A composite, its preparation and use

Abstract

The application discloses a kind of complex and its preparation method and application, including cysteine modified chitosan and peroxidase, and cysteine modified chitosan and peroxidase form stable complex;The complex provided by the application has good stability, and particle size is uniform;The complex described in the application has simple composition, and preparation and purification method are simple, production efficiency is high, it is helpful to batch production and popularization and application, cost can be controlled, and even with high concentration of the complex described in the application and eye drop is applied to mouse or rabbit eye surface, still cannot cause eyeball anomaly by safety experiment verification;The free thiol group on the surface of cysteine modified chitosan can increase the adhesion of complex on the ocular surface, so as to overcome the limitation of rapid clearance of ocular drug, significantly prolong the residence time of peroxidase on the ocular surface, greatly improve the feasibility of peroxidase in the field of dry eye treatment application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a complex, its preparation method, and its application. Background Technology

[0002] Dry eye syndrome, also known as keratoconjunctivitis sicca, refers to tear film instability and ocular surface damage caused by abnormalities in the quantity or quality of tears. It is a common, multifactorial ophthalmic disease affecting approximately 360 million people in my country, representing the largest patient group among ophthalmic diseases in the country, and has become a prevalent eye condition in modern society. Dry eye syndrome affects the cornea and conjunctiva, causing corneal epithelial damage, decreased corneal surface regularity and sensitivity, and impacting the refractive system, leading to blurred vision and significantly affecting patients' daily lives.

[0003] Although the pathogenesis of dry eye is diverse, a growing body of research suggests that oxidative stress damage is an upstream factor in its development and progression. Oxidative stress damage refers to the disruption of cellular metabolism and destruction of cellular components caused by the excessive production of reactive oxygen species (ROS), such as superoxide anions and hydrogen peroxide. The relationship between ROS production, lipid peroxidation-related membrane damage, protein oxidation, and inflammation has been confirmed in animal models and clinical studies. The eyeball is one of the most vulnerable parts of the human body. Direct exposure to ultraviolet radiation, drugs, air pollutants, and excessive use of electronic devices can lead to the accumulation of excessive ROS on the ocular surface, causing oxidative stress damage. This, in turn, leads to symptoms such as decreased tear film stability, tear hyperosmolarity, corneal epithelial cell apoptosis, goblet cell loss, and inflammation, creating a vicious cycle of dry eye.

[0004] Currently, treatments for dry eye primarily involve artificial tears to relieve symptoms and improve tear film stability, anti-inflammatory drugs to reduce ocular surface inflammation, and immunosuppressants. However, artificial tears only provide temporary relief by moisturizing and lubricating the ocular surface for a short period and cannot fundamentally cure dry eye. Steroidal anti-inflammatory drugs often cause serious adverse consequences such as glaucoma and cataracts, while immunosuppressants such as cyclosporine require continuous use for 3 to 6 months to be effective. Furthermore, due to frequent blinking and rapid tear metabolism, eye drops are quickly cleared from the ocular surface, resulting in low bioavailability of ocular medications. Therefore, there is an urgent need to develop safe and effective new strategies for treating dry eye. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the object of the present invention is to overcome the shortcomings of the prior art and provide a composite.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a complex comprising cysteine-modified chitosan and peroxidase, wherein the cysteine-modified chitosan and peroxidase form a stable complex, wherein the mass ratio of cysteine-modified chitosan to peroxidase in the complex is 1:4 to 4:1.

[0009] In a preferred embodiment of the complex described in this invention, the surface cysteine ​​modification degree of the cysteine-modified chitosan is on average 0.5-8%.

[0010] As a preferred embodiment of the complex described in this invention, the surface cysteine ​​modification degree of the cysteine-modified chitosan is on average 2%-7%.

[0011] In a preferred embodiment of the complex described in this invention, the surface cysteine ​​modification degree of the cysteine-modified chitosan is on average 2%-4.5%.

[0012] In a preferred embodiment of the complex described in this invention, the cysteine ​​contains a free thiol group and is modified on the surface of chitosan via an amide bond.

[0013] As a preferred embodiment of the complex described in this invention, the cysteine-modified chitosan has free thiol groups on its surface.

[0014] As a preferred embodiment of the complex described in this invention, the cysteine-modified chitosan has a surface free thiol content of 100–280 micromoles per gram of polymer.

[0015] In a preferred embodiment of the complex described in this invention, the cysteine-modified chitosan has an average molecular weight of 10–70 kDa.

