Mechanism And Pharmacokinetics Of Cyclodextrin-coated Oroxin B Eye Drops On Inhibition Of Corneal Neovascularization

Objective:Corneal neovascularization is a pathological change when the cornea is subjected to harmful conditions,threatening eyesight in severe cases.At present,clinical treatment is mainly composed of steroid-based topical anti-inflammatory drugs and off-label use of anti-VEGF antibodies.The former has weak anti-vascularization effects and a series of ocular side effects;whereas the efficacy of the latter is limited by poor corneal penetration.Oroxylum Indicum is a traditional Chinese medicine,and its main components include four flavonoids with the same parent nuclear structure,namely baicalein,oroxin A,chrysin,and oroxin B.Oroxin A,chrysin,and baicalein have been proven to have the dual effects of anti-inflammatory and anti-angiogenesis,particularly the application of chrysin and baicalein in ophthalmology has long been reported.Oroxin B has the most hydroxyl groups and best water solubility and has been proven to have anti-inflammatory and anti-tumor effects.Therefore,this study intended to develop oroxin B into eye drops and evaluate on the model of corneal neovascularization in rats,so as to assess its therapeutic effect and potential mechanism of angiogenesis inhibition.At the same time,the pharmacokinetic behavior of oroxin B in ocular tissues after topical instillation was investigated.This study provided findings in support of a potential new therapeutic candidate for the treatment of corneal neovascularization.Methods:1.The effect of anti-proliferation,anti-migration and tube formation of oroxin B on HUVEC induced by VEGF were evaluated by the assay of CCK8,cell wound scratch,transwell,and tube formation,respectively.2.To determine the inclusion concentration of HP-β-CD using phase solubility assay,0.2%,0.5%,and 1.0%oroxin B eye drops were prepared by saturated aqueous solution method,and the inclusion complex was characterized by differential scanning calorimetry,infrared spectra,and saturated solubility analyses.UPLC method was established to investigate the stability of oroxin B eye drops under different storage conditions.The ocular irritation and ocular surface toxicity of the oroxin B eye drops were also investigated in rabbits.3.180 male SD rats,180～200 g,were used to create a corneal neovascularization model for evaluation of the effect of oroxin B eye drops.30 SD rats were set as the blank control group without any treatment.The other 150 SD rats were for establishing a corneal neovascularization model induced by alkali burn.On the first day after the burn,150 SD rats were randomly divided into model group,low,medium,high dosing groups of oroxin B and positive drug control group according to weight,and the corresponding eye drops were given four times a day for 21 days.The corneal neovascularization growth,corneal transparency,and corneal staining with 0.1%fluorescein sodium solution were observed on 4,7,14,and 21 days after the alkali burn,respectively.The corneal tissues of each group were taken for HE staining and immunohistochemistry,and gene expression levels of Il-6、Il-1β、Vegf、Mmp-9、Mmp-2、Hif-1、Icam-1 were measured by RT-q PCR,and the protein levels of VEGF,MMP-9 and HIF-1 were determined by Western blot and IHC.To investigate the mechanism of oroxin B on the inhibition of inflammation and neovascularization,the protein levels of VEGFR,PI3K,AKT,and NF-κB were measured in VEGF treated HUVEC by Western blot.4.108 adult healthy rabbits,2～2.5kg,both male and female,were used to evaluate the ocular distribution and metabolism of oroxin B eye drops.The rabbits were randomly divided into 2 groups according to their weight.For the single dosing group（72）,50μL of low,medium and high concentrations of oroxin B eye drops were instilled into both eyes,and the animals were divided evenly into eight subgroups.In each subgroup,blood specimens were collected at 0.5,1,2,4,6,8,12,24 hours and the animals were sacrificed immediately to collect aqueous humor from the anterior chamber,and subsequently other ocular tissues.For the multiple dosing group（36）,the high concentration of oroxin B eye drop was instilled into both eyes four times per day for one week,the animals were divided evenly into 12 subgroups,and specimens in each subgroup were procured the same way as the single dosing group at 2 hours after consecutive dosing of 4,5 and 6 days,and at 0,0.5,1,2,4,6,8,12,24 hours after consecutive dosing of 7 days.The concentration of oroxin B in each specimen was measured by a validated UPLC-MS/MS method.The UPLC-Q-TOF-MS/MS assay was also used for the rapid identification of oroxin B ocular tissue metabolites,combined with the acquisition of precise molecular weights of the compounds and comparison of chromatographic behaviors and characteristics of secondary fragment ions to determine their possible metabolic pathways.Results:1.CCK-8 assay showed oroxin B was able to inhibit the proliferation of VEGF-induced HUVEC in a dose-dependent manner;a significant difference was observed when the concentration