New Application of ALW-II-41-27 in the Treatment of Ophthalmic Diseases

Abstract

The present invention relates to the application of an EphA2-specific tyrosine kinase inhibitor, ALW-II-41-27, in ophthalmic diseases. The present invention has confirmed that at a specific concentration, ALW-II-41-27 can inhibit the expression of EphA2 in the retina, reduce the damage of acute high intraocular pressure to retinal tissue, and improve the survival rate of retinal ganglion cells, thereby playing a neuroprotective role, and thus being used for the preparation of treatments for preventing and treating acute high intraocular pressure or glaucoma.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to the application of ALW-II-41-27 in the treatment of ophthalmic diseases. Background Art

[0002] Glaucoma is the second most common blinding disease in the world, which has a significant adverse impact on the quality of life of patients worldwide. According to the data of the World Health Organization, approximately 76 million people suffered from glaucoma in 2020. It is estimated that these numbers will exceed 111 million by 2040. Glaucoma is a neurodegenerative disease characterized by optic nerve atrophy and visual field defects. The death of retinal ganglion cells (RGCs) is the main pathological feature of glaucoma and the main cause of irreversible blindness. Current research suggests that the death process of RGCs involves an increase in abnormal intraocular pressure (IOP). The elevation of intraocular pressure plays a key role in the pathogenesis of glaucoma, and reducing intraocular pressure is the key method for the treatment of glaucoma. In recent years, with the development of medicine, the monitoring methods, diagnostic criteria, drug and surgical treatment regimens for high intraocular pressure have been gradually improved and shown beneficial effects in saving the vision of glaucoma patients. Nevertheless, clinical data indicate that approximately 10% of glaucoma patients do not respond to these treatments. Due to the loss and non-renewability of RGCs, a considerable number of glaucoma patients still experience visual field deterioration after intraocular pressure control. Therefore, in addition to implementing treatments related to reducing intraocular pressure, the development of durable and effective neuroprotective interventions that can slow down or prevent the loss of RGCs is very important in the treatment of glaucoma.

[0003] Erythropoietin-producing hepatocellular receptors (Eph receptors) constitute the largest subfamily of receptor tyrosine kinases. The Eph receptor signaling pathway is also of great significance in neuronal development, axon extension, and vascular growth remodeling. The Eph / ephrin system is closely associated with glaucomatous optic neuropathy. Some studies have shown that the expression of Eph receptors and their ligand ephrin proteins is upregulated after injury to the central nervous system, and Eph / ephrin has become a potential target for clinical treatment research of central nervous system injury. In a chronic high intraocular pressure rat model, the expression of Eph / ephrin family members is upregulated in Müller cells of the optic nerve head, and similar phenomena have been observed in experimental glaucoma models of rhesus monkeys and DBA / 2J pigmentary glaucoma mouse models.

[0004] EphA2 is an important member of the Eph family. It is located on chromosome 1p36.1 and encodes a polypeptide with a molecular weight of 130 KD and 976 amino acid residues, which is a type I glycoprotein. The EphA2 receptor is a bidirectional signal transduction molecule. After binding to the ligand, the molecular conformation of the binding region changes, inducing the receptor to migrate and aggregate to form a receptor-ligand complex, further activating the intracellular tyrosine kinase, causing autophosphorylation and phosphorylation of downstream substrate molecules, and initiating signal transmission. Research has shown that the increase in EphA2 expression is related to ischemic nervous system injury, disruption of the blood-brain barrier, exudation of inflammatory mediators, activation of glial cells, and further activation of the apoptotic signaling pathway in the central nerve ischemia model. However, the mechanism is still complex and uncertain, and further research is needed.

[0005] ALW-II-41-27 (Eph receptor tyrosine kinase inhibitor), whose molecular formula is C 32 H 32 F3N5O2S, and the structural formula is as follows:

[0006]

[0007] ALW-II-41-27 is a newly developed ATP-competitive EphA2 inhibitor. It was first discovered in 2009 and has been proven to reduce the phosphorylation of EphA2 in various cancers. Currently, it has no application in ophthalmic diseases. Summary of the Invention

[0008] Based on this, the purpose of the present invention is to provide a new application of ALW-II-41-27 in the prevention and treatment of ophthalmic diseases.

