Application of sakatinib in preparation of medicine for treating retinopathy

Abstract

The invention discloses an application of sakatinib in preparation of a medicine for treating retinopathy. The chemical formula of the saucatinib is C27H32ClN5O5, and the structural formula of the saucatinib is shown as a formula I in the specification. The invention provides a new application of the sakatinib in treatment of retinopathy, the research finds that the sakatinib achieves the purpose of treating the retinopathy by inhibiting the generation of retinal neovascularization and inflammatory response of the retina, and the safety of intraocular administration of the sakatinib to the retina is proved. A new and effective technical means is provided for treatment of retinopathy, existing clinical problems can be more effectively solved, the treatment effect is remarkably improved, complications and adverse reactions are reduced, and therefore the life quality of patients is improved. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a new drug for treating retinopathy, and particularly to the application of saracatinib in the preparation of a drug for treating retinopathy. The present invention belongs to the field of pharmaceutical technology. Background Art

[0002] Saracatinib (AZD0530) is an effective Src inhibitor, with IC50 values for inhibiting c-Src, Lck, c-YES, Lyn, Fyn, Fgr, and Blk being 2.7 - 11 nM, and having selectivity for other tyrosine kinases. The chemical formula is C 27 H 32 ClN5O5, and the structural formula is as shown below:

[0003]

[0004] Saracatinib is commonly used to treat tumors carrying RET gene fusions or mutations. It was approved for marketing by the FDA in 2020 (trade name Retevmo), and is applicable to solid tumors such as non-small cell lung cancer (NSCLC) and medullary thyroid cancer (MTC).

[0005] Retinopathy is a class of diseases involving abnormal retinal structure or function, covering various types such as diabetic retinopathy (DR), age-related macular degeneration (AMD), and retinal detachment. The main manifestations are blurred vision and visual field defects. Due to vitreous opacity, there may also be floating black shadows in the visual field. Currently, the types of anti-angiogenic VEGF drugs on the market are single, and the treatment effects for some patients are very poor. To solve this problem, the present invention proposes a new application of saracatinib in the treatment of retinopathy. Currently, the application of saracatinib in cancer and Alzheimer's disease has been reported, but its application in ophthalmic retinal diseases has not been seen. Summary of the Invention

[0006] The purpose of the present invention is to provide a new drug for treating retinopathy.

[0007] To achieve the above purpose, the present invention adopts the following technical means:

[0008] The present invention proposes the application of saracatinib in the preparation of a drug for treating retinopathy. The chemical formula of the saracatinib is C 27 H 32 ClN5O5, and the structural formula is as shown in Formula I below:

[0009]

[0010] Among them, preferably, the saracatinib achieves the purpose of treating retinopathy by inhibiting the generation of retinal neovascularization and the inflammatory reaction of the retina.

[0011] Among them, preferably, the drug is an injection drug.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] The present invention firstly proposes a new application of saracatinib in the treatment of retinopathy. Through research, it is found that saracatinib achieves the purpose of treating retinopathy by inhibiting the generation of retinal neovascularization and the inflammatory reaction of the retina, and proves the safety of intravitreal administration of saracatinib to the retina. The present invention provides a new and effective technical means for the treatment of retinopathy, can more effectively solve existing clinical problems, significantly improve the treatment effect, reduce complications and adverse reactions, and thus improve the quality of life of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Shows the results of the effect of Saracatinib on the retinal morphology and function of OIR mice;

[0015] Among them, (A) Representative figure of H&E staining of retinal sections, scale bar = 50 μm; The nuclei of neovascular endothelial cells in the anterior part of the inner limiting membrane (ILM) of the retina indicate the degree of RNV; Arrows highlight neovascular clusters; (B) Merged immunofluorescence staining of CD31 (red) and cell nuclei (blue), scale bar = 50 μm;

[0016] Figure 2 Shows the results of Saracatinib inhibiting retinal neovascularization in OIR mice;

[0017] Among them, (A) Representative figure of whole retina flat mount; (B) Quantitative analysis of the percentage of non-perfused area; (C) Quantitative analysis of the percentage of neovascular area; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no statistical significance; Scale bar = 200 μm;

[0018] Figure 3 Shows the results of Saracatinib inhibiting the inflammatory reaction of the retina in OIR mice;

[0019] Among them, (A) Representative figure of microglial activation in the OIR+Saline group in retinal flat mounts; (B) Representative figure of microglia transitioning from the activated state to the resting state in the OIR+Saracatinib group; Scale bar = 100 μm;

[0020] Figure 4Results of the inhibition of the expression of inflammation-related and angiogenesis-related mRNAs and proteins in the retinas of OIR mice by Saracatinib;

[0021] Among them, (A) protein expression levels of VEGFA, Albumin, MCP-1, and Iba-1 in the Control+Saline group, Control+Saracatinib group, OIR+Saline group, and OIR+Saracatinib group; (B) quantitative analysis of the protein expression levels of VEGFA, Albumin, MCP-1, and Iba-1 in the Control+Saline group, Control+Saracatinib group, OIR+Saline group, and OIR+Saracatinib group; (C) quantitative analysis of the mRNA expression levels of VEGFA, Iba-1, and MCP-1 in the Control+Saline group, Control+Saracatinib group, OIR+Saline group, and OIR+Saracatinib group; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001;

[0022] Figure 5 Results of the mechanism verification of Saracatinib in the treatment of retinal neovascularization;

[0023] Among them, (A) EPAS1 protein expression level and quantitative analysis in the Control+Saline group, Control+Saracatinib group, OIR+Saline group, and OIR+Saracatinib group; (B) HIF-1α protein expression level and quantitative analysis in the Control+Saline group, Control+Saracatinib group, OIR+Saline group, and OIR+Saracatinib group; (C) immunofluorescence analysis showing the co-localization of HIF-1α (red), EPAS1 (green), CD31 (gold), and Iba-1 (silver) in retinal tissues; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not statistically significant; scale bar = 50 μm;

[0024] Figure 6 Dose-response curve of Saracatinib in HUVECs;

[0025] Figure 7 Results of TUNEL staining of retinal sections after Saracatinib treatment;

[0026] Among them, TUNEL assay (green) shows apoptotic cells in the outer nuclear layer of the retina, and DAPI nuclear staining (blue); scale bar = 50 μm;

[0027] Figure 8 It is the HE staining result of retinal sections after Saracatinib treatment;

[0028] Among them, (A) H&E staining shows the retinal morphology of the Control+Saline group and the Control+Saracatinib group; (B-D) Retinal layer thickness (total thickness, inner nuclear layer, outer nuclear layer) measured at 500 μm and 1000 μm from the optic disc (P<0.05). Specific implementation manners

[0029] The present invention will be further described below in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0030] Example 1 Application of Saracatinib in treating retinopathy

[0031] 1 Experimental methods

[0032] 1.1 Experimental reagents

[0033] Table 1 Experimental reagents

[0034]

[0035] 1.2 Antibodies and drugs

[0036] Table 2 Antibodies and drugs

[0037]

[0038]

[0039] 1.3 Animal modeling and experimental grouping

[0040] C57BL / 6J mice, experimental grouping for the mouse retinal neovascularization model (OIR model). To verify whether the efficacy of Saracatinib drug will lead to the regression of physiological or pathological retinal neovascularization, neonatal mice were randomly divided into the following 4 groups according to the cage:

[0041] (1) Control + intravitreal injection (abbreviation: intravitreal injection) of normal saline group (Control+Saline): Neonatal mice were breastfed in a normal oxygen environment. At the 12th day (P 12), 1 μl of normal saline was injected into the right eye vitreous cavity of the mice, and erythromycin eye ointment was applied to the eye after the operation;

[0042] (2) Control + Saracatinib group: For the mice in the Control + Saline group, on the 12th day (P 12), 80 μM (1 μl) of Saracatinib solution was injected into the left eye. After the operation, erythromycin eye ointment was applied to the eye, and the mice were returned to the cage for observation until the 17th day (P 17) when samples were taken.

[0043] (3) OIR + Saline group: Newborn mice were first placed in a normal oxygen environment for breastfeeding for 7 days (P 7), then the mice and their mothers were placed in a sealed oxygen chamber for 5 days with the oxygen concentration maintained at 75%. On the 12th day (P 12), 1 μl of normal saline was injected into the vitreous cavity of the right eye of the mice. After the operation, erythromycin eye ointment was applied to the eye.

[0044] (4) OIR + Saracatinib group: For the mice in the OIR + Saline group, on the 12th day (P12), 80 μM (1 μl) of Saracatinib solution was injected into the left eye. After the operation, erythromycin eye ointment was applied to the eye, and the mice were returned to the cage for observation until the 17th day (P 17) when samples were taken.

[0045] 1.4 RNA extraction from tissues and cells

[0046] (1) Tissue samples: The retinal tissue was completely dissected and placed in a 1.5 ml RNase-Free centrifuge tube. 1 ml of Trizol solution was added, mixed well, and left standing on ice for 15 minutes.

[0047] (2) Lysing samples: Vortex to mix well and leave standing at room temperature for 5 minutes.

[0048] (3) Adding chloroform: 250 μl of chloroform was added to each sample, vortexed, and left standing at room temperature for 15 minutes.

[0049] (4) Centrifugation for layering: Centrifuge at 13,000 rpm at 4 °C for 15 minutes, and 400 μl of the supernatant was aspirated into a new centrifuge tube.

[0050] (5) Adding isopropanol: 400 μl of isopropanol was added and mixed well.

[0051] (6) Centrifugation to precipitate total RNA: Centrifuge at 13,000 rpm at 4 °C for 20 minutes, discard the supernatant, and leave the RNA precipitate.

[0052] (7) Rinsing RNA: 1 ml of 70% ethanol was added, centrifuge at 13,000 rpm at 4 °C for 5 minutes, and discard the supernatant.

[0053] (8) Rinsing RNA: 1 ml of absolute ethanol was added, centrifuge at 13,000 rpm at 4 °C for 5 minutes, and discard the supernatant.

[0054] (9) Volatile residual ethanol: Centrifuge instantaneously for 10 seconds and allow the ethanol to evaporate at room temperature;

[0055] (10) RNA solution: Add 10 - 20 μl of DEPC water to each tube and mix well;

[0056] (11) Determine the RNA concentration: Measure the RNA concentration using a UV spectrophotometer.

[0057] 1.5 Real-Time PCR

[0058] (1) Reverse transcription to synthesize cDNA: Reverse transcribe the RNA template into cDNA according to the method provided in the instruction manual.

[0059] The reaction system is prepared as follows:

[0060]

[0061] The reaction program is set as: 37°C for 15 minutes; 98°C for 5 minutes; 4°C for 5 minutes; Measure the cDNA concentration using a UV spectrophotometer, dilute the cDNA with DEPC water, and store it in a -20°C refrigerator.

[0062] (2) RT-qPCR: Prepare the reaction system according to the method provided in the instruction manual, as follows:

[0063]

[0064] The reaction program is set as:

[0065] Temperature-time cycle

[0066] 95°C for 10 minutes, 1 time

[0067] 95°C for 10 seconds, 40 times

[0068] 60°C for 30 seconds

[0069] 95°C for 15 seconds, 1 time

[0070] 60°C for 60 seconds, 1 time

[0071] 95°C for 15 seconds, 1 time

[0072] 4°C for 5 minutes, 1 time

[0073] After the reaction, analyze and calculate the melting curve data.

[0074] (3) The primer sequences are synthesized by Sangon Biotech Co., Ltd., and the specific sequences are shown in Table 3 below:

[0075] Table 3 Primer sequences

[0076]

[0077] 1.6 Western Blot

[0078] (1) Gel preparation: According to the molecular weight of the target protein, select a 7.5%-12.5% SDS-PAGE lower layer gel and a 5% upper layer gel, insert the corresponding 10-well or 15-well protein comb, and let it solidify at room temperature for later use;

[0079] (2) Electrophoresis: Place the gel plate in the electrophoresis tank, pour in the electrophoresis buffer, pull out the comb, and load the protein Marker and protein samples respectively. After electrophoresis at a constant voltage of 70V for 30 minutes, adjust the voltage to 110V until the electrophoresis is completed;

[0080] (3) Blotting: Adopt the wet blotting method. Activate the PVDF membrane with methanol, transfer the protein to the PVDF membrane, at a constant current of 300mA, and set the blotting time according to the molecular weight of the target protein;

[0081] (4) Blocking: Place the PVDF membrane in 5% milk, at room temperature, on a shaker, for 100 minutes;

[0082] (5) Primary antibody incubation: Prepare the primary antibody solution with the primary antibody diluent, incubate overnight at 4°C;

[0083] (6) Wash primary antibody: Wash 3 times with TBST, 10 minutes each time;

[0084] (7) Secondary antibody incubation: Prepare the secondary antibody solution with TBST, incubate at room temperature on a shaker for 90 minutes;

[0085] (8) Wash secondary antibody: Wash 3 times with TBST, 10 minutes each time;

[0086] (9) ECL development: Develop with a chemiluminescence imager, take pictures and record, and calculate the relative quantification of the target protein and β-Actin.

