| Part one:Study on the stability of a novel intraocular sustainedrelease preparation of artesunateObjectiveTo prepare three different concentrations of the novel intraocular sustained-release preparations of artesunate with excipients,and to investigate their stability under high temperature,strong light irradiation,freeze-thaw,acceleration and long-term storage.Methods1.Artesunate was included with special excipients to prepare intraocular sustained-release preparations of low(30%),medium(40%)and high(50%)concentrations of artesunate.2.High performance liquid chromatography was established to investigate the stability of three concentrations of intraocular sustained-release preparations of artesunate under high temperature,strong light irradiation,freeze-thaw,acceleration and long-term storage.ResultThe sustained-release preparations with low,middle and high concentrations of artesunate were unstable under high temperature and light conditions.the higher the temperature,the more obvious the decrease of the content and the increase of related substances,and the stability was good under the condition of avoiding light at room temperature.ConclusionThe intraocular sustained-release preparation of artesunate prepared by excipient inclusion technique has good stability under the condition of avoiding light at room temperature.Part two:Evaluation of Retinal Toxicity of a novel intraocular sustained-release preparation of artesunateObjectiveTo observe the effects of three concentrations of novel intraocular sustainedrelease preparations of artesunate on the retina after injection into the vitreous cavity of rabbit eyes,in order to evaluate the biocompatibility of this drug in rabbit eyes,and to provide preliminary information of ocular drug safety for subsequent experiments.Methods1.The selected chinchilla rabbits were divided into normal control group,blank matrix group,and groups with low,medium and high concentration of intraocular sustained-release preparations of artesunate.Slit-lamp microscopy,anterior segment photography,fundus photography and retinal OCT examination were performed before and at 7,14,21 and 28 days after vitreous cavity administration to evaluate the ocular condition.2.At 7,14,21 and 28 days after vitreous cavity administration,retinal patch and retinal HE staining were performed to observe the pathological changes of retina after administration.ResultNo obvious abnormal changes were observed in the eyes by slit lamp examination,anterior segment photography and fundus photography.No abnormal changes in the color,thickness and morphology of retinal layers were observed by retinal OCT examination,retinal patch and retinal HE staining.ConclusionBlank matrix and the novel intraocular sustained-release preparation of artesunate have no irritation and no obvious retinal toxicity after single intravitreal injection in rabbit eyes,and have good intraocular biocompatibility.Part three:Study on the inhibitory effect and mechanism of the novel intraocular sustained-release preparation of artesunate on experimental autoimmune uveitis in chinchilla rabbitsObjectiveTo explore the role of Notch signaling pathway in the occurrence and development of uveitis by blocking Notch signaling pathway with DAPT.To evaluate the inhibitory effect of the novel intraocular sustained-release preparation of artesunate on the inflammation of EAU chinchilla rabbits and explore the potential mechanism of action,so as to provide in vivo experimental basis for ART treatment of uveitis.Methods1.The EAU models were established by subcutaneous injection of FCA H37Ra and intravitreal injection of H37Ra in chinchilla rabbits.The rabbits were divided into normal control group,EAU model group,groups with low,medium and high concentration of intraocular sustained-release preparations of artesunate,TA group and DAPT blocking group.On the 1st,4th,7th,12th and 21st days after the modeling,slitlamp microscopy,anterior segment photography,anterior segment OCT,fundus photography and B-ultrasound were performed to evaluate ocular inflammation.2.On the 12th and 21st day after the modeling,chinchilla rabbit eye histopathological specimens were prepared and retinal HE staining was performed to observe the changes of retinal pathological tissue in different groups and histological scores were performed.3.ERG test was performed on the 12th and 21st day after modeling to evaluate the visual function of each group.4.Retinal tissue samples were obtained on the 12th and 21st day after modeling,and the protein levels of Notchl,TNF-α,IL-17,IL-13 and IL-4 in different groups were detected by Western-blot test.Results1.Clinical score of eye inflammation:The inflammation scores were performed according to the clinical manifestation records on the 1st,4th,7th,12th and 21st days after the modeling.The clinical scores of eye inflammation in EAU model group were significantly higher than those in normal control group(P<0.05),and the clinical