Biomacromolecular drug for ameliorating inflammation of ocular surface or superficial mucosa, preparation method therefor, and use thereof

Abstract

Provided are a biomacromolecular drug for ameliorating inflammation of the ocular surface or superficial mucosa, a preparation method therefor, and the use thereof. The biomacromolecular drug is mucoadhesive and can achieve prolonged retention on the ocular surface; the local oxidative stress level is ameliorated by removing reactive oxygen species from the ocular surface or superficial mucosa, thereby alleviating diseases caused by inflammation.

Description

Biological macromolecular drugs for improving superficial inflammation of the ocular surface or mucous membranes, preparation methods and applications Technical Field

[0001] This invention relates to the field of inflammatory disease treatment, and more particularly to a biomolecular drug for improving superficial inflammation of the ocular surface or mucous membrane, its preparation method, and its application. Background Technology

[0002] The human body's antioxidant enzyme system effectively eliminates reactive oxygen species (ROS) generated during metabolic activities, protecting biomolecules from oxidative damage caused by ROS free radicals. This system primarily consists of superoxide dismutase, glutathione peroxidase, catalase, thioredoxin, and other antioxidant enzymes. An imbalance between antioxidants and oxidants can lead to oxidative damage, and diseases can also cause oxidative damage, thus affecting the stability of the body's antioxidant enzyme system.

[0003] For example, dry eye syndrome can be caused by multiple factors. Existing research shows that reactive oxygen species (ROS) that the ocular surface tissue's antioxidant defense system cannot clear in time accumulate in large quantities in front of the eyes, leading to oxidative stress damage to the ocular surface. This further leads to an imbalance in the antioxidant defense system. Ocular surface damage and the imbalance of the antioxidant defense system mutually promote each other, jointly causing the ocular surface inflammation to worsen and exacerbate dry eye. Therefore, existing technologies disclose the use of superoxide dismutase or its mimics to directly clear ROS, directly reduce the level of reactive oxygen species on the ocular surface, and alleviate dry eye. In addition, acute inflammatory damage to the lungs can also cause the mucosal surface antioxidant enzyme system to work overtime or even fail to function normally. Furthermore, the large accumulation of reactive oxygen species on the mucosal surface has a counterproductive effect on the antioxidant defense system, which is detrimental to the treatment of inflammation.

[0004] Currently, there is a lack of technology that regulates the balance of local antioxidant enzyme systems to help these systems get back to work, alleviate dry eye symptoms, or even cure dry eye syndrome; or that this balancing technology can help treat and restore inflammatory damage to the mucous membranes of areas such as the lungs. Summary of the Invention

[0005] To address at least one of the problems existing in the prior art, this invention provides a biomolecular drug for improving superficial inflammation of the ocular surface or mucous membranes, its preparation method, and its application. The biomolecular drug can remain on the ocular surface for a relatively long time, reducing excess hydrogen peroxide on the ocular surface. The applicant unexpectedly discovered that the biomolecular drug for improving superficial inflammation of the ocular surface or mucous membranes of this application can promote a positive progression of redox balance, correct imbalances in the antioxidant defense system, thereby improving the level of reactive oxygen species in the superficial ocular or mucous membranes, improving the local inflammatory environment, and directly achieving a therapeutic effect.

[0006] On the one hand, this application provides a biological macromolecular drug for improving superficial inflammation of the ocular surface or mucosa. The biological macromolecular drug contains catalase, which is chemically grafted with adhesive groups. Catalase with adhesive groups is called mucosal adhesive catalase.

[0007] Furthermore, the adhesive group is selected from one or more of thiol, disulfide bond, thioether and maleimide groups.

[0008] Furthermore, the molar ratio W of the adhesive group to the catalase monomer is an integer, and 0 < W ≤ 20.

[0009] Furthermore, W can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0010] Furthermore, the molar ratio of the adhesive group to catalase is approximately (0–72):1.

[0011] Furthermore, the molar ratio of the adhesive group to catalase is approximately (0–72):1, and the molar ratio of the adhesive group to catalase is not 0:1.

[0012] Furthermore, the molar ratio of the adhesive group to catalase is set to N, where N is in the range of (0:1) < N ≤ (72:1). That is, 0 < N ≤ 72.

[0013] Furthermore, N is no greater than 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, or 72.

[0014] Furthermore, the mass fraction of the mucosal adhesive catalase in the biopharmaceutical is not less than 0.001 wt%.

[0015] Furthermore, the mass fraction of the mucosal adhesive catalase in the biopharmaceutical is not less than 0.001 wt%, 0.002 wt%, 0.004 wt%, 0.005 wt%, 0.006 wt%, 0.008 wt%, 0.009 wt%, 0.01 wt%, 0.015 wt%, 0.02 wt%, 0.05 wt%, 0.10 wt%, and 0.15 wt%. 0.20wt%, 0.30wt%, 0.45wt%, 0.50wt%, 0.60wt%, 0.80wt%, 0.90wt%, 1.00wt%, 1.20wt%, 1.40wt%, 1.50wt%, 1.60wt%, 1.80wt%, 1.90wt%, 2.00wt%, 2.10wt%, 2.30wt%, or 2.50wt%.

[0016] Furthermore, the concentration of the mucosal adhesive catalase in the biomolecular drug is not less than 0.001 mg / mL.

[0017] Furthermore, the mass fraction of the mucosal adhesive catalase in the biopharmaceutical is 0.001 wt% to 2.5 wt%.

[0018] Specifically, the mass concentration range of the mucosal adhesive catalase in the biopharmaceutical is 0.001 mg / mL to 10 mg / mL.

[0019] Furthermore, the concentration of the mucosal adhesive catalase in the biomolecular drug is approximately 0.001 mg / mL to 1 mg / mL.

[0020] Furthermore, the concentration of the mucosal adhesive catalase in the biopharmaceutical is not greater than 0.002 mg / mL, 0.004 mg / mL, 0.006 mg / mL, 0.008 mg / mL, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, or 0.10 mg / mL. 0.11mg / mL, 0.12mg / mL, 0.13mg / mL, 0.14mg / mL, 0.15mg / mL, 0.16mg / mL, 0.17mg / mL, 0.18mg / mL, 0.19mg / m L, 0.20mg / mL, 0.21mg / mL, 0.22mg / mL, 0.23mg / mL, 0.24mg / mL, 0.25mg / mL, 0.26mg / mL, 0.27mg / mL, 0.28mg / mL, 0.29mg / mL, 0.30mg / mL, 0.31mg / mL, 0.32mg / mL, 0.33mg / mL, 0.34mg / mL, 0.35mg / mL, 0.36mg / mL, 0.37m g / mL, 0.38mg / mL, 0.39mg / mL, 0.40mg / mL, 0.41mg / mL, 0.42mg / mL, 0.43mg / mL, 0.44mg / mL, 0.45mg / mL, 0.4 6mg / mL, 0.47mg / mL, 0.48mg / mL, 0.49mg / mL, 0.50mg / mL, 0.52mg / mL, 0.54mg / mL, 0.56mg / mL, 0.58mg / mL, 0 .60mg / mL, 0.65mg / mL, 0.70mg / mL, 0.75mg / mL, 0.80mg / mL, 0.85mg / mL, 0.90mg / mL, 0.95mg / mL or 1.00mg / mL.