[0016] In a preferred embodiment of the complex described in this invention, the peroxidase is one or both of catalase and superoxide dismutase.

[0017] As a preferred embodiment of the composite of the present invention, the particle size of the composite is 50 nm to 1000 nm.

[0018] As a preferred embodiment of the composite of the present invention, the average particle size of the composite is 220 nm to 500 nm.

[0019] Another objective of this invention is to overcome the shortcomings of the prior art and provide a method for preparing a complex, comprising: reconstitute cysteine-modified chitosan powder with ultrapure water, adjusting the pH of the solution to 6.4, to obtain solution A;

[0020] Catalase was dissolved in PBS, and the pH of the solution was adjusted to 9.0 to obtain solution B;

[0021] The above solutions A and B were mixed to prepare a complex.

[0022] As a preferred embodiment of the preparation method described in this invention, it includes mixing cysteine-modified chitosan and peroxidase.

[0023] As a preferred embodiment of the preparation method described in this invention, a purification process is also included.

[0024] In a preferred embodiment of the preparation method described in this invention, the mass ratio of the cysteine-modified chitosan to the peroxidase is 1:1 to 2:1.

[0025] Another object of the present invention is to overcome the shortcomings of the prior art and provide an application of a complex in the preparation of an eye drop formulation, wherein the eye drop formulation contains the complex.

[0026] In a preferred embodiment of the application described in this invention, the concentration of the complex in the eye drop preparation is 1–1.5 mg / mL.

[0027] As a preferred embodiment of the application described in this invention, the eye drop preparation contains one or more of the following: solvent, preservative, reducing agent, thickener, pH adjuster, antibacterial agent, and bactericide.

[0028] In a preferred embodiment of the application described in this invention, the pH of the eye drop preparation is 5.0 to 9.0.

[0029] Another objective of this invention is to overcome the shortcomings of the prior art and provide an application of a complex or eye drop formulation in the preparation of a drug for treating dry eye syndrome.

[0030] Beneficial effects of this invention:

[0031] 1. This invention develops a biomacromolecule delivery platform with excellent adhesion properties, overcoming the current limitation of rapid drug clearance, and for the first time verifies the therapeutic effect of catalase in dry eye syndrome. This strategy has broad prospects for clinical translation and application, and provides a new treatment approach for inhibiting local mucosal inflammatory diseases.

[0032] 2. The complex provided by this invention is obtained through self-assembly between cysteine-modified chitosan and peroxidase, exhibiting good stability and uniform particle size. Because the free thiol groups on the surface of cysteine-modified chitosan can increase the adhesion of the complex to the ocular surface, it can overcome the limitation of rapid drug clearance and significantly prolong the retention time of peroxidase on the ocular surface, greatly improving the feasibility of peroxidase application in the treatment of dry eye.

[0033] 3. The cysteine-modified chitosan of the present invention covalently binds to the abundant cysteine ​​in the tear film mucin layer by forming disulfide bonds, thereby increasing the adhesion to ocular surface mucin. In addition, the chitosan adhering to the tear film mucin layer can provide lubrication and protection to the ocular surface on the one hand, and significantly increase the retention time of the formulation on the ocular surface without causing significant blurring on the other hand.

[0034] 4. The peroxidase of the present invention is an endogenous biological macromolecule with good biocompatibility and is safer. Moreover, the peroxidase has high efficiency in decomposing hydrogen peroxide on the ocular surface and tear film, which can continuously improve the oxidative stress environment of the eye, effectively inhibit the initiation of inflammation and cell apoptosis, promote the recovery of eye health, and achieve the effect of treating dry eye syndrome. It has shown excellent therapeutic effects on dry eye syndrome in multiple models.

[0035] 5. The complex of the present invention has the advantages of simple composition, simple preparation and purification methods, high production efficiency, which facilitates mass production and widespread application, and controllable cost. Furthermore, safety experiments have verified that even when high concentrations of the complex and eye drops described in this invention are applied to the ocular surface of mice or rabbits, no ocular abnormalities are caused. Attached Figure Description

[0036] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0037] Figure 1 This is a synthetic pathway diagram for the preparation of L-cysteine-modified thiol chitosan in Example 1 of the present invention.

[0038] Figure 2 Fourier transform infrared spectra of thioglycolic chitosan with different degrees of L-cysteine ​​modification prepared in Example 2 of this invention.

