The Effect of Low pH on Breast Cancer Resistance Protein (ABCG2)-Mediated Transport of Methotrexate, 7- Hydroxymethotrexate, Methotrexate Diglutamate, Folic Acid, Mitoxantrone, Topotecan, and Resveratrol in In Vitro Drug Transport Models Pauline Breedveld, Dick Pluim, Greta Cipriani, Femke Dahlhaus, Maria A. J. van Eijndhoven, Cornelia J. F. de Wolf, Annemieke Kuil, Jos H. Beijnen, George L. Scheffer, Gerrit Jansen, Piet Borst, and Jan H. M. Schellens Departments of Experimental Therapy (P.Br., D.P., G.C., F.D., M.A.J.v.E., J.H.M.S.), Molecular Biology (C.J.F.d.W., A.K., P.Bo.), Pharmacy (J.H.B.), and Medical Oncology (J.H.B., J.H.M.S.), the Netherlands Cancer Institute, Amsterdam, the Netherlands; Departments of Rheumatology (G.J.) and Pathology (G.L.S.), VU University Medical Center, Amsterdam, the Netherlands; and Faculty of Pharmaceutical Sciences (J.B., J.H.M.S.), Utrecht University, Utrecht, the Netherlands Received June 25, 2006; accepted September 25, 2006 ABSTRACT Some cellular uptake systems for (anti)folates function opti- mally at acidic pH. We have tested whether this also applies to efflux from cells by breast cancer resistance protein (BCRP; ABCG2), which has been reported to transport folic acid, meth- otrexate, and methotrexate di- and triglutamate at physiological pH. Using Spodoptera frugiperda-BCRP membrane vesicles, we showed that the ATP-dependent vesicular transport of 1 M methotrexate by BCRP is 5-fold higher at pH 5.5 than at phys- iological pH. The transport of methotrexate was saturable at pH 5.5, with apparent K m and V max values of 1.3  0.2 mM and 44  2.5 nmol/mg of protein/min, respectively, but was linear with drug concentration at pH 7.3 up to 6 mM methotrexate. In contrast to recent reports, we did not detect transport of meth- otrexate diglutamate at physiological pH, but we did find trans- port at pH 5.5. We also found that 7-hydroxy-methotrexate, the major metabolite of methotrexate, is transported by BCRP both at physiological pH and (more efficiently) at low pH. The pH effect was also observed in intact BCRP-overexpressing cells: we found a 3-fold higher level of resistance to both methotrex- ate and the prototypical BCRP substrate mitoxantrone at pH 6.5 as at physiological pH. Furthermore, with MDCKII-BCRP monolayers, we found that resveratrol, which is a neutral com- pound at pH  7.4, is efficiently transported by BCRP at pH 6.0, whereas we did not detect active transport at pH 7.4. We conclude that BCRP transports substrate drugs more efficiently at low pH, independent of the dissociation status of the sub- strate. Uptake of weak acid and weak base chemotherapeutic drugs by tumors is greatly influenced by the dissociation properties of the drug itself and by the cellular pH gradient (i.e., the difference of extracellular pH in the tumor and the intracellular pH maintained by the cells) (Tannock and Ro- tin, 1989; Boyer and Tannock, 1992; Kozin et al., 2001; Ma- honey et al., 2003). Whereas the median pH value in normal tissues is 7.5, in many tumor tissues, the extracellular pH is more acidic and may be as low as 5.8 (Tannock and Rotin, 1989). This is a consequence of a high rate of lactic acid production in tumors even under aerobic conditions (Tannock and Rotin, 1989; Boyer and Tannock, 1992; Gatenby and This work has been presented previously in abstract form: Breedveld P, Pluim D, Cipriani G, Dahlhaus F, van Eijndhoven MAJ, Borst P, and Schellens JHM (2005) The effect of low pH on BCRP(ABCG2)-mediated transport of methotrexate, 7-hydroxymetotrexate, methotrexate diglutamate, folic acid and resveratrol in in vitro drug transport models. Proceedings of the American Association of Cancer Research LB-262. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.028167. ABBREVIATIONS: MTX, methotrexate; BCRP, breast cancer resistance protein; MRP, multidrug resistance-associated protein; 7-OH-MTX, 7-hydroxy-methotrexate; MTX-glu2, methotrexate diglutamate; Sf9, Spodoptera frugiperda; HPLC, high-performance liquid chromatography; LY335979, zosuquidar trihydrochloride; GF120918, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-me- thoxy-9-oxo-4-acridine carboxamide; SN-38, 7-ethyl-10-hydroxycamptothecin; solution A, formic acid and acetonitrile 5%; solution B, formic acid and acetonitril 23%; Ko143, 3-(6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-butyl ester. 0026-895X/07/7101-240 –249$20.00 MOLECULAR PHARMACOLOGY Vol. 71, No. 1 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 28167/3162706 Mol Pharmacol 71:240–249, 2007 Printed in U.S.A. 240 at Radboud Universiteit Nijmegen on July 18, 2012 molpharm.aspetjournals.org Downloaded from