Use of caffeic acid phenethyl ester in preparing medicine for treating myopia and pharmaceutical composition thereof

Abstract

The present invention belongs to the field of biomedicine and specifically relates to the use of caffeic acid phenethyl ester in the preparation of a drug for treating myopia and a pharmaceutical composition thereof. The present invention discovers for the first time that caffeic acid phenethyl ester can promote collagen synthesis in scleral fibroblasts, which are directly related to myopia, inhibit and slow axial elongation and increase in vitreous cavity length in myopic individuals, and increase scleral thickness, thereby delaying and treating myopia. This invention provides a new drug option for the clinical treatment of myopia and has promising application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to use of caffeic acid phenethyl ester in preparing a medicine for treating myopia and a pharmaceutical composition thereof. Background Art

[0002] Myopia is currently the most common eye disease, leading to visual impairment and blinding complications, and has become a major socioeconomic and public health problem worldwide. Its global prevalence is estimated to have increased from 28.3% to 34.0% between 2010 and 2020. A recent official report in my country indicates that the overall prevalence of myopia among primary school, middle school, and high school students is 36.0%, 71.6%, and 81.0%, respectively. Among urban Chinese children, the prevalence of myopia increases from 5.7% at 5 years old, 30.1% at 10 years old, to 78.4% at 15 years old, with a consistent upward trend. A study of 870,000 patients (aged 4-18 years) found a significant increase in the prevalence of myopia among children aged 4-6 years over the 16-year period from 2005 to 2021 (19.0% in 2005 vs. 51.1% in 2021). Myopia is developing at an earlier age and at a younger average age. Early onset of myopia may increase the likelihood of future vision impairment. The National Health and Nutrition Examination Survey in the United States found that the direct cost of correcting vision impairment caused by refractive error ranges from $3.9 billion to $7.2 billion annually. Myopia not only impairs vision-related quality of life but also increases the difficulty of vision-related tasks.

[0003] Myopia refers to the mismatch between refractive power and axial length of the eye, which converges in front of the retina to form a focus, causing distant objects to be blurred. The cause of myopia is still unclear, and scholars have proposed many hypotheses, mainly focusing on the environment and genetics. In an analysis combining the amount of outdoor activity and the amount of close work activity, children with short outdoor time and close work are two to three times more likely to develop myopia than children with low-distance work and high outdoor activity; similarly, some genetic factors can affect the composition of the sclera, for example, genes related to high myopia - TGF-β ; It has been confirmed in animal experimental myopia studies TGF-β In the scleral expansion caused by myopia, it can affect the growth, differentiation and migration of cells and is one of the cytokines that cause pathological changes in myopia tissues. Studies have also shown that TGF-βIt is possible that the growth process of the sclera is regulated by synthesizing and secreting myopia signaling molecules through retinal cells. Axial myopia is one of the common types of myopia, which is caused by the continuous remodeling of the extracellular matrix (ECM) of the sclera, resulting in decreased tissue hardness and increased elasticity. Myopia causes the sclera and choroid in the eyeball to become thinner. The sclera is mainly composed of collagen fibers, of which type I collagen fibers account for 50-70%, which play a supporting and protective role for the eye. Myopia can lead to a decrease in type I collagen fibers in the sclera. Studies have shown that matrix metalloproteinases (MMPs) promote extracellular matrix remodeling by degrading collagen-1 in the sclera. In a form deprivation myopia model, the lack of MMP-2 can reduce myopia progression by 59% compared with the control group.

[0004] Common clinical treatments include wearing glasses, surgery, and medication. Wearing glasses is not a non-curative method and cannot effectively prevent the occurrence of its complications. Surgery has its indications and is not suitable for everyone. There are not many drugs for treating myopia. Atropine, a common drug in my country, is a type of acetylcholine M receptor blocker that can slow the progression of myopia in children. At a dose of 0.1%-1.0%, the therapeutic effect is dose-dependent. However, as a ciliary muscle paralytic and mydriatic drug, atropine can cause photophobia, blurred near vision, loss of accommodation ability, and excessive pupil dilation, leading to inconvenience in life, and the effects vary greatly from person to person. Therefore, it is very necessary to seek new alternative drugs for the treatment of myopia.

[0005] Caffeic acid phenethyl ester (CAPE) has multiple pharmacological effects such as antioxidant, anti-inflammatory, antiviral and anti-tumor. It is mainly used in the treatment of cardiovascular diseases and tumors. It also has a protective effect on retinal damage. However, there are no reports on the use of caffeic acid phenethyl ester in the treatment of myopia. Summary of the Invention

[0006] In response to the problems of the prior art, the present invention provides use of caffeic acid phenethyl ester in preparing a drug for treating myopia and a pharmaceutical composition thereof.

[0007] Use of caffeic acid phenethyl ester or its optical isomers, or its racemates, or its solvates, or its pharmaceutically acceptable salts, or its prodrugs, or its metabolites, or its analogs, or its derivatives, or its crystalline compounds in the preparation of drugs for treating and / or preventing myopia.

[0008] Preferably, the myopia is at least one of low myopia, moderate myopia, and high myopia.

[0009] Preferably, the drug can promote the synthesis of collagen.

[0010] Preferably, the collagen is at least one of type I collagen, type III collagen, and type VI collagen.

[0011] Preferably, the drug can inhibit the elongation of the eyeball axis.

[0012] Preferably, the drug can inhibit the increase in vitreous cavity length.

[0013] Preferably, the drug is capable of increasing scleral thickness.

[0014] The present invention also provides a pharmaceutical composition for treating myopia, which is a preparation prepared by using caffeic acid phenethyl ester or its optical isomers, or its racemates, or its solvates, or its pharmaceutically acceptable salts, or its prodrugs, or its metabolites, or its analogs, or its derivatives, or its crystalline compounds as active ingredients, and adding pharmaceutically acceptable excipients.

[0015] Preferably, the preparation is an ophthalmic preparation, an oral preparation, an injectable preparation, or a skin and mucosal preparation.

[0016] Preferably, the ophthalmic administration preparation is at least one of eye drops, eye ointments, eye sprays, eye gels, eye patches, implants, eye microspheres, eye sustained-release preparations, and eye injections;

[0017] The oral administration preparation is at least one of a solution, a syrup, a granule, a capsule, a powder, a pill, and a tablet;

[0018] The injectable preparation is at least one of an injection and a lyophilized powder injection;

[0019] The skin and mucosal administration preparation is at least one of an aerosol, a liniment, a lotion, a cream, an ointment, and a patch.

[0020] The low myopia refers to a diopter of less than or equal to 300 degrees; the moderate myopia refers to a diopter of between 300 degrees and 600 degrees; and the high myopia refers to a diopter of more than 600 degrees.

[0021] The caffeic acid phenethyl ester of the present invention can promote the synthesis of myopia-related collagen, inhibit and slow down the elongation of the eye axis and the increase in the length of the vitreous cavity in myopic individuals, and increase the thickness of the sclera, thereby achieving the effect of delaying and treating myopia. It provides a new choice for drugs for clinical treatment of myopia and has good application prospects.

[0022] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0023] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The graph shows the results of CCK-8 measurement of the cytotoxic effect of different concentrations of CAPE.

[0025] Figure 2 Effects of CAPE on COL1A1 expression in HFSF cells. (A) Immunofluorescence staining of COL1A1 and F-actin expression in cells treated with atropine and CAPE; (B) Western blot analysis of COL1A1 protein expression in cells treated with different concentrations of CAPE; (C) Statistical analysis of COL1A1 protein expression in cells treated with different concentrations of CAPE.