[0039] Figure 3 This is an electron microscope image of the experiment in Example 2 of the present invention to prepare thiol chitosan / catalase nanocomposite.

[0040] Figure 4This is a statistical graph showing the fluorescence signal intensity of catalase labeled with Cy5.5 fluorescent substance loaded with thiol chitosan of different degrees of substitution on mouse cornea, obtained from the experiment of screening the optimal chitosan to L-cysteine ​​feeding ratio in Example 4 of the present invention.

[0041] Figure 5 This is a statistical graph showing the fluorescence signal intensity of Cy5.5 fluorescently labeled catalase on the mouse cornea over time, obtained from the residence time test of thiol chitosan / catalase eye drops on the mouse ocular surface in Example 5 of the present invention.

[0042] Figure 6 This is a line graph showing the changes in tear secretion in mice as obtained from the experiment of using the thiol chitosan / catalase nanocomposite eye drops in Example 6 of the present invention to treat benzalkonium chloride-induced evaporative dry eye in mice.

[0043] Figure 7 This is a line graph showing the changes in sodium fluorescein staining scores of mouse corneas obtained from the experiment of using thiol-chitosan / catalase nanocomposite eye drops to treat benzalkonium chloride-induced evaporative dry eye in mice, as described in Example 6 of this invention.

[0044] Figure 8 This is a line graph showing the changes in tear secretion in mice as obtained from the experiment of using the thiol chitosan / catalase nanocomposite eye drops in Example 6 of the present invention to treat scopolamine-induced dry eye syndrome in mice with insufficient secretion.

[0045] Figure 9 This is a line graph showing the changes in corneal fluorescein staining scores in mice obtained from the experiment of using the thiol-chitosan / catalase nanocomposite eye drops in Example 6 of the present invention to treat scopolamine-induced dry eye syndrome in mice with insufficient secretion.

[0046] Figure 10 This is a line graph showing the changes in tear secretion in New Zealand white rabbits obtained from the experiment of using the thiol chitosan / catalase nanocomposite eye drops in Example 6 of the present invention to treat atropine-induced dry eye in rabbits.

[0047] Figure 11 This is a line graph showing the changes in corneal fluorescein staining scores of New Zealand white rabbits obtained from the experiment of using thiol chitosan / catalase nanocomposite eye drops to treat atropine-induced dry eye in rabbits in Example 6 of this invention.

[0048] Figure 12 This is a line graph showing the changes in intraocular pressure in mice obtained from the ocular safety experiment of the eye drops prepared by the thiol chitosan / catalase nanocomposite in Example 7 of the present invention.

[0049] Figure 13This is a slit-lamp image of a mouse eyeball obtained from an ocular safety experiment of the eye drops prepared by the thiol chitosan / catalase nanocomposite in Example 7 of this invention.

[0050] Figure 14 This is a statistical chart of the sodium fluorescein staining scores of the eyes of mice in each group in Example 8 of the present invention.

[0051] Figure 15 This is a statistical graph showing the detection of tear secretion in the eyes of mice in each group in Example 8 of the present invention. The vertical axis represents the length of cotton thread soaking. Detailed Implementation

[0052] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0053] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0054] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0055] Unless otherwise specified, all raw materials and reagents used in this invention are commercially available.

[0056] Example 1

[0057] 1. Preparation and characterization of cysteine-modified chitosan

[0058] Using chitosan with a molecular weight of 50 kDa and a degree of deacetylation ≥92% as raw material, under nitrogen protection, an EDC / NHS activated cysteine ​​solution was added dropwise to the chitosan solution, and the mixture was stirred at room temperature in the dark for 5 hours. After the reaction, tris(2-carboxyethyl)phosphine (TCEP) was added in an equimolar amount to reduce the cross-linked cysteine. Figure 1 As shown; the product was then purified by dialyzing in 1 mM hydrochloric acid for 48 h, with the dialysis buffer being changed as needed during the process; after dialysis, the cysteine-modified chitosan solution was collected, freeze-dried, and then sealed for storage.