[0009] We found that ALW-II-41-27 can inhibit the death of retinal ganglion cells caused by high intraocular pressure, improve the survival rate of retinal ganglion cells, and has a good neuroprotective effect on glaucoma.

[0010] In order to achieve the above-mentioned invention purpose, the present study provides the following technical solutions:

[0011] Application of ALW-II-41-27 in the preparation of drugs for preventing or treating glaucoma or acute high intraocular pressure.

[0012] In some embodiments, the indicated application includes increasing the survival rate of retinal ganglion cells.

[0013] In some embodiments, the indicated application includes reducing retinal damage.

[0014] In some embodiments, the drug is an injection, and more preferably an injection for intravitreal injection.

[0015] In some of these embodiments, the concentration of ALW-II-41-27 in the drug is 0.2 μM - 1.0 μM. Preferably it is 0.3 μM - 0.8 μM, more preferably 0.45 μM - 0.55 μM, and even more preferably 0.5 μM. Preferably, the dosage of the drug is 0.9 μL - 1.1 μL, and more preferably 1 μL.

[0016] Through research, the present invention has found that the EphA2-specific tyrosine kinase inhibitor - ALW-II-41-27, especially at a specific concentration, can reduce retinal damage, increase the survival rate of retinal ganglion cells, and inhibit the expression of EphA2 during the treatment of glaucoma or acute ocular hypertension, providing neuroprotection for retinal ganglion cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Acute ocular hypertension causes loss of retinal tissue in mice. In glaucoma mice, the retinal structure is disordered and the cells are irregular, accompanied by a significant decrease in the thickness of the inner retina and a significant thinning of the full-thickness retina; (A) HE staining of central retinal sections at 1.1 mm on both sides of the optic nerve head in each group of mice 7 days after acute ocular hypertension injury (magnification ×20, scale bar, 100 μm), ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), outer plexiform layer (OPL), outer nuclear layer (ONL). (B) Bar graphs show the quantitative analysis of the thickness of the inner retina, full-thickness retina, and the number of RGCs in the central region in HE staining. The data are expressed as mean ± SEM (n = 5 mice per group); the P values were determined using one-way ANOVA and Tukey's test for multiple comparisons. ***P < 0.001, and ****P < 0.0001. Figure 2 Acute ocular hypertension causes death of retinal ganglion cells in mice; among them, (A) immunofluorescence staining of RGCs in retinal flat mounts using a specific RBPMS antibody (magnification ×20, scale bar, 50 μm); (B) bar graphs show the quantitative analysis of the density of RGCs in different groups. The data are expressed as mean ± SEM (n = 5 mice per group); the P values were determined using one-way ANOVA and Tukey's test for multiple comparisons. ****P < 0.0001.

[0018] Figure 3Injecting different concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM) of ALW-II-41-27 into the vitreous cavity affects the thickness of the mouse retinal tissue under non-acute and acute high intraocular pressure conditions; the results of the inner retinal thickness in the AOH group showed that compared with AOH+PBS, the degree of reduction in retinal thickness in the AOH+0.1 μM ALW group, AOH+0.2 μM ALW group, AOH+0.5 μM ALW group, and AOH+1.0 μM ALW group all decreased significantly, showing a significant protective effect; the results of the full-thickness retinal thickness also showed that compared with the AOH+PBS group, the degree of reduction in retinal thickness in the AOH+0.1 μM ALW group, AOH+0.2 μM ALW group, AOH+0.5 μM ALW group, and AOH+1.0 μM ALW group decreased. Among them, the AOH+0.5 μM ALW group showed a better retinal protective effect compared with other drug concentration groups; (A) HE staining of the central retinal section at 1.1 mm on both sides of the optic nerve papilla of mice in each group 7 days after acute high intraocular pressure injury (magnification ×20, scale bar, 100 μm); (B) Bar graph showing the quantitative analysis of the inner retinal thickness of different groups of retinal HE staining in the PBS and different concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM) of ALW-II-41-27 groups under non-acute and acute high intraocular pressure conditions. (C) Bar graph showing the quantitative analysis of the full-thickness retinal thickness of different groups of retinal HE staining in the PBS and different concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM) of ALW-II-41-27 groups under non-acute and acute high intraocular pressure conditions. Data are expressed as mean ± SEM (n = 5 mice per group); P values were determined using one-way ANOVA and Tukey's test for multiple comparisons. *P<0.01, **P<0.01, ***P<0.001, ****P<0.0001.

