Application of RGCs activity protection component in preparation of medicine for preventing and / or treating optic nerve injury

Abstract

The invention belongs to the field of medicines, and particularly relates to application of an RGCs activity protection component in preparation of a medicine for preventing and / or treating optic nerve injury, and the RGCs activity protection component is a compound with a structure # imgabs0 # as shown in a formula 1 and a crystal of the compound. In the formula 1, R1 to R3 are independently H or alkyl groups of C1 to C3. Researches show that the formula 1 can be used as an RGCs activity protection component, can effectively promote regeneration of injured optic nerves and improve the survival rate of optic ganglion cells after optic nerve injury, and is beneficial to prevention and / or treatment of optic nerve injury diseases caused by various causes.

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] Retinal ganglion cells (RGCs), which emit 1.2 million unmyelinated nerve axons at the posterior pole of the eye, are responsible for transmitting visual information received by the eye to the brain, allowing us to see the world around us. However, RGCs are susceptible to damage caused by trauma, elevated intraocular pressure, ischemia, optic neuritis, infection, tumor compression, and genetic factors.

[0003] When the optic nerve is damaged, axoplasmic flow—the transport of substances within the optic nerve—may be impaired. This can lead to the death of retinal ganglion cells and degeneration of optic nerve axons, ultimately affecting vision. Furthermore, once the optic nerve is damaged, it is difficult to repair itself, which means that if left untreated, we may permanently lose some or all of our vision.

[0004] Existing treatments for optic nerve damage include nutritional support therapy, neurotrophic drug therapy such as taking methylcobalamin, and visual function rehabilitation training, such as a series of activities aimed at improving visual clarity, contrast sensitivity, and visual field range. For another example, publication number CN114870023A discloses a sustained-release optic nerve protective drug nanosynthetic material, its preparation method, and application. The synthetic material is prepared with 7,8-dihydroxyflavone as the main body and lactic acid-polyglycolic acid copolymer as a sustained-release carrier. For another example, the Chinese patent document with publication number CN108853125A discloses a compound for preventing and / or treating optic nerve damage, its preparation method, and application, and specifically discloses the optic nerve damage protective effect of Centella asiatica.

[0005] In summary, although there are many methods, clinical studies have shown that their effects are limited, and different people have individual differences and different conditions. Therefore, there is still a lack of effective treatment methods, especially for glaucoma, a typical optic nerve damage disease. Therefore, we need to find drugs that can prevent retinal ganglion cell death and axonal degeneration, and promote axonal regeneration. Summary of the Invention

[0006] The first purpose of the present invention is to provide a novel RGCs active protective component for use in optic nerve damage protective drugs.

[0007] The second purpose of the present invention is to provide a protective drug for treating optic nerve damage using the RGCs active protective component.

[0008] A use of an RGCs active protective component in the preparation of a drug for preventing and / or treating optic nerve damage, wherein the RGCs active protective component is a compound having a structure of Formula 1 and a crystal thereof;

[0009]

[0010] Formula 1;

[0011] The R1 to R3 are independently H or C1 to C3 alkyl.

[0012] The present invention studies have shown that Formula 1 can be used as an active protective component for RGCs, can effectively promote the regeneration of damaged optic nerves, improve the survival rate of retinal ganglion cells after optic nerve damage, and is helpful for preventing and / or treating optic nerve damage diseases caused by various causes.

[0013] In Formula 1 of the present invention, R1 is a C1-C3 alkyl group, and R2 and R3 are H.

[0014] The application of the present invention is that the medicine is a medicine that can prevent and / or treat optic nerve damage caused by at least one of glaucoma, trauma, infection, tumor compression, and genetic factors.

[0015] The application of the present invention combines the RGCs active protective component with a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable medicine.

[0016] In the application of the present invention, the medicine is an ophthalmic medicine.

[0017] In the application of the present invention, the medicine is an eyeball injection medicine.

[0018] The present invention also provides an ophthalmic drug for preventing and / or treating optic nerve damage, comprising a pharmaceutically effective amount of the RGCs active protective component.

[0019] The ophthalmic medicine of the present invention further contains pharmaceutically acceptable excipients.

[0020] The ophthalmic medicine of the present invention is an eyeball injection medicine.

[0021] The ophthalmic medicine of the present invention is a powder injection or solution for eyeball injection.

