Gefitinib is an orally active inhibitor of the epidermal growth factor receptor 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 MadinDarby canine kidney II 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(Ϫ/Ϫ) Bcrp1(Ϫ/Ϫ) mice (ratio of approximately 7) compared with wild-type mice (ratio of approximately 0.1). The B/P ratio after oral administration increased significantly when gefitinib was coadministered with the dual P-gp and BCRP inhibitor elacridar. We investigated the integrity of tight junctions in the Mdr1a/b(Ϫ/Ϫ) Bcrp1(Ϫ/Ϫ) mice and found no difference in the brain inulin and sucrose space between the wild-type and Mdr1a/b(Ϫ/Ϫ) Bcrp1(Ϫ/Ϫ) 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 such as elacridar can increase delivery and thus enhance efficacy of gefitinib.Malignant gliomas account for approximately 70% of all new cases of malignant primary brain tumors diagnosed in the United States every year. Glioblastoma multiforme (GBM) is the most common type of glioma, accounting for approximately 60 to 70% of malignant gliomas (Wen and Kesari, 2008; CBTRUS, 2008) and claiming 12,000 lives every year (Davis et al., 2001). Epidermal growth factor receptor (EGFR) and its variant EGFRvIII play a critical role in the development of an aggressive phenotype of GBM; EGFR amplification, mutation, and overexpression are associated with poor prognosis and resistance to therapy (Brandes et al., 2008). Several therapeutic strategies targeting EGFR in GBM have been proposed, including the use of monoclonal antibodies against EGFR or EGFRvIII, vaccine therapies, bispecific antibodies, toxin-linked conjugates, and small molecule tyrosine kinase inhibitors (Omuro et al., 2007).

Spontaneous mouse models of cancer show promise to more accurately recapitulate human disease and predict clinical efficacy. Transgenic mice or viral vectors have been required to generate spontaneous models of glioma, a lethal brain tumor, because nonviral gene transfer is typically transient. To overcome this constraint, we used the Sleeping Beauty transposable element to achieve chromosomal integration of human oncogenes into endogenous brain cells of immunocompetent mice. Genetically engineered, spontaneous brain tumors were induced with plasmid DNA in a matter of weeks in three separate mouse strains. The phenotype of tumors was influenced by the combination of oncogenes delivered, resembling human astrocytoma or glioblastoma in the majority of cases. At least five different genes can be cotransfected simultaneously including reporters, allowing measurement of tumor viability by in vivo imaging. This model can accelerate brain tumor research in a variety of ways such as generation of ''humanized'' models for high throughput drug screening and candidate gene validation with exceptional speed and flexibility. [Cancer Res 2009;69(2):431-9]

The importance of the blood-brain barrier in preventing effective pharmacotherapy of glioblastoma has been controversial. The controversy stems from the fact that vascular endothelial cell tight junctions are disrupted in the tumor, allowing some systemic drug delivery. P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP) efflux drugs from brain capillary endothelial cells into the blood. We tested the hypothesis that although the tight junctions are “leaky” in the core of glioblastomas, active efflux limits drug delivery to tumor-infiltrated normal brain and consequently, treatment efficacy. Malignant gliomas were induced by oncogene transfer into wild-type (WT) mice or mice deficient for Pgp and BCRP (KO). Glioma-bearing mice were orally dosed with dasatinib, a kinase inhibitor and dual BCRP/PgP substrate that is being tested in clinical trials. KO mice treated with dasatinib survived over twice as long as WT mice. Microdissection of the tumor core, invasive rim, and normal brain revealed 2-3 fold enhancement in dasatinib brain concentrations in KO mice relative to WT. Analysis of signaling demonstrated that poor drug delivery correlated with the lack of inhibition of a dasatinib target, especially in normal brain. A majority of human glioma xenograft lines tested expressed BCRP or PgP and were sensitized to dasatinib by a dual BCRP/Pgp inhibitor, illustrating a second barrier to drug delivery intrinsic to the tumor itself. These data demonstrate that active efflux is a relevant obstacle to treating glioblastoma and provide a plausible mechanistic basis for the clinical failure of numerous drugs that are BCRP/Pgp substrates.

Taken overall, the results suggest compounds in this series may offer new strategies for the design of chemotherapeutics for treating brain cancers with high oral bioavailability and improved efficacy.