Use of recombinant type iii humanized collagen and composition comprising same in eye

Abstract

The present invention relates to use of a recombinant type III humanized collagen and a composition comprising the same in an eye. Provided are use of a recombinant type III humanized collagen in the preparation of a product for preventing and / or treating eye inflammation, a composition comprising the recombinant type III humanized collagen, and use of the composition in the preparation of a product for preventing and / or treating eye inflammation and / or eye dryness. The recombinant type III humanized collagen and the composition comprising the same can significantly promote the renewal and repair of corneal epithelial cells, and can also inhibit the release of inflammatory mediators, enhancing the ocular tissue barrier, enhancing tear film stability, and alleviating eye inflammation and eye dryness.

Description

Application of recombinant type III humanized collagen and compositions containing it in the ocular field

[0001] Citation of relevant applications

[0002] This disclosure claims priority to Chinese Patent Application No. 202510077256.0, filed on January 16, 2025, entitled "Application of Recombinant Type III Humanized Collagen and Compositions Containing the Thereof in the Eye," the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of biomedical materials, specifically to the application of recombinant type III humanized collagen and compositions containing therein in the ocular field. Background Technology

[0004] Contact lenses offer a convenient and aesthetically pleasing vision correction option for many people with myopia, hyperopia, or astigmatism; however, they can potentially cause damage to the eyes. For example, improper use of contact lenses can damage the corneal epithelium, leading to corneal edema, neovascularization, and other problems. Furthermore, since contact lenses adhere to the cornea, tears and oil can accumulate on the lens, eventually causing corneal hypoxia and abrasion. This weakens the corneal epithelial barrier function, making it easier for bacteria and pathogens to invade the cornea, leading to pathological eye inflammations such as keratitis. Long-term contact lens wear also reduces the eye's natural tear production, causing dryness and discomfort, and in severe cases, eye infections, corneal damage, and clouding. In addition, eye fatigue from prolonged use of electronic devices, decreased metabolic and immune function due to irregular sleep patterns, pathogen infections caused by poor eye hygiene, and complications from eye injuries and surgeries can all trigger eye inflammation, potentially leading to decreased vision and eye pain in severe cases.

[0005] Inflammation itself is a natural defense mechanism of the body against harmful substances. Typically, the activation of immune cells and other non-immune cells is the most common cause of inflammatory responses. In the eye, corneal epithelial cells participate in innate immunity and can directly respond to external stimuli, activating the expression of matrix metalloproteinases (MMPs), chemokines, and inflammatory cytokines. Metalloproteinases, including MMP-9, have the function of maintaining and remodeling tissue structure, participating in the physiological shedding of corneal epithelial cells, healing damage, and remodeling the extracellular matrix. Abnormal MMP-9 expression can reduce the tightness of corneal epithelial junctions and the basement membrane proteins connecting the cornea and conjunctiva; these changes can disrupt the integrity of corneal epithelial cells. The study "Changes and Significance of Inflammatory Cytokines and MMP-9 Expression in Tears of the Eye with Fungal Keratitis and the Uninfected Eye on the Contralateral Side" states that MMP-9 is mainly distributed in the corneal stroma, participating in the repair and remodeling of epithelial cells and playing an important role in normal immune function. Under pathological conditions, it is actively secreted by neutrophils and participates in corneal injury and inflammation. This enzyme is overexpressed in corneal epithelial cells and causes severe tissue damage. The study "Expression of MMP-2 and MMP-9 after Severe Thermal Burns of Rabbit Cornea and the Therapeutic Effect of Early Penetrating Keratokeratology" states that MMP-9 is mainly located in the corneal stroma and epithelial cell layer, and its expression is significantly increased in thermally burned corneal tissue. Inflammatory cytokines mainly include tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-10 (IL-10). Stimulation by inflammatory cytokines or infection by pathogens can trigger an excessive inflammatory response. This excessive inflammatory response causes deep cell damage, creating a cycle of inflammatory damage that further exacerbates the inflammatory response. Furthermore, literature indicates that increases in inflammatory cytokines such as IL-6, TNF-α, and IL-1β can significantly cause inflammation in different parts of the ocular surface, including the eyelids, conjunctiva, and cornea.