of oroxin B was above 30μM（p<0.01）.Cell wound scratch,transwell,and tube formation assays showed that 30μM and 60μM oroxin B significantly reduced the area of wound healing,the number of cell migration,and the number of cell tube formation of VEGF-induced HUVEC（p<0.05）.2.The phase solubility study showed that the solubility of oroxin B was increased linearly with the increasing concentration of HP-β-CD,which belonged to the A_L stable inclusion type;meanwhile,the optimal inclusion concentration of HP-β-CD was determined to be 25%（w/v）.Differential scanning calorimetry,infrared spectroscopy,and saturated water solubility assays confirmed the formation of oroxin B and HP-β-CD complex.A UPLC method was established for the determination of oroxin B in eye drops,which exhibited good specificity and a linear range of 7.06～35.6μg/m L.It has good precision and reproducibility;the average recoveries were 98.0～102%.The method was successfully applied to investigate the stability of oroxin B eye drops under conditions including light,high temperature,freeze-thaw,and storage.All the tests showed all three concentrations of oroxin B were stable,except for 1.0%oroxin B eye drops under 4℃.No irritation or corneal or conjunctival toxicity was observed in rabbit eyes after repeated administration of the blank solution and oroxin B drops for 21consecutive days.3.A model of corneal neovascularization in alkali-burned rats was established.Brush-like neovascular buds appeared at the corneal rim on the 4^th day after the burn;the treated eyes swelled significantly,the inflammatory reaction increased,and the neovascularization grew rapidly with large diameters on the 7^th day;the diameters of the neovascularization were decreased and the spreading area was increased on the 14^th day;the neovascularization covered the whole cornea on the 21^st day.The low,medium and high doses of oroxin B groups could reduce the area of corneal neovascularization and decrease the inflammatory response according to the results from slit lamps,which were consistent with the results of HE assay on the 7^th and 14^th days after the alkali burn.RT-q PCR showed that topical multiple administration of three doses of oroxin B eye drops could decrease m RNA expression of Il-6、Il-1β、Vegf、Mmp-9、Mmp-2、Hif-1、Icam-1,showing certain dose-dependence.There were significant differences between the medium and high dose groups compared with the model group（p<0.05）,and the high dose group did not show a difference from the positive drug control group.Western blot and immunohistochemical assay showed that topical multiple administration of three doses of oroxin B eye drops decreased the expression of VEGF and MMP-9 in the cornea.There were significant differences between the medium and high dose groups compared to the model group（p<0.05）,and no significant differences between the high dose group and the positive drug control group.Western blot of HUVEC results showed that60μM and 80μM oroxin B significantly down-regulated p-VEGFR2,p-PI3K,p-Akt,and p-NF-κB levels,and decreased the ratio of p-VEGFR2/t-VEGFR2,p-PI3K/t-PI3K,p-Akt/t-AKT,and p-NF-κB/t-NF-κB（p<0.05）.4.A rapid,simple,and sensitive UPLC-MS/MS method was developed for the determination of oroxin B in rabbit ocular tissues and plasma with a linear range of 5.00～500 ng/m L.The method was also validated according to the specifications for biological sample analysis,which could meet the requirements for preclinical pharmacokinetic studies of chemical drugs.After a single topical instillation,oroxin B reached a high concentration in anterior segment tissues,and its distribution was ranked in order from the highest to the lowest as follows:cornea,sclera,conjunctiva,iris-ciliary body≈aqueous humor,and lens.There was no significant difference among the concentrations of 2 hours after consecutive administration of 4 and 6 days.The AUC_0-24hafter multiple dosing was more than 3 times higher than that of the single dosing.Oroxin B levels were less than the lower limit of quantification in plasma at all time points.A UPLC-Q-TOF-MS/MS method was developed for the determination of the metabolism of oroxin B drops in ocular tissues.Only baicalein was found in the cornea and aqueous humor,with the highest conversion rates of 40%and 7%at 2 hours,respectively,followed by a gradual decrease.Conclusion:Oroxin B has the effect of anti-angiogenesis.In this study,topical ophthalmic formulation of oroxin B was prepared by cyclodextrin encapsulation technology,which was safe and stable,and could does-dependently inhibit alkali burn-induced corneal neovascularization and inflammatory response in rats,probably by inhibiting the VREGR/PI3K/AKT/NF-κB pathway.The oroxin B was mainly distributed in the target cornea tissue after topical administration,with a short peak time,slow elimination rate,and almost undetectable in the body circulation.Part of oroxin B could be metabolized into biologically active baicalin.Oroxin B has the potential to become a novel drug for neovascularization and possibly in other fields for neovascularization inhibition.