New application of ALW-II-41-27 in treatment of ophthalmic diseases

Abstract

The invention relates to an application of an EphA2 specific tyrosine kinase inhibitor ALW-II-41-27 in eye diseases, and proves that the ALW-II-41-27 can inhibit the expression of retina EphA2 at a specific concentration, alleviate the damage of acute ocular hypertension to retina tissues and improve the survival rate of retinal ganglion cells, thereby playing a role in neuroprotection, and having a good application prospect. Therefore, the traditional Chinese medicine composition is used for preventing and treating acute ocular hypertension or glaucoma.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of ALW-II-41-27 in treating 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 World Health Organization, approximately 76 million people suffer from glaucoma in 2020. It is estimated that by 2040, these numbers will exceed 11.1 million. Glaucoma is a neurodegenerative disease characterized by optic atrophy and visual field loss. The death of retinal ganglion cells (RGCs) is the main pathological feature of glaucoma and the main cause of irreversible blindness. Current studies believe that the death process of RGCs involves an increase in abnormal intraocular pressure (IOP). Increased intraocular pressure plays a key role in the pathogenesis of glaucoma, and lowering intraocular pressure is a key method for the treatment of glaucoma. In recent years, with the development of medicine, the monitoring methods, diagnostic criteria, drugs and surgical treatment plans for high intraocular pressure have been gradually improved and have shown beneficial effects in saving the vision of glaucoma patients. Despite this, clinical data show that about 10% of glaucoma patients still do not respond to these treatments. Since RGCs are lost and cannot be regenerated, a considerable number of glaucoma patients still experience visual field deterioration after controlling intraocular pressure. Therefore, in addition to implementing relevant treatments to lower intraocular pressure, it is very important to develop long-lasting and effective neuroprotective interventions that can slow down and prevent the loss of RGCs in the treatment of glaucoma.

[0003] Erythropoietin-producing hepatocellular receptors (Eph receptors) constitute the largest subfamily of receptor tyrosine kinases. Eph receptor signaling pathways are also important in neuronal development, axonal extension, and vascular growth and remodeling. The Eph / ephrin system is closely associated with glaucomatous optic neuropathy. Studies have shown that the expression of Eph receptors and their ligands, ephrin proteins, is upregulated after central nervous system injury, and Eph / ephrin has become a potential target for clinical treatment of central nervous system injury. In a rat model of chronic intraocular hypertension, Eph / ephrin family members are upregulated in Müller cells of the optic nerve head, and similar phenomena were observed in a rhesus monkey experimental glaucoma model and a DBA / 2J pigmented glaucoma mouse model.

[0004] EphA2 is an important member of the Eph family, located on chromosome 1p36.1, encoding a polypeptide with a molecular weight of 130KD and 976 amino acid residues, and 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 intracellular tyrosine kinase, causing autophosphorylation and phosphorylation of downstream substrate molecules, and initiating signal transduction. Studies have shown that increased EphA2 expression is associated with ischemic nervous system damage, destruction of the blood-brain barrier, infiltration of inflammatory mediators, activation of glial cells, and further activation of apoptotic signaling pathways in the central nervous system ischemia model. However, the mechanism remains complex and uncertain, and requires further research.

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

[0006]

[0007] ALW-II-41-27 is a newly developed ATP-competitive EphA2 inhibitor that was first discovered in 2009 and has been shown to reduce EphA2 phosphorylation in a variety of cancers. It is currently not used in ocular diseases. Summary of the invention

[0008] Based on this, the object 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 invention objectives, this study provides the following technical solutions:

[0011] Use of ALW-II-41-27 in preparing a drug for preventing or treating glaucoma or acute ocular hypertension.

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

[0013] In some of these embodiments, the use includes mitigating damage to the retina.

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

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

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

[0017] Figure 1 Acute high intraocular pressure caused retinal tissue loss in mice, among which the retina of glaucomatous mice had disordered structure and irregular cells, accompanied by a significant decrease in the thickness of the inner retina and a significant thinning of the thickness of the whole retina; (A) HE staining of retinal sections in the central area 1.1 mm on both sides of the optic nerve head of each group of mice 7 days after acute high intraocular pressure 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) The bar graph shows the quantitative analysis of the thickness of the inner retina in the central area, the thickness of the whole retina, and the number of RGCs in different groups in HE staining, and the data are expressed as mean ± SEM (n = 5 mice per group); P values ​​were determined using one-way analysis of variance and Tukey's test for multiple comparisons. ***P < 0.001, and ****P < 0.0001. Figure 2 Acute high intraocular pressure induces retinal ganglion cell death in mice; (A) Immunofluorescence staining of RGCs in retinal flat mounts was performed using a specific RBPMS antibody (magnification × 20, scale bar, 50 μm); (B) The bar graph shows the quantitative analysis of RGCs density in different groups, and the 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 < 0.0001.

