Cell experiment:

GL261 is an aggressive C57BL/6J-derived glioma line. This cell line is transfected with both green fluorescent protein (GFP) and luciferase (Luc) from separate plasmids. The resultant monoclonal GL261-GFP-Luc cells are maintained in Dulbecco's modified Eagle's medium supplemented with 10% FBS and Penicillin/Streptomycin (100 U/mL) and cultured at 5% oxygen. Cell selection used 4 mg/mL Puromycin and 4 mg/mL G418. Cellular viability assays are set up in a 96-well format with 2000 cells plated per well in the culture conditions. Cells are incubated in the presence of drug or vehicle for 48 hours, and viability was assessed by MTS assay. Absorbance at 490 nm is used to determine viability and at 650 nm to account for background using a Synergy Mx automated plate reader. Numerical values from drug-treated wells are normalized to the values of vehicle-treated wells to yield percent survival[1].

Animal experiment:

Mice[1] GL261-GFP-Luc cells are implanted into 7-week-old C57BL/6J mice. When tumors reach 5e7 photons/s/cm2/sr (radiance), animals are orally administered the maximum tolerated dose of GDC-0980 (7.5 mg/kg), GNE-317 (30 mg/kg) or vehicle once a day for 3 days. The maximum tolerated doses are defined as the greatest dose that could be administered to mice with<10% drop in bodyweight. Even at these different doses, both doses provide similar plasma concentrations and thus the same overall systemic exposure. At 1 or 6 hours after the third dose, mice are euthanized with carbon dioxide and perfused with 30 mL PBS. With the aid of GFP goggles, brains are dissected into tumor core, tumor rim, and normal brain tissue. Tissue samples and blood are processed, and tissue specimens from each group are analyzed for drug concentrations using LC-MS/MS.

GNE-317 is a potent inhibitor of PI3K [1].

Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) are a series of enzymes and play an important role in cell growth, proliferation, differentiation, survival, motility and intracellular trafficking.

In the GL261 cell line, GNE-317 showed cytotoxic activity [2].

In mouse brain, GNE-317(50 mg/kg) significantly inhibited pAkt, p4EBP1 and pS6 by 80%, 84%, and 92% respectively, which were downstream markers of the PI3K/mTOR pathway. In U87, GS2 and GBM10 tumor-bearing mice, GNE-317 inhibited tumor growth by 90%, 50% and a survival benefit, respectively [1]. In C57B6/J mice inoculated with GL261-GFP-Luc cells, the concentrations of GNE-317 in the normal brain, tumor core and rim were not significantly different. In tumor-bearing mice, GNE-317 significantly reduced the levels of p-AktSer473, p-S6Ser235/236 and p-4EBP1Thr37/46 within the tumor [2]. In U87 and GS2 glioblastoma multiforme (GBM) models, GNE-317 was uniformly distributed in the brain. The brain-to-plasma ratios for GNE-317 were greater than 1, in agreement with the brain penetration properties of GNE-317 [3].

References:
[1].  Salphati L, Heffron TP, Alicke B, et al. Targeting the PI3K pathway in the brain--efficacy of a PI3K inhibitor optimized to cross the blood-brain barrier. Clin Cancer Res, 2012, 18(22): 6239-6248.
[2].  Becker CM, Oberoi RK, McFarren SJ, et al. Decreased affinity for efflux transporters increases brain penetrance and molecular targeting of a PI3K/mTOR inhibitor in a mouse model of glioblastoma. Neuro Oncol, 2015, pii: nov081.
[3].  Salphati L, Shahidi-Latham S, Quiason C, et al. Distribution of the phosphatidylinositol 3-kinase inhibitors Pictilisib (GDC-0941) and GNE-317 in U87 and GS2 intracranial glioblastoma models-assessment by matrix-assisted laser desorption ionization imaging. Drug Metab Dispos, 2014, 42(7): 1110-1116.