[0021] On the one hand, the present invention also provides a method for preparing a biomacromolecule drug, including a step of chemically modifying the surface of catalase.

[0022] Specifically, the preparation method includes the step of chemically grafting an adhesive group donor with the peroxidase.

[0023] Further, the molar ratio of the adhesive group donor to catalase is approximately (0-300):1. Even further, the molar ratio of the adhesive group donor to catalase is approximately (0-300):1, and the molar ratio of the adhesive group donor to catalase is not 0:1.

[0024] Furthermore, the molar ratio of the adhesive group donor to catalase is set as M, and the range of M is: (0:1) < M ≤ (300:1). That is, 0 < M ≤ 300.

[0025] Further, the molar ratio of the adhesive group donor to the mucosal adhesive catalase is approximately (0-1):1, (1-5):1, (5-10):1, (10-20):1, (20-30):1, (30-50):1, (50-75):1, (75-90):1, (90-100):1, (100-120):1, (120-140):1, (120-160):1, (160-180):1, (180-200):1, (200-240):1, (240-280):1, or (280-300):1.

[0026] Specifically, M is no greater than 1, 5, 10, 20, 30, 40, 50, 60, 75, 80, 90, 100, 120, 150, 160, 180, 200, 220, 260, 280 or 300.

[0027] Furthermore, the preparation method includes a product purification step.

[0028] Furthermore, the preparation method includes the step of adding excipients.

[0029] On the one hand, the present invention also provides the application of a biomacromolecule drug containing catalase in the preparation of an ocular disease treatment agent, wherein the ocular disease includes any one or more of dry eye syndrome, corneal chemical burns, and allergic conjunctivitis.

[0030] On the one hand, the present invention also provides the application of a biomacromolecule drug containing catalase in the preparation of a therapeutic agent for pulmonary mucosal inflammation, wherein the pulmonary mucosal inflammation is selected from any one or more of acute lung injury, asthma, and chronic obstructive pulmonary disease.

[0031] According to the technical solution of the present invention, the following beneficial effects are achieved:

[0032] 1) The mucosal adhesion protein described in this invention can remain on the corneal surface for a longer period, prolonging the action time of catalase. This allows catalase to fully exert its function of clearing excess reactive oxygen species from the ocular surface, inhibiting oxidative stress damage from the upstream of dry eye pathogenesis, thereby suppressing inflammatory responses and ocular surface cell damage, and improving the ocular surface microenvironment. This not only improves tear secretion but also promotes corneal repair. Furthermore, the products of catalase's direct catalysis of hydrogen peroxide are non-toxic and harmless, with high scavenging efficiency, blocking the formation of active free radical reactive oxygen species. This blocks the damage of reactive oxygen species to the body from the upstream of the entire antioxidant enzyme system. Compared with the glutathione peroxide reduction mechanism, it does not require the participation of other substrates. In summary, the catalase used in this invention is lower in cost, safer, more efficient, and more effective than other antioxidant enzymes.

[0033] 2) Compared with existing hormone and steroid drugs used for dry eye treatment, it has the advantages of high efficiency and safety.

[0034] 3) The drug preparation method described in this invention is simple, produces few byproducts, and is relatively easy to purify, thus offering advantages such as controllable cost and ease of mass production. Furthermore, catalase, as a naturally occurring biomolecule protease in the human body, exhibits better biocompatibility and safety compared to non-natural substances such as biomimetic nanozymes.

[0035] 4) The mucosal adhesion protein described in this invention can improve a variety of inflammation-related eye diseases or lung mucosal inflammation-related diseases. It has a wide range of indications and applicable populations, and has great clinical demand and application prospects. Attached Figure Description

[0036] Figure 1 shows the SDS-PAGE electrophoresis bands of each group of samples in Example B1 after modification under different conditions;

[0037] Figure 2 is a statistical chart of enzyme activity detection in different samples in Example B2;

[0038] Figure 3 shows the mass spectra of different samples in Example B3, with the horizontal axis representing molecular weight (in Da) and the vertical axis representing intensity counts (in counts).

[0039] Figure 4 shows fluorescence micrographs of isolated corneas of mice in each group of Example C1;

[0040] Figure 5 is a statistical graph of fluorescence signal intensity of isolated mouse corneas in each group in Example C1;

[0041] Figure 6 is a statistical chart of the length of cotton thread soaked with tears in each group of mice in Example C2;

[0042] Figure 7 is a statistical chart of corneal fluorescein sodium staining scores of mice in each group in Example C2;

[0043] Figure 8 is a statistical chart of the length of cotton thread soaked with tears in mice in Example C3;

[0044] Figure 9 is a statistical chart of corneal fluorescein sodium staining scores in mice in Example C3;

[0045] Figure 10 is a statistical chart of the length of cotton thread soaked with tears in each group of mice in Example C4;

[0046] Figure 11 is a statistical chart of corneal fluorescein sodium staining scores of mice in each group in Example C4;

[0047] Figure 12 is a statistical chart of sodium fluorescein staining scores of five groups of mice on the second day after chemical corneal burns in Example D1.

[0048] Figure 13 is a statistical chart of the corneal opacity scores of each group of mice in Example D1.

[0049] Figure 14 is a statistical chart of corneal neovascularization scores in each group of mice in Example D1;

[0050] Figure 15 is a statistical chart of the incidence of cataracts in mice in each group in Example D1;

[0051] Figure 16 shows the OCT images of the anterior segment of mice in each group in Example D1;

[0052] Figure 17 is a line graph showing the changes in ocular symptom scores of mice in each group over time in Example D2;

[0053] Figure 18 is a statistical chart of the number of times mice in each group scratched their eyes within 10 minutes after receiving local stimulation in Example D2.

[0054] Figure 19 is a statistical chart of the relative serum OVA-SIgE content of mice in each group on day 7 in Example D2;

[0055] Figure 20 is a statistical graph of the fluorescence signal intensity (mean radiation intensity) in the lungs of mice in different groups in Example E1;

[0056] Figure 21 is a statistical graph of the concentration of IL-1β in the lung lavage fluid of mice in Example E2;

[0057] Figure 22 is a statistical graph of the concentration of TNF-α in the lung lavage fluid of mice in Example E2;

[0058] Figure 23 is a statistical graph of the concentration of IL-6 in the lung lavage fluid of mice in Example E2;

[0059] Figure 24 is a statistical chart of the percentage of macrophages in the lung lavage fluid of mice in Example E2;

[0060] Figure 25 is a statistical chart of the percentage of neutrophils in the lung lavage fluid of mice in Example E2;

[0061] Figure 26 is a micrograph of a section of mouse lung tissue from Example E2. Detailed Implementation

[0062] To further illustrate the technical means and effects of the present invention in order to achieve the intended purpose, the following detailed description, in conjunction with the accompanying drawings and preferred embodiments, describes the specific implementation methods, steps, structures, features and effects of the biopharmaceutical drugs proposed according to the present invention.

