur J Clin Pharmacol 56:81–87.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Williams WM,
    2. Chen TS,
    3. Huang KC
    (1984) Effect of penicillin on the renal tubular secretion of methotrexate in the monkey. Cancer Res 44:1913–1917.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Zhang L,
    2. Gorset W,
    3. Washington CB,
    4. Blaschke TF,
    5. Kroetz DL,
    6. Giacomini KM
    (2000) Interactions of HIV protease inhibitors with a human organic cation transporter in a mammalian expression system. Drug Metab Dispos 28:329–334.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Characterization of Methotrexate Transport and Its Drug Interactions with Human Organic Anion Transporters

Michio Takeda, Suparat Khamdang, Shinichi Narikawa, Hiroaki Kimura, Makoto Hosoyamada, Seok Ho Cha, Takashi Sekine and Hitoshi Endou
Journal of Pharmacology and Experimental Therapeutics August 1, 2002, 302 (2) 666-671; DOI: https://doi.org/10.1124/jpet.102.034330
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement