Optic nerve protective agent and application thereof in preparation of medicine for treating optic nerve injury

Abstract

The invention belongs to the field of medicines, and particularly discloses an application of an optic nerve protective agent in preparation of a medicine for treating optic nerve injury, and the optic nerve protective agent is a compound with a formula 1 # imgabs0 # structure and a pharmaceutically acceptable crystal thereof. According to the present invention, the component represented by the formula 1 can be adopted as the optic nerve protection active component to prepare the optic nerve injury treatment drug capable of improving the survival rate and / or the regeneration capacity of the RGCs cell.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the field of treatment for protection of optic nerve damage. Background Art

[0002] The eyeball is the main part of the visual organ, located in the orbit, and connected to the diencephalon by the optic nerve at the posterior end. The optic nerve is formed by approximately 1.2 million unmyelinated nerve axons emanating from retinal ganglion cells (RGCs), which converge at the nasal side of the posterior pole of the eyeball and are part of the central nervous system. Various diseases can cause optic nerve damage, such as glaucoma, trauma, infection, tumor compression, and genetic factors. The death of retinal ganglion cells and the degeneration of axons are the ultimate factors that lead to vision loss in many diseases. Glaucoma is a typical disease that causes optic nerve damage. It is the world's leading cause of blindness and the "thief of light." Glaucoma that develops to the level of low vision or disability will place a heavy burden on families and society. Therefore, promoting optic nerve regeneration and effectively rehabilitating the remaining visual function are of great significance.

[0003] However, the current effective measures for the clinical treatment of glaucoma are still limited to lowering intraocular pressure. How to promote the regeneration of damaged optic nerves is a major challenge in the treatment of optic nerve degenerative diseases such as glaucoma. On the one hand, the optic nerve has limited regenerative capacity and cannot regenerate spontaneously, so it is often difficult to fully recover after damage. On the other hand, optic nerve damage caused by different reasons has different pathological mechanisms, so personalized treatment plans need to be formulated for specific situations. Therefore, it is of great significance to find drugs that can prevent the death of retinal ganglion cells and axonal degeneration after optic nerve damage, and promote its axonal regeneration.

[0004] Publication number US20240108632A1 discloses an eye drop formulation containing cyclodextrin and adamantane, which can be used to prevent or treat ischemic optic neuropathy, for example. Chinese patent publication number CN112843085A discloses a complex for treating optic nerve diseases, tFNA-miR22, composed of DNA tetrahedron and miR-22 in a molar ratio of 1:(1-4); it can provide good protection for retinal ganglion cells.

[0005] In summary, although there are some optic nerve protection strategies in the existing technology, there are still common shortcomings such as complex ingredients and large side effects. The existing technology is still relatively lacking in safe and effective small molecule optic nerve protective agents. Summary of the Invention

[0006] In view of the problems faced by existing optic nerve damage protective drugs, the first object of the present invention is to provide an optic nerve protective agent for use in the preparation of optic nerve damage protective drugs.

[0007] A second object of the present invention is to provide a drug for protecting against optic nerve damage comprising the optic nerve protective agent.

[0008] A use of an optic nerve protectant in the preparation of a drug for treating optic nerve damage, wherein the optic nerve protectant is used as an optic nerve protective active ingredient in the preparation of a drug for treating optic nerve damage, wherein the optic nerve protectant is a compound having the structure of Formula 1 and a pharmaceutically acceptable crystal thereof;

[0009]

[0010] In the present invention, the component of formula 1 can be used as an optic nerve protective active ingredient to prepare a drug for treating optic nerve damage that can improve the survival rate and / or regeneration ability of RGCs cells.

[0011] A large number of studies have shown that the component of Formula 1 can be used as an active component for protecting the optic nerve, can protect RGCs cells, improve the survival rate and regeneration ability of RGCs cells, and reduce RGCs cell damage.

[0012] The application of the present invention can be to use the optic nerve protective agent as an optic nerve protecting active ingredient in the preparation of a drug for treating optic nerve damage caused by at least one of glaucoma, trauma, infection, tumor compression, and genetic factors.

[0013] In the application of the present invention, the content of the optic nerve protective agent of Formula 1 in the optic nerve protecting active ingredient is above 50 wt.%, preferably above 80 wt.%.