[0019] Figure 4, Inject PBS and ALW-II-41-27 solutions with different concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM) into the vitreous cavity to observe their effects on the survival rate of retinal RGCs in mice under non-acute and acute high intraocular pressure conditions; the results of the RBPMS+RGCs density in the AOH group showed that compared with AOH+PBS, the AOH+0.1 μM ALW group did not show an obvious protective effect on RGCs; the AOH+0.2 μM ALW group and the AOH+0.5 μM ALW group significantly increased the survival rate of RGCs, and the 0.5 μM ALW group had the best therapeutic effect. When the concentration of ALW-II-41-27 increased to 1.0 μM, the density of RGCs decreased, and there was no obvious protective effect on retinal RGCs; (A) Immunofluorescence staining of RGCs in retinal flat mounts using a specific RBPMS antibody (magnification ×20, scale bar, 50 μm); (B) Bar graph showing the quantitative analysis of the density of retinal RGCs in the PBS and ALW-II-41-27 groups with different concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM) under non-acute and acute high intraocular pressure conditions. Data are expressed as mean ± SEM (n = 5 mice per group); P values were determined using one-way ANOVA and Tukey's test for multiple comparisons. ***P < 0.001, ****P < 0.0001.

[0020] Figure 5 , ALW-II-41-27 inhibits the expression of EphA2 in the retina of mice with acute high intraocular pressure. Western blotting results showed that compared with the Control+PBS group, the expression of EphA2 protein in the retina of the AOH+PBS group increased significantly; under acute high intraocular pressure conditions, compared with the group injected with PBS alone, 0.5 μM ALW-II-41-27 reduced the expression of EphA2 in the retina of mice to a certain extent, while under non-high intraocular pressure conditions, ALW-II-41-27 had no obvious effect on the expression of EphA2 in the retina; ALW-II-41-27 inhibits the expression of EphA2 in the retina of mice with acute high intraocular pressure (A) The protein levels of EphA2 in the Control+PBS group, AOH+PBS group, Control+0.5 μM ALW group, and AOH+0.5 μM ALW group; (B) Analyzed by densitometry using ImageJ software to quantify the EphA2 / β-actin protein ratio. Data are expressed as mean ± SEM (n = 5 mice per group, the experiment was repeated more than 3 times); P values were determined using one-way ANOVA and Tukey's test for multiple comparisons. *P < 0.05, ****P < 0.0001. Detailed implementation methods

[0021] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0022] For the experimental methods without specific conditions noted in the following examples, they are generally in accordance with conventional conditions, such as the fourth edition of "Molecular Cloning: A Laboratory Manual" edited by Green and Sambrook, which was published in 2013, or in accordance with the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0023] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0024] AOH (Acute ocular hypertension, acute ischemia-reperfusion group / acute high intraocular pressure group), ALW (ALW-II-41-27, purchased from Sigma), PBS (Phosphate buffer saline).

[0025] The present invention focuses on studying the application of an EphA2-specific tyrosine kinase inhibitor (ALW-II-41-27) in ophthalmic diseases, exploring whether and how it can inhibit the death of RGCs cells caused by high intraocular pressure, thereby increasing the survival rate of RGCs cells and providing new treatment ideas for the treatment of glaucoma.

[0026] The above-mentioned ophthalmic diseases include ophthalmic diseases caused by high intraocular pressure, such as glaucoma.

