PEGylated HM-3 Polypeptide And Astragaloside ? In The Treatment Of Fundus Disease:experiment Research On Model Animals

Purpose:Age-related macular degeneration(AMD)and diabetic retinopathy(DR)is the leading cause of irreversible blindness in elderly people worldwide.Exudative AMD is typically associated with choroidal neovascularisation(CNV)beneath the fovea and it causes haemorrhage and macular oedema.HM-3 is a new anti-angiogenesis polypeptide that targets integrin ?v?3,which is a special site that is closely related with the angiogenic pathogenesis.However,in the in vivo pharmacokinetic studies,HM-3 has relatively short half-life,thus requiring administration frequently to achieve its optimal in vivo efficacy.Protein modification is an effective way to prolong its half-life.PEGylation is an attractive strategy to enhance the therapeutic efficacy of proteins with a short serum half-life.In this study,we sought to characterize the disposition of PEGylated HM-3 polypeptide after intravitreal administration in vivo and the protective effects of SC-mPEG20k-HM-3 in mice eyes with CNV.Besides,DR is a common diabetic eye disease which is well-known as the result of microvascular retinal changes.Although the potential biological functions of astragaloside IV have long been described in traditional system of medicine,its protective effect on DR remains unclear.This study also aims to investigate the function and mechanism of astragaloside IV on type 2 diabetic db/db mice.Methods:In the SC-mPEG20k-HM-3 study,mice were devided into 7 groups.Different dosages of mPEG-SC20k-HM-3 drug or control treatment was intravitreal injected in each group one week after laser-induced CNV.CNV leakage was then evaluated by fluorescein angiography.In vitro,Human Retinal Microvascular Endothelial Cells(HRMECs)stimulated with high glucose were treated with mPEG-SC20k-HM-3 to explore its function on cell viability and migration.To identify possible cellular responses to SC-mPEG20k-HM-3 in both the CNV model and in cells,VEGF,HIF-la and PI3K levels in RPE-choroid complexes and in HRMECs were quantified.Expression of PI3K,in choroid/sclera was also evaluated by immunohistochemistry.In the pharmacokinetic study,all the animals were randomized into the two groups with dosages of 260 ug/eye(1ul/eye)SC-mPEG20k-HM-3 and 20 ug/eye(1ul/eye)HM-3 mice were received mPEG-SC20k-HM-3 or HM-3 intravitreally.Ocular tissues and blood of each group were collected at 3,6,12,24,48,72,96,120,144,168,192 and 216h post dose under the microscope and concentrations of each polypeptide in retina,choroid/sclera and plasma were determined by high performance liquid chromatography methods.Pharmacokinetic properties in vivo,basic pharmacokinetic parameters were calculated.In the Astragaloside IV study,db/db mice were treated with astragaloside IV(4.5 mg/kg or 9 mg/kg)or physiological saline by oral gavage for 20 weeks along with db/m mice.In each group,retinal ganglion cell(RGC)function was measured by pattern electroretinogram(ERG)and apoptosis was determined by Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining.Blood and retina aldose reductase(AR)activity were quantified by chemiluminescence analysis.The expressions of phosporylated-ERK1/2,NF-kB were determined by western blot analysis.Furthermore,the expression of related downstream proteins were quantified by Label-based Mouse Antibody Array.Results:In the SC-mPEG20k-HM-3 study,the analysis of FA on day 14 after photocoagulation showed an obvious reduction in CNV area after SC-mPEG20k-HM-3 treatment.SC-mPEG20k-HM-3 inhibits cell proliferation stimulated by 20 mM hyperglycemia in CCK8 experiments.Compared with control group(0?M of SC-mPEG20k-HM-3),cell viability in 12?64?M SC-mPEG20k-HM-3 groups were significantly suppressed in day 3 and day 4.For cell migration,SC-mPEG20k-HM-3 as well as Avastin inhibited the cell migration rate in the transwell test compared with PEG or culture medium.Immunofluorescence staining of the frozen sections of mice choroid/sclera tissues revealed that strong PI3K-positive immunoreactivity was detected in the laser injury sites.VEGF and PI3K levels was significantly reduced compared with that in normal groups of both in vivo and in vitro experiments.For pharmacokinetics studies in mice,HM-3 and mPEG-SC20k-HM-3 were quickly absorbed after intravitreal administration.Compared with the HM-3 group,better AUC0-? and elimination half-life were obtained in the mPEG-SC20k-HM-3 group both in eye tissues and serum.In the Astragaloside IV study,administration of AS IV significantly improved the amplitude in pattern ERG and reduced the apoptosis of RGCs in db/db mice.Furthermore,downregulation of AR activity,ERK1/2 phosphorylation,NF-kB and related cytokine were observed in astragaloside IV treatment group.Conclusion:The intravitreal injections of SC-mPEG20k-HM-3 in comparison with intravitreal administration of HM-3,prolonged duration and increased local drug concentration,is a preferable approach to AMD.Furthermore,SC-mPEG20k-HM-3 mediated suppression of CNV formation or cell viability and migration observed in the present study is probably attributable to the inhibition of multiple angiogenic steps including the activation of PI3K and HIF-la.Therefore,we propose that intravitreal injection of SC-mPEG20k-HM-3 may serve as a therapeutic approach to the treatment of CNV in AMD.In the Astragaloside ? study,as an inhibitor of AR,Astragaloside IV could prevent the activation of ERK1/2 phosporylation and NF-kB and further relieve the RGCs disfunction in db/db mice with DR.It has provided a basis for investigating the clinical efficacy of AR inhibitors in preventing DR.