[0014] The application of the present invention can be to prepare an eyeball injection preparation for treating optic nerve damage using the neuroprotective agent. In the present invention, the eyeball injection preparation can be an injectable powder or an injectable solution.

[0015] The present invention also provides a drug for treating optic nerve damage, comprising a pharmaceutically effective amount of an optic nerve protecting active ingredient, wherein the optic nerve protecting active ingredient comprises more than 50 wt.%, preferably more than 80 wt.% of the optic nerve protecting agent of formula 1.

[0016] Preferably, the optic nerve protective active ingredient is the optic nerve protective agent.

[0017] The medicine of the present invention consists of the optic nerve protective agent and a diluent.

[0018] In the drug of the present invention, the diluent comprises at least one of DMSO, water, and PBS buffer. Further, the diluent is a mixed solution of DMSO and PBS buffer, and the concentration of DMSO is 0.1-1 wt%.

[0019] The medicine of the present invention is an eyeball injection preparation.

[0020] An optional eyeball injection preparation of the present invention is prepared by mixing Formula 1 with DMSO and then mixing with PBS buffer.

[0021] Beneficial effects

[0022] The present invention innovatively uses the component of Formula 1 as an active ingredient to improve the survival rate and regenerative capacity of RGCs cells, reduce RGCs cell damage, and can be used to treat various types of optic nerve damage diseases. In addition, the drug of the present invention has high biocompatibility, is safe, and has no side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For comparison in Example 1, Formula a, Formula 1 and DMSO have protective effects on RGCs cultured in vitro.

[0024] Figure 2 The compound of formula 1 in Example 2 can effectively promote optic nerve regeneration in mice with optic nerve clamp injury model.

[0025] Figure 3 The compound of formula 1 in Example 2 has a protective effect on retinal ganglion cells in mice with optic nerve clamp injury model. DETAILED DESCRIPTION

[0026] Example 1

[0027] The compound of formula 1 has a protective effect on the survival of RGCs cultured in vitro

[0028] Experimental procedures

[0029] RGC-specifically labeled vglut2-td Tomato gene mice (vgluta-Cre + / - Mice and ZsGreen flx / flx -STOP flx / flx -tdTomato mouse hybrid) were killed, and the eyeballs of both mice were removed after sacrifice. The cornea was cut off along the corneoscleral limbus with ophthalmic scissors, and the lens was taken out. The choroid of the outer layer of the retina was bluntly peeled off with toothless forceps, and then the vitreous body in the eyeball was cleaned with forceps, and the two retinas were taken out.

[0030] Place the retina in a preheated digestion solution at 37°C (6 ml of L-15CO3, 150 ul of papain, 28 ul of 1% BSA, 3 mg of L-cysteine), place the digestion tube in a 37°C incubator, and digest for 30 minutes. Carefully remove the upper layer of liquid that does not contain retinal cells with a pipette and discard it. Add 10 ml of freshly prepared L-15CO3 to the tube to wash the retina. Use a glass Pasteur pipette to blow and swirl dozens of times. After it stands for 30 seconds, aspirate the upper layer of liquid into a new 50 ml centrifuge tube (be careful not to aspirate the impurities at the bottom). Repeat this process three times, i.e., wash the retina three times. Centrifuge the treated suspension at 1000 rpm for 10 minutes, remove the supernatant, and add an appropriate amount of L-15 for resuspending.

[0031] Use culture medium (Leibovitz's L-15 Medium + 1% penicillin-streptomycin

[0032] +2% B27+1% N2) and dilute the resuspended cell solution to a density of 1.5×10 6 / ml cell suspension totaling 6.5ml, 150μL of cell suspension was taken from each well and inoculated into a 96-well plate. The experimental group selected 100uM concentration of formula 1 (which can also be expressed as ); control group: 100uM contrast The solvent for both the experimental and control groups was dimethylsulfoxide (DMSO). After seeding, the cells were cultured in a 37°C, 5% CO2 incubator for 3 days.

[0033] Data Analysis

[0034] Observation and photography were performed using an inverted fluorescence microscope. Nine preselected fields of view were photographed for each well, and the number of viable RCGs within each field of view was counted. Statistical analysis was performed using GraphPad Prism software. Data are presented as mean ± standard deviation (SEM). One-way anova analysis was used to analyze the data. P < 0.05 was considered statistically significant.

[0035] result

[0036] The results showed that compared with the control group, the compound of formula 1 had a protective effect on the survival of RGCs cultured in vitro.