[0087] 1.7 Immunofluorescence staining

[0088] Tissue sample: Fix the position of anesthetized mice. After perfusion through the heart, remove the eyeballs intact, trim the excess tissue, place the eyeballs in an eyeball fixative and fix them at room temperature overnight, then perform paraffin embedding and sectioning; bake the sections at 60 °C for 1 hour, and use a fully automatic research staining machine to dry and dewax the tissue sections; perform antigen retrieval by microwaving and boiling the AR buffer for 3 minutes, then transfer to medium-low heat and heat for 20 minutes, cool at room temperature, wash with distilled water once for 3 minutes; dry the moisture around the tissue, circle the tissue with an immunohistochemistry pen, place the sections in a humid chamber, add 3% H2O2 and incubate for 10 minutes, wash with PBS 2 times, 3 minutes each time; incubate with 5% BSA for 60 minutes for blocking, incubate with the primary antibody overnight at 4 °C, wash with PBS 3 times, 5 minutes each time; incubate with the secondary antibody for 60 minutes, wash with PBS 3 times, 5 minutes each time; mount the sections with an anti-fluorescence quencher containing DAPI, and take pictures with a fluorescence microscope.

[0089] 1.8 Whole retina flat mount staining

[0090] Fix the position of anesthetized mice. After perfusion through the heart, remove the eyeballs intact, trim the excess tissue, place the eyeballs in 4% paraformaldehyde and fix for 30 minutes; under the microscope, remove the retina intact, wash with PBS 3 times, 5 minutes each time; permeabilize with 0.5% triton X-100 overnight at 4 °C, wash with PBS 3 times, 5 minutes each time; add the Iba-1 primary antibody and incubate overnight at 4 °C, wash with PBS 3 times, 5 minutes each time; incubate with the Alexa Fluor 594-labeled goat anti-rabbit IgG secondary antibody for 60 minutes, wash with PBS 3 times, 5 minutes each time; add Isolectin B4 (IB4) staining solution (1:500), incubate overnight at 4 °C; wash with PBS 3 times, 5 minutes each time; place the retinal tissue on an adhesive-coated glass slide, cut the retina into a four-leaf clover shape with scissors and gently flatten and spread it out, drop an anti-fluorescence quencher on the retina, cover with a coverslip, and take pictures and record with a confocal microscope.

[0091] 1.9 Retinal cryosection

[0092] Retinal tissues were embedded in Tissue-Tek optimal cutting temperature compound and cut into 8-μm thick cryosections using a Leica CM1800 cryostat. The sections were fixed in 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% donkey serum. The following primary antibodies were incubated independently overnight at 4°C: rabbit anti-Iba1, rat anti-CD31, rabbit anti-HIF-1α, and rabbit anti-EPAS1. After washing with PBS, the sections were incubated with the corresponding secondary antibodies for 3 hours at room temperature: Alexa Fluor488-conjugated goat anti-rabbit IgG, Alexa Fluor594-conjugated goat anti-rabbit IgG, and Alexa Fluor 555-conjugated goat anti-rabbit IgG. The nuclei were counterstained with DAPI for 5 minutes, then mounted and imaged using a Nikon A1R confocal microscope and NIS-Elements AR 5.21 software. Image quantification was performed using ImageJ 1.53c.

[0093] 1.10 H&E staining

[0094] The anesthetized mice were fixed in position. After perfusion through the heart, the eyeballs were removed intact, excess tissues were trimmed, and the eyeballs were placed in an eyeball fixative and fixed overnight at room temperature. Paraffin embedding and sectioning were performed. First, the sections were dewaxed with xylene, then successively hydrated in 75%, 85%, and absolute ethanol. The sections were stained with hematoxylin solution, rinsed with tap water, then differentiated with 1% hydrochloric acid alcohol, rinsed with water to blue back, and finally stained with eosin solution and rinsed with running water. The stained sections were successively placed in 75% ethanol, 85% ethanol, absolute ethanol, and xylene for clearing, and then sealed with neutral resin and photographed.

