Application of Capilliposide A in preparation of medicine for preventing and treating age-related macular degeneration

Abstract

The invention discloses an application of Capilliposide A in preparation of a medicine for preventing and treating age-related macular degeneration. The Capilliposide A can be distributed to the retina through eye administration, mouse retina injury caused by sodium iodate modeling is improved, apoptosis of human retinal pigment epithelial cells (ARPE-19) and ROS accumulation can be inhibited, and the effect of treating and preventing age-related macular degeneration is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the effect of a pharmaceutical composition containing Capilliposide A as an active ingredient in treating and preventing age-related macular degeneration. Background Art

[0002] Age-related macular degeneration (AMD) is an eye disease that seriously threatens daily activities such as reading, driving and facial recognition for people over 55 years old. It is characterized by progressive damage to central vision and can lead to blindness in severe cases. However, there is currently no cure for this eye disease, which poses a huge risk to public health, and the cause and pathogenesis of AMD are not yet fully understood. It has been found to be related to factors such as aging, smoking, arteriosclerosis, oxidative stress, obesity, hypertension, hypercholesterolemia and high-fat diet. AMD can be divided into two types: wet AMD and dry AMD. Wet (exudative) AMD is a series of pathological changes caused by the invasion of new blood vessels from the choroid into the retina, and anti-vascular endothelial growth factor (VEGF) has a significant therapeutic effect on it; while dry (atrophic) AMD is characterized by geographic atrophy (GA) of the retina, characterized by irreversible loss of RPE cells, photoreceptors (PR) and choroidal capillaries.

[0003] RPE is a pigment epithelial cell located between the choroid and the neuroepithelium. Under normal circumstances, RPE cells maintain the homeostasis of the retina by exchanging substances (including nutrients and waste) between the choroid and the retinal photoreceptor cell layer, absorbing and reflecting light, and participating in the phagocytosis of the photoreceptor outer segment membrane. When the retina is in a high oxygen environment, the content of reactive oxygen species (ROS) increases significantly, causing RPE cells to be induced by oxidative stress of ROS, resulting in cell death, which in turn promotes the disease progression of dry AMD.

[0004] Capilliposide A, also known as capilliposide A, is a triterpenoid saponin of the oleanane family. Its Cas number is 897936-98-0. The compound structure is as follows:

[0005]

[0006] Patent CN202111683546.8 discloses the effects of Capilliposide A in treating and preventing proliferative vitreoretinopathy and proliferative diabetic retinopathy; Patent CN202210357402.1 discloses the use of Capilliposide A and its homologues in the preparation of drugs for treating rheumatoid arthritis. There is no relevant research on the therapeutic effect of Capilliposide A on dry AMD. Summary of the invention

[0007] The purpose of the present invention is to expand the use of Capilliposide A and provide an application of Capilliposide A in preparing a drug for preventing and treating age-related macular degeneration.

[0008] The present invention is implemented by the following technical solutions:

[0009] In a first aspect, the present invention provides the use of the above-mentioned Capilliposide A in the preparation of a drug for preventing and treating age-related macular degeneration.

[0010] As a preferred embodiment of the first aspect, the mechanism of action of the application is to improve retinal damage, inhibit apoptosis of retinal pigment epithelial cells and accumulation of ROS, thereby treating or preventing age-related macular degeneration.

[0011] In a second aspect, the present invention provides a pharmaceutical composition for treating and preventing age-related macular degeneration, wherein the active ingredient in the pharmaceutical composition is the above-mentioned Capilliposide A.

[0012] As a preference of the above second aspect, the pharmaceutical composition consists of the active ingredient and one or more pharmaceutically acceptable excipients.

[0013] As a preferred embodiment of the second aspect, the dosage form of the pharmaceutical composition is granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions.

[0014] As a preferred embodiment of the second aspect, the pharmaceutical composition is a solution prepared by the active ingredient and a liquid pharmaceutically acceptable carrier, and the liquid pharmaceutically acceptable carrier is one or more of saline, sterile water, Ringer's solution, buffered saline, glycerol, and ethanol.

[0015] As a preferred embodiment of the second aspect, the pharmaceutical composition further comprises one or more of an antioxidant, a buffer, an antibacterial agent, and a thickener.

[0016] In a third aspect, the present invention provides an eye drop for preventing and treating age-related macular degeneration, which contains the above-mentioned Capilliposide A as an active ingredient.

[0017] As a preferred embodiment of the third aspect, the composition is prepared from Capilliposide A, a thickener and physiological saline.

