Antitumour efficacy of VEGFR2 tyrosine kinase inhibitor correlates with expression of VEGF and its receptor VEGFR2 in tumour models

Abstract

During the development of indazolylpyrimidines as novel and potent inhibitors of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) tyrosine kinase, we observed that some human tumour xenografts are more sensitive to VEGFR2 kinase inhibitors than others. A better understanding of the basis for this differential response may help to identify a predictive marker that would greatly aid in the identification of a suitable patient population for treatment. One representative compound from the indazolylpyrimidine series is GW654652 that inhibited all three VEGFRs with similar potency. The inhibition of VEGFR2 kinase by GW654652 was about 150 to >8800 more potent than the inhibition of eight other kinases tested. GW654652 inhibited VEGF- and bFGF-induced proliferation in endothelial cells with an IC(50) of 110 and 1980 nM, respectively, and has good pharmacokinetic profile in mouse and dog. We investigated the association between VEGF and VEGFR2 expression and the antitumour efficacy of GW654652, in various xenograft models. Statistically significant associations were observed between the antitumour efficacy of GW654652 in xenografts and VEGF protein (P=0.005) and VEGFR2 expression (P=0.041). The oral dose of GW654652 producing 50% inhibition of tumour growth (ED(50)) decreased with increasing levels of VEGF (r=-0.94); and, in contrast, the ED(50) increased with the increased expression of VEGFR2 (r=0.82). These results are consistent with the observed inverse correlation between VEGF and VEGFR2 expression in tumours. These findings support the hypothesis that VEGF and VEGFR2 expression by tumours may predict the therapeutic outcome of VEGFR kinase inhibitors.

Figure 1

Growth inhibition of human tumour xenografts in mice treated with VEGFR2 kinase inhibitors, GW654652, GW612286, or GW695612. Animals with 100–200 mm³ tumour volume were randomly assigned to either vehicle or treatment group (n=8 mice group⁻¹) as described in Materials and Methods. All compounds were administered orally at 30 mg kg⁻¹ once daily, except GW695612 (30 mg kg⁻¹, twice day⁻¹). Data represent tumour growth inhibition (mean±s.e.m.) in drug-treated animals compared to vehicle-treated mice after 21 days of dosing.

Figure 2

Relationship between (A) inhibition of tumour growth by GW654652, (B) human VEGF expression, and (C) VEGFR2 expression in human tumour xenografts. All values of VEGF levels (ELISA) and VEGFR2 expression (represented as M_d: mean channel difference from FACS analysis) are mean±s.e. and were obtained by analysing 4–15 tumour samples.

Figure 3

Expression of VEGFR2 by human tumour xenografts. (A) Western blot analysis of VEGFR2 in tumour extracts. The amount of protein analysed for PC3=120 μg, A375P=200 μg, HCT116=180 μg, HT29=200 μg, and HN5=200 μg. (B) Expression of VEGFR2 by human tumour xenografts. FACS analysis of VEGFR2 expression in PC3, SW620, A375P, HN5, HCT116, and HT29 tumours dissociated as single-cell suspensions. The fluorescence profiles of cells treated with VEGFR2 antibody (solid line) and isotype controls (dotted line).

Figure 4

Modulation of VEGFR2 by VEGF in vivo. The VEGF₁₂₁ (15 μg/mouse) was administered via tail vein in mice, and their lungs harvested at the indicated time points. Values represent the ratio of VEGFR2 to β-tubulin as determined by densitometry and normalised to vehicle-treated mice. Human umbilical vein endothelial cells immunoprecipitated using anti-VEGFR2 antibody from 350 μg protein were used as controls.