ON123300 was administered to groups of brain tumor–bearing mice as an intravenous bolus at a dose of either 5 or 25 mg/kg via a tail vein

The cytotoxicity of ON123300 was determined using a colorimetric sulforhodamine B (SRB)-based assay. Suspensions of glioma cells (100 mL containing 2 x 10^3 cells) were seeded in 96-well plates and allowed to attach to the surface by overnight incubation. The cells were then treated with increasing concentrations of ON123300 for 72 hours. At the end of the treatment, cells were fixed with 10% (v/v) trichloroacetic acid (TCA) and stained with 0.4% SRB. The optical densities were measured with a SpectraMax M2 microplate reader (Molecular Devices) at a wavelength of 570 nm. A Sigmoid-Emax model (Win-Nonlin, Pharsight Corporation) was used to calculate IC50 values (mean of three independent studies), which were defined as the drug concentration that was required to reduce the number of viable cells to 50% compared with control treatment (vehicle alone). For combination studies, cells were treated with ON123300 (from 0.03 to 16 mkmol/L) and gefitinib (from 0.16 to 80 mkmol/L) alone or together at a fixed concentration ratio of 0.2, or treated with ON123300 (from 0.1 to 25 mkmol/L), temozolomide (from 10 to 2,500 mkmol/L), alone or together at a fixed concentration ratio of 0.01 for 72 hours and then processed using the SRB assay.