[0018] Furthermore, the thickener is preferably polyvinyl alcohol, and its mass percentage in the eye drops is preferably 0.01%.

[0019] Furthermore, the mass percentage of Capilliposide A in the eye drops is 0.01% to 0.05%.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] The present invention provides a new use of Capilliposide A, which can be used to improve retinal damage, inhibit apoptosis of human retinal pigment epithelial cells and the accumulation of ROS, thereby treating and improving age-related macular degeneration. Relevant experimental results show that Capilliposide A can be distributed to the retina after ocular administration, improve retinal damage in mice caused by sodium iodate modeling, and can also inhibit apoptosis of human retinal pigment epithelial cells (ARPE-19) and the accumulation of ROS, and has the effect of treating and preventing age-related macular degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The H&E staining photographs of the retina after administration of Capilliposide A to mice with sodium iodate-induced retinal damage;

[0023] Figure 2 The data are for the thickness of the retinal pigment epithelium after administration of Capilliposide A to mice with sodium iodate-induced retinal damage;

[0024] Figure 3 The data show that Capilliposide A inhibits sodium iodate-induced apoptosis in human retinal pigment epithelial cells.

[0025] Figure 4 Data showing that Capilliposide A inhibits sodium iodate-induced ROS accumulation in human retinal pigment epithelial cells. DETAILED DESCRIPTION

[0026] The present invention is further described below with reference to the accompanying drawings and specific embodiments.

[0027] The present invention is based on the compound Capilliposide A, whose CAS number is 897936-98-0 and the compound structure is as follows:

[0028]

[0029] The above-mentioned compound Capilliposide A is an existing compound and can be commercially available or prepared by itself. The preparation method of Capilliposide A can refer to the invention patent with application number CN202110321499.6 and invention name "A method for preparing capilliposide saponin A". The specific preparation method is as follows: (1) Take the cut capilliposide, add 10 to 20 times the mass of ethanol aqueous solution, reflux extract 2 to 4 times, each time for 1 to 3 hours, combine the extracts and concentrate until there is no ethanol, add water to make each 1 ml of the mixed solution contain 0.1 to 0.5 grams of capilliposide, stir well, centrifuge after precipitation, and collect the centrifuge; (2) pass the centrifuge of step (1) through a macroporous adsorption resin (non-polar, weakly polar or polar macroporous adsorption resin can be used, such as AB-8 macroporous adsorption resin, HP D450 macroporous adsorption resin, LX1180S macroporous adsorption resin), wash with water until the eluate color becomes significantly lighter, add alkaline solution for washing, then react at 10-50°C for 24-120 hours, wash with water until neutral, and then elute with ethanol gradient to collect part of the eluate containing pedunculate saponin A; (3) recover ethanol from the part of the eluate containing pedunculate saponin A in step (2), add it to a reverse phase chromatographic column for separation (the mobile phase A of the solvent system used is methanol or acetonitrile, and the mobile phase B is water), and collect the separated solution containing pedunculate saponin A; (4) recover the solvent from the separated solution containing pedunculate saponin A in step (3), and recrystallize to obtain high-purity pedunculate saponin A. The specific process parameters in the above preparation method can be optimized and adjusted according to actual conditions.

[0030] The present invention provides an application of Capilliposide A in preparing a medicine for preventing and treating age-related macular degeneration.

[0031] The mechanism of action of the above applications is to improve retinal damage, inhibit the apoptosis of retinal pigment epithelial cells and the accumulation of ROS, thereby treating or preventing age-related macular degeneration.

[0032] Based on the same inventive concept, the present invention also provides a pharmaceutical composition for treating and preventing age-related macular degeneration, wherein the active ingredient in the pharmaceutical composition is Capilliposide A.

[0033] In addition to the active ingredient, the above-mentioned pharmaceutical composition may also contain one or more pharmaceutically acceptable excipients, that is, Capilliposide A and pharmaceutical excipients may be used to prepare a pharmaceutical composition. The specific selection of pharmaceutical excipients may be selected according to the dosage form to be prepared, and the compatibility with the active ingredient and the requirements of safety and non-toxicity may be maintained. The dosage form of the pharmaceutical composition of the present invention may be selected from the form of granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions.

[0034] The pharmaceutical composition of the present invention can be administered orally or parenterally. For parenteral administration, the pharmaceutical composition can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, ocular administration, etc. The specific administration method needs to be adjusted accordingly according to the dosage form and the application site.