[0022] Beneficial effects

[0023] The present invention studies have shown that Formula 1 can be used as an active protective component for RGCs, can effectively promote the regeneration of damaged optic nerves, improve the survival rate of retinal ganglion cells after optic nerve damage, and is helpful for preventing and / or treating optic nerve damage diseases caused by various causes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Formula 1-1 and comparative formula A have a protective effect on RGCs cultured in vitro.

[0025] Figure 2 : Formula 1-1 can effectively promote optic nerve regeneration in mice with optic nerve clamp injury model.

[0026] Figure 3 Formula 1-1 has a protective effect on retinal ganglion cells in mice with optic nerve clamp injury model. DETAILED DESCRIPTION

[0027] In the present invention, the active ingredient of formula 1-1 can be listed, which is a compound of formula 1 wherein R1 is methyl, R2 and R3 are H, and the result is:

[0028]

[0029] Formula 1-1

[0030] Example 1

[0031] 1.1 Formula 1-1 has a protective effect on the survival of RGCs cultured in vitro

[0032] 1.1.1 Experimental steps

[0033] The RGC-specifically labeled vglut2-tdTomato 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.

[0034] Place the retina in a preheated digestion solution at 37°C (6ml L-15CO3, 150ul papain, 28ul 1% BSA, 3mg 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 10ml of newly prepared L-15CO3 to the tube to wash the retina. Use a glass Pasteur pipette to blow it dozens of times. After it stands for 30 seconds, aspirate the upper layer of liquid into a new 50ml 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.

[0035] Use culture medium (Leibovitz's L-15 Medium + 1% penicillin-streptomycin

[0036] +2% B27+1% N2) and dilute the resuspended cell solution to a density of 1.5×10 6 A total of 6.5 ml of cell suspension was prepared at 400 μg / ml. 150 μL of cell suspension was taken from each well and inoculated into a 96-well plate, with a total of 6 wells inoculated.

[0037] The experimental group selected 20uM concentration of formula 1-1

[0038] Control group: using the same concentration of comparative formula A Replace the above formula 1-1.

[0039] After seeding, the cells were cultured in a 37°C, 5% CO2 incubator for 3 days.

[0040] 1.1.2 Data Analysis

[0041] 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.

[0042] 1.1.3 Results

[0043] The results showed that compared with the control group, the concentrations of Formula 1-1 had a protective effect on the survival of RGCs cultured in vitro.

[0044] The experimental results are as follows Figure 1 :

[0045] A. The number of surviving specifically labeled RGCs cells cultured in vitro and treated with Dmso, comparative formula A-10uM, comparative formula A-50uM and formula 1-1-20uM was detected under an inverted fluorescence microscope. The scale bar is 50um.

[0046] B. Quantitative results of the number of surviving RGCs after 3 days of in vitro culture (Dmso: n=3; Comparative Formula A-10uM: n=3; Comparative Formula A-50uM: n=3; Formula 1-1-20uM: n=3).

[0047] The results showed that there was no significant difference in the number of surviving RGCs at either concentration of Comparative Formula A compared to the Dmso group. However, 20 μM of Formula 1-1 provided by the present invention increased the number of surviving RGCs by 1.51 times compared to Dmso, and by 1.71 and 1.88 times, respectively, compared to the similarly structured Comparative Formula A at concentrations of 10 μM and 50 μM. * indicates p < 0.05, ** indicates p < 0.01, and ns indicates p > 0.05.

[0048] Example 2

[0049] 1.2 Formula 1-1 can improve optic nerve regeneration and retinal ganglion cell survival in mice with optic nerve clamp injury model

[0050] 1.2.1 Experimental steps

[0051] 1.2.1.1 Establishment of the Optic Nerve Crush (ONC) Model

[0052] 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.

[0053] 1.2.1.2 Grouping and Dosing

[0054] Mice undergoing optic nerve clamp injury were randomly divided into three groups (n represents the number of optic nerves or retinas), using formula 1-1: n = 10 / 8 (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.