[0018] Figure 3, intravitreal injection of different concentrations (0.1μM, 0.2μM, 0.5μM, 1.0μM) of ALW-II-41-27 had an effect on the thickness of retinal tissue in mice under conditions of non-acute intraocular hypertension and acute intraocular hypertension; the results of inner retinal thickness in the AOH group showed that compared with AOH+PBS, the reduction degree of retinal thickness in the AOH+0.1μM ALW group, AOH+0.2μM ALW group, AOH+0.5μM ALW group, and AOH+1.0μM ALW group was significantly decreased, showing a significant protective effect; the results of full-layer retinal thickness also showed that compared with the AOH+PBS group, the reduction degree of retinal thickness in the AOH+0.1μM ALW group, AOH+0.2μM ALW group, AOH+0.5μM ALW group, and AOH+1.0μM ALW group was decreased. Among them, the AOH+0.5μM ALW group showed better retinal protection compared with other drug concentration groups; (A) HE staining of retinal sections in the central area 1.1mm on both sides of the optic nerve head of each group of mice 7 days after acute high intraocular pressure injury (magnification ×20, scale bar, 100μm); (B) The bar graph shows the quantitative analysis of the inner retinal thickness of different groups of retinal HE staining in PBS and different concentrations (0.1μM, 0.2μM, 0.5μM, 1.0μM) of ALW-II-41-27 under non-acute high intraocular pressure and acute high intraocular pressure. (C) The bar graph shows the quantitative analysis of the full-layer retinal thickness of different groups of retinal HE staining in PBS and different concentrations (0.1μM, 0.2μM, 0.5μM, 1.0μM) of ALW-II-41-27 under non-acute high intraocular pressure and acute high intraocular pressure. 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, intravitreal injection of PBS and different concentrations (0.1μM, 0.2μM, 0.5μM, 1.0μM) of ALW-II-41-27 solution had an impact on the survival rate of retinal RGCs in mice under conditions of non-acute intraocular hypertension and acute intraocular hypertension; the RBPMS+RGCs density results of the AOH group showed that compared with AOH+PBS, the AOH+0.1μM ALW group did not show obvious RGCs protective effect; the AOH+0.2μM ALW group and the AOH+0.5μM ALW group significantly improved the survival rate of RGCs, among which the 0.5μM 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 specific RBPMS antibodies (magnification × 20, scale bar, 50 μm); (B) The bar graph shows the quantitative analysis of the density of retinal RGCs 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 high intraocular pressure 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 ocular hypertension. Western blotting results showed that compared with the Control+PBS group, the expression of EphA2 protein in the retina of the AOH+PBS group was significantly increased; under the condition of acute ocular hypertension, compared with the group injected with PBS alone, 0.5μM ALW-II-41-27 reduced the expression of EphA2 in the mouse retina to a certain extent, while under the condition of non-ocular hypertension, ALW-II-41-27 had no significant effect on the expression of retinal EphA2; ALW-II-41-27 inhibits the expression of EphA2 in the retina of mice with acute ocular hypertension (A) EphA2 protein levels in the Control+PBS group, AOH+PBS group, Control+0.5μM ALW group, and AOH+0.5μM ALW group; (B) The EphA2 / β-actin protein ratio was quantified by densitometry analysis using ImageJ software. Data are expressed as mean ± SEM (n = 5 mice per group, experiments were repeated 3 times or more); P values ​​were determined using one-way ANOVA and Tukey's test for multiple comparisons. *P < 0.05, ****P < 0.0001. DETAILED DESCRIPTION

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

[0022] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0023] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification 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, 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 RGCs cell death caused by high intraocular pressure, thereby improving the survival rate of RGCs cells and providing a new treatment idea for the treatment of glaucoma.