[0035] In addition, in some embodiments of the present invention, the pharmaceutical composition can be a solution prepared by the active ingredient Capilliposide A and a liquid pharmaceutically acceptable carrier, wherein the liquid pharmaceutically acceptable carrier can be selected from one or more of saline, sterile water, Ringer's solution, buffered saline, glycerol, and ethanol. In addition, other conventional additives such as antioxidants, buffers, antibacterial agents, thickeners, etc. can be added to the pharmaceutical composition as needed.

[0036] Since age-related macular degeneration is an eye disease, in a preferred embodiment of the present invention, an eye drop for preventing and treating age-related macular degeneration is provided, which contains Capilliposide A as an active ingredient and can be directly dripped into the eye. The eye drop is preferably prepared from Capilliposide A, a thickener and physiological saline.

[0037] Capilliposide A plays a core role in the above-mentioned eye drops, and its mass percentage in the eye drops can be optimized according to actual conditions, preferably 0.01% to 0.05%.

[0038] The thickener in the above-mentioned eye drops can prolong the drug's action time, improve the user experience and protect the ocular surface tissue, and polyvinyl alcohol is preferably used.

[0039] In order to make the above-mentioned objects, features and advantages of the present invention more clearly understood, the technical solutions of the present invention are further described below in conjunction with embodiments. However, the present invention is not limited to the embodiments listed, and should also include any other known changes within the scope of the rights claimed by the present invention.

[0040] Example 1: Capilliposide A significantly improves retinal damage in mice after sodium iodate modeling

[0041] (1) C57 mice aged 6 to 8 weeks were ordered. After one week of acclimatization, some mice were injected with sodium iodate (25 mg / kg) via the tail vein to obtain a mouse model simulating age-related macular degeneration. The mice were then divided into three groups: model group (Model), capilliposide A high-dose group (LCA-high), and capilliposide A low-dose group (LCA-low), with 10 mice in each group. In addition, 10 mice that were not injected with sodium iodate via the tail vein were selected as the blank group (Blank).

[0042] (2) Capilliposide A was dissolved in physiological saline to prepare eye drops of two different concentrations. The concentration of Capilliposide A in the eye drops used in the high-dose group was 0.05wt.%, and the concentration of Capilliposide A in the eye drops used in the low-dose group was 0.01wt.%. Polyvinyl alcohol with a final concentration of 1wt.% was added as a thickener in both the high-dose group and the low-dose group. After filtering through a 0.22μm filter membrane, the final eye drops were used for administration to the two groups of mouse models for 14 days. The administration method was: administration twice a day after modeling, one drop per eye each time. The blank group and the model group were administered in the same manner using only the solvent of the eye drops (i.e., physiological saline containing 1wt.% of polyvinyl alcohol) to form a control.

[0043] (3) After the administration, the mouse eyeballs were removed and fixed in an eyeball fixative for 24 h. The eyeballs were dehydrated, embedded, and sliced. The slices were baked in a 65°C oven overnight, stained with H&E, and sealed. The images were scanned using a microscope. Figure 1 ImageJ and Adobe Photoshop software were used to analyze and process the thickness of the retinal pigment epithelium. Figure 2 Compared with the blank group, the retina of the mice in the sodium iodate group was damaged in all layers, the retinal pigment epithelium cells were significantly damaged, the outer nuclear layer was disordered and wave-like, and the thickness of the retinal pigment epithelium was significantly reduced; the damage to all layers of the retina in the high-dose and low-dose capilliposide A groups was improved, and the thickness of the retinal pigment epithelium was significantly increased compared with the sodium iodate group, and the improvement degree of the high-dose capilliposide A group was better than that of the low-dose group.