[0006] Currently, chemical drugs (such as trehalose, sodium hyaluronate, chondroitin sulfate, hexanediol, salicylic acid, allantoin, etc.) and plant extracts are commonly used to relieve eye inflammation. However, these drugs have various side effects. For example, chondroitin sulfate can easily cause gastrointestinal discomfort, hexanediol can cause irritation upon prolonged skin contact, and sodium hyaluronate may cause excessive inflammatory reactions such as rashes and itching. While some plant extracts have anti-inflammatory effects, these effects are relatively weak and difficult to maintain long-term. Therefore, there is still a need in the field for a safe, non-allergenic product that can effectively improve eye inflammation.

[0007] Reference 1 (ZL201811438582.6) discloses recombinant type III humanized collagen (rhCOL III) developed by Shanxi Jinbo Biopharmaceutical Co., Ltd., which possesses excellent biological properties, such as biodegradability and cell proliferation promotion, and has been widely used in the field of medical aesthetics. References 2 (CN202110960230.2) and 3 (CN202410340763.4) disclose the use of compositions containing recombinant type III humanized collagen from ZL201811438582.6, such as eye drops, in the prevention and / or treatment of dry eye syndrome, but no studies have explored its effects on ocular inflammation. Reference 4 (CN202311069893.0) discloses the recombinant type III humanized collagen developed by Shanxi Jinbo Biomedical Co., Ltd. and its use in preparing ophthalmic materials. Studies have shown that it can inhibit the amount of TNF-α secreted by macrophages and has soothing and anti-inflammatory effects. Summary of the Invention

[0008] The problem the invention aims to solve

[0009] Contact lenses are common devices used to correct vision, but improper use can easily cause problems such as dry eyes, eye irritation, and eye inflammation. While existing technologies have been researched for treating eye diseases or for drugs with anti-inflammatory effects, such research is still insufficient. For example, although cited references 2 and 3 disclose the role of recombinant type III humanized collagen in treating dry eye syndrome, they do not investigate other eye symptoms. Similarly, while cited reference 4 discloses the inhibitory effect of recombinant type III humanized collagen on TNF-α, it does not investigate eye symptoms. Therefore, there is still a need to develop safe and effective products with good therapeutic effects for eye conditions such as eye inflammation.

[0010] In the course of in-depth research on the function of the recombinant type III humanized collagen disclosed in ZL201811438582.6, this disclosure unexpectedly discovered that it can inhibit the release of inflammatory mediators, enhance the ocular tissue barrier, improve tear film stability, and relieve dry eyes and ocular inflammation. Based on this, the purpose of this disclosure is to provide the use of the recombinant type III humanized collagen in the prevention and / or treatment of ocular inflammation, particularly in the prevention and / or treatment of ocular surface inflammations such as blepharitis, keratitis, and conjunctivitis, which present with symptoms of tissue damage such as eyelid defects, conjunctival scarring, and corneal ulcers. The disclosure also provides compositions comprising the recombinant type III humanized collagen and their use in the prevention and / or treatment of ocular inflammation and / or dry eyes.

[0011] Solution for solving the problem

[0012] [1]. Use of recombinant type III humanized collagen in the preparation of products for the prevention and / or treatment of ocular inflammation; wherein the amino acid sequence of the recombinant type III humanized collagen comprises n repeats of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeat sequences are directly connected;

[0013] Preferably, the amino acid sequence of the recombinant type III humanized collagen includes any one of the following (a) to (d):

[0014] (a) A sequence as shown in SEQ ID NO.3;

[0015] (b) A sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO.3, and retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3;

[0016] (c) A sequence in which one or more amino acid residues are added, substituted or deleted in the sequence shown in SEQ ID NO.3, and which retains the cell adhesion effect of the sequence shown in SEQ ID NO.3;

[0017] (d) An amino acid sequence encoded by a nucleotide sequence hybridizing with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.3 under stringent conditions, and the amino acid sequence retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3, wherein the stringent conditions are moderately stringent, medium-high stringent, high stringent, or very high stringent.