[0063] The foregoing and other technical contents, features, and effects of the present invention will be clearly presented in the following detailed description of preferred embodiments with reference to the accompanying drawings. Through the description of the specific embodiments, a more in-depth and specific understanding can be gained of the technical means and effects adopted by the present invention to achieve its intended purpose. However, the accompanying drawings are for reference and illustration only and are not intended to limit the present invention.

[0064] To illustrate the technical solutions of the invention in more detail, specific embodiments are listed below to demonstrate the technical effects; it should be emphasized that these embodiments are used to illustrate the invention and not to limit the scope of the invention.

[0065] The experimental materials used in the examples are as follows:

[0066] The instruments and equipment used in this embodiment are as follows:

[0067] Microplate reader: Synergy H1

[0068] Laser confocal microscope: Zeiss 800

[0069] Slit-lamp microscope: SLM-KD4

[0070] LC-MS: Xevo G2-XS QTof

[0071] In this invention, the term "mucosal adhesive catalase" refers to catalase with adhesive groups, such as thiol-grafted catalase, maleimide-grafted catalase, disulfide-grafted catalase, and thioether-modified catalase.

[0072] In this invention, the term "and / or" should be understood to mean any one, two, or more of the alternatives or any combination thereof.

[0073] In this invention, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0074] Example A1:

[0075] A mucosal adhesive catalase a, wherein the adhesive group is a thiol group.

[0076] A method for preparing mucosal adhesive catalase a includes the following steps:

[0077] Take an appropriate amount of catalase, add 5 mmol / L EDTA, add 2 mg / mL Traut's reagent working solution, the molar ratio of catalase to Traut's reagent is 1:100, react at 25℃ for 4 hours to obtain mucosal adhesive catalase a.

[0078] Example A2:

[0079] A mucosal adhesive catalase b, wherein the adhesive group is a maleimide group.

[0080] A method for preparing mucosal adhesive catalase b includes the following steps:

[0081] Take an appropriate amount of catalase, add 5 mmol / L EDTA, add Sulfo-SMCC (sodium salt of 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid sulfonate succinimide ester), the mass ratio of catalase to Sulfo-SMCC is 20:1, react at 37℃ for 30 minutes, and mucosal adhesive catalase b.

[0082] Example A3:

[0083] A mucosal adhesive catalase C, the adhesive group being a disulfide bond.

[0084] A method for preparing mucosal adhesive catalase C includes the following steps:

[0085] Take an appropriate amount of catalase and add Sulfo-LC-SPDP (sulfosuccinimide-6-[3-(2-pyridyldithio)propionamide]hexanoic acid). The mass ratio of catalase to Sulfo-LC-SPDP is 5:1. React at 25℃ for 1 hour to obtain mucosal adhesive catalase c.

[0086] In the preparation methods of Examples A1 to A3, the degree of modification of different groups on the surface of catalase can be achieved by adjusting the initial reaction ratio of the raw materials.

[0087] Example B1: Molecular weight characterization of mucosal adhesive catalases prepared in Examples A1 to A3

[0088] The experimental method is as follows:

[0089] 1) BCA (disulfide bond thiol protein colorimetric method) is used to detect protein concentration and is used for quantitative sample loading.

[0090] 2) Mix the mucosal adhesive catalase a sample, mucosal adhesive catalase b sample, mucosal adhesive catalase c sample prepared in Examples A1 to A3, and the unmodified catalase control with loading buffer, heat at 100°C for 5 minutes to prepare SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) samples, and load 10 μg of protein for electrophoresis.

[0091] 3) After electrophoresis, the gel was stained with Coomassie Brilliant Blue for 30 minutes, destained with ultrapure water, and photographed for record.

[0092] The experimental groups are as follows:

[0093] Control Example B1.1: Unmodified catalase;

[0094] Example B1.2: Mucosal adhesive catalase a prepared in Example A1;

[0095] Example B1.3: Mucosal adhesive catalase b prepared in Example A2;

[0096] Example B1.4: Mucosal adhesive catalase c prepared in Example A3.

[0097] Figure 1 shows the SDS-PAGE electrophoresis bands of the samples in Example B1 after modification under different conditions. The results show that the protein samples described in Examples B1.2-B1.4 did not show significant changes in band position compared with Control Example B1.1, and there were no other impurities. This indicates that the mucosal adhesion proteins described in this application do not undergo aggregation, dissociation, or other effects on the structure of catalase itself due to surface adhesion protein modification.

[0098] Example B2: Enzyme activity detection of mucosal adhesive catalases prepared in Examples A1 to A3

[0099] The experimental procedure follows these steps:

[0100] The mucosal adhesive catalase a sample, mucosal adhesive catalase b sample, mucosal adhesive catalase c sample and catalase control prepared in Examples A1 to A3 were weighed quantitatively and prepared into 1 mg / mL sample solutions. The sample solutions were diluted 500 times and enzyme activity was detected according to the instructions of the detection kit.

[0101] The samples were grouped according to the scheme of Example B1, and numbered as Control Example B2.1, Example B2.2, Example B2.3, and Example B2.4, respectively. Figure 2 is a statistical chart of enzyme activity detection in different samples in Example B2. The results show that the enzyme activity data of the samples described in Examples B2.2-B2.4 are similar to those of Control Example B2.1, indicating that the mucosal adhesive catalase described in this application has catalase activity and is expected to be able to perform the original catalase function for further use in reactive oxygen species scavenging.

[0102] Example B3: Detection of surface modification groups of mucosal adhesive catalase

[0103] The experimental method is as follows:

[0104] Samples of mucosal adhesive catalase a, b, and c prepared in Examples A1 to A3, and an unmodified catalase control were weighed quantitatively and prepared into 1 mg / mL sample solutions. The sample solutions were analyzed using liquid chromatography-mass spectrometry (LC-MS). The samples were grouped according to the scheme in Example B1 and numbered as Control Example B3.1, Example B3.2, Example B3.3, and Example B3.4, respectively. Figure 3 shows the mass spectra of different samples in Example B3.

[0105] The results showed that the peak position of the unmodified catalase monomer in Control Example B3.1 was at 59754 Da, while the characteristic peaks of the three groups of catalases in Examples B3.2-B3.4 showed significant shifts. The shifted peaks represent catalase monomer subunits modified with different numbers of adhesive groups. The relative content of mucosal adhesive catalase at this grafting rate can be calculated from the peak area. Based on the data in Figure 3, it is shown that the corresponding adhesive groups were successfully grafted onto the surface of the three types of mucosal adhesive catalases. Mass spectrometry characterization can be used to estimate the ratio of surface-modified groups to catalase monomers, and further, the ratio of surface-modified groups to catalase can be estimated. The products are detectable, which is beneficial for large-scale production and quality control.