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

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

[0028] Example 1

[0029] Experimental methods:

[0030] 1. Animal grouping

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

[0032] 2. Establishment of acute ocular hypertension mouse model

[0033] C57BL / 6J mice with eye or systemic lesions or injuries, malnutrition, etc. were eliminated. The Normal group did not receive any treatment. After the remaining mice were weighed, 1% sodium pentobarbital solution was injected intraperitoneally at 50 mg / kg body weight for anesthesia. A heating pad was used to prevent hypothermia in anesthetized mice. One drop of compound tropicamide eye drops was instilled into the mouse's operated eye to dilate the pupil and then cover both eyes to avoid light. After the pupil was dilated, one drop of oxybuprocaine hydrochloride eye drops was instilled into the operated eye for surface anesthesia. The AOH group was used as the acute high intraocular pressure experimental group. A 33G sharp needle connected to a sterile saline perfusion tube was inserted into the anterior chamber of the surgical eye, the perfusion bottle was raised, and the intraocular pressure was measured with a TonoLab rebound tonometer to keep the intraocular pressure at 90 mmHg. The pale conjunctiva and iris, corneal edema, whitening of the scleral vein, and no liquid leakage from the puncture site were observed as signs of the success of the acute high intraocular pressure model. Carbomer eye gel was used to protect the cornea during modeling. After the model lasted for 60 minutes, the needle was removed, normal perfusion pressure was restored, and levofloxacin hydrochloride eye ointment was applied after surgery to avoid infection. The control group was used as the control group for non-acute ocular hypertension. After anesthesia and mydriasis, a 33G sharp needle was used to puncture the anterior chamber of the eye, without saline perfusion, and without increasing intraocular pressure. Levofloxacin hydrochloride eye ointment was applied after surgery to avoid infection.

[0034] 3. Intravitreal injection in mice

[0035] Within 12-24 hours after the establishment of the high intraocular pressure model, mice with obvious cataracts, eye infections, eyeball atrophy, eyeball hemorrhage, iris prolapse and other lesions were eliminated. The mice in the Control group or AOH group were anesthetized as described above and the pupils of the operated eyes were dilated. A heating pad was used to prevent hypothermia in the anesthetized mice. After the pupil was dilated, a drop of oxybuprocaine hydrochloride eye drops was dripped into the operated eye for surface anesthesia. The cornea was smeared with medical hyaluronic acid gel and covered with a 3mm round cover glass. After the fundus was clearly observed under a microscope, a 33G sharp needle was used to puncture the sclera 1mm behind the temporal corneoscleral limbus of the eyeball. The Hamilton microsyringe was inserted into the vitreous cavity of the mouse operated eye at the puncture site, and 1μL PBS or 1μL ALW-II-41-27 solution of different concentrations was slowly injected to make it evenly distributed in the vitreous body, and the concentrations reached 0.1μM, 0.2μM, 0.5μM, and 1.0μM (calculated based on the mouse vitreous volume of 5μL). After the injection, leave the needle in for 30 seconds and quickly remove it to avoid puncturing the lens and retina. Observe the mouse under a microscope for lens scratches, fundus hemorrhage, and retinal bulges to determine whether the injection was successful. Apply levofloxacin hydrochloride eye ointment after surgery to avoid infection.

[0036] 4. HE staining retinal thickness measurement

[0037] C57BL / 6J male mice in each group were collected on the 7th day after modeling. Mice with poor general health, eyeball atrophy, corneal lesions, eye infection and bleeding were eliminated. After death by anesthesia with an overdose of 1% sodium pentobarbital solution, the cervical vertebra was cut and the eyeball was removed. The eyeball was pretreated in FAS eyeball fixative for 48 hours, embedded in paraffin, and sliced ​​from the plane of the optic nerve at 6 o'clock to 12 o'clock (thickness 7μm). 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 1.1mm on both sides of the optic nerve head was photographed (magnification x20, scale 100μM), and the inner retina (ganglion cell layer GCL to inner nuclear layer IPL thickness), full-layer retinal thickness, 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] C57BL / 6J male mice in each group were collected according to the above method on the 7th day after modeling. The eyeballs were removed and fixed in 4% PFA paraformaldehyde fixative at room temperature for 2 hours. The complete retina was quickly peeled out under a microscope, and the retina was cut into a regular four-leaf clover shape, placed in a 24-well plate, placed on a shaker, and washed 3 times with PBS, 5 minutes each time. 1.5μL of anhydrous ethanol was added to each well, and incubated on a shaker at 50 rpm for 30 minutes. Washed 3 times with PBS, 5 minutes each time. The retina was transferred to a slide, keeping the ganglion cell layer superimposed, and the immunohistochemistry pen drew a closed circle around the retina. 150μL of 1% TritonX-100 solution was added, and the retina was soaked for 20 minutes, and washed 3 times with PBS, 5 minutes each time. 150μL of 10% donkey serum solution was added to block overnight at 4℃. Washed 4 times with PBS, 5 minutes each time. Add RBPMS antibody solution diluted 1:1000 and incubated overnight in a refrigerator at 4℃. Wash with PBS 4 times, 5 minutes each time. Add 150 μL of Alexa Fluor 488-labeled goat anti-rabbit IgG (H+L) secondary antibody solution diluted 1:1000 at room temperature. Incubate in the dark for 2 hours. Wash with PBS 4 times, 5 minutes each time. Use DAPI anti-fluorescence quenching mounting agent to seal the slides. The retina was photographed using a multifunctional microplate imaging detection and analysis system, and RGCs can be seen in green using the GFP channel. Four quadrants of the retina were photographed at 1.1 mm from the optic nerve (magnification x20, scale 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 film.

