GNE-317, a PI3K/mTOR inhibitor, can pass through the blood-brain barrier (BBB).

GNE-317, an oxetane derivative synthesized by GDC-0980, is aimed at reducing substrate affinity for efflux transporters. In vitro, GDC-0980 demonstrate similar profiles with GNE-317 in MTS cytotoxicity experiments using the GL261 cell line.

Mice, which are i.c. inoculation with GL261-GFP-Luc cells Seven days later,are treated once daily with the maximum tolerated dose of GDC-0980 (7.5 mg/kg), GNE-317 (30 mg/kg), or vehicle. Tumor growth is tracked in GL261 through bioluminescence imaging on a weekly basis. There are no significant differences in GL261 tumor growth among the 3 groups treated by GDC-0980, GNE-317 or vehicle. The data show that the drugs have limited efficacy in inducing cell death in the GL261 cell line. Although GNE-317 has greater delivery and enhanced therapeutic targeting efficacy, it is not effective in the treatment of the GL261 tumor.

Cellular viability assays are set up in a 96-well format with 2000 GL261-GFP-Luc cells plated per well in the culture conditions. GL261, an aggressive C57BL/6J-derived glioma line, is transfected with both green fluorescent protein (GFP) and luciferase (Luc) from separate plasmids.GL261-GFP-Luc cells are cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS and Penicillin/Streptomycin (100 U/mL) at 5% oxygen, and are selected by 4 mg/mL Puromycin and 4 mg/mL G418. Suspend GNE-317 in DMSO and then diluted with the medium.GL261-GFP-Luc cells are incubated in the presence of drug or vehicle for 48 hours, and viability was assessed by MTS assay. Results were detected using a Synergy Mx automated plate reader,which are set up absorbance at 490 nm and used to determine viability and at 650 nm to account for the background. Numerical values from drug-treated wells are normalized to the values of vehicle-treated wells to yield percent survival.

7-week-old C57BL/6J mice are implanted GL261-GFP-Luc cells. When tumors reach 5e7 photons/s/cm2/sr (radiance), mice are orally administered the maximum tolerated the dose,which is defined as <10% drop in mice bodyweight dose, GDC-0980 for 7.5 mg/kg, GNE-317 for 30 mg/kg or vehicle once daily for 3 days. At 1 or 6 hours after the third dose, mice are euthanized with carbon dioxide and perfused with 30 mL PBS.