[0026] Figure 3 Figure 3. Effects of CAPE on the refractive power, axial length, vitreous length, and scleral thickness of myopic mice. (A) Statistical results of refractive measurement in the WT and FDM groups before FDM myopia model establishment; (B) Statistical results of refractive measurement in the WT and FDM groups after FDM myopia model establishment; (C) Statistical results of refractive power measurement in myopic mice under different treatment groups; (D) Statistical results of axial length measurement in myopic mice under different treatment groups; (E) Statistical results of vitreous length measurement in myopic mice under different treatment groups; (F) Statistical results of scleral thickness measurement in myopic mice under different treatment groups. DETAILED DESCRIPTION

[0027] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available.

[0028] Example 1 In vitro investigation of the therapeutic effect of caffeic acid phenethyl ester on myopia

[0029] 1. Experimental Methods

[0030] 1. Cell culture

[0031] Human scleral fibroblasts (HFSF) were purchased from Procell Life Science & Technology Co., Wuhan, China, and cultured in a fibroblast-specific culture medium (CM-H242, Procell Life Science & Technology Co., Ltd.) containing 10% fetal bovine serum, penicillin (100 U / mL), and streptomycin (100 µg / mL). Cells were cultured at 37°C in a 5% CO2 incubator.

[0032] 2. CCK8 cytotoxicity assay

[0033] HFSF cells were seeded at 5,000 cells / well in a 96-well culture plate and placed in an incubator overnight. The cells were starved for 4 hours in serum-free medium. Caffeic acid phenethyl ester (CAPE) was then added to the cells at varying concentrations (10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM) for 24 and 48 hours. CCK-8 reagent was then added to each well and incubated for 2 hours. The absorbance was measured at 450 nm, and the proliferation rate was calculated.

[0034] 3. Western blot detection

[0035] (1) HFSF cells were cultured at a rate of 2×10 5 Cells were seeded at a density of 100 cells / well in six-well culture plates and cultured overnight in an incubator. Cells were starved for 4 hours in serum-free DMEM high-glucose medium and then treated with different concentrations of caffeic acid phenethyl ester (5 μM, 10 μM, and 20 μM) for 24 hours.

[0036] (2) Collect protein samples: discard the culture medium in the culture plate, wash twice with 1×PBS, and 6 Each cell pellet was added with 200 μL of pre-chilled RIPA lysis buffer (1× PBS, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, 100 μg / mL PMSF), the cells were evenly dispersed, lysed on ice for 30 min, centrifuged (10,000 rpm, 10 min), and the supernatant was collected. The protein concentration was determined using a BCA protein concentration assay kit. A standard curve of protein concentration and OD value was drawn using Excel to calculate the protein concentration of the sample.

[0037] (3) Electrophoresis: Add an appropriate amount of concentrated SDS-PAGE protein loading buffer to the collected protein sample and heat in a boiling water bath for 5 min. After cooling to room temperature, load the protein sample into the SDS-PAGE gel loading well. Electrophoresis is performed at 100 V for 3 h.

[0038] (4) Transfer: Immerse the PVDF membrane of the same size as the gel in electrophoresis buffer and electrotransfer buffer, soak for 5 minutes and balance for 15 minutes respectively. Transfer the separated proteins to the nitrocellulose membrane at 350 mA, 4°C, for 120 minutes. Use the sandwich method to place the sponge, filter paper, gel, nitrocellulose membrane, filter paper, and sponge in order and fix them with a sieve plate. Insert the gel into the electrophoresis tank facing the cathode, pour in transfer buffer, and transfer at 200 mA constant voltage for 2 hours. Stain the gel with Coomassie Brilliant Blue. After decolorization with destaining solution, identify the transfer effect.

[0039] (5) Blocking: Place the protein membrane in Western washing solution, rinse for 2 minutes, and absorb the washing solution: Place the nitrocellulose membrane in 5% skimmed milk powder, place it on a shaker and shake it slowly, and block it at room temperature for 60 minutes.