[0040] 6. Retinal tissue protein extraction

[0041] On the 7th day after modeling, the C57BL / 6J male mice in each group were collected according to the above method and placed in a 1.5mL EP tube, placed in an ice box, and the retina was quickly peeled under a microscope. After obtaining the retina, it was placed in a new 1.5mL EP tube, and an appropriate amount of Inhabit protease inhibitor and RIPA Lysis and Extraction Buffer mixture was added to each tube at a ratio of 1:100, and 2 2mm tissue grinding steel beads were added. The total protein was ground and extracted in a tissue grinder. 90 seconds each time, a total of 4 times. Place it in a 4℃ centrifuge and centrifuge at 13000rpm for 15 minutes, and the supernatant was aspirated into a new 1.5mL 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. After adding 25% volume of the total solution NuPAGE LDS Sample Buffer (4×) and mixing well, it was heated in a 95℃ dry heat bath for 5 minutes. The prepared protein and original protein solution were placed in a -80℃ refrigerator for standby use.

[0042] 7. Immunoblotting analysis

[0043] Assemble the electrophoresis tank and Plus PAGE precast gel (Tris-Gly, 4-20%), remove the comb, put it into the electrophoresis tank, add enough 1× electrophoresis solution, add each group protein sample (25μg) and Prestained Protein Ladder (10μL), electrophoresis at 120V voltage, remove the gel after the end, transfer all the bands to the polyvinylidene fluoride (PVDF) membrane activated by methanol in 1x fast transfer solution, 30mA current, 30 minutes. Add PVDF membrane to 5% skim milk powder solution, place on a shaker, and block at room temperature for 1 hour. Wash 4 times with 1x TBST, 5 minutes each time. Add 1:1000 EphA2 antibody dilution, incubate overnight on a shaker at 4℃, and wash 4 times with 1x TBST, 5 minutes each time. Add 1:1000 secondary antibody Anti-rabbit IgG, HRP-linked Antibody secondary antibody dilution, incubate at room temperature on a shaker for 1 hour, wash 4 times with 1x TBST, 5 minutes each time. Ultrasensitive ECL chemiluminescence kit was used for color development, and the images were taken in the ImageQuant LAS 500 imaging system. Each group (n = 5 mice) was 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 GrahPad Prism 9.0 software. All data were quantitative data, and the values ​​were expressed as mean ± standard error (X ± SEM). P values ​​were determined by one-way ANOVA and Tukey test for multiple testing. When P < 0.05, the difference was considered statistically significant.

[0046] Experimental results:

[0047] 1. Acute high intraocular pressure causes retinal tissue loss and ganglion cell death

[0048] 1.1 Acute intraocular hypertension induces retinal tissue loss in mice

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

[0050] 1.2 Acute high intraocular pressure induces retinal ganglion cell death in mice

[0051] Seven days after acute ocular hypertension, immunofluorescence staining of RGCs in retinal flat mounts was performed using a specific RBPMS antibody to quantify changes in retinal RGCs density. There was no significant difference in the RBPMS+RGCs density in the Normal group (5871±176.6 / mm 2) compared with the Control+PBS group (5617±136.4 / mm 2) (P>0.05). The RBPMS+RGCs density in the AOH+PBS group (5871±176.6 / mm 2) was significantly reduced compared with the Control+PBS group (P<0.0001) (see Figure 2 AB).

