Proprietary LMW VEGF receptor inhibitors (PTK787, BAW2881, BFH772 and UF-61-QB443) that inhibit VEGF receptor-2 or a proprietary antibody that neutralizes VEGF (4G3) were used as positive controls.
11,12 In a fluorescence resonance energy transfer human enzymatic assay, compounds BFH772 and BAW2881 inhibited VEGF receptor-2 enzymatic activity with a 50% inhibitory concentration (IC
50) of 4 nM and compound UF-61-QB443 with an IC
50 of 8 nM (LanthaScreen kinase activity assay; LifeTechnologies, Grand Island, NY, USA). In a scintillation proximity assay of human enzymatic activity, PTK787 inhibited VEGF receptor-2 kinase activity with an IC
50 of 37 nM.
10 Compound 4G3 is a human Fab converted to a full-length IgG antibody with a mouse IgG1 Fc tail. The 4G3 binds to mouse VEGF
164 with a K
d of 10 pM, as measured by Biacore (GE Healthcare Bio-Sciences Pittsburgh, PA, USA; data not shown) and neutralizes mouse VEGF binding to human VEGFR-2 with an EC
50 of 0.15 nM in a binding assay (ELISA; MSD, Rockville, MD, USA). To validate our in vivo methodology, we assessed the dose-response of pazopanib, a LMW VEGF receptor inhibitor,
9,13,14 in three independent experiments. Compared to vehicle-treated mice that served as negative controls, the ED
50 of orally administered pazopanib was 63 mg/kg (
Fig. 1, and see
Supplementary Figs. S2–S4). Similar dose-response studies were performed for the proprietary LMW VEGF receptor inhibitors that we used as positive controls. LMW VEGF receptor inhibitors BFH772, BAW2881, and PTK787 have ED
50 values of ~2, 4, and 60 mg/kg, respectively, when administered orally once a day.