[0027] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0028] Example 1

[0029] Experimental method:

[0030] 1. Animal grouping

[0031] According to the random number table method, the mice were randomly divided into a Normal group, a Control group, and an AOH group. The mice in the Normal group were not treated. The mice in the Control group underwent simple anterior chamber paracentesis without anterior chamber perfusion to keep the intraocular pressure normal. The eyes of the mice in the AOH group were perfused with normal saline in the anterior chamber to construct an acute high intraocular pressure model. Intravitreal drug injection was performed within 12 - 24 hours after modeling. Again, according to the random number table method, the mice in the Control group and the AOH group were randomly and evenly divided into a Control + PBS group, a Control + ALW-II-41-27 groups with different drug concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM), an AOH + PBS group, and an AOH + ALW-II-41-27 groups with different drug concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM).

[0032] 2. Establishment of an acute high intraocular pressure mouse model

[0033] C57BL / 6J mice with ocular or systemic lesions or injuries, malnutrition, etc. were excluded. The Normal group was not treated with anything. After weighing the remaining mice, they were anesthetized by intraperitoneal injection of 1% sodium pentobarbital solution at a dose of 50 mg / kg body weight. A heating pad was used to prevent the body temperature of the anesthetized mice from being too low. One drop of compound tropicamide eye drops was instilled into the operative eye of the mice to dilate the pupils, and then both eyes were covered to avoid light. After the pupils were dilated, one drop of oxybuprocaine hydrochloride eye drops was instilled for surface anesthesia. As the experimental group of acute high intraocular pressure, the AOH group inserted a 33G sharp needle connected to a sterile normal saline perfusion tube into the anterior chamber of the operative eye, raised the perfusion bottle, and measured the intraocular pressure with a TonoLab rebound tonometer to keep the intraocular pressure at 90 mmHg. When it was observed that the conjunctiva and iris of the mice were pale, the cornea was edematous, the scleral veins turned white, and there was no liquid leakage at the puncture site, it was a sign of successful establishment of the acute high intraocular pressure model. Carbomer ophthalmic gel was used to protect the cornea during modeling. After 60 minutes of the model, the needle was removed, the normal perfusion pressure was restored, and levofloxacin hydrochloride eye ointment was applied after the operation to avoid infection. As the control group of non-acute high intraocular pressure, after anesthesia and pupil dilation, the Control group was given anterior chamber paracentesis operation with a 33G sharp needle in the anterior chamber of the operative eye, without normal saline perfusion operation, without increasing the intraocular pressure, and levofloxacin hydrochloride eye ointment was applied after the operation to avoid infection.

[0034] 3. Intravitreal injection in mice

[0035] Within 12 - 24 hours after the establishment of the ocular hypertension model, mice with obvious cataracts, ocular infections, eyeball atrophy, eyeball hemorrhage, iris prolapse and other lesions were excluded. Mice in the Control group or AOH group were anesthetized according to the above method and the pupil of the operative eye was dilated. A heating pad was used to prevent the body temperature of the anesthetized mice from being too low. After the pupil was dilated, a drop of oxybuprocaine hydrochloride eye drops was instilled on the operative eye for surface anesthesia. Medical transparent hyaluronic acid gel was applied to the cornea and a 3 - mm round coverslip was covered. After seeing the fundus clearly under the microscope, a 33G sharp needle was used to puncture the sclera 1 mm behind the limbus corneoscleral at the temporal side of the eyeball. A Hamilton microsyringe was inserted into the vitreous cavity of the operative eye of the mouse at the puncture site, and 1 μL of PBS or 1 μL of ALW - II - 41 - 27 solution with different concentrations was slowly injected to make it evenly distributed in the vitreous body, and the concentrations reached 0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM (calculated according to the vitreous volume of 5 μL in mice). After the injection, the needle was left in place for 30 s and then quickly withdrawn to avoid puncturing the lens and retina during this process. Whether the injection was successful was judged by observing whether there were lens scratches, fundus hemorrhage, and retinal elevation in the mice under the microscope. Levofloxacin hydrochloride eye ointment was applied after the operation to avoid infection.

