Received for publication March 13, 2006.
Revised April 19, 2006.
Accepted for publication May 11, 2006.

Interaction Of Methotrexate With OATP1A2 And Its Genetic Variants

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 a human organic anion transporting polypeptide transporter, OATP1A2, 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 X. laevis oocytes expressing OATP1A2, the uptake of the model substrate, estrone-3-sulfate (ES), was enhanced 30-fold compared to uninjected oocytes. MTX uptake in oocytes expressing OATP1A2 was saturable (K_m= 457 ± 118 µM; V_max= 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, p. Ile13Thr, was hyperfunctional for ES and MTX and showed a 2-fold increase in the V_max for ES. The common variant, p. Glu172Asp exhibited reduced maximal transport capacity for ES and MTX. p. Arg168Cys was hypofunctional and p. Asn277DEL was non-functional. 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.

Key words: cancer, membrane transporter, methotrexate, nephrotoxicity, organic anion transporting polypeptide, single nucleotide polymorphism