[0040] (6) Incubation with primary antibody: Aspirate the blocking solution, add the diluted primary antibody (COL1A1 diluted 1:1000; GAPDH diluted 1:5000), place on a side-swing shaker and shake slowly, incubate at 4°C for 12 hours, recover the primary antibody, add TBST washing solution, shake slowly and wash once every 10 minutes.

[0041] Given that COL1A1 is one of the most authoritative and recognized scleral marker proteins in the progression of myopia (see PMIDs: 22690110 and 32652319), and is also commonly used as a detection marker in the development of existing myopia-related therapeutic drugs and viruses (see PMID: 36723926), this patented test primarily uses this protein as a detection indicator.

[0042] (7) Incubation with secondary antibody: Add horseradish peroxidase-labeled secondary antibody, place on a side-swing shaker and shake slowly, incubate at room temperature for 1 hour; recover the secondary antibody, add TBST washing solution, shake slowly and wash once every 5 minutes.

[0043] (8) Add horseradish peroxidase exposure solution and perform exposure.

[0044] 4. Immunofluorescence staining

[0045] HFSF cells were seeded on slides and placed in an incubator overnight. They were then starved for 4 hours before drug addition. After treatment with 250 μM atropine and 20 μM caffeic acid phenethyl ester (CAPE), the cells were fixed with paraformaldehyde for 20 minutes, blocked at room temperature for 2 hours, and incubated with primary antibodies overnight at 4°C. The cells were then incubated with fluorescently labeled secondary antibodies for 2 hours at 37°C, and fluorescence images were captured and analyzed using a fluorescence microscope.

[0046] 2. Experimental Results

[0047] The CCK-8 method was used to detect the cytotoxicity of HFSF cells at different concentrations of caffeic acid phenethyl ester (CAPE) at 24h and 48h. Figure 1 As shown, CAPE had no toxic effect on HFSF cells at concentrations of 10 μM, 20 μM, 40 μM, and 80 μM; CAPE had toxic effects on HFSF cells at concentrations of 160 μM and 320 μM.

[0048] The sclera and cornea contain a large amount of collagen and collagen fibers. The synthesis of collagen and the arrangement of collagen fibers can directly control the growth of the eye axis. In patients with high myopia, the sclera becomes thinner and type I collagen decreases. By detecting the expression level of α-1 collagen (COL1A1), the therapeutic effect of CAPE on myopia was investigated. First, the expression of COL1A1 and F-actin was detected by immunofluorescence staining. The results are as follows: Figure 2 As shown in (A), CAPE treatment of HFSF cells significantly increased the expression of COL1A1 (green fluorescence for COL1A1, red fluorescence for F-actin); at the same time, compared with the positive control group (atropine group), the green fluorescence in the CAPE group was higher, indicating that the effect of CAPE in promoting COL1A1 protein synthesis in HFSF cells is comparable to that of atropine. Figure 2 As shown in (B, C), CAPE at both 10 μM and 20 μM significantly increased the expression level of COL1A1 in a dose-dependent manner. These results suggest that CAPE can promote the expression of COL1A1 in HFSF cells, thereby promoting the synthesis of type I collagen in the sclera and treating myopia.

[0049] Example 2 In vivo investigation of the therapeutic effect of caffeic acid phenethyl ester on myopia

[0050] 1. Experimental Methods

[0051] This example tests the effect of CAPE on the visual acuity of myopia model mice. Methods: CAPE tests the refractive error of the myopia model mice; CAPE tests the axial length of the eyeballs of the myopia model mice; CAPE tests the vitreous length of the eyeballs of the myopia model mice; CAPE tests the scleral thickness of the eyeballs of the myopia model mice. The specific experimental procedures are as follows:

[0052] 1. Construction and drug administration of form deprivation myopia model (FDM myopia model)

[0053] Three-week-old, inbred, SPF-grade, healthy male C57BL / 6 mice were used. Their necks were measured, and collars were cut using 3M tape to prevent them from scratching their eyes. Based on the size of the mouse's eye area, a translucent eye mask of the same size, approximately 8 mm in diameter, and with a 1 mm edge was made from a translucent plastic sheet to induce form deprivation. The mice's eyes were examined twice daily, morning and evening, for abnormal secretions, infection, and any detachment of the eye mask to ensure that the experimental eye remained in a form deprived state. Three and four weeks after induction (i.e., six and seven weeks after birth), the mice's refractive power and axial length were measured, and myopic mice were selected to establish the FDM myopia model.