[0036] 4. Measurement of retinal thickness by HE staining

[0037] Male C57BL / 6J mice in each group were sacrificed on the 7th day after modeling. Mice with poor general health, eyeball atrophy, corneal lesions, ocular infections and hemorrhage were excluded. After being anesthetized with an overdose of 1% sodium pentobarbital solution and sacrificed, the cervical vertebrae were dissected and the eyeballs were removed, then immersed in FAS eyeball fixative for pretreatment for 48 hours, and the eyeballs were paraffin - embedded. Sections (7 μm thick) were made from the plane passing through the optic nerve at 6 o'clock to the 12 - o'clock direction. At least 10 retinal sections were made for each group (n = 5 mice). The sections were dried and stained with conventional HE. Under a Leica white - light microscope, the central retina at 1.1 mm on both sides of the optic nerve head was photographed (magnification x20, scale bar 100 μM). The thickness of the inner retina (from the ganglion cell layer GCL to the inner plexiform layer IPL), the thickness of the whole - layer retina, and the number of RGCs at the same position were measured using ImageJ software. Each section was measured twice by the same experimenter and the average value was taken.

[0038] 5. Immunofluorescence counting of retinal ganglion cells

[0039] On the 7th day after modeling, male C57BL / 6J mice in each group were sacrificed and samples were taken according to the above method. The eyeballs were removed and placed in 4% paraformaldehyde fixative at room temperature for 2 hours. Under a microscope, the intact retina was quickly dissected out and cut into a regular four-leaf shape, then placed in a 24-well plate and shaken on a shaker. The retina was washed 3 times with PBS, 5 minutes each time. 1.5 μL of absolute ethanol was added to each well, and the plate was incubated on the shaker at 50 rpm for 30 minutes. The retina was washed 3 times with PBS, 5 minutes each time. The retina was transferred onto a glass slide with the ganglion cell layer facing up, and an immunocytochemistry pen was used to draw a closed circle around the retina. 150 μL of 1% TritonX-100 solution was added to soak the retina for 20 minutes, and the retina was washed 3 times with PBS, 5 minutes each time. 150 μL of 10% donkey serum solution was added and incubated at 4°C overnight for blocking. The retina was washed 4 times with PBS, 5 minutes each time. The RBPMS antibody solution diluted 1:1000 was added and incubated in a 4°C refrigerator overnight. The retina was washed 4 times with PBS, 5 minutes each time. At room temperature, 150 μL of Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) secondary antibody solution diluted 1:1000 was added. The sample was incubated in the dark for 2 hours. The retina was washed 4 times with PBS, 5 minutes each time. The sample was mounted with an anti-fluorescence quenching mounting medium containing DAPI. The retina was photographed using a multi-functional microplate imaging detection and analysis system, and RGCs could be seen as green under the GFP channel. Four quadrants of the retina were photographed at a distance of 1.1 mm from the optic nerve (magnification x20, scale bar 50 μM), and 4 films were taken from each retina. At least 20 films were taken for each group (n = 5 mice). The average number of RGCs was counted using Image J software, and the average RGCs density was calculated based on the area of the membrane.

[0040] 6. Extraction of retinal tissue protein

[0041] On the 7th day after modeling, male C57BL / 6J mice in each group were sacrificed according to the above method, and the tissues were placed in 1.5 mL EP tubes and kept on an ice box. The retinas were quickly dissected under a microscope. After obtaining the retinas, they were placed in new 1.5 mL EP tubes. An appropriate amount of Inhibit protease inhibitor and RIPA Lysis and Extraction Buffer mixture was added to each tube at a ratio of 1:100, and 2 steel beads for tissue grinding (2 mm) were added. Total protein was extracted by grinding in a tissue grinder. Grind for 90 seconds each time, for a total of 4 times. Centrifuge at 13,000 rpm for 15 minutes in a 4°C centrifuge, and transfer the supernatant to a new 1.5 mL EP tube. Protein quantification was performed according to the PierceTM BCA Protein Assaykit protein quantification kit. The protein concentration of each group was calculated and adjusted according to the protein quantification results. Add 25% volume of NuPAGE LDS Sample Buffer (4×) of the total solution, mix well, and heat in a 95°C dry heat bath for 5 minutes. The prepared protein and the original protein solution were stored at -80°C for later use.