[0095] 1.11 TUNEL staining

[0096] A one-step TUNEL apoptosis detection kit was used to detect apoptotic cells in the retina.

[0097] After harvesting at P17, the samples were fixed in 4% PFA for 1 hour, then transferred to 30% sucrose dissolved in PBS at 4°C overnight, embedded in OCT, and frozen at -80°C. Sections near the optic nerve were selected, washed 3 times in PBS, and incubated in 3% BSA, 0.3% Triton X-100 PBS at room temperature for 5 minutes. Then, the equilibration buffer containing terminal deoxynucleotidyl transferase (TdT) was placed in a 37°C incubator for 1 hour of staining in the dark. The nuclei were stained with DAPI, mounted, photographed, and recorded using a confocal microscope.

[0098] 1.12 CCK-8 assay

[0099] (1) Seed 5000 HUVECs cells per well (100 μl / well) in a 96-well plate and set up 5 replicate wells.

[0100] (2) According to the experimental needs, culture the cells and stimulate them with Saracatinib at concentrations of 0 μM, 40 μM, 80 μM, and 160 μM for 24 hours respectively.

[0101] (3) Add 10 μl of CCK-8 solution to each well and continue to incubate in a cell culture incubator for 2 hours.

[0102] (4) Measure the absorbance at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader and record the results.

[0103] 1.13 Statistical analysis method

[0104] All experiments were repeated 5 times. Data analysis and graph production were performed using GraphPad Prism 9.0, ImageJ 1.8.0, and FlowJo 10.9.0. The data results were expressed as mean ± standard deviation. ANOVA or t-tests were used for comparative analysis. A P value < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0105] 2. Experimental results

[0106] 2.1 Effects of Saracatinib on the retinal morphology and function of OIR mice

[0107] To verify the effects of Saracatinib on the retinal morphology and function of OIR mice, we used H&E staining to observe the tissue structure and cell morphology of the retina. The most prominent feature of the OIR model is neovascularization, which helps us explore the effect of Saracatinib on retinal neovascularization. We injected saline into the right vitreous cavity and Saracatinib solution into the left vitreous cavity of P12-day OIR and control mice. Samples were taken on P17, sectioned, and stained. The H&E staining results showed that there were no retinal neovascularizations in the control mice regardless of whether they were injected with Saracatinib or saline Figure 1 A); obvious neovascularizations protruding from the inner limiting membrane were visible in the retina of OIR mice injected with saline; while the number of retinal neovascularizations in mice treated with Saracatinib was significantly reduced, and no neovascularizations protruding from the inner limiting membrane were observed. Similar results were also observed in CD31-stained sections, confirming the reduction of pathological neovascularization Figure 1 B). This also indicates that intravitreal injection of Saracatinib can inhibit the formation of retinal neovascularization.

[0108] 2.2 Saracatinib inhibits neovascularization in the retina of OIR mice

[0109] To more comprehensively evaluate the inhibitory effect of Saracatinib on retinal neovascularization, we performed whole retina flat-mount IB4 staining on the retinas of the normal control + Saline group, control + Saracatinib group, OIR + Saline group, and OIR + Saracatinib group, respectively. We took pictures of the complete retinal tissue using a confocal microscope and calculated the area of retinal neovascularization and the area of retinal non-perfused area in each group. The results are as Figure 2 shown. The results showed that the retinal tissue morphology of the control + Saline group and the control + Saracatinib group was good, and no obvious neovascularization and non-perfused area were seen ([[]] Figure 2 A); a large number of neovascular buds were formed at the junction of the peripheral vascular area and the central non-perfused area of the retina in the OIR + Saline group; while the retina of the OIR + Saracatinib group had significantly fewer neovascularization and non-perfused area compared with the retina of the OIR + Saline group ([[]] Figure 2 B, C). This also suggests that Saracatinib has a significant inhibitory effect on retinal neovascularization and can also improve the non-perfused area of the retina to a certain extent, which makes us more convinced of the therapeutic potential of Saracatinib in retinal diseases.

[0110] 2.3 Saracatinib inhibits the inflammatory response in the retina of OIR mice

[0111] Under hypoxic conditions, microglia activation releases inflammatory factors and promotes retinal neovascularization, while removing microglia reduces neovascularization. To study the effect of Saracatinib on retinal inflammation, we performed double immunofluorescence staining of Iba-1 and IB4 in the OIR + Saline group and the OIR + Saracatinib group. The results are as Figure 3 shown. Treatment with Saracatinib led to significant changes in the morphology of microglia, changing from an amoeboid state to a branched state, indicating a reduction in microglia activation.

