Foretinib

MET, KDR

632, 65

B16F10 tumor cells (2, 10 5) are implanted via i.v. tail vein injection into athymic nude mice (NCr or BALB/c) 5 to 8 weeks old

A Phase II study of XL880 about Recurrent or Metastatic Squamous Cell Cancer of the Head and Neck has been completed.

100 mg/kg

0.4 nM, 0.4 nM, 0.4 nM, 0.4 nM, 0.4 nM, 0.4 nM

A single 100 mg/kg oral gavage dose of XL880 results in substantial inhibition of phosphorylation of B16F10 tumor Met and ligand (e.g., HGFor VEGF)–induced receptor phosphorylation of Met in liver and Flk-1/KDR in lung, which both persisted through 24 hours. Treatment with XL880 (30-100 mg/kg, once daily, oral gavage) results in reduction in tumor burden. The lung surface tumor burden is reduced by 50% and 58% following treatment with 30 and 100 mg/kg XL880, respectively. XL880 treatment of mice bearing B16F10 solid tumors also results in dose-dependent tumor growth inhibition of 64% and 87% at 30 and 100 mg/kg, respectively. For both studies, administration of XL880 is well tolerated with no significant body weight loss. [1] XL880 is developed to target abnormal signaling of HGF through Met and simultaneously target several receptors tyrosine kinase involved in tumor angiogenesis. XL880 caused tumor hemorrhage and necrosis in human xenografts within 2 to 4 hours, and maximal tumornecrosis is observed at 9hours (after five daily doses), resulting in complete regression. [3]

Kinase Inhibition Assay, Kinase inhibition is investigated using one of three assay formats: [33P]phosphoryl transfer, luciferase-coupled chemiluminescence, or AlphaScreen tyrosine kinase technology. IC50s are calculated by nonlinear regression analysis using XLFit.33P -Phosphoryl Transfer Kinase Assay Reactions are performed in 384-well white, clear bottom, high-binding microtiter plates (Greiner, Monroe, NC). Plates are coated with 2 ug/well of protein or peptide substrate in a 50 uL volume of coating buffer contained 40 ug/mL substrate (poly(Glu, Tyr) 4:1, 22.5 mM Na2CO 3, 27.5 mM NaHCO 3, 50 mM NaCl and 3 mM NaN 3. Coated plates are washed once with 50 uL of assay buffer following overnight incubation at room temperature (RT). Test compounds and enzymes are combined with 33P-gamma-ATP (3.3 uCi/nmol) in a total volume of 20 uL. The reaction mixture is incubated at RT for 2 hours and terminated by aspiration. The microtiter plates are subsequently washed 6 times with 0.05% Tween-PBS buffer (PBST). Scintillation fluid (50 uL/well) is added and incorporated 33P is measured by liquid scintillation spectrometry using a MicroBeta scintillation counter (Perkin Elmer, Wellesley, MA).Luciferase-Coupled Chemiluminescence Assay Reactions are conducted in 384-well white, medium binding microtiter plates (Greiner). In a first step enzyme and compound are combined and incubated for 60 minutes, reactions are initiated by addition of ATP and peptide substrate (poly(Glu, Tyr) 4:1) in a final voume of 20 uL, and incubated at RT for 2-4 hours. Following the kinase reaction, a 20 uL aliquot of Kinase Glo (Promega, Madison, WI) is added and luminescence signal is measured using a Victor plate reader (Perkin Elmer). Total ATP consumption is limited to 50%. AlphaScreenTM Tyrosine Kinase Assay Donor beads coated with streptavidin and acceptor beads coated with PY100 anti-phosphotyrosine antibody are used (Perkin Elmer). Biotinylated poly(Glu, Tyr) 4:1 (Perkin Elmer) is used as the substrate. Substrate phosphorylation is measured by addition of donor/acceptor beads by luminescence following donor-acceptor bead complex formation. Kinase and test compounds are combined and preincubated for 60 minutes, followed by addition of ATP, and biotinylated poly(Glu, Tyr) in a total volume of 20 uL in 384-well white, medium binding microtiter plates (Greiner). Reaction mixtures are incubated for 1 hour at room temperature. Reactions are quenched by addition of 10 uL of 15-30 ug/mL AlphaScreen bead suspension containing 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20. After 2-16 hours incubation at room temperature plates are read using an AlphaQuest reader (Perkin Elmer).

DMSO 127 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL

N-(3-fluoro-4-(6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yloxy)phenyl)-N-(4-fluorophenyl)cyclopropane-1, 1-dicarboxamide