[0042] 7. Immunoblot analysis

[0043] Assemble the electrophoresis tank and Plus PAGE precast gel (Tris-Gly, 4-20%). Pull out the comb, place it in the electrophoresis tank, add enough 1× electrophoresis buffer, add the protein samples of each group (25 μg) and Prestained Protein Ladder (10 μL), and perform electrophoresis at 120 V. After completion, remove the gel and transfer all the bands to a polyvinylidene fluoride (PVDF) membrane that has been activated with methanol in 1× rapid transfer buffer at 30 mA for 30 minutes. Add the PVDF membrane to a 5% skim milk solution, place it on a shaker, and block at room temperature for 1 hour. Wash 4 times with 1× TBST, 5 minutes each time. Add 1:1000 EphA2 antibody dilution, incubate overnight on a shaker at 4°C, wash 4 times with 1× TBST, 5 minutes each time. Add 1:1000 secondary antibody Anti-rabbit IgG, HRP-linked Antibody dilution, incubate on a shaker at room temperature for 1 hour, wash 4 times with 1× TBST, 5 minutes each time. Visualize with a SuperSignal West Pico PLUS Chemiluminescent Substrate kit and take pictures in an ImageQuant LAS 500 imaging system. Each group (n = 5 mice), and the results were repeated at least 3 times. The intensity of the bands was quantified using Image J image processing software.

[0044] 8. Statistical analysis

[0045] Statistical analysis was performed using the software GraphPad Prism 9.0. All data were measurement data, and the values were expressed as mean ± standard error of the mean (X ± SEM). The P-value was determined by one-way analysis of variance (one-way ANOVA) and Tukey's test for multiple comparisons. A P-value < 0.05 was considered statistically significant.

[0046] Experimental results:

[0047] 1. Acute ocular hypertension causes retinal tissue loss and ganglion cell death

[0048] 1.1 Acute ocular hypertension causes retinal tissue loss in mice

[0049] HE staining of the central retina at 1.1 mm on both sides of the optic nerve head of C57BL / 6J mice in each group on the 7th day after acute ocular hypertension injury in mice was constructed. The retinal layers of the Normal group and the Control + PBS group were clear, with uniform thickness and regular cell arrangement. There was no significant difference between the Normal group (inner retinal thickness, 71.82 ± 1.70 μm, full retinal thickness, 198.4 ± 9.25 μm) and the Control + PBS group (inner retinal thickness, 67.7 ± 1.71 μm, full retinal thickness, 192.1 ± 1.80 μm) (P > 0.05). Compared with the Control + PBS group, the structure of the retina in the AOH + PBS group was disordered, the cells were less regular, accompanied by a significant decrease in the inner retinal thickness (18.61 ± 1.05 μm) (P < 0.0001) and a significant thinning of the full retinal thickness (119.5 ± 6.144 μm) (P < 0.001). There was no difference in the number of RGCs between the Control + PBS group and the Normal group (74.80 ± 2.72 / vs 89.10 ± 9.73 / , P < 0.05). The number of RGCs in the AOH + PBS group (23.90 ± 2.67 / ) was significantly decreased compared with that in the Control + PBS group (P < 0.001) (see Figure 1 A - B).

[0050] 1.2 Acute ocular hypertension causes death of retinal ganglion cells in mice

[0051] Seven days after acute high intraocular pressure injury, specific RBPMS antibodies were used for immunofluorescent staining of RGCs in retinal flat mounts to quantify the changes in retinal RGC density. There was no significant difference in the density of RBPMS+RGCs between the Normal group (5871±176.6 / mm 2) and the Control+PBS group (5617±136.4 / mm 2) (P>0.05). The density of RBPMS+RGCs in the AOH+PBS group (5871±176.6 / mm 2) was significantly decreased compared with that in the Control+PBS group (P<0.0001) (see Figure 2 A-B).

[0052] This model has the advantages of simple operation, short modeling time, high repeatability, and obvious retinal damage. It can be seen from the experimental results that there was no significant difference in the central inner retinal thickness, full-thickness retinal thickness, and retinal RGC density between the mice in the Control+PBS group with simple anterior chamber puncture and normal mice, excluding the influence of anterior chamber surgery and intravitreal injection of PBS on the retina. The central inner retinal thickness and full-thickness retinal thickness in the high intraocular pressure group were significantly decreased, and the number of RGCs was significantly reduced. It indicates that the pathological increase in intraocular pressure in mice causes obvious damage to the retina.

