Distribution of gefitinib to the brain is limited by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2)-mediated active efflux

Abstract

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 Madin-Darby 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(-/-) Bcrp1(-/-) 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(-/-) 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 such as elacridar can increase delivery and thus enhance efficacy of gefitinib.

Fig. 1.

Intracellular accumulation of [¹⁴C]gefitinib in MDCKII cells. A, accumulation in wild-type (black bar) and Bcrp1-transfected (gray bar) cells. Gefitinib accumulation in the Bcrp1-transfects was approximately 30-fold lower compared with that in wild-type cells (*, p < 0.001). This difference in accumulation was abolished when BCRP was inhibited using the specific inhibitor Ko143. B, accumulation in wild-type (black bar) and MDR1-transfected (gray bar) cells. Gefitinib accumulation was significantly lower in the MDR1-transfected cells compared with wild type (*, p < 0.001). Treatment with LY335979 reduced the difference in accumulation between the two cell types. Results presented as mean ± S.E., n = 18.

Fig. 2.

Flux of [¹⁴C]gefitinib across MDCKII cell monolayers. A, gefitinib permeability across wild-type and Bcrp1-transfected cells. In the Bcrp1-transfected cells, the B-to-A permeability (gray bar) of gefitinib was significantly greater than the A-to-B permeability (black bar) (*, p < 0.001). This directionality in transport was abolished when cells were treated with the BCRP-specific inhibitor Ko143. B, permeability across wild-type and MDR1-transfected cells. The B-to-A permeability (gray bar) was significantly greater than the A-to-B permeability (black bar) in the MDR1- transfected cells (*, p < 0.001). P-gp inhibition by LY335979 abolished this directionality in transport, such that there was no difference in permeability between the two directions. Results presented as mean ± S.E., n = 9.

Fig. 3.

Brain and plasma concentrations of gefitinib in FVB wild-type mice after a 25 mg/kg oral dose of gefitinib. Brain gefitinib concentrations (○) were significantly lower than the plasma concentrations (●), demonstrating the limited brain penetration of gefitinib (mean ± S.E., n = 4, *, p < 0.05 compared with wild-type control).

Fig. 4.

Brain distribution of gefitinib in wild-type and Mdr1a/b(−/−) Bcrp1(−/−) FVB mice after an oral dose of 25 mg/kg gefitinib. A and B, brain concentrations (A) and corresponding B/P concentration ratios (B) of gefitinib in wild-type (●) and Mdr1a/b(−/−) Bcrp1(−/−) (▴) mice. Brain concentrations in the Mdr1a/b(−/−)Bcrp1(−/−) mice were significantly greater than the wild type. The B/P ratios increased by greater than 16-fold in the Mdr1a/b(−/−) Bcrp1(−/−) mice, indicating the enhancement in brain distribution of gefitinib due to absence of P-gp and BCRP at the BBB. Results are represented as mean ± S.E. (**, p < 0.05, *, p < 0.001 compared with wild-type control).

Fig. 5.

Effect of P-gp and BCRP inhibition on brain distribution of gefitinib. A and B, brain concentrations (A) and corresponding B/P concentration ratios (B) of gefitinib in the vehicle-treated (●) and elacridar-treated (▴) wild-type mice. Brain concentrations increased in the wild-type mice when elacridar was administered before gefitinib. The B/P ratios were also significantly greater in the elacridar-treated group, suggesting that dual inhibitors such as elacridar can be used to improve brain penetration of substrate drugs. Results are represented as mean ± S.E. (**, p < 0.05, *, p < 0.001 compared with vehicle control).

Fig. 6.

A, effect of pharmacological inhibition of drug efflux transporters on brain distribution of gefitinib in wild-type FVB mice. Wild-type mice received 25 mg/kg gefitinib via oral gavage 30 min after intravenous administration of 25 mg/kg LY335979, 10 mg/kg Ko143, or 10 mg/kg GF120918 (elacridar). B, effect of genetic deletion of drug efflux transporters on brain distribution of gefitinib in FVB mice. Wild-type, Mdr1a/b(−/−), Bcrp1(−/−), and Mdr1a/b(−/−) Bcrp1(−/−) mice were administered 25 mg/kg gefitinib. Whole-brain tissue was collected at 90 min after dose (n = 4 at each time point) and analyzed for gefitinib. The values are presented as mean ± S.E. (**, p < 0.05, compared with wild-type control).

Fig. 7.

Integrity of the blood-brain barrier in Mdr1a/b(−/−) Bcrp1(−/−) mice. Brain spaces of sucrose and inulin in wild-type (black bar) and Mdr1a/b(−/−) Bcrp1(−/−) (gray bar) mice. The brain spaces of sucrose or inulin in the Mdr1a/b(−/−) Bcrp1(−/−) mice were similar to those in the wild-type mice, indicating that the BBB is intact in these mice. The values are presented as mean ± S.E. (n = 4).