scores of eye inflammation in TA group at day 4,7,12 and 21 after the completion of the modeling were significantly lower than those in EAU model group(P<0.05).The clinical scores of eye inflammation in ART high-concentration group were significantly lower than those in EAU model group at 4,7 and 12 days after modeling(P<0.05).The clinical score of eye inflammation in DAPT blocking group was significantly lower than that in EAU model group at day 12 after modeling(P<0.05).There was no statistical significance between other groups and EAU model group(P≥0.05).2.Retinal histology score:The retinal histological scores of chinchilla rabbits in EAU model group were significantly higher than those in normal control group at the 12th and 21st days after modeling(P<0.05),and those in TA group were significantly lower than those in EAU model group at the 12th and 21st days after modeling(P<0.05).The retinal histological score of ART high-concentration group was significantly lower than that of EAU model group on day 12 after modeling(P<0.05).There was no statistical significance between other groups and EAU model group(P≥0.05).3.ERG test:On the 12th day after modeling,the ERG test results showed that the ERG of the right eye of 3 chinchilla rabbits randomly selected from EAU model group,groups with low and medium concentration of intraocular sustained-release preparations of artesunate and DAPT blocking group were all extinguish,while ERG in ART high-concentration group and TA group were reduced and the peak time was prolonged.Compared with the normal control group,The amplitude differences of dark adaptation 0.01 ERG b wave,dark adaptation 3.0ERG a wave,dark adaptation 3.0ERG b wave,dark adaptation 3.0 shock potential P2 wave,open adaptation 3.0ERG a wave and open adaptation 3.0 scintillation light response P1 wave in high concentration ART sustained-release preparation group and TA group were statistically significant(P<0.05).The peak time of dark adaptation 0.01 ERG b wave,dark adaptation 3.0ERG a wave,light adaptation 3.0ERG a wave and light adaptation 3.0ERG b wave in high concentration ART sustained-release preparation group and TA group was prolonged,and the difference was statistically significant compared with the normal control group(P<0.05).On the 21st day after modeling,ERG test results showed that except for the normal control group and the TA group,the ERG of the right eye of 3 rabbits randomly selected in the other groups was extinguish type,and the ERG of the TA group was decreased type,and the difference was statistically significant compared with the normal control group(P<0.05),and the peak time was prolonged,some results had significant differences(P<0.05).4.Western-blot test:Compared with normal control group,the level of TNF-αprotein in EAU model group was significantly increased on the 12th after modeling(P<0.05).The expression of protein in groups with medium and high concentration of intraocular sustained-release preparations of artesunate decreased in a concentration dependent manner,which was significantly lower than that in EAU model group(P<0.05).The expression of protein in the positive TA group and DAPT blocking group was significantly lower than that in EAU model group(P<0.05).There was no significant difference in protein level between EAU model group and each group on the 21th after modeling.The level of IL-17 protein in EAU model group was significantly higher than that in normal control group on the 12th after modeling(P<0.05).The expression of protein in group with high concentration of intraocular sustained-release preparations of artesunate and TA group was significantly lower than that in EAU model group(P<0.05);There was no statistical difference between the other groups.On the 21st day after modeling,the protein expression level in the EAU model group was significantly higher than that in the normal control group(P<0.05),while the protein expression level in the TA group was significantly lower than that in the EAU model group(P<0.05);There was no statistical difference between the other groups.The level of IL-13 protein in the EAU model group was significantly higher than that in normal control group on the 12th after modeling(P<0.05).The expression of protein in group with high concentration of intraocular sustained-release preparations of artesunate and TA group was significantly lower than that in EAU model group(P<0.05);There was no statistical difference between the other groups.On the 21st day after modeling,the protein expression level in the EAU model group was significantly lower than that in the normal control group(P<0.05),the protein expression level in TA group was significantly higher than that in EAU model group(P<0.05);There was no statistical difference between the other groups.The level of IL-4 protein in the EAU model group was significantly higher than that in normal control group on the 12th after modeling(P<0.05).The expression of protein