[0037] The experimental results are as follows Figure 1 :

[0038] Figure 1A. The number of surviving specifically labeled RGCs cultured in vitro and treated with Dmso, Comparative Formula a-100 μM, and Formula 1-100 μM, respectively, was measured under an inverted fluorescence microscope. Scale bar, 50 μm. B. Quantification of surviving RGCs after 3 days of in vitro culture (Formula 1-100 μM: n=3; Comparative Formula a-100 μM: n=3; Dmso: n=3). The results showed that there was no significant difference in the number of surviving RGCs in the Comparative Formula a group compared to the Dmso group. However, the 100 μM Formula 1 provided by the present invention increased the number of surviving RGCs by 75% compared to Dmso and by 2.36 times compared to the structurally similar Comparative Formula a. ** represents p<0.01, *** represents p<0.001, and ns represents p>0.05.

[0039] Example 2

[0040] The compound of formula 1 can improve the optic nerve regeneration and the survival rate of retinal ganglion cells in mice with optic nerve clamp injury model

[0041] Experimental procedures

[0042] (1) Establishment of the optic nerve crush injury model (ONC)

[0043] Wild-type C57BL / 6 mice (8 weeks, 18-20 grams) were used, and after weighing, 1% sodium pentobarbital was injected intraperitoneally at a dose of 10 ml / kg for anesthesia. After the mice were anesthetized, local anesthesia was performed with oxybuprocaine hydrochloride eye drops. The mice were placed in a suitable position under the microscope, the outer canthus was cut open with tissue scissors, and then a 1 mm small incision was made at the temporal border of the bulbar conjunctiva with ophthalmic scissors. The incision was bluntly enlarged to 2-3 mm with forceps, and the fat and other tissues were peeled down until the eye muscles could be clearly seen. Then, the tissue around the optic nerve was peeled through the eye muscles to expose the optic nerve. The mouse optic nerve was clamped with reverse forceps 2 mm behind the eyeball and held for 3 seconds (be careful to avoid blood vessels during this process). After clamping, the forceps were removed, the eyeball was repositioned, and tobramycin dexamethasone eye ointment was applied to the ocular surface. Repeat on the contralateral side. After completion, the mice were placed on a heated workbench and placed in a cage after waking up.

[0044] (2) Grouping and drug administration

[0045] Mice subjected to optic nerve clamp injury were randomly divided into three groups (n represents the number of optic nerves or retinas): formula 1: n = 4 / 4 (optic nerve / retina), PBS: n = 25 / 15 (optic nerve / retina), and DMSO: n = 4 / 4 (optic nerve / retina). Drugs were injected intraocularly, and cholera toxin subunit B (CTB) was injected intraocularly on day 13 to clearly visualize optic nerve axons. Mice were sacrificed on day 14, and the optic nerves and retinas were harvested and processed appropriately to assess optic nerve regeneration and retinal ganglion cell survival. Formula 1: 600 μM (prepared in PBS, containing 0.40% DMSO) of the compound of Formula 1 was injected into the vitreous of the mouse eyeball on day 0 (after model establishment) and day 5, respectively; PBS: Phosphate buffered saline (PBS) was injected into the vitreous of the mouse eyeball on day 0 (after model establishment) and day 5, respectively; DMSO: DMSO was injected into the vitreous of the mouse eyeball on day 0 (after model establishment) and day 5, respectively;

[0046] After weighing the mice, they were anesthetized with 1% sodium pentobarbital injected intraperitoneally at a dose of 10 ml / kg. After the mice were anesthetized, topical anesthesia was performed with oxybuprocaine hydrochloride eye drops, followed by dilation of the pupil with compound tropicamide eye drops. The mice were placed in a suitable position under a microscope, and a needle was used to puncture the sclera near the limbus and in front of the eyeball at the temporal superior border (to reduce intraocular pressure). A pre-assembled 5ul glass microneedle syringe (first extracting 1ul of air, then extracting 3ul of Formula 1 compound) was used, at a 60° angle to the optic nerve. The needle was carefully inserted through the needle hole at the limbus into the vitreous cavity and injected (careful to avoid damaging the lens). After 15 seconds, the needle was slowly removed, the eyeball was repositioned, and tobramycin dexamethasone eye ointment was applied to the ocular surface. Repeat on the contralateral side. After completion, the mice were placed on a heated workbench and placed in a cage after waking up. The control group mice were injected with the same volume of PBS, and the steps were the same as above.

