YN968D1 is a novel and selective inhibitor of vascular endothelial growth factor receptor-2 tyrosine kinase with potent activity in vitro and in vivo

Abstract

Angiogenesis is an important process in cell development, especially in cancer. Vascular endothelial growth factor (VEGF) signaling is an important regulator of angiogenesis. Several therapies that act against VEGF signal transduction have been developed, including YN968D1, which is a potent inhibitor of the VEGF signaling pathway. This study investigated the antitumor activity of YN968D1 (apatinib mesylate) in vitro and in vivo. YN968D1 potently suppressed the kinase activities of VEGFR-2, c-kit and c-src, and inhibited cellular phosphorylation of VEGFR-2, c-kit and PDGFRβ. YN968D1 effectively inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells induced by FBS, and blocked the budding of rat aortic ring. In vivo, YN968D1 alone and in combination with chemotherapeutic agents effectively inhibited the growth of several established human tumor xenograft models with little toxicity. A phase I study of YN968D1 has shown encouraging antitumor activity and a manageable toxicity profile. These findings suggest that YN968D1 has promise as an antitumor drug and might have clinical benefits.

Figure 1

Chemical structure of YN968D1.

Figure 2

Effects of YN968D1 on various growth factor‐stimulated receptor phosphorylation at the cellular level detected by western blot analysis. VEGFR, ERK1/2, c‐kit and PDGFR phosphorylation was suppressed by YN968D1 in a dose‐dependent manner, which were comparable with sunitinib (A–C). YN968D1 had no effect on the phosphorylation of EGFR and Her‐2, and lapatinib served as a positive control (D,E). Tubulin was used as a loading control. VEGF, vascular endothelial growth factor. VEGFR, vascular endothelial growth factor receptors; PDGF, platelet derived growth factor; PDGFR, PDGF receptors; SCF, stem cell factor.

Figure 3

Inhibition of vascular endothelial growth factor (VEGF)‐stimulated HUVEC proliferation, HUVEC tubule formation, HUVEC migration and microvessel outgrowth from rat aortic ring by YN968D1. (A) HUVEC were incubated with test agents together with 20% FBS or 20 ng/mL VEGF. The inhibitory activity was expressed as IC₅₀. (B) HUVEC suspended in M199 medium with 20% FBS were added into solidified matrigel, with different test agents for 8 h, and the cells were imaged using a high magnification field. The tube formation ability of HUVEC was impaired by 1 μM YN968D1 and sunitinib. (C) Transwell migration assays using the Corning chamber showed that the migration of HUVEC induced by 20% FBS was inhibited by 1 μM YN968D1 and sunitinib. (D) The aortic rings were embedded in matrigel and incubated in supplemented media at 37°C, 5% CO₂ for 6 days. Images taken through an inverse microscope showed YN968D1 and sunitinib suppressed vessels sprouting from the rat aorta rings. Columns, mean; bars, SE. *P < 0.05 versus vehicle control.

Figure 4

Antitumor activity of YN968D1 against human tumor xenografts in nude mice. (A) YN968D1 inhibited growth of established NCI‐H460 human lung tumors, HCT 116 human colon tumors, or SGC‐7901 human gastric tumors in nude mice. Tumor volume was measured on the indicated days, with the mean tumor volume indicated for groups of 6 (treated) or 12 (vehicle control) animals. *P < 0.05 versus vehicle control. (B) YN968D1 in combination with docetaxel or oxaliplatin showed synergistic tumor growth inhibition effects against NCI‐H460 and Ls174t xenografts, respectively. *P < 0.05 versus YN968D1 alone. **P < 0.05 versus cytotoxic drugs alone. Data are represented as mean ± standard error. (C) Angiogenesis was inhibited markedly by YN968D1 within NCI‐H460 xenograft tumor tissues as evidenced by CD31 staining (positive cells, brown color, indicated by arrows). Columns, mean; bars, SE. *P < 0.05 versus vehicle control.

Figure 5

Pulmonary computed tomography (CT) images of a patient with metastatic rectal cancer involving the liver and lung who was treated with YN968D1 and obtained a partial response.^{( 21 )} (A) Baseline CT image. (B) CT image 53 days after treatment.