Title Page Distribution of Gefitinib to the Brain is Limited by P-glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) Mediated Active Efflux

factor receptor mutant vIII; Mdr1, gene encoding the murine p-glycoprotein; MDR1, gene encoding the human p-glycoprotein; Bcrp1, gene encoding the murine breast cancer resistance protein. This article has not been copyedited and formatted. The final version may differ from this version. Abstract Gefitinib is an orally active inhibitor of the epidermal growth factor receptor (EGFR) approved for use in patients with locally advanced or metastatic non-small-cell lung cancer. It has also been evaluated in several clinical trials for treatment of brain tumors such as high-grade glioma. In this study, we investigated the influence of p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) on distribution of gefitinib to the central nervous system. In vitro studies conducted in MDCKII cells indicate that both P-gp and BCRP effectively transport gefitinib limiting its intracellular accumulation. In vivo studies demonstrated that transport of gefitinib across the blood-brain barrier (BBB) is significantly limited. Steady-state brain-to-plasma (B/P) concentration ratios were 70-fold higher in the Mdr1a/b-/-Bcrp1-/-mice (ratio ~7) compared with wild-type mice (ratio ~ 0.1). The B/P ratio after oral administration increased significantly when gefitinib was co-administered with the dual P-gp and BCRP inhibitor elacridar. We investigated the integrity of tight junctions in the Mdr1a/b-/-Bcrp1-/-mice and found no difference in the brain inulin and sucrose space between the wild-type and Mdr1a/b-/-Bcrp1-/-mice. This suggested that the dramatic enhancement in the brain distribution of gefitinib is not due to a leakier BBB in these mice. These results show that brain distribution of gefitinib is restricted due to active efflux by P-gp and BCRP. This finding is of clinical significance for therapy in brain tumors such as glioma, where concurrent administration of a dual inhibitor like elacridar can increase delivery and thus enhance efficacy of gefitinib. This article has not been copyedited and formatted. The final version may differ from this version.