[0059] Cysteine-modified chitosan with different feed ratios (molar ratio, cysteine:chitosan) was prepared according to the above process. The cysteine-modified chitosan powder was mixed with potassium bromide, ground, and compressed into tablets. The chemical bonds of the products were detected by Fourier transform infrared spectroscopy, and the products were grouped as follows:

[0060] Control group (cs): Unmodified chitosan;

[0061] Experimental group 1 (cs-cys1): The ratio of chitosan to cysteine ​​was 1:1;

[0062] Experimental group 2 (cs-cys2): The ratio of chitosan to cysteine ​​was 1:2;

[0063] Experimental group 3 (cs-cys3): The ratio of chitosan to cysteine ​​was 1:3;

[0064] Figure 2 These are the Fourier transform infrared (FTIR) spectra of each group. The results show that, compared to the control group, experimental groups 1-3 showed higher FTIR values ​​at 1710 cm⁻¹. -1 Significant changes in absorption intensity were observed at all locations, and this characteristic peak proved the presence of amide bonds, indicating that cysteine ​​was successfully grafted onto chitosan.

[0065] The degree of cysteine ​​substitution in cysteine-modified chitosan was quantitatively determined using 5,5'-dithio-bis-(2-nitrobenzoic acid). The free thiol content in samples from experimental groups 1 to 3 was detected according to the test method provided by the supplier. The degree of cysteine ​​substitution was calculated using the following formula, and the results are shown in Table 1.

[0066] Degree of substitution = (Molar amount of free thiol groups / Molar amount of free amino groups in chitosan) * 100%

[0067] Table 1

[0068]

[0069] The results showed that as the amount of cysteine ​​added increased, its degree of substitution on chitosan also increased. In practical applications, the degree of substitution of cysteine ​​in the product can be controlled by adjusting the amount of cysteine ​​added, thereby controlling the adhesion properties of the product.

[0070] Example 2

[0071] 1. Preparation and characterization of the complex

[0072] The cysteine-modified chitosan powder was reconstituted with ultrapure water, and the pH of the solution was adjusted to 6.4 to obtain solution A.

[0073] Catalase was dissolved in PBS, and the pH of the solution was adjusted to 9.0 to obtain solution B;

[0074] The above solutions A and B were mixed, with the mass ratio of cysteine-modified chitosan to catalase being 1:1. The mixture was stirred in a vortex mixer for 10 minutes to prepare the complex.

[0075] Complexes of chitosan modified with cysteine ​​and catalase with different degrees of substitution were prepared according to the above method, and their average particle size distribution and surface potential were tested. The results are shown in Table 2.

[0076] Table 2

[0077]

[0078]

[0079] Particle size test results showed that the composite particles in control group 2 were larger. This is because the chitosan molecule contains a large number of amino and acetamino groups, which form hydrogen bonds inside the molecule, enhancing intramolecular interactions and making chitosan more prone to aggregation. As a result, the solubility in water is low and the composite particles are larger. In contrast, the assembly effect of the composites in experimental groups 2-2 to 2-3 was more ideal, with an average particle size of about 200 nm.

[0080] Based on the surface potential test results, it can be seen that the complex formed by the combination of negatively charged catalase and chitosan carries a large amount of positive charge (control group 2). In experimental groups 2-2 to 2-3, the amount of positive charge of the complex decreased significantly, indicating that catalase and cysteine-modified chitosan self-assembled through electrostatic interaction, with good assembly effect and uniform particle size distribution.

[0081] In experimental groups 2 and 3, the complexes rapidly precipitated after assembly because the cysteine ​​modification of chitosan was too high, and oxidative cross-linking between free thiol groups led to the precipitation of the complexes, which is not conducive to practical use.

[0082] The morphology of the complexes in control group 1 and experimental groups 2-2 to 2-3 was characterized by transmission electron microscopy. Figure 3 As shown, the results indicate that the complex structure in control group 1 is relatively loose, while the complexes in experimental groups 2-2 to 2-3 exhibit a uniform spherical morphology, further verifying the good assembly effect of cysteine-modified chitosan and catalase.

[0083] Example 3

[0084] 1. Statistical analysis of particle size and surface potential of composites prepared with different feed ratios.

[0085] The complex was prepared according to the method described in Example 2, wherein the degree of cysteine ​​modification on the chitosan was 4.2%. The difference was that the mass ratio of cysteine-modified chitosan to catalase was different. The test results of its average particle size distribution and surface potential are shown in Table 3.

[0086] Table 3

[0087]

[0088]

[0089] All complexes carried positive charges, verifying the successful self-assembly process. However, the complex in experimental group 3-1 had a larger particle size and was easily precipitated from the solution, while the complexes in experimental groups 3-2 to 3-5 had uniform particle size and showed good self-assembly effect.

