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

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 K m values for hOAT1-, hOAT3-, and hOAT4-mediated methotrexate uptake were 553.8 M, 21.1 M, and 17.8 M, 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. The K i 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 K i 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...

Chemosensitization of Plasmodium falciparum by Probenecid In Vitro

“…As a result, probenecid treatment leads to an increase in the steady-state concentration of methotrexate in the cell. As an organic anion, methotrexate could be expected to be a multiresistance-associated protein substrate (4,11,32,37); however, this chemical classification does not extend to the drugs that we have used in this study. Therefore, it is unlikely that these drugs are targets of multiresistance-associated proteins.…”

A Novel Transporter of SLC22 Family Specifically Transports Prostaglandins and Co-localizes with 15-Hydroxyprostaglandin Dehydrogenase in Renal Proximal Tubules

“…The procedure to establish S 2 cell lines stably expressing exogenous genes has been described elsewhere (28). Empty vectors (i.e.…”

Screening of genetic variations of SLC15A2, SLC22A1, SLC22A2 and SLC22A6 genes

“…11,12 Located on 11q13.1-q13.2, organic anion transporter, SLC22A6, has been shown to transport methotrexate (cytotoxic antimetabolite used for rheumatoid arthritis, psoriasis and cancer of various types) and to be inhibited by non-steroidal anti-inflammatory drugs. 13,14 The R50H variant in SLC22A6 is associated with kinetic differences for the nucleoside phosphonate analogs adefovir, cidofovir and tenofovir. 15 Because the genetic variants associated with impaired transport function may have an influence on substrate disposition, it is of importance to identify the polymorphisms and ethnic diversity of the four SLC genes.…”