[0053] 2. Histological evaluation of the effects of different drug concentrations of ALW-II-41-27 on the retina

[0054] 2.1 Retinal thickness evaluation

[0055] At 12 - 24 hours after acute high - intraocular pressure injury in mice, 1 μL of PBS or 1 μL of ALW - II - 41 - 27 solution with different concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM) was injected into the vitreous cavity of each group of mice. Histological evaluation was performed by HE staining of the central retina section at 1.1 mm on both sides of the optic nerve papilla 7 days after high - intraocular pressure modeling. The results of the inner retinal thickness (GCL + IPL) in the Control group showed that there was no significant difference between the Control + PBS group (65.96 ± 1.86 μm) and the Control + 0.1 μM ALW group (62.01 ± 0.77 μm), the Control + 0.2 μM ALW group (61.92 ± 0.56 μm), and the Control + 0.5 μM ALW group (61.94 ± 0.44 μm) (P > 0.05). The results of the total retinal thickness also showed that there was no significant difference between the Control + PBS group (189.8 ± 1.62 μm) and the Control + 0.1 μM ALW group (187.0 ± 2.75 μm), the Control + 0.2 μM ALW group (185.0 ± 1.89 μm), and the Control + 0.5 μM ALW group (188.6 ± 0.94 μm) (P > 0.05). However, when the concentration of ALW - II - 41 - 27 was 1.0 μM, both the inner retinal thickness (56.49 ± 0.36 μm) and the total retinal thickness (119.5 ± 6.144 μm) decreased significantly. The results of the inner retinal thickness in the AOH group showed that compared with AOH + PBS (18.61 ± 1.05 μm), the degree of reduction in retinal thickness in the AOH + 0.1 μM ALW group (38.83 ± 2.09 μm), the AOH + 0.2 μM ALW group (40.09 ± 1.10 μm), the AOH + 0.5 μM ALW group (44.37 ± 1.92 μm), and the AOH + 1.0 μM ALW (32.97 ± 1.62 μm) all decreased significantly (P < 0.0001), showing a significant protective effect. The results of the total retinal thickness also showed that compared with the AOH + PBS group (119.5 ± 6.14 μm), the degree of reduction in retinal thickness in the AOH + 0.1 μM ALW group (144.9 ± 4.10 μm), the AOH + 0.2 μM ALW group (141.7 ± 1.95 μm), the AOH + 0.5 μM ALW group (155.9 ± 5.05 μm), and the AOH + 1.0 μM ALW (144.9 ± 5.51 μm) decreased. Among them, the AOH + 0.5 μM ALW group showed better retinal protection compared with other drug - concentration groups ( Figure 3 A - C).

[0056] 2.2 Evaluation of the number of surviving retinal ganglion cells

[0057] At 12 - 24 hours after acute high - intraocular pressure injury in mice, 1 μL of PBS or 1 μL of ALW - II - 41 - 27 solution with different concentrations (0.1 μM, 0.2 μM, 0.5 μM, 1.0 μM) was injected into the vitreous cavity of each group of mice. On the 7th day after high - intraocular pressure modeling, the retinas of mice were taken, and the RGCs in retinal flat mounts were immunofluorescently stained with a specific RBPMS antibody to quantify the density of retinal RGCs. The results showed that there was no significant difference in the density of RBPMS+RGCs between the Control+PBS group (6053±100.3 / mm 2) and the Control+0.1 μM ALW group (6016±132.4 / mm 2), the Control+0.2 μM ALW group (5651±74.21 / mm 2), and the Control+0.5 μM ALW group (5968±109.5 / mm 2) (P>0.05). When the concentration continued to increase, the density of RGCs in the Control+1.0 μM ALW group (3154±234.2 / mm 2) began to decrease significantly (P<0.0001). The results of the density of RBPMS+RGCs in the AOH group showed that compared with AOH+PBS (456.9±53.4 / mm 2), the AOH+0.1 μM ALW group (835.5±136.4 / mm 2) did not show an obvious protective effect on RGCs. The AOH+0.2 μM ALW group (1289±73.84 / mm 2) and the AOH+0.5 μM ALW group (1925±114.5 / mm 2) significantly increased the survival rate of RGCs, and the 0.5 μM ALW group had the best therapeutic effect (P<0.0001). When the concentration of ALW - II - 41 - 27 increased to 1.0 μM, the density of RGCs decreased to 946.3±34.13 / mm 2, and there was no obvious protective effect on retinal RGCs. (See Figure 4 A - B).