[0090] Example 4: Ocular surface adhesion test of the complex

[0091] A complex of fluorescently labeled catalase and cysteine-modified chitosan was prepared according to the method described in Example 2, wherein the catalase was labeled with fluorescein Cy5.5.

[0092] Specifically, after anesthetizing mice with isoflurane, 5 μL of sample solution (7.5 μg / eye based on catalase) was instilled into the eye. Two hours later, eyeball samples were collected and fixed with paraformaldehyde for one hour. The mouse cornea was dissected under an optical microscope, and cut from the outer edge towards the center to allow it to lie flat on a glass slide, preparing a mounting sample. Fluorescence signals from the mounting samples were acquired using a Zeiss confocal microscope and quantitatively analyzed using ImageJ.

[0093] The embodiments are grouped as follows:

[0094]

[0095] Figure 4 The results show the quantitative statistical results of the fluorescence signal intensity of the cornea in each group of mice. In experimental groups 4-1 to 4-3, the Cy5.5 fluorescence signal in the cornea of ​​mice was significantly enhanced compared with that in control group 1, while there was no significant change in control group 2. This indicates that the cysteine-modified chitosan has a significant corneal adhesion effect, and its assembly can help the protein remain in the anterior segment of the mouse eye. The fluorescence signal in experimental group 4-2 was slightly higher than that in the other two groups, which is presumably because the particle size of the complex in this group is more uniform and easier to remain on the ocular surface.

[0096] Example 5: Retention time experiment of the complex

[0097] A complex of Cy5.5-labeled catalase and cysteine-modified chitosan was prepared according to the method described in Example 2, wherein the degree of substitution of cysteine ​​on the chitosan was 4.2%, and the mass ratio of catalase to cysteine-modified chitosan was 1:1.

[0098] The specific experimental steps are as follows:

[0099] After mice were anesthetized with isoflurane, 5 μL of sample solution was instilled into the eye (the dosage was 7.5 μg / eye based on catalase). After instillation, the mouse eyeballs were collected and corneal sections were prepared at different time points. The fluorescence signal of the mounted samples was acquired using a Zeiss confocal microscope and the fluorescence signal was quantitatively analyzed using ImageJ.

[0100] Figure 5 The results are quantitative statistical results of fluorescence intensity signals on the mouse cornea at different time points after drug administration. The results show that the fluorescence intensity in the mouse cornea gradually decreases with increasing time. At 12 hours, a certain amount of Cy5.5 fluorescence signal is still present on the mouse cornea, indicating that the complex can achieve long-term corneal retention. Compared to existing eye drop formulations that are rapidly cleared, the complex described in this application can reduce the frequency of drug administration, improve drug bioavailability, thereby improving patient compliance, and simultaneously control costs.

[0101] Example 6: Therapeutic efficacy experiment of the compound

[0102] 1. Treatment of a benzalkonium chloride-induced model of tear hyperevaporation-type dry eye syndrome

[0103] 1.1 Modeling Methods:

[0104] A dry eye model of excessive tear evaporation was established in mice by administering 5 μL of 0.2% benzalkonium chloride eye drops twice daily, morning and evening, with an interval of approximately 12 hours between drops. This treatment was continued for one week to establish the dry eye model. This modeling method induced clinical manifestations and related histopathological changes, including impaired tear film stability, ocular surface inflammation, corneal epithelial cell apoptosis, and goblet cell loss.

[0105] Experimental Groups:

[0106] Control Example A1.0: Healthy mice that did not undergo dry eye modeling;

[0107] Control Example A1.1: Dry eye mice, treated with PBS eye drops daily;

[0108] Control Example A1.2: Dry eye mice were given daily eye drops of catalase solution at a concentration of 1.5 mg / mL;

[0109] Control Example A1.3: Dry eye mice were given daily eye drops of a chitosan solution modified with cysteine, with a cysteine ​​substitution degree of approximately 4.2% and a concentration of 1.5 mg / mL;

[0110] Example A1.4: Dry eye mice were given eye drops of the complex solution obtained in Example B1.3 daily at a concentration of 1.5 mg / mL (calculated as catalase);

[0111] Control Example A1.5: Dry eye mice were given commercially available cyclosporine preparation (trade name: Zirun) eye drops daily at a concentration of 0.05% (commercial concentration);

[0112] Control Example A1.0: Healthy mice that did not undergo dry eye modeling;

[0113] Control Example A1.1: Dry eye mice, treated with PBS eye drops daily;