[0018] [2]. According to the use described in [1], the ocular inflammation includes any one or more of blepharitis, keratitis, conjunctivitis, scleritis, meibomianitis, dacryocystitis, optic neuritis and uveitis;

[0019] Preferably, the eye inflammation is caused by wearing contact lenses.

[0020] [3]. According to the use described in [1] or [2], wherein the ocular inflammation is an ocular surface inflammation, which includes any one or more of blepharitis, keratitis and conjunctivitis;

[0021] Preferably, the ocular surface inflammation is blepharitis, keratitis, or conjunctivitis;

[0022] More preferably, the blepharitis has symptoms including eyelid defects, the keratitis has symptoms including corneal ulcers, and the conjunctivitis has symptoms including conjunctival scarring.

[0023] [4]. The use according to any one of [1] to [3], wherein the prevention and / or treatment of ocular inflammation includes any one or more of the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more cells of eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues of the eyelids, cornea and / or conjunctiva and enhancing the stability of the tear film.

[0024] [5]. According to the use described in [4], wherein the inflammatory mediator includes any one or more of MMP-1, MMP-9, TNF-α, and IL-1β;

[0025] Preferably, the inflammatory mediators include MMP-1, MMP-9, TNF-α, and IL-1β;

[0026] More preferably, the inflammatory mediator is a combination of MMP-1, MMP-9, TNF-α and IL-1β.

[0027] [6]. According to the use described in [4] or [5], wherein the enhancement of tear film stability includes any one or more of increasing the mucin content in the tear and reducing the osmotic pressure of the tear.

[0028] [7]. A composition comprising the following components in weight percentage: 0.001%-0.1% recombinant type III humanized collagen, 0.5%-1.5% osmotic pressure regulator, 0.01%-0.5% buffer salt, 0.02%-1% sodium hyaluronate and the balance being water;

[0029] Preferably, the composition is an eye drop.

[0030] [8]. Use of the composition according to [7] in the preparation of products for the prevention and / or treatment of ocular inflammation and / or dry eyes.

[0031] [9]. According to the use described in [8], the ocular inflammation includes any one or more of blepharitis, keratitis, conjunctivitis, scleritis, meibomianitis, dacryocystitis, optic neuritis and uveitis;

[0032] Preferably, the eye inflammation and / or dryness are caused by wearing contact lenses.

[0033]

[0010] . According to the use described in [8] or [9], wherein the ocular inflammation is an ocular surface inflammation, which includes any one or more of blepharitis, keratitis and conjunctivitis;

[0034] Preferably, the ocular surface inflammation is blepharitis, keratitis, or conjunctivitis;

[0035] More preferably, the blepharitis has symptoms including eyelid defects, the keratitis has symptoms including corneal ulcers, and the conjunctivitis has symptoms including conjunctival scarring.

[0036]

[0011] . The use according to any one of [8] to

[0010] , wherein the prevention and / or treatment of ocular inflammation and / or dry eyes includes any one or more of the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more cells among eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues in the eyelids, cornea and / or conjunctiva and enhancing the stability of the tear film.

[0037]

[0012] . According to the use described in

[0011] , the inflammatory mediator includes any one or more of MMP-1, MMP-9, TNF-α, and IL-1β;

[0038] Preferably, the inflammatory mediators include MMP-1, MMP-9, TNF-α, and IL-1β;

[0039] More preferably, the inflammatory mediator is a combination of MMP-1, MMP-9, TNF-α and IL-1β.