[0058] Therefore, in this experiment, 1 μL of four different concentrations of ALW-II-41-27 solution was slowly injected into the vitreous cavity of mice to make it evenly distributed in the vitreous cavity, and the concentrations in the vitreous cavity of mice reached 0.1 μM, 0.2 μM, 0.5 μM, and 1.0 μM respectively. The results of histological evaluation showed that when 0.1 μM, 0.2 μM, and 0.5 μM ALW-II-41-27 were injected into the vitreous cavity of the control group mice, compared with the group simply injected with PBS, there were no obvious changes in the number of retinal RGCs and the thickness of the central retinal tissue. When 1.0 μM ALW-II-41-27 was injected, certain degrees of damage or loss occurred. Therefore, it is speculated that ALW-II-41-27 with a concentration of 1.0 μM is toxic to the retinal tissue and RGCs to a certain extent, and its specific mechanism remains to be further studied. In the AOH animal model, ALW-II-41-27 plays a retinal protective role in a concentration-dependent manner. The main manifestations are to relieve the thinning and loss of the retinal tissue after the action of high intraocular pressure, and reduce the mortality of retinal RGCs. Among them, 0.5 μM ALW-II-41-27 shows the best protective effect. When the concentration is further increased to 1.0 μM, the above protective effect begins to decline. It is speculated that this may be related to the toxic effect of the drug on the retina due to too high a concentration, and the specific mechanism is still unclear.

[0059] 3. ALW-II-41-27 inhibits the expression of EphA2 in the retina of mice with acute high intraocular pressure

[0060] Seven days after the establishment of the mouse model of acute high intraocular pressure injury, retinal tissue proteins were extracted and Western blotting was performed. The results showed that compared with the Control+PBS group, the expression of EphA2 protein in the retina of the AOH+PBS group (1.93±0.18) increased significantly (P < 0.0001). Under the condition of acute high intraocular pressure, compared with the group simply injected with PBS, 0.5 μM ALW-II-41-27 (1.47±0.12) reduced the expression of EphA2 in the retina of mice to a certain extent (P < 0.05). Under the condition of non-high intraocular pressure, ALW-II-41-27 had no obvious effect on the expression of EphA2 in the retina. (See Figure 5 A - B).

[0061] Therefore, in this experiment, the expression of EphA2 protein in the retina of different groups was further detected. Intravitreal injection of 0.5 μM ALW-II-41-27 significantly reduced the overactivation of EphA2 in the retina after high intraocular pressure injury in the AOH mouse model. In the eyes of the control group mice, intravitreal injection of 0.5 μM ALW-II-41-27 had no obvious effect on the expression of EphA2 in the retina.

[0062] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

Use of ALW-II-41-27 in the preparation of a medicament for preventing or treating glaucoma or acute ocular hypertension, wherein the medicament is an injection for intravitreal injection, and the concentration of ALW-II-41-27 in the medicament is 0.2 μM - 0.5 μM.

2. The application according to claim 1, wherein The use includes increasing the survival rate of retinal ganglion cells.

3. The application according to claim 1, wherein The use includes reducing retinal damage.

4. The application according to claim 1, wherein, The concentration of ALW-II-41-27 in the medicament is 0.2 μM.

5. The application according to claim 1, wherein, The concentration of ALW-II-41-27 in the medicament is 0.5 μM.

6. The application according to any one of claims 1-5, wherein, The dosage of the medicament is 0.9 μL - 1.1 μL.

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