[0114] Control Example A1.2: Dry eye mice were given daily eye drops of catalase solution at a concentration of 1.5 mg / mL;

[0115] Control Example A1.3: Dry eye mice were given daily eye drops of a chitosan solution modified with cysteine, with a cysteine ​​substitution degree of approximately 4.2% and a concentration of 1.5 mg / mL;

[0116] Example A1.4: Dry eye mice were given eye drops of the complex solution obtained in Example B1.3 daily at a concentration of 1.5 mg / mL (calculated as catalase);

[0117] Control Example A1.5: Dry eye mice were given commercially available cyclosporine preparation (trade name: Zirun) eye drops daily at a concentration of 0.05% (commercial concentration);

[0118] Mice in each group were administered the drug twice daily, with an interval of approximately 12 hours. The drug volume was 5 μL per eye, and the administration was continued for 7 days.

[0119] 1.2 Efficacy evaluation methods / testing methods:

[0120] Corneal fluorescein staining score: After anesthetizing the mice, fluorescein stain was applied to the eyes, rinsed with physiological saline, dried with cotton balls, and the mice's eyeballs were photographed under slit lamp blue light mode. The scores were then based on the stained area.

[0121] Tear secretion test: After anesthetizing the mice, the mouse eyelids were opened, and phenol red cotton thread was placed at the outer 1 / 3 of the mouse eyelid with tweezers. It was left for 30 seconds and then removed. The length of the cotton thread that was wetted by tears was measured.

[0122] Experimental results: The corneal epithelial defect status of mice was evaluated by corneal fluorescein staining on days 0, 3, 5, and 7 after drug administration. Figure 6The corneal epithelium of healthy mice (control group A1.0) showed almost no defects, and the fluorescein staining was negligible. In contrast, the scores of mice in control group A1.1 were all greater than 3, indicating severe dry eye symptoms. The scores of control groups A1.2 and A1.3 were reduced, indicating that catalase or cysteine-modified chitosan alone can alleviate dry eye symptoms such as corneal epithelial damage as an indicator of fluorescein staining. The score of experimental group A1.4 decreased significantly after 3 days of treatment, which was better than that of commercial reagents (control group A1.5). After 5 days of treatment, the score recovered to a level similar to that of healthy mice, confirming the superior efficacy of the complex formed by catalase and cysteine-modified chitosan.

[0123] Tear secretion in mice was evaluated on days 0, 3, 5, and 7 after drug administration. Figure 7 The results showed that control groups A1.2 and A1.3 did not significantly help restore tear secretion in mice. This may be because catalase has a short residence time on the ocular surface, and cysteine-modified chitosan does not have the effect of reducing inflammation, thus failing to effectively improve ocular oxidative stress symptoms. In contrast, after 3 days of treatment, experimental group A1.4 significantly increased tear secretion in mice, restoring it to levels similar to healthy mice, and demonstrated better therapeutic efficacy compared to commercially available drugs.

[0124] Based on the above test results, cysteine-modified chitosan can form disulfide bonds with the abundant cysteine ​​on the ocular surface, thereby adhering to the ocular surface for a long time, playing a lubricating and symptom-relieving role. Furthermore, after assembling with catalase to form the complex described in this application, it can help it stay on the ocular surface, effectively clear ROS, relieve ocular inflammation, inhibit cell apoptosis, and effectively relieve benzalkonium chloride-induced evaporative dry eye syndrome.

[0125] Because commercially available cyclosporine drugs require a longer duration of action to treat dry eye by modulating T-cell-related immune responses (the instructions suggest a treatment cycle of three months), the control group A1.5 did not show a significant therapeutic effect 7 days after administration. This further comparison demonstrates that the complex described in this application can rapidly relieve dry eye symptoms in a short period of time and has high therapeutic efficiency.

[0126] 2. Treatment of a scopolamine-induced model of dry eye with insufficient tear secretion

[0127] 2.1 Modeling Methods:

[0128] A dry eye model with insufficient tear secretion was established in mice by subcutaneous injection of scopolamine hydrobromide solution, 0.5 mg / mouse, at a dose of 0.2 mL, four times daily for one week. This modeling method competitively inhibits lacrimal gland M receptors, significantly reducing tear secretion.

[0129] The mice were grouped and treated according to the protocol in 1.1 of Example 6, and were numbered as Control Example A2.0, Control Example A2.1, Control Example A2.2, Control Example A2.3, Example A2.4, and Control Example A2.5, respectively. The corneal damage and tear secretion of the mice were evaluated on days 0, 3, 5, and 7 after administration.

