| Purpose:To investigate the effects of HM-3on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms and the pharmacokinetics of HM-3after intravitreal administration.Methods:The experiment was conducted in two parts. The first part, CNV was induced by laser photocoagulation on Day0in C57BL/6J mice. In HM-3groups, each mouse received single bilateral intravitreal injection with doses of0,10,20and40μg/eye HM-3. In the positive control group, Avastin was administrated with intravitreal injection. But in vehicle group, the same dose of physiological saline was given. The CNV area was analyzed by fluorescein-labeled dextran angiography of retinal pigment epithelium (RPE)-choroid flat mounts on day7after laser photocoagulation, and CNV leakage was also evaluated by fluorescein angiography (FA). The infiltration of neovascular endothelial cell was evaluated by immunohistochemistry on RPE-choroid flat mounts on day3. The VEGF expression in RPE-choroid complex was quantified by real-time PCR. RPE-choroid levels of vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)-a were examined by ELISA on day3. The expression of ERK1/2and p-ERKl/2in the RPE-choroid was determined by Western blotting. The second part,288C57BL/6J mice were evaluated and divided into4groups. In HM-3group, each mouse received single bilateral intravitreal administration of doses of0,10,20and40μg/eye HM-3. The vehicle group was not given any treatment. The concentrations of HM-3in choroid/sclera, retina and serum were determined by indirect competitive enzyme-linked immunosorbent assay (ELISA).Results:In the first part, HM-3-treated mice had significantly less CNV area (P< 0.05) and CNV leakage (P<0.001) than vehicle-treated mice, and there is no significant difference between20ug/eye HM-3and Avastin group. HM-3treatment led to significant inhibition of infiltration of neovascular endothelial cell (P<0.05). VEGF mainly expressed in laser injury sites, which was suppressed by HM-3treatment (P<0.05). HM-3inhibited the RPE-choroid levels of TNF-a (P<0.05), and suppressed the activation of ERK1/2(P<0.05) and the activation of p-ERK1/2(P<0.05).In the second part, after intravitreal administration of doses of0,10,20and40μg/eye HM-3, the elimination half-life (T1/2) in retina was104.85±36.90,107.42±35.25and101.12±15.82h, respectively; the mean residence time (MRT) was172.46±63.80,164.70±52.72and181.32±26.01h, respectively. In choroid/sclera, the values were54.04±25.99,59.33±24.46and47.10±10.00h, respectively;. MRT was139.98±23.93,155.43±17.81and136.45±18.17h, respectively. But in serum, the results were were48.39±14.89,47.96±12.97and49.98±30.07h, respectively; MRT were108.6±47.17,159.76±18.82and125.33±21.41h, respectively.Conclusion:HM-3treatment could inhibit the occurrence and development of laser-induced choroidal neovascularization, which would though specific binding with integrin αvβ3, activating ERK1/2signaling pathway by reducing the expression of inflammation and down-regulation of angiogenesis factor. The pharmacokinetics of HM-3in the retina, choroid/sclera tissue and serum in mice with all preparations were fitted to non-compartmental model. The half-life of HM-3was prolonged and the concentration of HM-3in ocular tissues was significantly increased via intravitreal injection in mice. The pharmacokinetic profiles of intravitreal administration HM-3provide the basis for the development of reasonable dosing regimens of clinical CNV treatment. Carrying out the research of HM-3provides the information for conversion to clinical application of CNV. |
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