Pazopanib Hydrochloride (Votrient HCl) is a novel multi-target inhibitor of VEGFR1, VEGFR2, VEGFR3, PDGFR, FGFR, c-Kit and c-Fms with IC50 of 10 nM, 30 nM, 47 nM, 84 nM, 74 nM, 140 nM and 146 nM in cell-free assays, respectively.

PDGFR:84 nM, c-Kit:140 nM, VEGFR3:47 nM, FGFR:74 nM, VEGFR2:30 nM, VEGFR1:10 nM, c-Fms:146 nM

Pazopanib以8 nM的IC50强效抑制VEGF诱导的VEGFR2在HUVEC细胞中的磷酸化。[1] Pazopanib对包括SYO-1和HS-SY-II细胞在内的所有滑膜肉瘤细胞系显示出剂量依赖性的生长抑制。即使在Pazopanib浓度为1μg/mL时，SYO-1和HS-SY-II细胞的增殖也受到抑制，并且在5μg/mL时完全停止。Pazopanib引发G1阶段停滞，从而抑制滑膜肉瘤细胞的生长。与对照组相比，Pazopanib处理的SYO-1细胞中Akts、GSK-3β、JNKs、p70 S6 Kinase和mTOR的磷酸化受到抑制。[2] 当Pazopanib浓度在20μg/mL到22.5μg/mL之间时，RPE细胞存活率呈现增加的减少。[3]

经30mg/kg或100mg/kg Pazopanib处理的小鼠与接受安慰剂或10mg/kg Pazopanib处理的小鼠相比，肿瘤负担显著减少。Pazopanib的治疗具有良好的耐受性，各组小鼠之间的体重无显著差异。[2]

Kinase enzyme assays: VEGFR enzyme assays for VEGGR1, VEGFR2, and VEGFR3 are run in homogeneous time-resolved fluorescence (HTRF) format in 384-well microtiter plates using a purified, baculovirus-expressed glutathione-S-transferase (GST) fusion protein encoding the catalytic c-terminus of human VEGFR receptor kinases 1, 2, or 3. Reactions are initiated by the addition of 10 μL of activated VEGFR2 kinase solution [final concentration, 1 nM enzyme in 0.1 M HEPES, pH 7.5, containing 0.1 mg/mL bovine serum albumin (BSA), 300 μM dithiothreitol (DTT)] to 10 μL substrate solution [final concentration, 360 nM peptide, (biotin-aminohexyl-EEEEYFELVAKKKK-NH2), 75 μM ATP, 10 μM MgCl2], and 1 μL of titrated Pazopanib in DMSO. Plates are incubated at room temperature for 60 min, and then the reaction is quenched by the addition of 20 μL of 100 mM ethylene diamine tetraacetic acid (EDTA). After quenching, 20 μL HTRF reagents (final concentration, 15 nM Streptavidin-linked allophycocyanin, 1 nM Europium-labeled antiphosphotyrosine antibody diluted in 0.1 mg/mL BSA, 0.1 M HEPES, pH 7.5) is added and the plates incubated for a minimum of 10 min. The fluorescence at 665 nM is measured with a Wallac Victor plate reader using a time delay of 50 μs.

Phosphorylation of VEGFR2 is assessed in HUVEC stimulated with VEGF. HUVEC are plated in type-I collagen-coated 10 cm plates in Clonetics EGM-MV medium at 1.0-1.5 × 106 cells/plate. After 24 hours, the confluent cells are serum starved overnight by replacing the growth medium with Clonetics EBM medium containing 0.1% BSA, 500 μg/mL hydrocortisone. Cells are treated with Pazopanib at various concentrations for 1 hour, followed by addition of 10 ng/mL VEGF or vehicle for 10 min. Cells are solubilized in lysis buffer. VEGFR2 is immunoprecipitated using antiflk-1 antibody and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting and detection with antiflk-1 or with antiphosphotyrosine (anti-P-tyr-biotin) antibody. The VEGFR2 phosphorylation level is quantified by densitometry and normalized to the total VEGFR2 level. (Only for Reference)