[0106] Example B4:

[0107] Samples with different feed ratios were prepared according to the preparation method of Example A1, and the mass spectra of the samples were detected using the method of Example B3. The molar ratio of thiol groups to catalase monomers was estimated, and the results are shown in the table below. The degree of modification (i.e., the molar ratio of thiol groups to catalase) can be further calculated. The degree of modification is approximately 4 times the ratio of thiol groups to catalase monomers.

[0108] Table 1: Statistical table of Calculation of Thiol Modification Degree on Catalase Surface Obtained by Changing Feed Ratio in Example A1

[0109] The results showed that the number of thiol groups modified by catalase increased with the increase of the feed ratio, indicating that within a certain range, products with different degrees of modification can be obtained by adjusting the feed ratio. Generally, the more adhesive groups modified, the easier and more stable the binding of mucosal adhesive catalase to mucosal surface proteins. However, considering the cost of raw materials and the final therapeutic effect, it is necessary to balance the relationship between the degree of modification and the dosage, and select a mucosal adhesive catalase with an appropriate degree of modification for the treatment of specific diseases as needed.

[0110] Example C1: Detection of corneal adhesion by catalase in mucosal adhesiveness

[0111] The experimental method is as follows:

[0112] Catalase was modified according to the methods of Examples A1 to A3, except that the catalase was labeled with fluorescein Cy5.5 before chemical grafting, resulting in the preparation of fluorescently labeled mucosal adhesive catalase a', mucosal adhesive catalase b', and mucosal adhesive catalase c'. Sample solutions of mucosal adhesive catalase a', mucosal adhesive catalase b', and mucosal adhesive catalase c', as well as a sample solution of unmodified catalase control, were prepared quantitatively at a concentration of 1.5 mg / mL. Mice were anesthetized with isoflurane, and 5 μL of the sample solution was instilled into the eye. Two hours after instillation, eyeball samples were collected, fixed with paraformaldehyde for 1 hour, and the cornea was dissected under a microscope. The cornea was then flattened on a glass slide to prepare a mounting sample. The fluorescence signal of the mounting sample was acquired and quantitatively analyzed using a Zeiss confocal microscope.

[0113] The sample information for each experimental group's eye drop administration is as follows:

[0114] Control example C1.1: None (no medication administered);

[0115] Control example C1.2: Catalase labeled with fluorescein Cy5.5 and without modification;

[0116] Example C1.3: Mucosal adhesion protein a' prepared by labeling with fluorescein Cy5.5 and following the method of Example A1;

[0117] Example C1.4: Mucosal adhesion protein b' prepared by labeling with fluorescein Cy5.5 and following the method of Example A2;

[0118] Example C1.5: Mucosal adhesion protein c' prepared by labeling with fluorescein Cy5.5 and following the method of Example A3.

[0119] Figure 4 shows fluorescence micrographs of isolated corneas from mice in each group of Example C1. Figure 5 is a statistical graph of fluorescence signal intensity of isolated corneas from mice in each group of Example C1. Since no drug was administered to control example C1.1, the fluorescence intensity should be 0. The higher the fluorescence signal intensity in other samples, the higher the catalase retention. The results show that the retention of mucoadhesive catalase a', mucoadhesive catalase b', and mucoadhesive catalase c' on the corneal surface of mice in Examples C1.3-C1.5 is significantly higher than that in control example C1.2. Even two hours after eye drop administration, a large amount of catalase remains, indicating that the mucoadhesive catalase described in this application can significantly prolong the retention of catalase on the corneal surface and prolong the action time of catalase on the ocular surface, thereby maintaining the efficacy of clearing hydrogen peroxide for a longer period of time. It has the beneficial effect of lower administration frequency and higher clearance efficiency in clinical use.

[0120] Example C2: Treatment efficacy for dry eye syndrome

[0121] The experimental method is as follows:

[0122] Dry eye model establishment: Mice were given 0.2% benzalkonium chloride eye drops once in the morning and once in the evening to establish a dry eye model. The amount of drops was 5 μL per eye, and the interval between the two drops was about 12 hours. The dry eye model was established by continuous drops for one week.

[0123] Mucosal adhesive catalase a was prepared according to the method in Example A1, and eye drop samples were prepared in two specifications: 1 mg / mL and 0.1 mg / mL. Healthy mice were not used for modeling.

[0124] Mice that successfully modeled the dry eye were randomly divided into groups and treated with eye drops. The dosage was 5 μL per eye, twice a day, morning and evening, with an interval of approximately 12 hours between treatments. 0.2% benzalkonium chloride was administered once daily to consolidate the dry eye model. The commercially available drugs used in Examples C2.4 to C2.8 were administered directly as eye drops without dilution or concentration.

[0125] The treatment drugs used on each group of mice are as follows:

[0126] Control C2.0: Healthy mice, without any eye treatment;

[0127] Control Example C2.1: Use PBS (phosphate buffered saline) eye drops as a solvent control;

[0128] Example C2.2: A sample solution of 1 mg / mL mucosal adhesive catalase a, in PBS;

[0129] Example C2.3: A sample solution of 0.1 mg / mL mucosal adhesive catalase a, in PBS;

[0130] Example C2.4: Cyclosporine eye drops, Zirun, drug name: Cyclosporine eye drops;

[0131] Example C2.5: Dexamethasone eye drops, Tobramycin Dexamethasone Eye Drops;

[0132] Example C2.6: Reproxalap, Manufacturer: MCE, Part No.: HY-107150;

[0133] Example C2.7: Cyclosporine in perfluorobutylpentane solution, i.e., cyclosporine (CAS No.: 59865-13-3) was dissolved in perfluorobutylpentane (manufacturer: Shanghai Bid Pharmaceutical Technology Co., Ltd., CAS No.: 1190430-21-7), with a cyclosporine concentration of 0.1%;

[0134] Example C2.8: Perfluorohexyloctane (manufacturer: Shanghai Haohong Biomedical Technology Co., Ltd., CAS No.: 133331-77-8).

[0135] Detection time and method: On the 3rd day after treatment, the tear secretion of mice was detected using phenol red cotton thread. Specifically, after anesthetizing the mice, the phenol red cotton thread was gently placed in the lower eyelid near the outer 1 / 3 of the outer canthus, left for 30 seconds, and then removed. The length of the cotton thread soaked with tears was measured with vernier calipers.

[0136] Figure 6 is a statistical chart of the length of cotton thread soaked in tears in each group of mice in Example C2. The results show that in Examples C2.2, C2.3, and C2.8, the length of cotton thread soaked in tears was significantly higher than in other groups, approaching or even exceeding the tear secretion of healthy mice in Control Example C2.0. In contrast, the tear secretion of mice in Control Example C2.1 was significantly reduced. This indicates that the mucosal adhesion protein described in this invention helps improve ocular surface moisture, increases tear secretion in mice, and alleviates dry eye symptoms. Furthermore, compared with other commercially available and clinically investigated dry eye treatments, such as cyclosporine and dexamethasone, the mucosal adhesion catalase described in this invention has a more significant improving effect and shows good clinical application prospects.

