Cilengitide (EMD 121974) is a potent integrin inhibitor for the αvβ3/5 receptor (IC50: 4.1/79 nM, in cell-free assays); ~10-fold selectivity against gpIIbIIIa.

Cilengitide is supplied as an apyrogenic sterile infusion solution in physiological saline. Cilengitide is diluted in saline to a concentration of 1 mM[2]. The cytotoxicity of the two drugs, Belotecan and Cilengitide, is measured by the Cell Counting Kit-8 (CCK-8). U87 mg and U251 mg cells are seeded in 96 well plates at a density of 4×103 cells per well to allow for adhesion overnight. After this, the cells are treated with Cilengitide at a concentration of 0, 0.1, 0.5, 1, 5 and 25 μM and Belotecan at a concentration of 0, 6.25, 12.5, 25, 50 and 100 nM. All possible combinations of concentrations are used to assess the combined therapeutic effect of Cilengitide and Belotecan. After 3 days, 10 μL of the CCK-8 solution is added to each well of the plate, and the plate is incubated for 3 hr in the incubator (37°C; 5% CO2). The optical density (OD) of the sample plate is measured at 450 nm in a microplate reader. The viability of tumor cells is assessed by calculating the OD ratio of the specific OD in each sample to that of the control. Each experiment is conducted in quadruplicate. The values are averaged and normalized against the controls to generate dose-response curves[2].