[0044] Example 2: Inhibitory effect of capilliposide A on apoptosis of human retinal pigment epithelial cells Human retinal pigment epithelial cell line (Adult Retinal Pigment Epithelial cell line-19, ARPE-19) was used as a retinal pigment epithelial model. When ARPE-19 cells grew to about 60%-70%, five groups of different treatments were performed: a blank control group (Blank) without any treatment, a sodium iodate group (NAIO 3 ) Sodium iodate with a final concentration of 20 mM was added to ARPE-19 cells for modeling treatment. N-acetylcysteine ​​with a final concentration of 500 μM was added to the positive drug group (NAC) for drug treatment on the basis of the modeling of the sodium iodate group. Capilliposide A group was treated with capilliposide A with final concentrations of 6.25 μM and 12.5 μM, respectively, on the basis of the modeling of the sodium iodate group (the two drug concentrations of capilliposide A groups were recorded as LCA6.25 μM and LCA12.5 μM, respectively). Each group was then treated as follows: ARPE-19 cells were treated with drugs for 48 hours, digested with trypsin, transferred to a 5 mL centrifuge tube, resuspended with PBS and counted, and the cells were collected; about 100,000 cells were added with 195 μL Annexin V-FITC binding solution and mixed, and then 5 microliters of Annexin V-FITC were added and mixed; then 10 microliters of PI dye were added and mixed, wrapped in tin foil at room temperature and protected from light for 20 minutes, and then placed on ice; finally, the cells were tested on the machine, and the cell concentration was adjusted before loading, and the loading speed was kept as low as 1000 cells per minute to prevent clogging of the flow cytometer pipeline. The results were input into FlowJo for analysis and plotted using GraphPad Prism.

[0045] The results obtained in this example are as follows Figure 3 As shown, the results showed that the apoptosis rate of cells in the sodium iodate group increased significantly, and the apoptosis rate decreased after the administration of Capilliposide A. The apoptosis of cells was improved after the addition of Capilliposide A, indicating that Capilliposide A has an inhibitory effect on sodium iodate-induced apoptosis of human retinal pigment epithelial cells.

[0046] Example 3: Inhibitory effect of Capilliposide A on ROS accumulation in human retinal pigment epithelial cells

[0047] ARPE-19 cells were used as a retinal pigment epithelial model. When the ARPE-19 cells grew to about 60%-70%, five different treatments were performed. The blank control group (Blank) did not receive any treatment, and the sodium iodate group (NAIO 3) Sodium iodate with a final concentration of 20 mM was added to ARPE-19 cells for modeling. N-acetylcysteine ​​with a final concentration of 500 μM was added to the positive drug group (NAC) for drug administration based on the modeling of the sodium iodate group. Capilliposide A group was treated with capilliposide A with final concentrations of 12.5 μM and 25 μM, respectively, based on the modeling of the sodium iodate group (the two drug concentrations of capilliposide A groups were recorded as LCA12.5 μM and LCA25 μM, respectively). Each group was then treated as follows: ARPE-19 cells were treated with drugs for 24 hours and then detected using a ROS detection kit. DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μM. After cell digestion, the cells were resuspended with the diluted DCFH-DA and cultured in a 37°C incubator for 20 minutes. The cells were mixed every 5 minutes and washed three times with serum-free culture medium. The cells were detected by flow cytometry, and the results were input into FlowJo for analysis and plotted using GraphPad Prism.

[0048] The results obtained in this example are as follows Figure 4 As shown, the results showed that the ROS content of the cells in the sodium iodate group increased, and after the administration of Capilliposide A, the ROS content decreased compared with the sodium iodate group. The accumulation of cellular ROS was improved after the addition of Capilliposide A, indicating that Capilliposide A has the effect of inhibiting the accumulation of ROS in human retinal pigment epithelial cells.

[0049] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of Capilliposide A in the preparation of drugs for preventing and treating age-related macular degeneration.

2. The use according to claim 1, characterized in that By improving retinal damage, inhibiting the apoptosis of retinal pigment epithelial cells and the accumulation of ROS, age-related macular degeneration can be treated or prevented.

3. A pharmaceutical composition for treating and preventing age-related macular degeneration, wherein the active ingredient in the pharmaceutical composition is Capilliposide A.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition consists of the active ingredient and one or more pharmaceutically acceptable excipients.

5. The pharmaceutical composition according to claim 4, characterized in that The dosage form of the pharmaceutical composition is granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops or injectable solutions.

6. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is a solution prepared by the active ingredient and a liquid pharmaceutically acceptable carrier, and the liquid pharmaceutically acceptable carrier is one or more of saline, sterile water, Ringer's solution, buffered saline, glycerol, and ethanol.

7. The pharmaceutical composition according to claim 6, characterized in that The pharmaceutical composition also contains one or more of an antioxidant, a buffer, an antibacterial agent, and a thickener.

8. An eye drop for preventing and treating age-related macular degeneration, characterized in that: With Capilliposide A as the active ingredient.

9. The eye drops according to claim 7, characterized in that It is prepared from Capilliposide A, a thickener and physiological saline; preferably, the thickener is polyvinyl alcohol.

10. The eye drops according to claim 8 or 9, characterized in that The mass percentage of the Capilliposide A in the eye drops is 0.01% to 0.05%.

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