[0137] In particular, the applicant unexpectedly discovered that although the concentration of mucosal adhesive catalase a in Example C2.2 was higher than that in Example C2.3, Example C2.3 had a better effect on improving dry eye symptoms. The lower concentration was actually more beneficial for the treatment of dry eye, which is contrary to common knowledge. Considering the cost of protein drugs, the mucosal adhesive protein described in this application is more conducive to future clinical promotion and use than traditional protein drugs.

[0138] In addition, on the third day after treatment, corneal fluorescence staining analysis was used to detect the extent of ocular epithelial defects in mice. The specific experimental method was as follows: after anesthetizing the mice, sodium fluorescein dye was instilled onto the eyes, stained for 1-2 minutes, rinsed with physiological saline, dried with cotton balls, and the corneal staining was detected under a slit lamp with cobalt blue light. Figure 7 is a statistical chart of the sodium fluorescein staining scores of the corneas of each group of mice in Example C2.

[0139] Sodium fluorescein staining is a method to examine corneal epithelial defects. 1% to 2% sodium fluorescein is instilled into the conjunctival sac and observed after 1 to 2 minutes. Yellow-green staining areas indicate corneal epithelial defects. For specific experimental methods and scoring methods, refer to the SICCA-OSS scoring criteria in the "Chinese Dry Eye Expert Consensus: Definition and Classification (2020)".

[0140] The results showed that in Examples C2.4 to C2.8, treatment with commercially available or clinically investigated drugs such as cyclosporine, Reproxalap, perfluorobutylpentane solution of cyclosporine, and perfluorohexyloctane did not significantly differ from control example C2.1 in terms of corneal epithelial defects in mice. In fact, in Example C2.5, treatment with dexamethasone significantly increased the degree of corneal epithelial defects in mice, indicating that existing clinical drugs cannot treat ocular surface damage in mice with dry eye. However, in Examples 2.2 and 2.3, the corneal epithelial defects in mice significantly improved, indicating that 0.1 mg / mL of mucosal adhesive catalase, administered for 3 days, effectively reduced the degree of local epithelial cell apoptosis in mice with dry eye, while other clinical drugs did not improve the degree of local eye defects in mice. Dexamethasone treatment even exacerbated the local defects, demonstrating that the mucosal adhesive catalase described in this application has a better overall therapeutic effect on dry eye.

[0141] Example C3: The therapeutic effect of different mucosal adhesive catalases on dry eye in mice

[0142] The experimental procedure follows these steps:

[0143] The mucosal adhesive catalase a, mucosal adhesive catalase b, and mucosal adhesive catalase c prepared in Examples A1 to A3 were diluted to form eye drop samples of 0.1 mg / mL.

[0144] The experimental method described in Example C2 was used for modeling, administration, and evaluation of efficacy, with the difference being that different drug samples were used in each group.

[0145] The specific groups are as follows:

[0146] Control C3.0: Healthy mice, without any eye treatment;

[0147] Control Example C3.1: PBS eye drops were used as a solvent control;

[0148] Example C3.2: Sample solution of 0.1 mg / mL mucosal adhesive catalase a;

[0149] Example C3.3: Sample solution of 0.1 mg / mL mucosal adhesive catalase b;

[0150] Example C3.4: Sample solution of 0.1 mg / mL mucosal adhesive catalase c.

[0151] Figure 8 is a statistical chart of tear secretion in mice in Example C2, and Figure 9 is a statistical chart of corneal fluorescein staining scores in mice in Example C3. As shown in Figure 8, in Examples C3.2 to C3.4, the tear secretion levels in the mice were close to those in Control Example 3.0 and significantly higher than those in Control Example C3.1. This indicates that the mucosal adhesion peroxidase described in this application can improve dry eye symptoms after 3 days of use and help dry eye mice restore tear secretion to normal levels in a short period. Meanwhile, the corneal fluorescein staining results in Figure 9 show that the corneal damage in mice in Examples C3.2 to C3.4 was significantly lower than that in Control Example C3.1. This means that the mucosal adhesion peroxidase described in this application can also significantly improve corneal epithelial defects in mice, and compared to only improving tear secretion levels, it also has the effect of promoting corneal damage repair, and is expected to be further applied to the treatment of other corneal injuries.

[0152] Example C4: Comparison of therapeutic effects of other peroxidases involved in the oxidative stress pathway related to the pathogenesis of dry eye

[0153] Using the method of Example A1, mucosal adhesive catalase a and thiol-modified superoxide dismutase were prepared with the same feed ratio and reaction conditions.

[0154] A mouse dry eye model was established according to the method in Example C2, and the mice were randomly divided into groups and treated according to the following grouping method:

[0155] Control C4.1: Healthy mice without dry eye syndrome;

[0156] Control Example C4.2: Dry eye mice were given 5 μL of PBS eye drops twice daily, once in the morning and once in the evening.

[0157] Example C4.3: Dry eye mice were given 0.1 mg / mL mucosal adhesive catalase a instillation daily, twice a day, morning and evening, 5 μL each time;

[0158] Example C4.4: Dry eye mice were given eye drops of thiol-modified superoxide dismutase at a concentration of 0.1 mg / mL, twice a day, morning and evening, 5 μL each time;

[0159] Example C4.5: Dry eye mice were given 5 μL of a mixture of 0.1 mg / mL mucosal adhesive catalase a and thiol-modified superoxide dismutase as eye drops twice a day, morning and evening, with the concentration of both proteins being 0.1 mg / mL.

[0160] The first administration was recorded as day 0. On days 3, 5, and 7, the degree of corneal damage in mice was scored using sodium fluorescein staining. The length of the cotton thread soaked with tears was measured over 30 seconds using phenol red cotton thread. The length of the cotton thread soaked with tears and the sodium fluorescein staining scores for each group were statistically analyzed. Figure 10 shows the statistical chart of the length of the cotton thread soaked with tears in each group of mice in Example C4, and Figure 11 shows the statistical chart of the sodium fluorescein staining scores for each group of mice in Example C4. The experimental results, as shown in Figure 10, indicate that the length of the cotton thread soaked with tears in Example C4.5 was slightly higher than that in Example C4.3 on days 5 and 7 of treatment. In Control Example C4.1, the length of the cotton thread soaked with tears remained at 6.4 ± 0.35 mm over 7 days, and in Control Example C4.2, the length of the cotton thread soaked with tears remained at 3.1 ± 0.33 mm over 7 days, demonstrating the effectiveness of the dry eye modeling and that physiological buffer solution did not improve dry eye symptoms. In Example C4.3, mice treated with mucosal adhesive catalase a showed increased tear secretion, with the length of a cotton thread soaked with tears reaching 6.0 ± 0.15 mm on day 7, close to the length of a cotton thread soaked with tears in healthy mice. However, in Example C4.4, mice treated with thiol-modified superoxide dismutase showed an average tear thread length of only 4.5 ± 0.36 mm on day 7. While this slightly improved dry eye symptoms, the effect was far less than that of catalase.