[0040]

[0013] . According to the use described in

[0011] or

[0012] , the enhanced tear film stability is characterized by any one or more of increasing the mucin content in the tear and reducing the osmotic pressure of the tear.

[0041] The effects of the invention

[0042] This disclosure provides the use of recombinant type III humanized collagen in the preparation of products for the prevention and / or treatment of ocular inflammation. Research data show that recombinant type III humanized collagen can significantly promote the renewal and repair of corneal epithelial cells, while inhibiting the release of inflammatory mediators, strengthening the ocular tissue barrier, enhancing tear film stability, and alleviating ocular inflammation, thus providing a new approach for the prevention and / or treatment of ocular conditions such as ocular inflammation caused by improper contact lens wear.

[0043] This disclosure also provides a composition comprising recombinant type III humanized collagen, and its use in the preparation of products for the prevention and / or treatment of ocular inflammation and / or dry eyes. Research data shows that the composition provided in this disclosure has hydrating, moisturizing, repairing, and anti-inflammatory effects, and can relieve or treat ocular inflammation and / or dry eyes, and repair corneal damage. Furthermore, the composition provided in this disclosure can replenish tear film components, alleviate tear hypertonicity, and enhance tear film stability, thereby relieving symptoms such as dry eyes and eye fatigue caused by wearing contact lenses, and it can also act as a lubricant when wearing contact lenses. Attached Figure Description

[0044] Figure 1: Results of cytotoxicity experiment in Example 1.

[0045] Figure 2: Results of cell adhesion experiment in Example 1.

[0046] Figure 3: Results of cell scratch experiment in Example 1.

[0047] Figure 4: Results of tear crystallization in each group of animals in Example 2.

[0048] Figure 5: Gross photographs of the eyes of the animals in each group in Example 4.

[0049] Figure 6: Staining results of pathological sections of the cornea and conjunctiva of animals in each group in Example 4. Detailed Implementation

[0050] The following describes the implementation schemes of this disclosure, but this disclosure is not limited thereto. Various modifications can be made within the scope of protection claimed in this disclosure, and implementation schemes and embodiments obtained by appropriately combining the technical means disclosed in different implementation schemes and embodiments are also included in the technical scope of this disclosure.

[0051] I. Terminology Definition

[0052] In this disclosure, references to “some specific / preferred embodiments,” “other specific / preferred embodiments,” “implementation,” etc., mean that a particular element (e.g., feature, structure, property, and / or characteristic) related to that embodiment is included in at least one of the embodiments described herein, and may or may not be present in other embodiments. Furthermore, it should be understood that the elements may be combined in any suitable manner in various embodiments.

[0053] In this disclosure, the word "may" has both the meaning of performing a certain process and the meaning of not performing a certain process.

[0054] In this disclosure, the terms “comprising,” “having,” “including,” or “containing” can mean inclusive or open-ended, and do not exclude additional, uncited ingredients or method steps. At the same time, “comprising,” “having,” “including,” or “containing” can also mean closed-ended, excluding additional, uncited ingredients or method steps.

[0055] In this disclosure, the terms “optional” or “optionally” mean that the event or condition described below may or may not occur, or that the steps or ingredients described below may or may not be included in the embodiments described herein, and the description includes both cases where the event occurs and cases where the event does not occur, as well as cases where the embodiments include or do not include the steps or ingredients.

[0056] In this disclosure, the terms “individual,” “patient,” or “subject” include mammals. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., human and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0057] In this disclosure, the term "prevention" means reducing the probability of developing a disease (such as eye inflammation) and / or alleviating symptoms after developing a disease by exposing the subject to the recombinant type III humanized collagen or composition described in this disclosure before such development, compared to when no exposure is made, and does not imply the necessity of completely suppressing the development of the disease. Developing a disease means the appearance of symptoms of a disease.

