Eye drops containing l-ergothioneine and preparation method therefor

Abstract

The present invention discloses eye drops containing L-ergothioneine and a preparation method therefor. The eye drops comprise 0.0015-15 parts by weight of L-ergothioneine, 0.001-0.01 parts by weight of a bacteriostatic agent, 0.05-15 parts by weight of a buffer, and normal saline to make up 100 parts by weight; the pH of the eye drops is 5.0-7.0. In the present invention, it is found through experiments that L-ergothioneine can prevent and reverse crystallins denaturation. The eye drops containing L-ergothioneine prepared in the present invention can prevent and reduce the opacification of the crystallins.

Description

An eye drop containing L-ergothioneine and its preparation method

[0001] Cross-referencing related applications

[0002] This patent application claims priority to Chinese Patent Application No. 202410697185X, filed on May 31, 2024, entitled “An eye drop containing L-ergothioneine and a method for preparing the same,” the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of pharmaceutical preparations, specifically to an eye drop containing L-ergothioneine and its preparation method. Background Technology

[0004] Cataracts are a general term for various diseases that cause partial or complete clouding of the lens structure inside the eye, leading to visual impairment. Cataracts have consistently been the leading cause of blindness worldwide, accounting for over 45% of global blindness, and their incidence rate is very high. With the increasing aging of the global population, the trend of rising cataract incidence rates is expected to intensify.

[0005] Currently, cataract extraction combined with intraocular lens implantation remains the only effective treatment for cataracts. However, surgery is not suitable for all types of cataracts, and despite the relative maturity of cataract surgery, complications are still unavoidable, leading to decreased vision or even blindness. These complications include shallow anterior chamber, epithelial ingrowth into the anterior chamber, persistent corneal edema, anterior chamber and vitreous hemorrhage, uveitis, purulent endophthalmitis, glaucoma, abnormal intraocular lens placement, and posterior capsule opacification. These complications are also the main reasons why vision may not fully recover after surgery. Surgical treatment alone cannot solve the increasingly serious problem of cataracts; early detection and early treatment will become one of the main directions for cataract prevention and treatment in the future.

[0006] Although eye drops for cataracts are available on the market, no medication has yet been proven to be effective in preventing or treating cataracts. Given the limitations of current cataract treatments, there is an urgent need for eye drops that can effectively prevent and treat cataracts.

[0007] L-Ergothione is a water-soluble amino acid primarily found in mushrooms. It cannot be synthesized by humans (or other vertebrates) and must be obtained through diet. It is a naturally occurring thiol / thion derivative of the essential amino acid histidine, with the molecular formula C9H10. 15 N3O2S. To date, there is no precedent for using L-ergothioneine to treat cataracts. Summary of the Invention

[0008] The purpose of this invention is to address the current situation where existing cataract medications are not very effective, and to provide an eye drop containing L-ergothioneine and its preparation method.

[0009] To achieve the above objectives, the present invention provides an eye drop containing L-ergothioneine, comprising the following components:

[0010] The eye drops contain 0.0015-15 parts by weight of L-ergothioneine, 0.001-0.01 parts by weight of antibacterial agent, 0.05-15 parts by weight of buffer, and physiological saline to make up to 100 parts by weight. The pH of the eye drops is 5.0-7.0.

[0011] Furthermore, the L-ergothionein is 2 parts by weight.

[0012] Furthermore, the pH of the eye drops is 6.0.

[0013] Furthermore, the antibacterial agent is composed of one or more of thimerosal, benzalkonium chloride, and p-hydroxybenzoate mixed in any proportion.

[0014] Furthermore, the buffer is any one of the following: sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate buffer pair, boric acid and borax buffer pair, and citric acid and disodium hydrogen phosphate buffer pair.