[0161] Furthermore, as shown in Figure 11, the fluorescein sodium staining scores of Example C4.3 on days 3, 5, and 7 were significantly lower than those of Example C4.4, indicating that mucosal adhesive catalase helps in the recovery of corneal damage, while the thiol-modified superoxide dismutase of Example C4.4 has a poor effect on the recovery of corneal damage.

[0162] Based on the experimental results, the inventors unexpectedly discovered that catalase and superoxide dismutase, which are also modified with thiol groups and have mucosal adhesion effects, showed that catalase had a significantly better dry eye relief effect than superoxide dismutase, completely breaking the conventional understanding in existing technologies that prioritize the use of superoxide dismutase to improve the local inflammatory environment.

[0163] Furthermore, the applicant discovered that the human body's antioxidant defense system includes both enzymatic and non-enzymatic antioxidant systems. The enzymatic antioxidant system includes superoxide dismutase, catalase, glutathione peroxidase, myelin-derived peroxidase, peroxide reductase, and thioredoxin. The cascade reaction of oxygen free radicals begins with the generation of superoxide anions, which are then rapidly dismutated into hydrogen peroxide by superoxide dismutase. Hydrogen peroxide can be catalyzed by catalase to produce non-toxic water and oxygen, or, in the presence of glutathione, by glutathione peroxidase to produce water, or by metal catalysis to form active hydroxyl radicals that damage various biological macromolecules, or by myelin-derived peroxidase to produce more toxic hypohalite ions. Among the cascade reactions of various reactive oxygen species, hydrogen peroxide is a non-radical reactive oxygen species with a long half-life, thus allowing for wider diffusion and distribution within cells and tissues. The substances produced by the decomposition of hydrogen peroxide catalyzed by catalase are non-toxic and harmless. It can directly and effectively block the generation of active reactive oxygen free radicals by removing hydrogen peroxide, thereby blocking the oxidative damage of reactive oxygen to biological macromolecules from the upstream of the reaction. Unlike glutathione peroxidase, it does not require the participation of other substrates.

[0164] Existing technologies utilize superoxide dismutase to help scavenge oxygen free radicals, which, in principle, can directly reduce the damage of reactive oxygen species to the eyes. However, the increased content of downstream products such as hydrogen peroxide hinders the positive progress of the reactive oxygen species scavenging reaction. The applicant unexpectedly discovered in experiments that catalase's therapeutic effect is far superior to that of superoxide dismutase. Compared to current technologies, catalase is a more direct and cost-effective pathway for converting reactive oxygen species into non-toxic substances. Furthermore, the modified catalase achieved unexpected technical results in experiments treating dry eye syndrome.

[0165] Example D1: Treatment Experiment of a Corneal Chemical Burn Model

[0166] A mouse ocular surface alkali chemical burn model was established. Specifically, a 2mm diameter circular sterile filter paper strip soaked in a 1M sodium hydroxide solution was placed on the right eye surface of a healthy mouse for 20 seconds to burn the eye. The modeling mice were randomly divided into 5 groups of 7 mice each, and received different subsequent treatments. The specific grouping and treatment methods are as follows:

[0167] Control Example D1.1: Blank control group, no treatment was given to the chemically burned eyes of the mice;

[0168] Control case D1.2: A catalase solution with a concentration of 0.1 mg / mL was applied dropwise to the surface of the chemically burned cornea, 5 μL each time, twice a day, for a total of 28 days;

[0169] Example D1.3: A solution of mucosal adhesive catalase a at a concentration of 0.1 mg / mL was dripped onto the surface of the chemically burned cornea, 5 μL each time, twice a day, for a total of 28 days;

[0170] Comparative Example D1.4: Using commercially available Bepanthen Bovine basic fibroblast growth factor eye drops were applied to the corneal surface of mice with chemical burns, 5 μL each time, twice a day, for a total of 28 days.

[0171] Compare with Example D1.5: Using commercially available Dompeix. Tobramycin and dexamethasone eye drops were applied to the corneal surface of mice with chemical burns, 5 μL each time, twice a day, for a total of 28 days.

[0172] The corneal condition of mice was assessed before and during treatment. The treatment effect was evaluated using several indicators, including corneal fluorescein staining score, corneal opacity score, corneal neovascularization score, cataract incidence, and changes in anterior chamber depth. The statistical results are shown in Figures 12-16.

[0173] Figure 12 is a statistical chart of fluorescein sodium staining scores on the second day after chemically burned corneas in five groups of mice in Example D1 (score criteria: 0 points no staining, 1 point punctate staining, 2 points <1 / 3 area, 3 points 1 / 3-2 / 3 area, 4 points >2 / 3 area). The results show that the fluorescein sodium staining score of the control mice was significantly higher than that in Example D1.3. A higher score indicates a larger area of ​​corneal tissue stained with fluorescein sodium, and the stained area represents the corneal damage area. The results in Figure 12 demonstrate that the use of mucosal adhesive catalase solution eye drops for one day in Example D1.3 can significantly repair corneal damage and reduce the area of ​​chemically burned corneas.

[0174] During the treatment period after modeling, the opacity of the cornea in mice with chemical burns was observed using slit-lamp imaging. Figure 13 shows the statistical graphs of corneal opacity scores for each group of mice in Example D1. As time progressed, the opacity of the untreated mice in Control Example D1.1 continued to increase. Furthermore, white opacity was observed on the surface of the chemically burned corneas in this control group, possibly due to corneal scarring caused by epithelial fibrosis. The pupil and iris texture were not visible, resembling symptoms of cataracts. By day 28, the corneal opacity symptoms in all four control mice with chemical burns worsened. Only the corneal opacity in mice treated with mucosal adhesive catalase in Example D1.3 did not increase significantly, remaining at a relatively stable level, demonstrating the efficacy of alleviating corneal chemical burn symptoms or slowing the progression of corneal chemical burns.

[0175] Corneal neovascularization in mice was assessed on days 7, 14, 21, and 28 post-treatment. The scoring criteria were as follows: each of the five corneal regions (superior, inferior, left, right, and central) was scored from 0 to 4 points based on the area occupied by new blood vessels: 0 points - no new blood vessels; 1 point - less than 30% of the area; 2 points - more than 30% but less than 70%; 3 points - more than 70% but less than 100%; 4 points - 100% of the area; the total score ranged from 0 to 20. Corneal neovascularization is not an independent corneal disease but a pathological change; a higher score indicates a more severe condition. Figure 14 shows the statistical chart of corneal neovascularization scores in each group of mice in Example D1. The results showed that the neovascularization scores of the injured cornea in Example D1.3 were significantly lower than those in the other control groups, indicating that mucosal adhesive catalase can effectively inhibit the formation of corneal neovascularization in mice and reduce corneal damage.