[0052] This model has the advantages of simple operation, short modeling time, high repeatability, and obvious retinal damage. From the experimental results, it can be seen that there is no significant difference in the central inner retinal thickness, full-thickness retinal thickness, and retinal RGCs density between the Control+PBS group mice with simple anterior chamber puncture and normal mice, which excludes the effects of anterior chamber surgery and intravitreal injection of PBS on the retina. The central inner retinal thickness and full-thickness retinal thickness of the high intraocular pressure group decreased significantly, and the number of RGCs decreased significantly. This shows that pathological increase in intraocular pressure in mice causes significant 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 Assessment of retinal thickness

[0055] After acute high intraocular pressure injury in mice, 1 μL PBS or 1 μL ALW-II-41-27 solution of 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 within 12-24 hours. On the 7th day after high intraocular pressure modeling, retinal sections at 1.1 mm on both sides of the optic nerve head were stained with HE for histological evaluation. The results of the inner retinal thickness (GCL+IPL) of the Control group showed that there was no significant difference between the Control+PBS group (65.96±1.86μm) and the Control+0.1μM ALW group (62.01±0.77μm), the Control+0.2μM ALW group (61.92±0.56μm), and the Control+0.5μM ALW group (61.94±0.44μm) (P>0.05). The results of full-thickness retinal thickness also showed no significant difference between the Control+PBS group (189.8±1.62μm) and the Control+0.1μM ALW group (187.0±2.75μm), Control+0.2μM ALW group (185.0±1.89μm), and Control+0.5μM ALW group (188.6±0.94μm) (P>0.05). However, when the concentration of ALW-II-41-27 was 1.0μM, the inner retinal thickness (56.49±0.36μm) and full-thickness retinal thickness (119.5±6.144μm) were significantly decreased. The results of the inner retinal thickness in the AOH group showed that compared with AOH+PBS (18.61±1.05μm), the reduction in retinal thickness in the AOH+0.1μM ALW group (38.83±2.09μm), AOH+0.2μM ALW group (40.09±1.10μm), AOH+0.5μM ALW group (44.37±1.92μm), and AOH+1.0μM ALW (32.97±1.62μm) were significantly decreased (P<0.0001), indicating a significant protective effect. The results of full-thickness retinal thickness also showed that compared with the AOH+PBS group (119.5±6.14μm), the AOH+0.1μM ALW group (144.9±4.10μm), AOH+0.2μM ALW group (141.7±1.95μm), AOH+0.5μM ALW group (155.9±5.05μm), and AOH+1.0μM ALW (144.9±5.51μm) showed a decreased degree of retinal thickness reduction. Among them, the AOH+0.5μM ALW group showed better retinal protection than the other drug concentration groups ( Figure 3 AC).

[0056] 2.2 Assessment of retinal ganglion cell survival

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

[0058] Therefore, in this experiment, 1 μL of four different concentrations of ALW-II-41-27 solutions were slowly injected into the vitreous cavity of mice to make them 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, there was no significant change in the number of retinal RGCs and the thickness of central retinal tissue compared with the group injected with PBS alone. However, when 1.0 μM ALW-II-41-27 was injected, a certain degree of damage or loss occurred. Therefore, it is speculated that ALW-II-41-27 with a concentration of 1.0 μM is toxic to retinal tissue and RGCs, and its specific mechanism needs further study. In the AOH animal model, ALW-II-41-27 exerts a concentration-dependent retinal protective effect. The main manifestations were to reduce the thinning and loss of retinal tissue after high intraocular pressure, and reduce the mortality of retinal RGCs, among which 0.5μM ALW-II-41-27 showed the best protective effect. When the concentration was further increased to 1.0μM, the above protective effect began to decline. It is speculated that this may be related to the toxic effect of the drug concentration on the retina, 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 intraocular hypertension

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

[0061] Therefore, this experiment further detected the expression of retinal EphA2 protein in different groups. Intravitreal injection of 0.5 μM ALW-II-41-27 significantly reduced the overactivation of retinal EphA2 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 significant effect on retinal EphA2 expression.

[0062] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of ALW-II-41-27 in the preparation of drugs for preventing or treating glaucoma or acute ocular hypertension.

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

3. The use according to claim 1, wherein: The uses include alleviating damage to the retina.

4. The use according to claim 1, wherein: The medicine is an injection.

5. The use according to claim 4, wherein: The medicine is an injection for intravitreal injection.

6. The use according to claim 1, wherein: The concentration of ALW-II-41-27 in the drug is 0.2 μM-1.0 μM.

7. The use according to claim 6, wherein: The concentration of ALW-II-41-27 in the drug is 0.3 μM-0.8 μM.

8. The use according to claim 7, wherein: The concentration of ALW-II-41-27 in the drug is 0.45 μM-0.55 μM.

9. The use according to claim 7, wherein: The concentration of ALW-II-41-27 in the drug was 0.5 μM.

10. The use according to any one of claims 7 to 9, wherein: The dosage of the drug is 0.9 μL-1.1 μL.

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