[0058] In this disclosure, the term "treatment" means, after suffering from a disease (such as eye inflammation), exposing (e.g., administering medication) a subject to the recombinant type III humanized collagen or composition described in this disclosure, thereby alleviating the symptoms of the disease compared to when not exposed, and does not imply the necessity of completely suppressing the symptoms of the disease.

[0059] In this disclosure, the terms “application,” “giving,” and “treatment,” when applied to animals, humans, experimental subjects, cells, tissues, or organs, refer to contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and an animal, human, experimental subject, cell, tissue, or organ.

[0060] In this disclosure, the term "effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical disease. The effective amount for a particular subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity.

[0061] In this disclosure, the term "identity" refers to the percentage of identical amino acids among two or more polypeptides. Sequence identity between two or more polypeptides can be determined by aligning the amino acid sequences of the polypeptides and scoring the number of positions containing identical amino acid residues in the aligned polypeptides, comparing this to the number of positions containing different amino acid residues in the aligned polypeptides. Sequence identity can be calculated by dividing the number of positions containing identical amino acid residues by the total number of amino acid residues in the polypeptide.

[0062] In this disclosure, amino acid addition can refer to adding one, two, or three or more amino acids at any position on the C-terminus, N-terminus, or between the C-terminus and N-terminus of an amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0063] In this disclosure, amino acid substitution refers to the replacement of an amino acid at a certain position in an amino acid sequence with another amino acid, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence. Amino acid substitution can be conserved amino acid substitution, meaning that compared to the original amino acid sequence, several amino acids are replaced by amino acids with similar or related properties to form a peptide (conserved variant peptide). For example, these conserved variant peptides can be generated based on the following amino acid substitutions: substitution of Ala with Val, Leu, or Ile; substitution of Arg with Lys, Gln, Asn, or His; substitution of Asn with Gln, His, Lys, or Arg; substitution of Asp with Glu or Asn; substitution of Cys with Ser or Ala; substitution of Gln with Asn or Glu; substitution of Glu with Asp or Gln; substitution of Gly with Ala; substitution of His with Asn, Lys, Gln, or Arg; substitution of Cys with Leu, Met, Ala, Val, Phe, or leucine. Substitutions include: Ile substitutions; substitutions of Leu with Ile, Met, Ala, Val, Phe, or leucine; substitutions of Lys with Asn, Gln, or Arg; substitutions of Met with Ile, Leu, or Phe; substitutions of Phe with Leu, Val, Ile, Ala, or Tyr; substitutions of Pro with Ala; substitutions of Ser with Thr; substitutions of Thr with Ser or Val; substitutions of Trp with Phe or Tyr; substitutions of Tyr with Trp, Phe, Thr, or Ser; and substitutions of Val with Phe, Ala, Met, Ile, Leu, or leucine. Amino acid substitutions can also be non-conserved amino acid substitutions.

[0064] In this disclosure, amino acid deletion may refer to the deletion of one, two, or three or more amino acids from an amino acid sequence, as long as the altered sequence completely or partially retains the activity of the original amino acid sequence.

[0065] In this disclosure, "hybridization" means the ability of a polynucleotide or oligonucleotide to bind to a substantially complementary sequence under stringent conditions, without nonspecific binding to noncomplementary sequences. The terms "medium stringent conditions," "medium-high stringent conditions," "high stringent conditions," or "very high stringent conditions" as used in this disclosure describe the conditions for nucleic acid hybridization and washing. For example, specific hybridization conditions are as follows: (1) Low-toughness hybridization conditions: 6× sodium chloride / sodium citrate (SSC) at about 45°C, then at at least 50°C, washed twice in 0.2× SSC, 0.1% SDS (for low-toughness conditions, the washing temperature can be increased to 55°C); (2) Medium-toughness hybridization conditions: 6× SSC at about 45°C, then at 60°C, washed once or more in 0.2× SSC, 0.1% SDS; (3) High-toughness hybridization conditions: 6× SSC at about 45°C, then at 65°C, washed once or more in 0.2× SSC, 0.1% SDS, preferably; (4) Very high-toughness hybridization conditions: 0.5M sodium phosphate, 7% SDS at 65°C, then at 65°C, washed once or more in 0.2× SSC, 1% SDS.