[0047] (3) Remove the optic nerve and retina

[0048] After weighing, mice were anesthetized with 1% sodium pentobarbital injected into the abdominal cavity at a dose of 10 ml / kg. After anesthesia, mice were fixed, the chest cavity was cut open with tissue scissors to expose the heart. After cutting the right atrial appendage, a 20 ml syringe was used to insert a needle into the apex of the heart, and 15 ml of pbs solution was slowly injected. The syringe was pulled out from the needle (be careful not to remove the needle tip from the apex of the heart). A 20 ml syringe containing 15 ml 4% paraformaldehyde was replaced and slowly injected for lavage. After withdrawing the needle and syringe, perfusion was completed. All skin and hair around the top of the head were removed with tissue scissors, the back of the head was removed, and the skin and hair were carefully cut along the sagittal and squamous sutures to expose the cortex. The olfactory bulb and the cortex were bluntly separated with tweezers. The brain was peeled off from the skull to expose the optic nerve at the base of the skull. The optic nerve was cut off with ophthalmic scissors close to the eyeball. The eyeball was separated and the cornea was cut open. After being placed in 4% paraformaldehyde and fixed at room temperature for 1 hour, it was respectively replaced with 30% sucrose solution and PBS solution at 4 ° C for preservation.

[0049] (4) Processing the optic nerve

[0050] After soaking in 30% sucrose, the optic nerve was placed on a coverslip, adjusted under a microscope, and the surrounding solution was aspirated. The optic nerve was then placed on the freezing stage of a cryostat. Optimal Cutting Temperature (OCT) cryosection embedding medium was applied, and another coverslip was placed. The tissue was pressed with a heat absorber until the embedding medium and tissue froze into a white, icy mass. The tissue block was carefully removed and placed on a specimen holder containing cryosection embedding medium. Another layer of OCT was applied to completely cover the block. Once frozen, the block was clamped firmly onto the microtome holder. The tissue was flattened and sectioned at a 14 μm thickness. The sections were carefully attached to slides, washed three times with phosphate-buffered saline-tween 20 (PBST) for 10 min, and then mounted with mounting medium. Fluorescence microscopy was used to image the optic nerve using a tiled mosaic mode, capturing images from the optic nerve clamping point to a point 500 μm from the point of the clamping point. Three sections were collected for each optic nerve.

[0051] (5) Processing the retina

[0052] The fixed eyeball was placed in a culture dish containing PBS solution, and the cornea was cut off along the corneoscleral margin with ophthalmic scissors, and the lens was removed. The choroid on the outer layer of the retina was bluntly peeled off with toothless forceps, and then the vitreous body in the eyeball was cleaned with forceps. Four incisions evenly spaced 2-3 mm were made from the edge of the retina toward the optic disc with ophthalmic scissors to divide the retina into four quadrants.

[0053] Retinas were washed three times in PBST on a shaker at room temperature for 15 minutes each. After blocking in 10% BSA for 1 hour at room temperature, the retinas were incubated with the primary antibody (Mouse Anti-beta III Tubulin) diluted 1:500 in 10% BSA overnight at 4°C. Retinas were then washed three times in PBST on a shaker at room temperature for 20 minutes each. They were then stained with the secondary antibody (Mouse Anti-Alexa Fluor 488) diluted 1:400 in 10% BSA. After incubation for 2 hours at room temperature, the retinas were washed three times in 0.1% PBST on a shaker at room temperature for 15 minutes each. Retinas were transferred to slides and mounted with mounting medium. Images were obtained under a fluorescence microscope at 40x magnification. Each retina was divided into four quadrants, and 1-2 images were acquired per quadrant, resulting in 4-8 images per retina.

[0054] Data processing

[0055] optic nerve

[0056] The number of CTB-positive optic nerve axons at 500 μm from the pinch point was counted using Image J. The width of the nerve (R) was measured at the counting point (d) and used together with the thickness of the section (t = 14 μm) to calculate the number of axons per optic nerve using the following formula: ∑ad = πr 2 =R × number of axons / (R × t). The average number of optic nerve regeneration is the mean value calculated from three different sections of the optic nerve. Statistical analysis was performed using GraphPad Prism software. Experimental data are presented as mean ± standard deviation (SEM). Differences between groups were compared using the Student's t-test. P < 0.05 was considered statistically significant.