[0112] 2.4 Saracatinib inhibits the expression of inflammation-related and angiogenesis-related mRNAs and proteins in the retina of OIR mice

[0113] Newborn mice and their lactating mother mice were kept in room air from birth to P7 days, and the retinal vessels developed normally. At P7 days, the mice were exposed to 75% oxygen, which could significantly inhibit the growth of retinal vessels. When the mice returned to room air at P12 days, the avascular retina became hypoxic, triggering normal angiogenesis and pathological neovascularization. The neovascularization reached its peak at P17 days and then gradually regressed, and the retina almost returned to normal by P25 days. RT-qPCR and WB techniques were used to detect the expression levels of mRNA and proteins related to retinal inflammation and angiogenesis in OIR mice. We evaluated the expression levels of VEGFA, MCP-1, Iba-1, and Albumin. The results are as Figure 4 shown in A and B. These results showed that Saracatinib significantly reduced the expression of these proteins, suggesting an improvement in endothelial barrier integrity and inhibition of microglial activation. To verify these findings, we performed RT-qPCR analysis, and the results are as Figure 4 shown in C. These results confirmed the downregulation of VEGFA, MCP-1, and Iba-1 at the transcriptional level, which was consistent with the observations at the protein level.

[0114] 2.5 Mechanism of Saracatinib in improving pathological retinal neovascularization

[0115] Western Blot analysis showed that compared with the OIR control group injected with normal saline, the protein levels of HIF-1α and EPAS1 in the retinas of OIR mice treated with Saracatinib were significantly reduced ( Figure 5 A, B). Immunofluorescence staining confirmed the spatial localization of these proteins. In the OIR retina, HIF-1α was expressed in all retinal cells, with stronger CD31 expression, while EPAS1 was mainly co-localized with Iba-1 ( Figure 5 C). These findings revealed the specific mechanism by which Saracatinib improved pathological retinal neovascularization by targeting endothelial cells and downregulating HIF-1α, reducing the expression of EPAS1 in microglia, thus enhancing its ability to treat retinal vascular diseases.

[0116] 2.6 Toxicity test of Saracatinib and retinal safety assessment

[0117] To evaluate the potential retinal toxicity of Saracatinib, a comprehensive assessment was conducted. For in vitro testing, we used CCK8 to detect the cytotoxicity of Saracatinib on HUVECs and set up three drug concentration gradients, namely 40 μmol / L, 80 μmol / L, and 160 μmol / L. After co-culturing Saracatinib with HUVECs for 24 hours, CCK8 detection was performed. The absorbance values of each well were measured at a wavelength of 450 nm using a microplate reader, and the cell viability was calculated. The results are as Figure 6 shown. The experimental results showed that when the drug concentration was 40 μmol / L and 80 μmol / L, Saracatinib had no obvious cytotoxicity on HUVECs; while when the drug concentration increased to 160 μmol / L, the cytotoxicity of Saracatinib on HUVECs was significant. Therefore, in this study, a drug concentration of 80 μmol / L was selected for subsequent experimental research.

[0118] For in vivo evaluation, intravitreal injection of Saracatinib was performed on normal mice and OIRP12 mice. Five days later, TUNEL detection was carried out to observe apoptotic cells. The results are as Figure 7 shown. The experimental results showed that 80 μM of Saracatinib did not increase the number of TUNEL+ cells in the retina of the control group or the OIR group.

[0119] The quantitative analysis results of HE-stained sections of the entire retina, inner nuclear layer (INL), and outer nuclear layer (ONL) of the Saracatinib treatment group are as Figure 8 shown in A - D. The results showed no significant difference compared with the control group, demonstrating the safety of intravitreal administration of Saracatinib to the retina.

Claims

1. Use of Saracatinib in the preparation of a drug for treating retinopathy, wherein the chemical formula of Saracatinib is C 27 H 32 ClN5O5, the structural formula is shown in the following formula I:

2. The use according to claim 1, characterized in that Said saracatinib achieves the purpose of treating retinopathy by inhibiting the formation of retinal neovascularization and retinal inflammatory response.

3. The use according to claim 1, characterized in that The medicine is an injectable medicine.

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