[0015] To achieve the above objectives, the present invention also provides a method for preparing the above-mentioned eye drops containing L-ergothioneine, comprising the following steps:

[0016] (1) Dissolve the antibacterial agent in physiological saline and filter it through a 0.22 μm microporous membrane to remove bacteria, thus obtaining solution I;

[0017] (2) Add sterile L-ergothioneine to solution I and sonicate for 30 min to obtain solution II;

[0018] (3) Dilute solution II to 100 parts by weight with physiological saline to obtain solution III;

[0019] (4) Adjust the pH of solution III to 5.0-7.0 with a buffer to obtain solution IV;

[0020] (5) Sterilize solution IV at 121-134℃ and 102.9-137.2kPa for 15-30 min to obtain eye drops containing L-ergothioneine.

[0021] To achieve the above objectives, the present invention also provides the use of the above-mentioned eye drops containing L-ergothioneine in the preparation of drugs for the prevention and treatment of cataracts.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] (1) The eye drops containing L-ergothioneine of the present invention have a significant improvement effect on the H2O2-induced oxidative damage model of human lens epithelial cells, and its improvement effect is reflected in protecting the activity of human lens epithelial cells.

[0024] (2) The eye drops containing L-ergothioneine of the present invention have a significant improvement effect on the lens organoid cataract model, and the improvement effect is reflected in slowing down the turbidity progression of the age-related cataract lens organoid model.

[0025] (3) The eye drops containing L-ergothioneine of the present invention have a significant effect on UVB-induced mouse cataract model. The improvement effect is reflected in preventing the occurrence of anterior subcapsular opacity of the mouse lens, and can reverse the early anterior subcapsular opacity that has already occurred, and alleviate the later progression of anterior subcapsular opacity to a certain extent.

[0026] (4) According to the research results of this invention, eye drops containing L-ergothioneine have significant preventive and therapeutic effects on cataracts. They have significant improvement effects on in vitro, organoid, and in vivo cataract models. The improvement effect is manifested in protecting the activity of human lens epithelial cells, slowing the progression of lens organoid opacity, and preventing and alleviating subcapsular opacity in mice. The results of this invention indicate that eye drops containing L-ergothioneine will have good clinical efficacy for age-related cataracts, and can play a role in prevention and early reversal, and to a certain extent control the later progression of cataracts;

[0027] (5) The eye drops containing L-ergothioneine of the present invention have high stability and significant efficacy;

[0028] (6) The eye drops containing L-ergothioneine of the present invention are safe and have no side effects. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 is a schematic diagram of the cell activity results of the eye drops containing L-ergothioneine prepared in this invention in the H2O2-induced oxidative damage model of human lens epithelial cells.

[0031] Figure 2 is a schematic diagram of the slit lamp results of the eye drops containing L-ergothioneine prepared in this invention for the purpose of preventing cataracts in a UVB-induced mouse cataract model.

[0032] Figure 3 is a schematic diagram of the slit lamp results of the eye drops containing L-ergothioneine prepared in this invention for the purpose of reversing early cataracts in a UVB-induced mouse cataract model.

[0033] Figure 4 is a schematic diagram of the slit lamp results of the eye drops containing L-ergothioneine prepared in this invention for the purpose of treating cataracts in a UVB-induced mouse cataract model. Detailed Implementation

[0034] To enable those skilled in the art to better understand the present invention, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0035] The preparation method of the eye drops containing L-ergothioneine of the present invention comprises the following steps:

[0036] (1) Dissolve 0.001-0.01 parts by weight of antibacterial agent in 70 parts by weight of physiological saline (0.9% sodium chloride injection), filter and sterilize with a 0.22 μm microporous membrane to obtain solution I; wherein the antibacterial agent is composed of one or more of thimerosal, benzalkonium chloride and p-hydroxybenzoate in any ratio.

[0037] (2) Add 0.0015-15.0 parts by weight of sterile L-ergothioneine to solution I and sonicate for 30 min to obtain solution II;

[0038] (3) Dilute solution II to 100 parts by weight with physiological saline (0.9% sodium chloride injection) to obtain solution III;

[0039] (4) Adjust the pH of solution III to 5.0-7.0 with 0.05-15 parts by weight of buffer to obtain solution IV; wherein the buffer is any one of the following: sodium dihydrogen phosphate monohydrate and disodium hydrogen phosphate buffer pair, boric acid and borax buffer pair, citric acid and disodium hydrogen phosphate buffer pair;

[0040] (5) Sterilize solution IV at 121-134℃ and 102.9-137.2kPa for 15-30 minutes to obtain eye drops containing L-ergothioneine.

