Axitinib is an orally bioavailable tyrosine kinase inhibitor with IC50s of 0.1, 0.2, 0.1-0.3, 1.7, 1.6 nM for VEGFR1, VEGFR2, VEGFR3, c-kit, and PDGFRβ, respectively.
In transfected or endogenous RTK-expressing cells, axitinib potently blocked growth factor-stimulated phosphorylation of VEGFR-2 and VEGFR-3 (IC50s: 0.2 and 0.1 to 0.3 nmol/L, respectively). Cellular activity against VEGFR-1 was 1.2 nmol/L. Axitinib rapidly and dose-dependently reduced the phosphorylation of Akt, endothelial nitric oxide synthase (eNOS), and extracellular signal-regulated kinase 1/2 (ERK1/2), key downstream signaling molecules of VEGF . Axitinib reduced cell viability in a dose-dependent manner with IC50 doses of >10,000, 849 and 274 nmol/l for IGR-N91, IGR-NB8, and SH-SY5Y, respectively. the sensitivity to axitinib of neuroblastoma cell lines appeared to be in a similar range as non-VEGF stimulated HUVEC (IC50: 573 nmol/l) .
Acute axitinib treatment rapidly and significantly reduced retinal vascular VEGFR-2 phosphorylation. One hour after the second dose, retinal VEGFR-2 phosphorylation was reduced by 80% to 90% compared with that of the control tissues. Six and 24 to 32 h post-dose, the phospho-VEGFR-2 levels returned to ～50% and 100% of the control, respectively. The EC50 value for the inhibition of VEGFR-2 phosphorylation was 0.49 nmol/L . Mice were next treated for a period of 2 weeks with either fractionated radiation (5 × 2 Gy/wk) or AG-013736 (25 mg/kg/d) and 1 to 3 weeks for the combination. Tumor volume at the end of 2 weeks was significantly reduced for either single or combination treatments. Percentage increases in tumor volume were similar between radiotherapy (40 ± 9.8%) and AG-013736 (48 ± 9.2%), and the combination was markedly reduced versus controls (12 ± 5.7% versus 77 ± 11%) .
Porcine aorta endothelial (PAE) cells overexpressing full-length VEGFR-2, PDGFR-β, KIT, and NIH-3T3 overexpressing murine VEGFR-2 (Flk-1) or PDGFR-α were generated as described previously. The ELISA capture plates were prepared by coating 96-well ReactiBind plates with 100 μL/well of 2.5 μg/mL anti-VEGFR-2 antibody, 0.75 μg/mL anti-PDGFR-β antibody, 0.25 μg/mL anti-PDGFR-α antibody, 0.5 μg/mL anti-KIT antibody, or 1.20 μg/mL anti-Flk-1 antibody. Measurement of RTK phosphorylation by ELISA was done as described previously .
Endothelial or tumor cells were starved for 18 h in the presence of either 1% FBS (HUVEC) or 0.1% FBS (tumor cells). Axitinib was added and cells were incubated for 45 min at 37°C in the presence of 1 mmol/L Na3VO4. The appropriate growth factor was added to the cells, and after 5 min, cells were rinsed with cold PBS and lysed in the lysis buffer and a protease inhibitor cocktail. The lysates were incubated with immunoprecipitation antibodies for the intended proteins overnight at 4°C. Antibody complexes were conjugated to protein A beads and supernatants were separated by SDS-PAGE. The Super Signal West Dura kit was used to detect the chemiluminescent signal .
AG-013736, a receptor kinase inhibitor of VEGFRs and, at higher doses, PDGFRs (IC50 = 0.1 nmol/L for VEGFR-1, 0.2 nmol/L for VEGFR-2, 0.1–0.3 nmol/L for VEGFR-3, and 1.6 nmol/L for PDGFRβ; ref. 18), was provided by Pfizer Global Research and given once daily by gavage in a volume of 0.13 mL. Control animals received 0.5% carboxymethylcellulose drug carrier. Irradiations were done on nonanesthetized mice using a 137Cs source operating at 2.4 Gy/min. Mice were confined to plastic jigs with tumor-bearing legs extended through an opening in the side, allowing local irradiations. Fractionated doses were given in five daily 2 Gy fractions per week (omitting weekends). For combination treatments, radiotherapy was delivered first, and AG-013736 was given within ～4 h. Mice were sacrificed, and tumors were excised and then quick frozen (using liquid nitrogen) following 1, 2, or 3 weeks of treatment .