[0066] In this disclosure, the unit of "parts by mass" can be any weight unit such as g, mg, kg or t.

[0067] Unless otherwise defined, other technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0068] II. Recombinant Type III Humanized Collagen and its Preparation Method

[0069] The recombinant type III humanized collagen described in this disclosure is recorded in the patent application number 201811438582.6, entitled "Polypeptide, its production method and use".

[0070] In some embodiments, the amino acid sequence of the recombinant type III humanized collagen comprises n repeats of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, and n is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20, 24 or 32, wherein when n is an integer greater than or equal to 2, the repeat sequences are directly connected.

[0071] In some embodiments, the amino acid sequence of the recombinant type III humanized collagen includes or does not contain the sequence shown in SEQ ID NO.2; preferably, the amino acid sequence of the recombinant type III humanized collagen does not contain the sequence shown in SEQ ID NO.2.

[0072] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen includes any one of the following (a) to (d):

[0073] (a) A sequence as shown in SEQ ID NO.3;

[0074] (b) A sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO.3, and retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3;

[0075] (c) A sequence in which one or more amino acid residues are added, substituted or deleted in the sequence shown in SEQ ID NO.3, and which retains the cell adhesion effect of the sequence shown in SEQ ID NO.3;

[0076] (d) An amino acid sequence encoded by a nucleotide sequence hybridizing with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.3 under stringent conditions, and the amino acid sequence retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3, wherein the stringent conditions are moderately stringent, medium-high stringent, high stringent, or very high stringent.

[0077] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen includes the sequence shown in SEQ ID NO.3.

[0078] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen is as shown in SEQ ID NO.3.

[0079] In some embodiments, the amino acid sequence of the recombinant type III humanized collagen further includes an amino acid sequence that can be cleaved by the TEV protease, the amino acid sequence that can be cleaved by the TEV protease being the sequence shown in SEQ ID NO.4.

[0080] In some specific embodiments, the amino acid sequence that can be cleaved by the TEV protease is located at the N-terminus of the amino acid sequence of the recombinant type III humanized collagen.

[0081] In some specific embodiments, the amino acid sequence of the recombinant type III humanized collagen includes the sequence shown in SEQ ID NO.5.

[0082] In some specific implementations, the amino acid sequence of the recombinant type III humanized collagen is as shown in SEQ ID NO.5.

[0083] The recombinant type III humanized collagen described herein can be prepared by conventional methods in the art. For example, it can be prepared by the following steps: (1) construction of Escherichia coli genetically engineered bacteria; (2) fermentation culture of Escherichia coli genetically engineered bacteria; (3) induced expression of recombinant type III humanized collagen; and (4) purification and optional enzymatic digestion of recombinant type III humanized collagen.

[0084] III. Composition and its preparation method

[0085] This disclosure provides a composition comprising the following components: recombinant type III humanized collagen, an osmotic pressure regulator, a buffer salt, sodium hyaluronate, and water; wherein the recombinant type III humanized collagen is as described in Part II above.

[0086] In some embodiments, the composition comprises the following components in weight percentage: 0.001%-0.1% recombinant type III humanized collagen, 0.5%-1.5% osmotic pressure regulator, 0.01%-0.5% buffer salt, 0.02%-1% sodium hyaluronate, and the balance being water.

[0087] To achieve good preventive and / or therapeutic effects on ocular inflammation and / or dry eyes, in some embodiments, the composition contains 0.005%-0.05% recombinant type III humanized collagen, preferably 0.01%, by weight percentage.

