title: Interaction of methotrexate with organic-anion transporting polypeptide 1A2 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 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 microM; 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 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 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.
authors.affiliation: Department of Biopharmaceutical Sciences, University of California, 1550 4th Street, Box 2911, San Francisco, CA 94158, USA.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Badagnani
authors.forename: Richard A
authors.initials: RA
authors.lastname: Castro
authors.forename: Travis R
authors.initials: TR
authors.lastname: Taylor
authors.forename: Claire M
authors.initials: CM
authors.lastname: Brett
authors.forename: Conrad C
authors.initials: CC
authors.lastname: Huang
authors.forename: Douglas
authors.initials: D
authors.lastname: Stryke
authors.forename: Michiko
authors.initials: M
authors.lastname: Kawamoto
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Johns
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Ferrin
authors.forename: Elaine J
authors.initials: EJ
authors.lastname: Carlson
authors.forename: Esteban G
authors.initials: EG
authors.lastname: Burchard
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Giacomini
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D005493:Folic Acid Antagonists / Q000493:pharmacokinetics / Q000494:pharmacology*; D014644:Genetic Variation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008727:Methotrexate / Q000031:analogs & derivatives / Q000493:pharmacokinetics / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D027361:Organic Anion Transporters / Q000187:drug effects* / Q000235:genetics*; D014982:Xenopus laevis
chemical_list: D005493:Folic Acid Antagonists; D027361:Organic Anion Transporters; C512988:SLCO1A2 protein, human; C011864:7-hydroxymethotrexate; D008727:Methotrexate