Decreased affinity for efflux transporters increases brain penetrance and molecular targeting of a PI3K/mTOR inhibitor in a mouse model of glioblastoma

Abstract

Background: Targeting drug delivery to invasive glioma cells is a particularly difficult challenge because these cells lie behind an intact blood-brain barrier (BBB) that can be observed using multimodality imaging. BBB-associated efflux transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) influence drug distribution to these cells and may negatively impact efficacy. To test the hypothesis that efflux transporters influence brain pharmacokinetics/pharmacodynamics of molecularly targeted agents in glioma treatment, we assessed region-specific penetrance and molecular-targeting capacity for a PI3K/mTOR kinase inhibitor that has high substrate affinity for efflux transporters (GDC-0980) and an analog (GNE-317) that was purposely designed to have reduced efflux.

Methods: Brain tumor penetrance of GDC-0980 and GNE-317 was compared between FVB/n wild-type mice and Mdr1a/b(-/-)Bcrp(-/-) triple-knockout mice lacking P-gp and BCRP. C57B6/J mice bearing intracranial GL261 tumors were treated with GDC-0980, GNE-317, or vehicle to assess the targeted pharmacokinetic/pharmacodynamic effects in a glioblastoma model.

Results: Animals treated with GNE-317 demonstrated 3-fold greater penetrance in tumor core, rim, and normal brain compared with animals dosed with GDC-0980. Increased brain penetrance correlated with decreased staining of activated p-Akt, p-S6, and p-4EBP1 effector proteins downstream of PI3K and mTOR.

Conclusions: GDC-0980 is subject to active efflux by P-gp and BCRP at the BBB, while brain penetrance of GNE-317 is independent of efflux, which translates into enhanced inhibition of PI3K/mTOR signaling. These data show that BBB efflux by P-gp and BCRP is therefore an important determinant in both brain penetrance and molecular targeting efficacy in the treatment of invasive glioma cells.

Keywords: blood-brain barrier; brain tumors; efflux transport; molecularly targeted agents; p-glycoprotein.

Fig. 1.

Different imaging modalities visualize tumors residing behind an intact blood-brain barrier. (A) Patient exhibiting multifocal glioblastoma with large tumors displayed through ¹⁸F-DOPA PET imaging. (B) Another patient 3 weeks post surgical resection exhibits obvious tumor via ¹⁸F-DOPA PET that was missed during surgery.

Fig. 2.

Characterization of the GL261 murine model. (A) A macroscopic view of GL261-GFP-Luc tumor-bearing brain demonstrates discernible core, rim, and normal brain sections. Scale bar represents 1 mm. (B) and (C) Images of GFP+ tumors and Texas Red dextran infiltration were overlaid to colocalize tumors and heterogeneous BBB disruption. Scale bars represent 1 mm.

Fig. 3.

Cytotoxic and pharmacokinetic comparison of GDC-0980 and GNE-317. (A) Chemicals highlight the common molecular backbone and differentiating side chains. (B) Cytotoxicity of each drug in the GL261 cell line was assessed using the MTS assay. (C) Mdr1/b^−/− Bcrp^−/− triple knockout (TKO) mice treated with GDC-0980 display a significantly higher (P < .0005) brain-to-plasma ratio than wild-type (WT) mice. (D) Mdr1/b^−/− Bcrp^−/− TKO and WT mice treated with GNE-317 display comparable brain-to-plasma ratios.

Fig. 4.

Regional distribution of GDC-0980 and GNE-317 in a GL261-GFP-Luc tumor-bearing brain. (A) and (C) Regional brain and plasma distributions of GDC-0980 and corroborating brain-to-plasma ratios. (B) and (D) regional brain and plasma distributions of GNE-317 and corroborating brain-to-plasma ratios.

Fig. 5.

Immunohistochemistry 1 hour after drug treatment. Scale bars associated with whole slices represent 100 μm, while scale bars associated with close-up insets represent 65 μm. (A) p-Akt^Ser473 staining is reduced in the presence of GNE-317 and, to a lesser extent, GDC-0980. (B) p-S6^Ser235/236 staining is reduced in the presence of GNE-317 but not GDC-0980. (C) p-4EBP1^Thr37/46 staining is reduced in the presence of GNE-317 but not GDC-0980.

Fig. 6.

In vivo efficacy studies. (A) Tumor progression tracked through bioluminescent imaging shows no demonstrable impact of either GDC-0980 or GNE-317 over vehicle-treated animals. (B) Kaplan-Meier survival curve indicates that neither GDC-0980 nor GNE-317 provides survival benefit over vehicle.