[0088] In some embodiments, the osmotic pressure regulator comprises at least one of sodium nitrate, potassium nitrate, sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, glycerol, propylene glycol, mannitol, sorbitol, and glucose.

[0089] In some embodiments, the osmotic pressure regulator comprises sodium chloride.

[0090] In some specific implementations, the osmotic pressure regulator is sodium chloride.

[0091] In some embodiments, the buffer salt comprises at least one of phosphate, carbonate, bicarbonate, borate, citrate, and tartrate.

[0092] In some embodiments, the buffer salt comprises sodium dihydrogen phosphate monohydrate and anhydrous disodium hydrogen phosphate.

[0093] In some specific implementations, the buffer salt is sodium dihydrogen phosphate monohydrate and anhydrous disodium hydrogen phosphate.

[0094] Considering the intended use of the composition, such as its application as an eye drop in the human physiological environment, the pH adjuster in the composition needs to ensure that the pH of the finished product is 6-8, preferably 6.5-7.5. In some specific embodiments, the composition contains 0.02%-0.1% buffer salt, preferably 0.03%, by mass percentage. In some more specific embodiments, the composition contains 0.005%-0.02% sodium dihydrogen phosphate monohydrate and 0.01%-0.05% anhydrous disodium hydrogen phosphate, preferably 0.01% sodium dihydrogen phosphate monohydrate and 0.02% anhydrous disodium hydrogen phosphate, by total mass.

[0095] In some more specific embodiments, the composition contains 0.05%-0.5% sodium hyaluronate, preferably 0.1%, by weight percentage.

[0096] In some embodiments, the composition is an eye drop.

[0097] This disclosure also provides a method for preparing the composition, which includes the following steps: dissolving the prescribed amount of osmotic pressure regulator, buffer salt, and sodium hyaluronate in the prescribed amount of water, mixing until homogeneous, sterilizing, adjusting the pH value to 6-8, adding recombinant type III humanized collagen filtered through a filter membrane in proportion, mixing evenly, and thus obtaining the composition.

[0098] In some embodiments, the preparation of the composition is carried out in a sterilized environment; preferably, the preparation environment is sterilized by ultraviolet irradiation, and the equipment required for preparation is sterilized by treatment at 121°C for 15 minutes.

[0099] In some implementations, the sterilization conditions for the homogeneous mixture of osmotic pressure regulator, buffer salt, sodium hyaluronate and water are 121°C for 15 min.

[0100] IV. Medical Uses

[0101] In some embodiments, this disclosure provides the use of the recombinant type III humanized collagen in the preparation of products for the prevention and / or treatment of ocular inflammation; wherein the recombinant type III humanized collagen is as described in Part II above.

[0102] In some embodiments, this disclosure provides for the use of the composition in the preparation of products for the prevention and / or treatment of ocular inflammation and / or dry eyes; wherein the composition is as described in Part III above.

[0103] In some embodiments, the recombinant type III humanized collagen of this disclosure is used for the prevention and / or treatment of ocular inflammation; wherein the recombinant type III humanized collagen is as described in Part II above.

[0104] In some embodiments, the compositions described in this disclosure are used to prevent and / or treat ocular inflammation and / or dry eyes; wherein the compositions are as described in Part III above.

[0105] In some embodiments, this disclosure provides a method for preventing and / or treating ocular inflammation, the method comprising administering an effective amount of the recombinant type III humanized collagen to an individual in need; wherein the recombinant type III humanized collagen is as described in Part II above, and the composition is as described in Part III above.

[0106] In some embodiments, this disclosure provides a method for preventing and / or treating ocular inflammation and / or dry eyes, the method comprising administering an effective amount of the composition to an individual in need; wherein the composition is as described in Part III above.

[0107] In some implementations, the ocular inflammation includes any one or more of blepharitis, keratitis, conjunctivitis, scleritis, meibomianitis, dacryocystitis, optic neuritis, and uveitis.

