Preclinical characterization of anlotinib, a highly potent and selective vascular endothelial growth factor receptor-2 inhibitor

Abstract

Abrogating tumor angiogenesis by inhibiting vascular endothelial growth factor receptor-2 (VEGFR2) has been established as a therapeutic strategy for treating cancer. However, because of their low selectivity, most small molecule inhibitors of VEGFR2 tyrosine kinase show unexpected adverse effects and limited anticancer efficacy. In the present study, we detailed the pharmacological properties of anlotinib, a highly potent and selective VEGFR2 inhibitor, in preclinical models. Anlotinib occupied the ATP-binding pocket of VEGFR2 tyrosine kinase and showed high selectivity and inhibitory potency (IC50 <1 nmol/L) for VEGFR2 relative to other tyrosine kinases. Concordant with this activity, anlotinib inhibited VEGF-induced signaling and cell proliferation in HUVEC with picomolar IC50 values. However, micromolar concentrations of anlotinib were required to inhibit tumor cell proliferation directly in vitro. Anlotinib significantly inhibited HUVEC migration and tube formation; it also inhibited microvessel growth from explants of rat aorta in vitro and decreased vascular density in tumor tissue in vivo. Compared with the well-known tyrosine kinase inhibitor sunitinib, once-daily oral dose of anlotinib showed broader and stronger in vivo antitumor efficacy and, in some models, caused tumor regression in nude mice. Collectively, these results indicate that anlotinib is a well-tolerated, orally active VEGFR2 inhibitor that targets angiogenesis in tumor growth, and support ongoing clinical evaluation of anlotinib for a variety of malignancies.

Keywords: VEGF; VEGFR2; angiogenesis; anlotinib; tyrosine kinase inhibitor.

© 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Figures

Figure 1

Characterization of anlotinib as a vascular endothelial growth factor receptor‐2 (VEGFR2) inhibitor. A, Chemical structure of anlotinib. B, Molecular modeling of the VEGFR2–anlotinib/sunitinib complex. Hydrogen bonds are presented as yellow dashed lines, and critical residues are presented as maroon sticks. Anlotinib (cyan, docked pose with structure PDB code 4ASD) and sunitinib (orange, from crystallographic structure PDB code 4AGD). C, Molecular modeling of the c‐Kit–anlotinib complex (orange) and VEGFR2–anlotinib complex (cyan). Hydrogen bonds are presented as yellow dashed lines, and critical residues are presented as maroon sticks (VEGFR2) and dark green sticks (c‐Kit)

Figure 2

Effects of anlotinib on growth factor‐stimulated receptor phosphorylation. Serum‐starved (A) HUVEC, (B) Mo7e, (C) U‐87MG and (D) A431 cells were treated with different concentrations of test agents for 1.5 h and then stimulated with vascular endothelial growth factor (VEGF; 20 ng/mL), stem cell factor‐1 (SCF‐1; 2.5 ng/mL), platelet‐derived growth factor‐BB (PDGF‐BB; 10 ng/mL), or epidermal growth factor (EGF; 10 ng/mL) for 10 min, respectively. E, BT‐474 cells which have constitutive HER2 autophosphorylation and downstream signaling activation were treated with test agents for 1.5 h. Cell lysates were probed with the indicated antibodies

Figure 3

Inhibitory effects of anlotinib on cell proliferation. A, Inhibitory effect of anlotinib on vascular endothelial growth factor (VEGF)‐ or FBS‐stimulated HUVEC proliferation. HUVEC were incubated with different concentrations of drugs together with FBS (20%) or VEGF (20 ng/mL). B, Effects of anlotinib on tumor cell proliferation. Tumor cells were cultured with 10% FBS and then treated with anlotinib. Cell viability was determined by sulforhodamine B (SRB) assay. IC 50 values are presented as means ± SD of 3 independent experiments

Figure 4

Inhibitory effects of anlotinib on angiogenesis in vitro. A, Effect of anlotinib on HUVEC migration induced by vascular endothelial growth factor (VEGF)‐A. B, Effect of anlotinib on FBS‐stimulated HUVEC tube formation. C, Effect of anlotinib on VEGF‐stimulated microvessel sprouting from rat aortic rings. Representative images are shown and data are presented as the mean ± SD of 3 independent experiments. *P < .05, **P < .01 compared with VEGF‐ or FBS‐treated groups

Figure 5

In vivo antitumor efficacy of anlotinib in SW620 tumor xenografts. (A,B) SW620 tumor‐bearing mice were orally given vehicle (n = 12) or the indicated doses of anlotinib or sunitinib (n = 6) daily for 18 d. A, Tumor volumes and (B) mouse bodyweights were determined twice weekly during the course of the experiment. C, Photographs of tumors on the final day. D, Immunohistochemical detection of the endothelial cell‐specific marker, CD31, in tumor tissue sections of SW620 xenografts. Data are presented as means ± SEM. *P < .05, **P < .01 vs vehicle

Figure 6

In vivo antitumor activity of anlotinib against a panel of tumor xenografts. A, U‐87MG, (B) Caki‐1, (C) SK‐OV‐3, and (D) Calu‐3 tumor‐bearing mice were orally given vehicle (n = 12) or the indicated doses of anlotinib or sunitinib (n = 6) daily. In SK‐OV‐3 and Calu‐3 xenograft models, anlotinib at 6 mg/kg was orally given daily for only 9 d. Data are presented as means ± SEM. **P < .05, **P < .01 vs vehicle