[0041] Preferably, solution IV is sterilized at a temperature of 121.3°C and a pressure of 103.4 kPa for 20 min to obtain eye drops containing L-ergothioneine.

[0042] (6) The L-ergothioneine content, osmotic pressure and pH value of the prepared eye drops were tested, and the eye drops were dispensed into 0.4-10 mL in a sterile environment.

[0043] It should be understood that cataracts are characterized by clouding caused by various factors such as aging, genetics, local nutritional deficiencies, immune and metabolic abnormalities, trauma, poisoning, and radiation, which lead to denaturation of lens proteins.

[0044] Example 1: Preparation of L-ergothioneine eye drops

[0045] Weigh the following components in parts by weight: 0.2 parts L-ergothioneine, 0.003 parts antibacterial agent (benzalkonium chloride), 0.1 parts buffer (citric acid), 0.3 parts buffer (disodium hydrogen phosphate), and 99.397 parts physiological saline. Adjust the pH to 5.0 with buffer. Prepare eye drops containing L-ergothioneine according to the preparation method in the above specific implementation scheme.

[0046] Example 2: Preparation of L-ergothioneine eye drops

[0047] Weigh the following components in parts by weight: 2 parts L-ergothioneine, 0.005 parts antibacterial agent (para-hydroxybenzoate), 2.4 parts buffer (sodium dihydrogen phosphate monohydrate), 0.3 parts buffer (disodium hydrogen phosphate), and 95.295 parts physiological saline. Adjust the pH to 6.0 with buffer. Prepare eye drops containing L-ergothioneine according to the preparation method in the above specific implementation scheme.

[0048] Example 3: Preparation of L-ergothioneine eye drops

[0049] Weigh the following components in parts by weight: 12 parts L-ergothioneine, 0.001 parts antibacterial agent (thimerosal), 9.9 parts buffer (boric acid), 1.1 parts buffer (borax), and 76.999 parts physiological saline. Adjust the pH to 7.0 with buffer. Prepare eye drops containing L-ergothioneine according to the preparation method in the above specific implementation scheme.

[0050] Example 4: Application of L-ergothioneine eye drops in a H2O2-induced oxidative damage model of human lens epithelial cells

[0051] Human lens epithelial cells were used in this embodiment and divided into a blank control group, a model group, and an experimental group. Cells were cultured until the cell density reached 80% before the experiment. The experimental group cells were pretreated by incubating with eye drops containing L-ergothioneine prepared in Example 1 for 1-72 hours. Subsequently, all three groups of cells (blank control group, model group, and experimental group) were washed with Hanks' solution and then incubated with medium containing 600 μM H2O2 for 24 hours. Finally, the cell viability of each group was detected using a Cell Counting Kit-8 (CCK8).

[0052] Figure 1 shows that in the H2O2-induced oxidative damage model of human lens epithelial cells, the eye drops containing L-ergothioneine of the present invention have a protective effect on human lens epithelial cells. Pretreatment with the eye drops for 24h, 36h and 72h can significantly enhance cell activity.

[0053] Example 5: Application of L-ergothioneine eye drops in a lens organoid cataract model

[0054] In this embodiment, the "fried egg" method was used to induce a lens organoid model, which was divided into a model group (n=3), a positive control group (n=3), and an experimental group (n=3). The lens organoids of the positive control group were incubated with commercially available lanosterol, while the lens organoids of the experimental group were incubated with eye drops containing L-ergothioneine prepared in Example 1. All three groups (model group, positive control group, and experimental group) of organoids were cultured for a long time to simulate the lens opacity process of age-related cataracts. The opacity of the lens organoids in each group was observed and compared under a microscope.

[0055] Experimental results show that the eye drops containing L-ergothioneine of the present invention can slow down the progression of turbidity in an age-related cataract lens organoid model.

