Inhibitory Effect And Mechanism Of A Novel VEGF-target Antibody On Corneal Neovascularization

Purpose To evaluate the pharmacological characteristics and efficacy of FD006in corneal neovascularization (CoNV). To varify the safety application of FD006in the treatment of corneal disease both in vivo and vitro. To evaluate the ocularpharmacological characteristics of the FD006when given subconjunctivally.Method The binding specificity and affinity of FD006was measured by ELISAand binding kinetics assays; besides, VEGF-induced proliferation of HUVECassay was used to detect the in vitro antiangiogenic activity of FD006;furthermore, rat corneal neovascularization model was set up. Whatman filterpapers3mm in diameter were soaked in NaOH and touched to the central corneaof the right eyes for40s to induce corneal neovascularization. On the next day,rats were randomly divided into five groups:0.9%NaCl control group, solventgroup,0.05ml (25mg/ml) bevacizumab group,0.05ml (25mg/ml) FD006groupand dexamethasone (5mg/mL) group, and the rats in each group received a singlesubconjunctival injection of0.05ml of solution. Corneal neovascularization wasanalyzed by slit-lamp microscopy and digital photographs were used to analyzethe length and area of the corneal neovasculature on the third, seventh, fourteenth,twenty-first and twenty-eight days. Immunohistochemical staining and westernblot were used to analyze the expression of VEGF, VEGFR-1, VEGFR-2, MMP-9and ICAM-1.Use CCK8to test the toxicity of FD006on cultured human cornealepithelial cells. FACs was used to analyze the apotosis of HCE after treated withFD006. Also evaluate the effect of FD006on corneal epithelial healing model inrat. After subconjunctival injection of FD006, take aqueous, vitrous and serum toanalyze the concentration of FD006with ELISA.Results FD006was predicted to have similar binding mode to bevacizumab. Experimental analysis showed that the binding ability of FD006was about5-foldstronger than bevacizumab, for the EC50of FD006to bind VEGF analyzed byELISA was about0.037μg/mL while that of bevacizumab was0.18μg/mL.Binding kinetics assays showed similar results that FD006possessed2-fold higheraffinity to bind VEGF than bevacizumab due to slower dissociation rate of FD006;meanwhile, FD006inhibited the VEGF-induced proliferation of HUVEC with anIC50value of0.031±0.0064μg/ml, which was better than bevacizumab (0.047±0.0081μg/ml). The subconjunctival administration of FD006, bevacizumab ordexamethasone could significantly inhibit the growth of CoNV contrasting to N.S(p <0.01). At the early stage, FD006showed better inhibitory effect on thegrowth of CoNV compared with bevacizumab (p <0.05) however, at the laterstage, the length and area were equal to that of the bevacizumab group (p>0.05).Western blot analysis showed that FD006could inhibit the expression of VEGF,VEGFR-1, VEGFR-2, MMP-9and ICAM-1, which could explain its favorableanti-angiogenic activity. FD006does no harm on the proliferation of HCE and theaptosis of HCE, and has no effect on corneal epithelial healing in rats. Aftersubconjunctivally administration, FD006can penetrate into ocular, and it canmaintain at an effective concentration in the aqueous or vitreous for about10.7or6.8days. Also FD006can be found in the uninjected eye through bloodtransportion.Conclusions The subconjunctival injection of FD006significantly inhibitedalkali burn-induced corneal neovascularization in rats. The pharmacologicalcharacteristics of FD006were better than bevacizumab in inhibiting cornealneovascularization at the early stage. It was safe with ocular application; also itcan maintain a therapeutic concentration in the injected eyes. The administrationof FD006may have a broad application for inhibiting human cornealneovascularization in the near future.