Abstract
Methotrexate (MTX) is used in patients with malignant and autoimmune diseases. This drug is primarily excreted unchanged in the urine, and its net excretion occurs via active secretory and reabsorptive processes. We characterized the interaction of MTX with human organic-anion transporting polypeptide transporter (OATP) 1A2, which is expressed in tissues important for MTX disposition and toxicity, such as the intestine, kidney, liver, and endothelial cells of the blood-brain barrier. In Xenopus laevis oocytes expressing OATP1A2, the uptake of the model substrate, estrone-3-sulfate (ES), was enhanced 30-fold compared with uninjected oocytes. MTX uptake in oocytes expressing OATP1A2 was saturable (Km = 457 ± 118 μM; Vmax = 17.5 ± 4.9 pmol/oocyte/60 min) and sensitive to extracellular pH. That is, acidic pHs stimulated MTX uptake by as much as 7-fold. Seven novel protein-altering variants were identified in 270 ethnically diverse DNA samples. Four protein-altering variants in OATP1A2 exhibited altered transport of ES and/or MTX. The common variant, protein reference sequence (p.) Ile13Thr, was hyperfunctional for ES and MTX and showed a 2-fold increase in the Vmax for ES. The common variant, p. Glu172Asp, exhibited reduced maximal transport capacity for ES and MTX. p. Arg168Cys was hypofunctional, and p. Asn277DEL was nonfunctional. Because of its expression on the apical membrane of the distal tubule and in tissues relevant to MTX disposition and toxicity, these findings suggest that OATP1A2 may play a role in active tubular reabsorption of MTX and in MTX-induced toxicities. Furthermore, genetic variation in OATP1A2 may contribute to variation in MTX disposition and response.
Footnotes
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This work was supported by the National Institutes of Health Grants GM61390 and GM36780 and by the General Clinical Research Center at San Francisco General Hospital, funded by the National Center for Research Resources, National Institutes of Health (Grant MO1-RR00083-42). I.B. was supported in part by a National Science Foundation Graduate Research Fellowship.
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Data in this manuscript were deposited in the Pharmacogenetics and Pharmacogenomics Knowledgebase (www.pharmgkb.org). Some parts of this study were presented as an abstract; Interaction of methotrexate with SLCO1A2 (OATP-A) and its 12 protein-altering variants: clinical implications for delayed excretion and nephrotoxicity; International Society for the Study of Xenobiotics Annual Meeting; 2005 October 23-27; Maui, HI. International Society for the Study of Xenobiotics, Washington, DC.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.104364.
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ABBREVIATIONS: MTX, methotrexate; 7-OH-MTX, 7-hydroxymethotrexate; SLC, solute carrier; OATP, organic-anion transporting polypeptide; ES, estrone-3-sulfate; p., protein reference sequence; HEK293, human embryonic kidney cell line; cRNA, complementary RNA; FCCP, carbonylcyanide p-trifluoromethoxyphenyl hydrazone; ANOVA, analysis of variance; c., coding DNA reference sequence; TMD, transmembrane domain.
- Received March 10, 2006.
- Accepted May 11, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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