[0176] The condition of the mouse eyeballs and ocular surface was observed using slit-lamp imaging, and the incidence of cataracts in each group was statistically analyzed. Figure 15 shows the statistical chart of the incidence of cataracts in each group of mice in Example D1. The results showed that the mice treated with mucosal adhesive catalase a in Example D1.3 had the lowest incidence of cataracts, while the other control groups all developed cataracts with a high probability. This indicates that mucosal adhesive catalase can effectively inhibit the occurrence of cataracts after corneal chemical burns.

[0177] The changes in corneal morphology, thickness, and anterior chamber depth in mice were observed using anterior segment OCT imaging (Optical Coherence Tomography). Figure 16 shows the anterior segment OCT images of mice in each group in Example D1. The corneas of healthy mice were semi-circular with intact structure, and the anterior chamber depth was approximately 330 μm. The results showed that the corneal thickness of mice in Example D1.3 remained almost at a thin level, and the morphology was the most intact. In the control cases, most mice showed varying degrees of morphological damage to their corneas, with different degrees of deformation of the corneal epithelium and stroma, a significant decrease in anterior chamber depth, and adhesion between the cornea and the iris and lens.

[0178] The evaluation results of five indicators demonstrate that the mucosal adhesive catalase described in this invention can reduce the damage of chemical substances to the cornea and has the potential to treat corneal chemical injuries. Furthermore, the modified catalase with mucosal adhesive properties fully achieves the therapeutic effect of treating corneal chemical burns, and at the same dosing frequency, it has better efficacy than commercially available eye drops such as Bepanthen and Tobradex, possessing significant market potential.

[0179] Example D2: Prevention of Allergic Conjunctivitis

[0180] Allergic conjunctivitis, also known as allergic conjunctivitis, is one of the most common types of ocular allergic reactions. Its main symptoms include itchy eyes, tearing, and redness, and it is often treated with topical antihistamines.

[0181] In this embodiment, ovalbumin (OVA) was used as the allergen to establish a mouse model of allergic conjunctivitis. Simultaneously, the mucosal adhesive catalase a described in this invention was used for treatment, specifically following these steps:

[0182] Basic sensitization: Mice were intraperitoneally sensitized by injecting 200 μL of a suspension containing 50 μg OVA and 2 mg Al(OH)3 antigen adjuvant prepared with physiological saline into the peritoneum on day -14, day -7, and day 0. Mice in the Healthy group were intraperitoneally injected with an adjuvant suspension without OVA.

[0183] Challenge phase: After basal sensitization, from day 1 to day 10, 10 μL of 5% OVA saline solution was instilled into the conjunctival sacs of both eyes of mice for challenge, once daily for a total of 10 times. The control mice (D2.1) received an equal volume of saline solution in their eyes.

[0184] Treatment phase: From day 1 to day 10 of the experiment, 10 μL of the corresponding drug was instilled into the left and right conjunctival sacs of mice 1 hour before OVA challenge each day. The treatments received by each group of mice were as follows:

[0185] During the study, the mice's ocular symptoms were observed and evaluated daily, and scores were recorded. Evaluation indicators included conjunctival edema, conjunctival hyperemia, eyelid hyperemia and edema, and the degree of tearing. A double-blind experiment was conducted to reduce the influence of subjective factors on the scoring results. The evaluation criteria for each indicator are as follows:

[0186] The four scores for each mouse were summed daily to create an ocular symptom score chart. Figure 17 is a line graph showing the changes in ocular symptom scores over time for each group of mice in Example D2. The results showed that the ocular symptom scores of mice in Control Example D2.1 remained consistently low, while the scores in Control Example D2.2 indicated effective modeling and significant allergic conjunctivitis caused by subsequent local stimulation. In Control Examples 2.4 and 2.5, the ocular symptom scores initially increased and then decreased, indicating that catalase and commercially available olopatadine eye drops could alleviate allergic conjunctivitis. However, with prolonged stimulation, symptom improvement was limited and could not return to normal levels. In Example D2.3, the use of the mucosal adhesive catalase a described in this invention continuously improved the ocular symptoms of allergic conjunctivitis in mice, and approached the normal level, showing a significant difference compared with Controls D2.4 and D2.5. Mucosal adhesive catalase a has the potential to rapidly relieve the symptoms of allergic conjunctivitis and help patients relieve eye discomfort and prevent further aggravation of allergic conjunctivitis symptoms under continuous external allergen stimulation.

[0187] Figure 18 is a statistical chart of the number of eye-scratching events in each group of mice in Example D2 within 10 minutes after receiving local stimulation. A higher number indicates higher ocular sensitivity and stronger discomfort in the mice. Except for the healthy mice in Control Example D2.1, all groups of mice showed an increase in eye-scratching events during the basal sensitization process. Subsequently, with the simultaneous implementation of local stimulation and treatment, the number of eye-scratching events significantly decreased. Only Example D2.3 maintained a low level; the other control group mice did not show a sustained reduction in eye-scratching events. This indicates that the mucosal adhesive catalase a described in this invention can effectively alleviate the discomfort of allergic conjunctivitis in mice, consistent with the results of the ocular symptom scoring.

[0188] On day 7, serum samples were collected from mice to detect the OVA-SIgE content. Higher OVA-SIgE levels indicated a more severe allergic reaction to OVA in the mice. Figure 19 shows the relative OVA-SIgE content in the serum of each group of mice in Example D2 on day 7. The results show that the relative OVA-SIgE content in the mice was significantly lower than that in Control Example D2.2, and close to that in Control Example D2.1. This demonstrates that the mucosal adhesive catalase a described in this invention can indeed alleviate allergic symptoms and play a role in preventing allergies, rather than merely improving local symptoms.

[0189] Based on the data from Example D2, it is demonstrated that the mucosal adhesive catalase a described in this invention can be used to improve symptoms related to allergic conjunctivitis and even prevent the occurrence of allergic conjunctivitis. It has advantages over existing small molecule drugs in terms of preventive effect and maintaining the stability of the body's immune indicators.

[0190] Example E1: Retention of mucosal adhesive catalase in the lungs

[0191] An acute lung injury model was induced in mice using the LPS spray lung modeling method. The mice were then randomly divided into three groups and inhaled different samples. The grouping and inhaled samples are described below:

[0192] Control Example E1.1: No sample inhalation was performed;

[0193] Control Example E1.2: Inhalation of Cy5.5 fluorescently labeled catalase;

[0194] Example E1.3: Inhalation of Cy5.5 fluorescently labeled mucosal adhesive catalase;

[0195] The inhaled samples in Examples E1.2 and E1.3 contained equal amounts of catalase. At 12 and 24 hours post-administration, mouse lungs were removed, and the fluorescence signal was detected using a small animal fluorescence imaging system. Figure 20 shows the statistical graph of the fluorescence signal intensity (mean radiation intensity) in the lungs of mice in different groups in Example E1. The results showed that no fluorescence signal was detected in the lungs of mice in Control Example E1.1, while fluorescence signals were detected in the lungs of mice in Control Example E1.2 and Example E1.3. However, the fluorescence signal intensity in Example E1.3 was significantly higher than that in Control Example 1.2, indicating that mucosal adhesive catalase can significantly increase the amount and duration of retention in the lungs. Under the same enzyme activity, a higher local catalase concentration leads to a better effect on improving local hydrogen peroxide concentration, promoting positive redox reactions in the lungs, thereby reducing the content of upstream peroxides, reducing local ROS, alleviating the irritation of harmful peroxides on the lung mucosa, and prolonging the local retention time of catalase. Through continuous and efficient removal of downstream products of the redox pathway, it can significantly improve the symptoms of lung inflammation and even achieve a therapeutic effect. Therefore, mucosal adhesive catalase can enter and remain in the lungs via inhalation, showing potential for further application in the treatment of lung diseases.