[0057] retina

[0058] Beta III tubulin-positive cells were counted using Image J to determine the RGC density (RGCs per square millimeter) in each retina. Statistical analysis was performed using GraphPad Prism software. Data are presented as mean ± standard deviation (SEM). Differences between groups were compared using the Student's t-test. P < 0.05 was considered statistically significant.

[0059] Experimental results

[0060] The compound of formula 1 promotes optic nerve regeneration in mice with optic nerve clamp injury model

[0061] The results showed that compared with the control group, DMSO had no effect on the regeneration of optic nerve axons in the optic nerve clamp injury model mice (p>0.05). The compound of formula 1 provided by the present invention can promote the regeneration of the optic nerve in the experimental group model mice. The compound of formula 1 can be used as a therapeutic drug to protect the eyes from optic nerve damage.

[0062] The experimental results are as follows Figure 2 :

[0063] A. Axon regeneration of the optic nerve was observed in optic nerve sections using immunofluorescence staining against CTB. Asterisks indicate the site of optic nerve clamping. Scale bar, 100 μm. B. Quantification of the number of regenerated axons in the optic nerve two weeks after optic nerve clamping (PBS: n=25; Formula 1: n=4). Results showed that the number of regenerated axons in the optic nerve of mice treated with Formula 1 increased by 5.82-fold. **** indicates p<0.0001.

[0064] The compound of formula 1 has a protective effect on retinal ganglion cells in mice with optic nerve clamp injury model

[0065] Compared with the control group, DMSO had no effect on the number of surviving RGCs in the optic nerve clamp injury model mice (p>0.05). Therefore, DMSO has no protective effect on the retinal ganglion cells of the mice with optic nerve clamp injury model. However, the compound of formula 1 provided by the present invention can increase the survival rate of RGCs in the experimental group model mice. The compound of formula 1 can be used as a therapeutic drug to protect the eyes with optic nerve damage.

[0066] The experimental results are as follows Figure 3 :

[0067] The compound of Formula 1 has a protective effect on retinal ganglion cells in mice subjected to an optic nerve clamp injury model. A. Surviving RGCs were assessed on retinal flat mounts by immunofluorescence staining against TUJ1. Scale bar, 50 μm. B. Quantification of surviving RGCs two weeks after optic nerve clamp (PBS: n=15; Formula 1: n=4). Surviving RGCs increased by 49% in mice treated with the compound of Formula 1. *** indicates p<0.0001.

Claims

1. Use of an optic nerve protective agent in the preparation of a drug for treating optic nerve damage, characterized in that: The optic nerve protective agent is used as an optic nerve protective active ingredient for preparing a drug for treating optic nerve damage, wherein the optic nerve protective agent is a compound having the structure of Formula 1 and a pharmaceutically acceptable crystal thereof; 2. The use according to claim 1, characterized in that The optic nerve protective agent is used as an optic nerve protecting active ingredient to prepare a drug for treating optic nerve damage that can improve the survival rate and / or regeneration ability of RGCs cells.

3. The use according to claim 1, characterized in that The optic nerve protective agent is used as an optic nerve protective active ingredient to prepare a medicine for treating optic nerve damage caused by at least one of glaucoma, trauma, infection, tumor compression and genetic factors.

4. The use according to claim 1, wherein The content of the optic nerve protective agent of Formula 1 in the optic nerve protective active ingredient is above 50 wt.%, preferably above 80 wt.%.

5. The use according to any one of claims 1 to 4, characterized in that The medicine is an eyeball injection preparation.

6. A drug for treating optic nerve damage, characterized in that: The invention comprises a pharmaceutically effective amount of an optic nerve protective active ingredient, wherein the optic nerve protective active ingredient comprises 50 wt.% or more, preferably 80 wt.% or more of the optic nerve protective agent according to claim 1.

7. The drug for treating optic nerve damage according to claim 6, wherein The invention consists of the optic nerve protective agent and a diluent.

8. The drug for treating optic nerve damage according to claim 7, wherein The diluent includes at least one of DMSO, water, and PBS buffer.

9. The drug for treating optic nerve damage according to any one of claims 6 to 8, characterized in that Preparations for eye injection.

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