[0130] Experimental results:

[0131] Figure 8 The results are the results of sodium fluorescein staining of mouse corneas. The complex described in this application also rapidly promoted the recovery of damaged corneal epithelium in the scopolamine-induced dry eye model. Three days after treatment, the sodium fluorescein score of A2.4 mice in the experimental group was significantly reduced and remained at a level close to that of the control group A2.0.

[0132] Figure 9 The results of the tear secretion test showed that the tear secretion of the A2.4 mice in the experimental group increased significantly after treatment, and recovered to a level similar to that of the healthy group after 7 days, which was significantly better than that of other control groups. This further illustrates the function of the compound described in this application in lubricating the ocular surface, improving inflammatory response and tear secretion, and showing a more ideal therapeutic effect in a dry eye model with severe tear secretion deficiency.

[0133] 3. Treatment of atropine-induced dry eye model in rabbits

[0134] 3.1 Modeling Methods:

[0135] A dry eye model was established in New Zealand white rabbits by administering 50 μL of 2% atropine eye drops twice daily, morning and evening, with an interval of approximately 12 hours between drops. This treatment was continued for 14 days. During subsequent treatment, 50 μL of 2% atropine was administered once daily to delay the rabbits' natural recovery.

[0136] 3.2 Efficacy evaluation method: Corneal fluorescein sodium staining was performed by two ophthalmologists in a double-blind professional scoring process.

[0137] Experimental Groups:

[0138] Control Example A3.1: Rabbits with dry eye syndrome were given PBS eye drops daily;

[0139] Example A3.2: Rabbits with dry eye syndrome were given eye drops of the complex solution obtained in Example B1.3 daily at a concentration of 1.5 mg / mL (calculated as catalase).

[0140] Example A3.3: Rabbits with dry eye syndrome were given commercially available cyclosporine preparation (trade name: Zirun) eye drops daily at a concentration of 0.05%;

[0141] The rabbits were administered the drug twice a day, with an interval of approximately 12 hours, and the drug volume was 50 μL / eye for 14 days.

[0142] Experimental results:

[0143] The efficacy was evaluated on days 0, 3, 7, 10, and 14 after drug administration. Results showed that in the control group (A3.1), dry eye symptoms remained significant after 14 days of treatment, tear secretion did not increase significantly, and the sodium fluorescein staining score showed no significant change. Figure 10 This indicates that the corneal epithelial damage in the rabbits remained severe; after 3 days of treatment with compound eye drops, the fluorescein sodium staining score of the rabbits significantly decreased, the corneal epithelial damage was significantly alleviated, and tear secretion also increased. Figure 11 After 14 days of treatment, tear secretion further increased, and corneal epithelial damage was further alleviated. The complex described in this application contains high-molecular-weight polymer components that can lubricate the ocular surface and protect the ocular mucosa, demonstrating significant efficacy advantages compared to commercially available cyclosporine preparations.

[0144] This embodiment further validated the efficacy of the compound described in this application in a rabbit dry eye model with a more complex structure and larger eyeball volume. Rapid relief was achieved 3 days after administration, and it still showed significantly better efficacy than commercially available cyclosporine preparations after 14 consecutive administrations, further demonstrating its potential in the treatment of dry eye.

[0145] Example 7 Security Verification

[0146] 1. Experimental Groups:

[0147] Control Example B1.1: Healthy mice, treated with PBS eye drops daily;

[0148] Example B1.2: Healthy mice were given eye drops of the complex solution obtained in Example B1.3 daily at a concentration of 1.5 mg / mL (calculated as catalase).

[0149] The mice were administered the drug twice daily, with an interval of approximately 12 hours, at a dose of 5 μL per eye, for 30 days.

[0150] 2. Safety evaluation methods:

[0151] Intraocular pressure (IOP) measurement: IOP was measured in mice at fixed time points every 3 days under unanesthesia using an iCareTONOLAB mouse and rat tonometer. During measurement, the probe momentarily contacted the mouse's cornea to obtain the IOP value. IOP values ​​were displayed in millimeters of mercury (mmHg).

[0152] Corneal condition examination: Mice were anesthetized every 7 days, and eye photos were recorded using a slit lamp. Subsequently, sodium fluorescein dye was applied to the eyes, and after rinsing with saline, the eyes were dried with cotton balls, and eye photos were recorded under slit lamp blue light mode.