[0056] Example 6: The preventive effect of L-ergothioneine eye drops on a UVB-induced mouse cataract model

[0057] In this study, 36 male C57BL / 6 mice weighing 16-18g and aged 6 weeks were used. Before the study began, each eye of each mouse was examined with a slit lamp through the dilated pupil (1% atropine eye drops) to rule out any pre-existing abnormalities.

[0058] Mice were randomly divided into four groups: (1) blank control group (n=9); (2) model group (n=9); (3) positive control group (n=9); and (4) prevention group (n=9).

[0059] From day 1, except for the blank control group, all mice in the other groups were subjected to pupil dilation (1% atropine eye drops) for 5 minutes, followed by continuous irradiation of both eyes for 30 minutes at a distance of 15 cm using a 310 nm ultraviolet lamp (TL20w / 01RS, Philips, Germany), once a day for 12 consecutive days.

[0060] The blank control group and model group were given PBS (phosphate-buffered saline) eye drops starting from day (-1), the positive control group was given commercially available Shapusi eye drops starting from day (-1), and the prevention group was given eye drops containing L-ergothioneine prepared in Example 2 starting from day (-1). Each mouse received eye drops in both eyes, at a dose of 5 μL / eye / time, at a frequency of 3 times / day.

[0061] The lens opacity was observed using a slit lamp on days 0, 4, 8, 10, and 12.

[0062] Figure 2 shows that the eye drops containing L-ergothioneine of the present invention can effectively prevent the occurrence of cataracts in mice, and the effect is significantly better than that of the eye drops in the positive control group. In Figure 2, the first row is the blank control group, the second row is the model group, the third row is the positive control group, and the fourth row is the prevention group; the column names represent the days before the construction of the UVB-induced mouse cataract model (day 0) and the 4th, 8th, 10th, and 12th days after the model construction.

[0063] Example 7: Early reversal effect of L-ergothioneine eye drops in a UVB-induced mouse cataract model

[0064] In this study, 36 male C57BL / 6 mice weighing 16-18g and aged 6 weeks were used. Before the study began, each eye of each mouse was examined with a slit lamp through the dilated pupil (1% atropine eye drops) to rule out any pre-existing abnormalities.

[0065] Mice were randomly divided into four groups: (1) blank control group (n=9); (2) model group (n=9); (3) positive control group (n=9); and (4) early reversal group (n=9).

[0066] From day 1, except for the blank control group, all mice in the other groups were subjected to pupil dilation (1% atropine eye drops) for 5 minutes, followed by continuous irradiation of both eyes for 30 minutes at a distance of 15 cm using a 310 nm ultraviolet lamp (TL20w / 01RS, Philips, Germany), once a day for 12 consecutive days.

[0067] The blank control group and model group were given PBS (phosphate-buffered saline) eye drops starting from day 9, the positive control group was given commercially available Shapusi eye drops starting from day 9, and the early reversal group was given eye drops containing L-ergothioneine prepared in Example 2 starting from day 9. Each mouse received eye drops in both eyes, at a dose of 5 μL / eye / time, at a frequency of 3 times / day.

[0068] The lens opacity was observed using a slit lamp on days 8, 10, 11, and 12.

[0069] Figure 3 shows that the eye drops containing L-ergothioneine of the present invention can effectively reverse early cataracts in mice, and the effect is significantly better than that of the eye drops in the positive control group. In Figure 3, the first row is the blank control group, the second row is the model group, the third row is the positive control group, and the fourth row is the early reversal group; the column names represent days 8, 10, 11, and 12 of constructing the UVB-induced mouse cataract model, respectively.

[0070] Example 8: The therapeutic effect of L-ergothioneine eye drops on a UVB-induced mouse cataract model

[0071] In this study, 36 male C57BL / 6 mice weighing 16-18g and aged 6 weeks were used. Before the study began, each eye of each mouse was examined with a slit lamp through the dilated pupil (1% atropine eye drops) to rule out any pre-existing abnormalities.

[0072] Mice were randomly divided into four groups: (1) blank control group (n=9); (2) model group (n=9); (3) positive control group (n=9); and (4) treatment group (n=9).