[0196] Example E2:

[0197] Mice were randomly divided into 5 groups, 4 of which were treated with LPS inhalation to establish an acute lung injury model. The specific grouping and treatment methods are as follows:

[0198] Control Example E2.1: Healthy Mice

[0199] Control Example E2.2: 3 hours after modeling, mice were given nebulized phosphate buffer.

[0200] Example E2.3: Three hours after modeling, mice were given catalase solution via nebulization at a dose of 4 mg / kg.

[0201] Example E2.4: Three hours after modeling, mice were given a nebulized catalase solution with a dose of 4 mg / kg.

[0202] Twenty-four hours after treatment, lung tissue was collected from mice to obtain bronchoalveolar lavage fluid. The levels of IL-1β, TNF-α, and IL-6 in the lavage fluid were detected using an ELISA kit, and the levels of neutrophils and macrophages in the lavage fluid were detected by flow cytometry. The lung tissue was then stained with hematoxylin and eosin (HE) and eosin (H&E) and sectioned. Figure 21 shows the concentration of IL-1β in the mouse bronchoalveolar lavage fluid in Example E2; Figure 22 shows the concentration of TNF-α in the mouse bronchoalveolar lavage fluid in Example E2; Figure 23 shows the concentration of IL-6 in the mouse bronchoalveolar lavage fluid in Example E2; Figure 24 shows the percentage of macrophages in the mouse bronchoalveolar lavage fluid in Example E2; and Figure 25 shows the percentage of neutrophils in the mouse bronchoalveolar lavage fluid in Example E2. The results showed that the inflammatory factor indices of mice in Examples E2.3 and E2.4 were closer to those of mice in the blank control example E2.1 than those in control example E2.2, indicating that the lung inflammation level decreased after mice with acute lung injury inhaled a therapeutic agent containing catalase. Furthermore, the effect of mucosal adhesive catalase treatment in Example E2.4 was more significant than that of catalase treatment in Example E2.3. Figure 26 is a micrograph of lung tissue sections from mice in Example E2. As can be seen from Figure 26, the lung tissue of mice in Example E2.4 is closer to that of healthy mice (control example E2.1), indicating a significant reduction in lung inflammation.

[0203] This indicates that the mucosal adhesive hydrogen peroxide described in this invention has an ameliorative effect on lung inflammation and has the potential to treat lung mucosal inflammation such as acute lung injury.

[0204] Obviously, the above embodiments are merely examples for clear illustration and are not intended to limit the implementation.

[0205] While the foregoing disclosure has discussed some inventive embodiments that are currently considered useful through various examples, it should be understood that such details are for illustrative purposes only, and the appended claims are not limited to the disclosed embodiments. Rather, the claims are intended to cover all modifications and equivalent combinations that conform to the substance and scope of the embodiments of this application.

[0206] Similarly, it should be noted that, in order to simplify the description of the present application and thus aid in the understanding of one or more embodiments of the invention, the foregoing description of the embodiments of the present application sometimes combines multiple features into a single embodiment, drawing, or description thereof. However, this disclosure method does not imply that the subject matter of the application requires more features than those mentioned in the claims. In fact, the embodiments contain fewer features than all the features of the single embodiments disclosed above.

[0207] In some embodiments, numbers describing the quantity of components and attributes are used. It should be understood that such numbers used in the description of embodiments are sometimes modified by the terms "approximately," "approximately," or "generally." Unless otherwise stated, "approximately," "approximately," or "generally" indicates that the numbers are allowed to vary by ±. Accordingly, in some embodiments, the numerical parameters used in the specification and claims are approximate values, which may be changed according to the characteristics required by individual embodiments.

[0208] Finally, it should be understood that the embodiments described in this application are merely illustrative of the principles of the embodiments of this application. Other modifications may also fall within the scope of this application. Therefore, alternative configurations of the embodiments of this application are considered as examples and not limitations, and are regarded as consistent with the teachings of this application. Accordingly, the embodiments of this application are not limited to the embodiments explicitly described and illustrated in this application.

Claims

1. A biological macromolecular drug for improving superficial inflammation of the ocular surface or mucous membrane, characterized in that, Biological macromolecular drugs contain catalase, which is chemically grafted with adhesive groups. Catalase with adhesive groups is called mucosal adhesive catalase.

2. The biological macromolecular drug according to claim 1, characterized in that, The adhesive group is selected from one or more of thiol, disulfide bond, thioether and maleimide groups.

3. The biological macromolecular drug according to claim 1, characterized in that, The molar ratio of the adhesive group to the mucosal adhesive catalase is (0-72):

1.

4. The biological macromolecular drug according to any one of claims 1-3, characterized in that, The concentration of the mucosal adhesive catalase in the biomacromolecule drug is not less than 0.001 wt%.

5. The biological macromolecular drug according to any one of claims 1-3, characterized in that, The concentration of the mucosal adhesive catalase in the biomacromolecule drug is not less than 0.001 mg / mL.

6. The biological macromolecular drug according to any one of claims 1-3, characterized in that, The mass concentration range of the mucosal adhesion catalase in the biopharmaceutical is 0.001 wt% to 2.5 wt%.

7. The biological macromolecular drug according to any one of claims 1-3, characterized in that, The concentration range of the mucosal adhesive catalase in the biopharmaceutical is 0.001 mg / mL to 10 mg / mL.

8. A method for preparing a biomacromolecule drug according to any one of claims 1-5, characterized in that, Chemical modification of the surface of catalase.

9. The preparation method according to claim 6, characterized in that, This includes the step of chemically grafting an adhesive group donor with the catalase.

10. The preparation method according to claim 6, characterized in that, This includes the step of product purification.

11. The use of a biomolecular drug according to any one of claims 1-5 in the preparation of an ocular disease treatment formulation, characterized in that, The eye diseases mentioned include one or more of the following: dry eye syndrome, corneal chemical burns, and allergic conjunctivitis.

12. The use of a biomolecular drug according to any one of claims 1-5 in the preparation of a therapeutic agent for pulmonary mucosal inflammation, characterized in that, The lung mucosal inflammation is selected from one or more of acute lung injury, asthma, and chronic obstructive pulmonary disease.

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