[0153] Experimental results:

[0154] Figure 12 The results are the intraocular pressure measurements in mice. The monitoring results show that the intraocular pressure changes in mice were all within the normal range during the administration period, indicating that long-term administration of the compound via eye drops will not increase intraocular pressure or cause glaucoma or other related side effects.

[0155] Figure 13 The images are slit-lamp images of mouse eyes. The results show that compared with the control group B1.1, there was no significant difference in the corneal and limbal condition of mice in the experimental group B1.2. The anterior chamber maintained high clarity, and the results of corneal fluorescein sodium staining were all negative during the dosing period. This indicates that the complex described in this application has no obvious ocular irritation and can be used for a long time.

[0156] In summary, the compound described in this invention has high safety for eye drop treatment and will not cause side effects such as increased intraocular pressure or eye irritation.

[0157] Example 8: Effective Concentration Exploration Experiment

[0158] 1. Modeling and Treatment: A mouse dry eye model was established by administering 0.2% benzalkonium chloride eye drops twice daily, 5 μL / eye, at 9 am and 9 pm for 7 consecutive days. Then, different samples were administered as eye drops twice daily, 5 μL each time, while maintaining dry eye stimulation with 5 μL / eye of 0.2% benzalkonium chloride once daily.

[0159] 2. Experimental Groups:

[0160] Control group C1.1: Healthy mice;

[0161] Control group C1.2: Modeling mice, sample was PBS;

[0162] Experimental group C1.3: Modeling mice, the sample was a 1.5 mg / mL compound eye drop formulation;

[0163] Experimental group C1.4: Modeling mice, the sample was a 0.5 mg / mL compound eye drop formulation;

[0164] Experimental group C1.5: Modeling mice, the sample was a 0.15 mg / mL compound eye drop formulation.

[0165] 3. Detection method:

[0166] On days 2, 4, and 7 of the start of treatment, the degree of corneal damage in mice was evaluated using sodium fluorescein staining, and the amount of tear secretion in mice was detected using phenol red cotton thread.

[0167] 4. Experimental Results:

[0168] Figure 14 The statistical charts of sodium fluorescein staining scores in the eyes of mice in each group show that different concentrations of the complex can significantly improve corneal damage in mice, resulting in a stable decrease in sodium fluorescein staining scores, which are close to the sodium fluorescein staining scores of healthy mice. This indicates that the complex described in this invention can improve corneal damage in dry eye at different concentrations.

[0169] Figure 15 The graph shows the statistical analysis of tear secretion in mice across different groups, with the vertical axis representing the length of cotton thread soaked in the liquid. The results indicate that the compound increases tear secretion in mice, showing a positive correlation with the compound concentration. Higher concentrations result in more significant improvement, even leading to higher tear secretion than in healthy mice, thus greatly alleviating dry eye symptoms. Therefore, the compound described in this invention can treat dry eye in mice at different concentrations, demonstrating significant application potential.

[0170] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A complex, characterized in that: The mixture includes cysteine-modified chitosan and peroxidase, wherein the cysteine-modified chitosan and peroxidase form a stable complex, and the mass ratio of cysteine-modified chitosan to peroxidase in the complex is 1:4 to 4:

1.

2. The complex according to claim 1, characterized in that: The surface cysteine ​​modification degree of the cysteine-modified chitosan is on average 0.5-8%.

3. The complex according to claim 1, characterized in that: The cysteine-modified chitosan has a surface free thiol content of 100–280 micromoles per gram of polymer.

4. The complex according to claim 1, characterized in that: The average particle size of the composite is 50–1000 nm.

5. The complex according to claim 1, characterized in that: The peroxidase is one or both of catalase and superoxide dismutase.

6. The use of the complex according to claim 1 in the preparation of eye drop formulations, characterized in that: The eye drop formulation contains the complex.

7. The application as described in claim 6, characterized in that: The concentration of the complex in the eye drop formulation is 0.15–1.5 mg / mL.

8. The application as described in claim 6, characterized in that: The eye drop formulation contains one or more of the following: solvent, preservative, reducing agent, thickener, pH adjuster, antibacterial agent, and bactericide.

9. The application as described in claim 6, characterized in that: The pH of the eye drops formulation is 5.0–9.

0.

10. The application as described in claim 6, characterized in that: Application of a compound or eye drop formulation in the preparation of a drug for treating dry eye syndrome.

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