[0055] Formula 1-1: 600 μM (prepared with PBS containing 0.2% DMSO) of Formula 1-1 was injected into the vitreous of the mouse eyeball on day 0 (after model establishment) and day 5, respectively;

[0056] PBS: Phosphate buffered saline (PBS) was injected into the vitreous of the mouse eyes on day 0 (after model establishment) and day 5, respectively;

[0057] DMSO: DMSO was injected into the vitreous of the mouse eyes on day 0 (after model establishment) and day 5 respectively;

[0058] After weighing, mice were anesthetized with 1% sodium pentobarbital injected intraperitoneally at a dose of 10 ml / kg. After anesthesia, topical anesthesia was administered with oxybuprocaine hydrochloride eye drops, followed by dilation of the pupil with compound tropicamide eye drops. The mice were positioned appropriately under a microscope, and a needle was used to puncture the sclera near the limbus and anteriorly at the superior temporal border of the eyeball (to reduce intraocular pressure). Using a pre-assembled 5 μl glass microneedle syringe (first withdraw 1 μl of air, then withdraw 3 μl of formula 1-1), the needle was carefully inserted through the limbus hole into the vitreous cavity at a 60-degree angle to the optic nerve (taking care to avoid damaging the lens). After a 15-second pause, the needle was slowly removed, the eyeball repositioned, and tobramycin-dexamethasone ointment was applied to the ocular surface. This was repeated on the contralateral side. After completion, the mice were placed on a heated workbench and, upon awakening, placed in their cages. A control group of mice was injected with the same volume of PBS, following the same procedure as above.

[0059] 1.2.1.3 Optic Nerve and Retina Removal

[0060] 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.

[0061] 1.2.1.4 Processing the Optic Nerve

[0062] 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.

[0063] 1.2.1.5 Processing the retina

[0064] Place the fixed eyeball in a culture dish containing PBS solution, cut the cornea along the corneoscleral margin with ophthalmic scissors, remove the lens, and bluntly peel off the choroid on the outer layer of the retina with toothless forceps. Then clean the vitreous body in the eyeball with forceps, and use ophthalmic scissors to make four evenly spaced 2-3 mm incisions from the edge of the retina toward the optic disc to divide the retina into four quadrants.

[0065] 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.

[0066] 1.2.2 Data Processing

[0067] 1.2.2.1 Optic nerve

[0068] 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.

[0069] 1.2.2.2 Retina

[0070] 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.

[0071] 1.2.3 Experimental Results

[0072] 1.2.3.1 Formula 1-1 promotes optic nerve regeneration in mice with optic nerve clamp injury

[0073] 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 formula 1-1 provided by the present invention can promote the regeneration of the optic nerve in the experimental group model mice, and formula 1-1 can be used as a therapeutic drug to protect eyes with optic nerve damage.

[0074] The experimental results are as follows Figure 2 :

[0075] A. Axon regeneration in optic nerve sections was observed by 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 1-1: n=10). Results showed that the number of regenerated axons in the optic nerve of mice treated with Formula 1-1 increased by 3.44-fold. **** indicates p<0.0001.

[0076] 1.2.3.2 Formula 1-1 has a protective effect on retinal ganglion cells in mice with optic nerve clamp injury model

[0077] 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, Formula 1-1 provided by the present invention can increase the survival rate of RGCs in the experimental group model mice. Formula 1-1 can be used as a therapeutic drug to protect eyes with optic nerve damage.

[0078] The experimental results are as follows Figure 3 :

[0079] A. Surviving RGCs were assessed on retinal flat mounts using immunofluorescence staining for TUJ1. Scale bar, 50 μm. B. Quantification of surviving RGCs two weeks after optic nerve clamping (PBS: n=15; Formula 1-1: n=8). Surviving RGCs increased 1.46-fold in mice treated with Formula 1-1. **** indicates p<0.0001.

Claims

1. Use of an RGCs active protective component in the preparation of a drug for preventing and / or treating optic nerve damage, characterized in that: The RGCs active protective component is a compound having the structure of Formula 1 and its crystal; The R1 to R3 are independently H or C1 to C3 alkyl.

2. The use according to claim 1, characterized in that The R1 is a C1-C3 alkyl group, and the R2 and R3 are H.

3. The use according to claim 1 or 2, characterized in that The medicine is a medicine capable of preventing and / or 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 RGCs active protective component is combined with a pharmaceutically acceptable excipient to prepare a pharmaceutically acceptable drug.

5. The use according to claim 4, characterized in that The medicine is an ophthalmic medicine.

6. The use according to claim 5, characterized in that The medicine is an eyeball injection medicine.

7. An ophthalmic drug for preventing and / or treating optic nerve damage, characterized in that: The invention comprises a pharmaceutically effective amount of the RGCs active protective component described in the application according to claims 1 to 6.

8. The ophthalmic drug according to claim 7, wherein It also contains pharmaceutically acceptable excipients.

9. The ophthalmic drug according to claim 8, wherein Injecting medication into the eye.

10. The ophthalmic drug according to claim 9, wherein It is a powder injection or solution for eye injection.

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