in group with high concentration of intraocular sustained-release preparations of artesunate and TA group was significantly lower than that in EAU model group(P<0.05);There was no statistical difference between the other groups.On the 21st day after modeling,the protein expression level in the EAU model group was significantly lower than that in the normal control group(P<0.05).The expression of protein in group with medium concentration of intraocular sustained-release preparations of artesunate and TA group was significantly higher than that in EAU model group(P<0.05);There was no statistical difference between the other groups.The level of Notch1 protein in the EAU model group was significantly higher than that in normal control group at 12 and 21 days after modeling(P<0.05).The protein levels in ART high-concentration group,TA group and DAPT blocking group were significantly lower than those in EAU model group(P<0.05);There was no statistical difference between the other groups.Conclusions1.The chinchilla rabbit EAU model induced by Mycobacterium tuberculosis H37Ra has the advantages of simple operation,high specificity,good stability and strong repeatability.2.The low and middle concentrations of ART sustained-release preparation had no obvious inhibitory effect on the inflammation of EAU chinchilla rabbits,while the high concentration of ART sustained-release preparation could significantly inhibit the inflammatory response and protect the visual function of EAU chinchilla rabbits,and there were no obvious side effects.3.DAPT significantly inhibits the inflammatory response of uveitis by blocking Notch signal pathway,which proves that the activation of Notch signal pathway plays an important role in the occurrence and development of uveitis.4.ART can significantly reduce the expression levels of Notchl,TNF-α,IL-17,IL-13 and IL-4 protein,and promote the polarization balance of M1/M2 macrophages.The anti-inflammatory mechanism may be related to the inhibition of Notch signal pathway and macrophage activation.Part four:Study on the distribution and metabolism of the novel intraocular sustained-release preparation of artesunate in rabbit plasma and eye tissueObjectiveTo investigate the pharmacokinetic characteristics of ART in eye tissue after intravitreal injection of the intraocular sustained-release preparation of artesunate,and to provide basic data for pharmacodynamic experiments.Methods1.The chinchilla rabbits were randomly divided into 15 groups.The corresponding groups were injected with 10μL of ART intraocular sustained-release preparation at low,medium and high concentrations in the vitreous cavity of the right eye,respectively.Blood was collected at 3h,5h,17h,Id and 3d after vitreous cavity injection,and rabbits were sacrificed by air embolization.Plasma,iris-ciliary body,aqueous humor,vitreous body and retina/choroid of the right eye were collected.2.The biological samples were processed and the concentrations of artesunate and dihydroartemisinin in rabbit plasma and eye tissues were determined by ultra-high performance liquid chromatography coupled with tandem mass spectrometry.Results1.UPLC-MS/MS method was established for the determination of ART and DHA concentrations in rabbit plasma and eye tissue.The quantification ranges were 20.04000ng/g or 2.00-400ng/mL,40.0-8000ng/g or 4.00-800ng/mL,respectively.The systematic applicability,specificity,interference coefficient,residue,linear range,stability,precision,accuracy,sensitivity,matrix effect and recovery rate of the method were verified,which all conform to the parameters of preclinical pharmacokinetic study of chemical drugs.2.The distribution of ART in eye tissues after intravitreal injection of low,medium and high concentrations of intraocular sustained-release of ART was as follows:vitreous body>aqueous humor>retina/choroid>iris-ciliary body,but no distribution was found in plasma.The distribution of DHA,the active metabolite of ART,in the eye tissues was as follows:iris-ciliary body>retina/choroid>vitreous body>aqueous humor,but not in plasma.3.AUC0-3d distribution of ART and DHA in vitreous body,aqueous humor,retina/choroid and iris-ciliary body showed dose-dependent increase.The peak time of DHA in the iris-ciliary body and aqueous humor was later than that of ART.Conclusions1.After intravitreal injection of ART sustained-release preparation,ART was gradually absorbed in the vitreous cavity,reached the peak quickly,and eliminated slowly;part of it could be metabolized into its active product DHA,which could be eliminated gradually;ART and DHA could be completely eliminated in about 3 days.2.The distribution of ART and DHA in vitreous,aqueous humor,iris-ciliary body and retina/choroid showed a dose-dependent increase,and almost did not enter the systemic circulation.3.The elimination time of DHA in iris-ciliary body and aqueous humor was later than that of ART. |
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