Optimal enzyme, ATP, and substrate (gastrin peptide) concentrations are established for each enzyme using homogeneous time-resolved fluorescence (HTRF) assays. Motesanib is tested in a 10-point dose-response curve for each enzyme using an ATP concentration of two-thirds K_m for each. Most assays consist of enzyme mixed with kinase reaction buffer [20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 5 mM MnCl₂, 100 mM NaCl, 1.5 mM EGTA]. A final concentration of 1 mM DTT, 0.2 mM NaVO₄, and 20 μg/mL BSA is added before each assay. For all assays, 5.75 mg/mL streptavidin-allophycocyanin and 0.1125 nM Eu-PT66 are added immediately before the HTRF reaction. Plates are incubated for 30 minutes at room temperature and read on a Discovery instrument. IC₅₀ values are calculated using the Levenberg-Marquardt algorithm into a four-parameter logistic equation.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cells are preincubated for 2 hours with different concentrations of Motesanib, and exposed with 50 ng/mL VEGF or 20 ng/mL bFGF for an additional 72 hours. Cells are washed twice with DPBS, and plates are frozen at -70°C for 24 hours. Proliferation is assessed by the addition of CyQuant dye, and plates are read on a Victor 1420 workstation. IC₅₀ data are calculated using the Levenberg-Marquardt algorithm into a four-parameter logistic equatio.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

A431 cells are cultured in DMEM (low glucose) with 10% FBS and penicillin/streptomycin/glutamine. Cells are harvested by trypsinization, washed, and adjusted to a concentration of 5×10⁷/mL in serum-free medium. Animals are challenged s.c. with 1×10⁷ cells in 0.2 mL over the left flank. Approximately 10 days thereafter, mice are randomized based on initial tumor volume measurements and treated with either vehicle (Ora-Plus) or Motesanib. Tumor volumes and body weights are recorded twice weekly and/or on the day of sacrifice. Tumor volume is measured with a Pro-Max electronic digital caliper and calculated using the formula length (mm)×width (mm)×height (mm) and expressed in mm³. Data are expressed as mean±SE. Repeated measures ANOVA followed by Scheffe post hoc testing for multiple comparisons is used to evaluate the statistical significance of observed differences^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Polverino A, et al. AMG 706, an oral, multikinase inhibitor that selectively targets vascular endothelial growth factor, platelet-derived growth factor, and kit receptors, potently inhibits angiogenesis and induces regression in tumor xenografts. Cancer Res. 2006 Sep 1;66(17):8715-21.  [Content Brief]

  [2]. Kruser TJ, et al. Augmentation of radiation response by motesanib, a multikinase inhibitor that targets vascular endothelial growth factor receptors. Clin Cancer Res, 2010, 16(14), 3639-3647.  [Content Brief]

  [3]. Coxon A, et al. Broad antitumor activity in breast cancer xenografts by motesanib, a highly selective, oral inhibitor of vascular endothelial growth factor, platelet-derived growth factor, and Kit receptors. Clin Cancer Res, 2009, 15(1), 110-118.  [Content Brief]