PF-04217903

Immunodeficient nude mice (nu/nu) subcutaneously implanted with tumor cell lines B16F1, EL4, LLC, or Tib6

2-(4-(3-(quinolin-6-ylmethyl)-3H-[1, 2, 3]triazolo[4, 5-b]pyrazin-5-yl)-1H-pyrazol-1-yl)ethanol

Dissolved in DMSO, final concentrations ca.2 uM

45 mg/kg

4.8 nM [1], 4.8 nM [1], 4.8 nM [1], 4.8 nM [1], 4.8 nM [1], 4.8 nM [1]

Although unable to inhibit tumor growth in the sunitinib-sensitive B16F1 and Tib6 tumor models, the combination of PF-04217903 and sunitinib significantly inhibits tumor growth in sunitinib-resistant EL4, and LLC tumor models compared with sunitinib or PF-04217903 alone by significantly blocking vascular expansion, indicating a functional role for HGF/c-Met axis in the sunitinib-resistant tumors. [2]

Cellular c-Met phosphorylation ELISA, A549 cells with endogenous human WT c-Met are seeded in 96-well plates in growth medium and cultured overnight. On the second day of the assay, the growth medium is replaced with serum-free medium (with 0.04% BSA). Serial dilutions of PF-04217903 are added to each well, and cells are incubated at 37 C for 1 hour. Then 40 ng/mL HGF is added to the cells for 20 minutes. The cells are washed once with HBSS supplemented with 1 mM Na3VO4, and protein lysates are generated from cells using lysis buffer. Phosphorylation of c-Met is assessed by an ELISA method utilizing capture antibodies specific for c-Met and a detection antibody specific for phosphorylated tyrosine residues. Antibody-coated plates are incubated in the presence of protein lysates at 4 C overnight and washed with 1% Tween 20 in PBS seven times. HRP-PY20 (horseradish peroxidase-conjugated anti-phosphotyrosine) is diluted 1:500 in blocking buffer and added to each plate for 30 minutes. Plates are then washed again, and TMB peroxidase substrate is added to initiate the HRP-dependent colorimetric reaction and the reaction stopped by addition of 0.09 N H2SO4. ELISA end points are the absorbance measured at 450 nm using a spectrophotometer. IC50 value is calculated by concentration-response curve fitting utilizing a Microsoft Excel-based four-parameter analytical method.

372, 38

Data independently produced by, , , Dr. Zhang of Tianjin Medical University, PF-04217903purchased from Selleck, Western blot analysis of c-Met, MAPK and Akt. 0-100nM PF04217903 was added.

2 years -20CPowder, 2 weeks4Cin DMSO, 2 months-80Cin DMSO