[0054] The experiment involved four groups of five mice each: 0.9% saline (WT), FDM-0.9% saline, FDM-CAPE, and FDM-atropine. CAPE was prepared in saline to a final concentration of 0.2 mM, pH 7.0-7.6; atropine was prepared in saline to a concentration of 0.01%. Drugs were administered twice daily, morning and evening, with 4 μL of drug dripped into each eye. One hour after drug instillation, the mice were collared and fed normally, and their eyes were rinsed with saline.

[0055] 2. Mouse refraction measurement

[0056] Mouse refractive error was measured using an infrared autorefractor. Mice were acclimated to a darkened room before measurement and the refractive error was measured according to the instrument's operating instructions. A small animal infrared keratometer was used to measure the corneal radius and curvature of mice. This keratometer automatically and conveniently measures the corneal radius of conscious animals by analyzing the reflection points of eight infrared LEDs on the corneal surface.

[0057] 3. Axial length measurement

[0058] The axial length of each mouse was measured using an OCT small animal ophthalmic biometrics system (VisonX, AOCT-1000M, Shanghai Aikonte Medical Technology Co., Ltd.). During scanning, the optical axis of the incident light passed perpendicularly through the corneal apex. Real-time images were displayed on vertical and horizontal image monitoring screens, and researchers ensured that the iris was horizontally aligned with reference lines on both screens. Each eye was measured three times, and biometrics were manually completed by a single researcher after an image was captured for each measurement. The raw OCT images were used to calculate corrected geometric lengths based on optical path length, including central corneal thickness, anterior chamber depth, lens thickness, vitreous cavity depth, and axial length (AL).

[0059] 2. Experimental Results

[0060] The statistical results of refractive measurement before and after modeling are as follows: Figure 3As shown in (A, B), the refractive power of mice in the FDM group was significantly reduced, indicating that the FDM myopia model was successfully established; the physiological indicators of myopia in FDM mice after 15 days of CAPE treatment, as shown in Figure 3 As shown in (C), compared with the refractive index of the FDM group, the refractive index of the FDM-atropine and FDM-CAPE groups recovered, indicating that CAPE treatment has a control effect on myopia; Figure 3 As shown in (D, E), the axial length and vitreous length of the FDM group increased significantly, while the axial length and vitreous length of the FDM-atropine and FDM-CAPE groups recovered, indicating that CAPE treatment has an inhibitory effect on the reduction of refractive power and axial length growth in myopic mice. Figure 3 As shown in (F), compared with the scleral thickness of the FDM group, the scleral thickness of the FDM-atropine and FDM-CAPE groups was restored, indicating that CAPE treatment can increase scleral thickness.

[0061] In summary, the caffeic acid phenethyl ester of the present invention can promote myopia-related collagen synthesis, inhibit and slow down the elongation of the eye axis and the increase in vitreous cavity length in myopic individuals, and increase the thickness of the sclera, thereby achieving the effect of delaying and treating myopia. It provides a new choice for clinical drugs for the treatment of myopia and has good application prospects.

Claims

1. Use of caffeic acid phenethyl ester or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating and / or preventing myopia.

2. The use according to claim 1, characterized in that: The myopia is at least one of low myopia, moderate myopia, and high myopia.

3. The use according to claim 1, characterized in that: The drug can inhibit the elongation of the eyeball's axial length.

4. The use according to claim 1, characterized in that: The drug can increase scleral thickness.

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