[0073] From day 1, except for the blank control group, all mice in the other groups were subjected to pupil dilation (1% atropine eye drops) for 5 minutes, followed by continuous irradiation of both eyes for 30 minutes at a distance of 15 cm using a 310 nm ultraviolet lamp (TL20w / 01RS, Philips, Germany), once a day for 24 consecutive days.

[0074] The blank control group and model group were given PBS (phosphate-buffered saline) eye drops starting from day 13, the positive control group was given commercially available Shapusi eye drops starting from day 13, and the treatment group was given eye drops containing L-ergothioneine prepared in Example 2 starting from day 13. Each mouse received eye drops in both eyes, at a dose of 5 μL / eye / time, at a frequency of 3 times / day.

[0075] The lens opacity was observed using a slit lamp on days 12, 14, 16, 18, 20, 22, and 24.

[0076] Figure 4 shows that the eye drops containing L-ergothioneine of the present invention can alleviate existing mouse cataracts to a certain extent and effectively control the progression of cataracts, and the overall therapeutic effect is significantly better than that of the eye drops in the positive control group. In Figure 4, the first row is the blank control group, the second row is the model group, the third row is the positive control group, and the fourth row is the treatment group; the column names represent days 12, 14, 16, 18, 20, 22, and 24 of constructing the UVB-induced mouse cataract model, respectively.

[0077] Although the present invention has been described in detail above with general description and specific embodiments, some modifications or improvements can be made to it based on the present invention, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. An eye drop solution comprising L- ergothioneine, characterized in that, The eye drops comprising L-ergothioneine are prepared by the following steps: The L-ergothioneine is 2 parts by weight.

2. The eye drop according to claim 1, characterized by, The eye drops comprising L-ergothioneine are prepared by the following steps:

3. The eye drops according to claim 1, characterized in that, The bacteriostatic agent is composed of one or more of thiomersal, benzalkonium chloride, and parabens in any ratio.

4. The eye drop according to claim 1, characterized by, The buffer is any one of sodium phosphate monobasic monohydrate and sodium phosphate dibasic buffer pair, boric acid and borax buffer pair, citric acid and sodium phosphate dibasic buffer pair.

5. The eye drop according to claim 1, wherein The eye drops comprising L-ergothioneine are prepared by the following steps:

6. A method of preparing the eye drop of claim 1, characterized by, (1) dissolving the bacteriostatic agent in physiological saline, filtering sterilization with 0.22 μm microporous filter membrane to obtain solution I; (2) adding sterile L-ergothioneine to solution I, ultrasonic stirring for 30 min to obtain solution II; (3) adding physiological saline to solution II to make up to 100 parts by weight to obtain solution III; (4) adjusting the pH value of solution III to 5.0-7.0 with the buffer to obtain solution IV; (5) sterilizing solution IV at 121-134 ℃, 102.9-137.2 kPa for 15-30 min to obtain the eye drops comprising L-ergothioneine.

7. Use of the eye drops comprising L-ergothioneine for: (i) preparing a drug for preventing and treating cataract; and / or (ii) preventing and treating cataract; and / or (iii) improving H2O2-induced oxidative damage of human lens epithelial cells in vitro non-therapeutically; The eye drops comprising L-ergothioneine are prepared by the following steps: wherein The L-ergothioneine is 2 parts by weight.

8. Use according to claim 7, characterized in that, The pH value of the eye drops is 6.

0.

9. Use according to claim 7, characterized in that, The bacteriostatic agent is composed of one or more of thiomersal, benzalkonium chloride, and parabens in any ratio.

10. Use according to claim 7, characterized in that, The buffer is any one of sodium phosphate monobasic monohydrate and sodium phosphate dibasic buffer pair, boric acid and borax buffer pair, citric acid and sodium phosphate dibasic buffer pair.

11. Use according to claim 7, characterized in that, The eye drops comprising L-ergothioneine are prepared by the following steps:

12. Use according to claim 7, characterized in that, The cataract is UVB-induced cataract.

13. Use according to any one of claims 7 to 12, characterized in that, The cataract is lens organoid cataract.

14. Use according to any one of claims 7-12, characterized in that, The cataract is age-related cataract.

15. Use according to any one of claims 7-12, characterized in that, ​

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