Simultaneous use of nonsteroidal anti-inflammatory drugs (NSAIDs), probenecid, and other drugs has been reported to delay the plasma elimination of methotrexate in patients. Previously, we have reported that inhibition of the uptake process cannot explain such drug-drug interactions using rats. The present study quantitatively evaluated the possible role of the transporters in such drug-drug interactions using human kidney slices and membrane vesicles expressing human ATP-binding cassette (ABC) transporters. The uptake of methotrexate by human kidney slices was saturable with a K m of 45 to 49 M. Saturable uptake of methotrexate by human kidney slices was markedly inhibited by p-aminohippurate and benzylpenicillin, but only weakly by 5-methyltetrahydrofolate. These transport characteristics are similar to those of a basolateral organic anion transporter (OAT) 3/SLC22A8. NSAIDs and probenecid inhibited the uptake of methotrexate by human kidney slices, and, in particular, salicylate, indomethacin, phenylbutazone, and probenecid were predicted to exhibit significant inhibition at clinically observed plasma concentrations. Among ABC transporters, such as BCRP/ABCG2, multidrug resistance-associated protein (MRP) 2/ABCC2, and MRP4/ABCC4, which are candidates for the luminal efflux of methotrexate, ATPdependent uptake of methotrexate by MRP4-expressing membrane vesicles was most potently inhibited by NSAIDs. Salicylate and indomethacin were predicted to inhibit MRP4 at clinical plasma concentrations. Diclofenac-glucuronide significantly inhibited MRP2-mediated transport of methotrexate in a concentration-dependent manner, whereas naproxen-glucuronide had no effect. Inhibition of renal uptake (via OAT3) and efflux processes (via MRP2 and MRP4) explains the possible sites of drug-drug interaction for methotrexate with probenecid and some NSAIDs, including their glucuronides.Drug-drug interactions involving metabolism and/or excretion processes prolong the plasma elimination half-lives leading to the accumulation of drugs in the body and potentiate pharmacological/adverse effects. Recent progress in molecular biological research has shown that many types of transporters play important roles in the tissue uptake and/or subsequent secretion of drugs in the liver and kidney, and such transporters exhibit a broad substrate specificity with a degree of overlap, suggesting the possibility of transportermediated drug-drug interactions with other substrates (Shitara et al., 2005;Li et al., 2006). Methotrexate (MTX) is an analog of natural folate and has been widely and successfully used for the treatment of neoplastic diseases and autoimmune diseases, including rheu-

The activities of renal multispecific organic anion transporters (OATs) 1 and 3 have not been fully evaluated in human kidneys. In the present study, the uptake of some organic anions was characterized in kidney slices from human intact renal cortical tissues: hOAT1 and hOAT3 substrates [p-aminohippurate (PAH) and 2,4-dichlorophenoxyacetate (2,4-D)] and hOAT3 substrates [benzylpenicillin (PCG), dehydroepiandrosterone sulfate (DHEAS), and estrone sulfate (ES)]. Despite large interbatch differences, hOAT1 and hOAT3 mRNA levels correlated well, and there was a good correlation between the uptake of PAH and PCG by kidney slices. The uptake of organic anions by kidney slices was saturable with K m values of 31 to 48 M for PAH, 0.73 to 4.9 M for 2,4-D, 14 to 90 M for PCG, and 9.2 to 11 M for ES. These parameters were comparable with those for hOAT1 and/or hOAT3. The uptake of DHEAS consists of two saturable components with K m values of 2.2 to 3.9 and 1300 M, and the K m value of the high-affinity component was close to that for hOAT3. Furthermore, PAH more potently inhibited the uptake of 2,4-D than that of PCG and DHEAS. PCG had a weaker effect on the uptake of PAH and 2,4-D than expected from its K m value. Taken together, it is likely that the uptake of PAH and 2,4-D is due to OAT1, and the uptake of PCG and ES and part of DHEAS uptake are due to OAT3 in human kidney slices. Human kidney slices are useful tools for characterizing the renal uptake of drugs.

ABSTRACT:Olmesartan, a novel angiotensin II AT1-receptor antagonist, is excreted into both bile and urine, with minimal metabolism. Because olmesartan is a hydrophilic anionic compound, some transporters could be involved in its hepatic and renal clearance. In this study, we characterized the role of human drug transporters in the pharmacokinetics of olmesartan and determined the contribution of each transporter to the overall clearance of olmesartan. Olmesartan was significantly taken up into human embryonic kidney 293 cells expressing organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, organic anion transporter (OAT) 1, and OAT3. We also observed its saturable uptake into human hepatocytes and kidney slices. Estimated from the relative activity factor method and application of specific inhibitors, the relative contributions of OATP1B1 and OATP1B3 to the uptake of olmesartan in human hepatocytes were almost the same, whereas OAT3 was predominantly involved in its uptake in kidney slices. The vectorial transport of olmesartan was observed in OATP1B1/multidrug resistance-associated protein (MRP) 2 double transfectants, but not in OATP1B1/multidrug resistance (MDR) 1 and OATP1B1/ breast cancer resistance protein (BCRP) transfectants. ATP-dependent transport into membrane vesicles expressing human MRP2 and MRP4 was clearly observed, with K m values of 14.9 and 26.2 M, respectively, whereas the urinary excretion of olmesartan in Mrp4-knockout mice was not different from that of control mice. We also investigated the transcellular transport of olmesartan medoxomil, a prodrug of olmesartan. Vectorial basal-to-apical transport was observed in OATP1B1/MRP2, OATP1B1/MDR1 double, and OATP1B1/BCRP double transfectants, suggesting the possible involvement of MRP2, MDR1, and BCRP in the limit of intestinal absorption of olmesartan medoxomil. From these results, we suggest that multiple transporters make a significant contribution to the pharmacokinetics of olmesartan and its prodrug.

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