[0108] In some implementations, the eye inflammation and / or dryness is caused by wearing contact lenses.

[0109] In some implementations, the ocular inflammation is ocular surface inflammation, which includes any one or more of blepharitis, keratitis, and conjunctivitis.

[0110] In some implementations, the ocular inflammation is keratitis, blepharitis, or conjunctivitis.

[0111] In some embodiments, the blepharitis has symptoms including eyelid defects, the keratitis has symptoms including corneal ulcers, and the conjunctivitis has symptoms including conjunctival scarring.

[0112] In some implementations, the prevention and / or treatment of ocular inflammation includes any one or more of the following: reducing the levels of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more cells among eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues in the eyelids, cornea and / or conjunctiva, and enhancing tear film stability.

[0113] In some implementations, the prevention and / or treatment of ocular inflammation also includes the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more cells among eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues in the eyelids, cornea and / or conjunctiva, and enhancing tear film stability.

[0114] In some implementations, the prevention and / or treatment of ocular inflammation also includes the following manifestations: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more cells among eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, and maintaining the normal structural morphology of any one or more tissues in the eyelids, cornea and / or conjunctiva.

[0115] In some implementations, the prevention and / or treatment of dry eyes includes enhancing tear film stability.

[0116] In some implementations, the product reduces the level of ocular inflammatory mediators by inhibiting the generation and / or release of inflammatory mediators.

[0117] In some embodiments, the inflammatory mediator includes any one or more of MMP-1, MMP-9, TNF-α, and IL-1β. In some specific embodiments, the inflammatory mediator includes MMP-1, MMP-9, TNF-α, and IL-1β simultaneously. In some specific embodiments, the inflammatory mediator is a combination of MMP-1, MMP-9, TNF-α, and IL-1β.

[0118] Previous studies on cultured corneal / conjunctival epithelial cells have shown that the inflammatory cytokine TNF-α can promote the production of MMPs, which in turn can activate potential inflammatory cytokines such as IL-1β, forming a cycle of corneal / conjunctival epithelial inflammation. Furthermore, research has indicated that the inflammatory cytokine IL-1β participates in the pathogenesis of various ocular inflammations. In addition, studies have shown that the inflammatory mediator MMP-9 plays an important role in the pathogenesis of keratitis / conjunctivitis, particularly in infectious keratitis / conjunctivitis caused by bacterial biofilms. However, this disclosure reveals that the recombinant type III humanized collagen and the composition described herein exhibit inhibitory effects on MMP-1, MMP-9, TNF-α, and IL-1β, with a particularly unexpected inhibitory effect on MMP-9.

[0119] In some embodiments, enhancing tear film stability includes any one or more of increasing the mucin content in the tear fluid and decreasing the osmotic pressure of the tear fluid. In some embodiments, enhancing tear film stability includes both increasing the mucin content in the tear fluid and decreasing the osmotic pressure of the tear fluid.

[0120] In some implementations, the product includes a pharmaceutical composition, a medical device, or a combination of pharmaceutical and medical devices.

[0121] V. The amino acid sequence involved in this disclosure

[0122] SEQ ID NO. 1: GERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0123] SEQ ID NO.2: GPPGPCCGGG.

[0124] SEQ ID NO.3: GERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGE RGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERG APGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGA PGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0125] SEQ ID NO.4: ENLYFQ.

[0126] SEQ ID NO.5: ENLYFQGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKG PAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAG ERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERG APGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAPGERGAPGFRGPAGPNGIPGEKGPAGERGAP.

[0127] Example

[0128] The embodiments of this disclosure will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of this disclosure. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials or instruments used in the embodiments are all commercially available conventional products.

[0129] The amino acid sequence of the recombinant humanized type III collagen (rhCol III) used in the examples is shown in SEQ ID NO.3.

[0130] Example 1: rhCol III promotes corneal epithelial cell renewal and repair

[0131] (1) Cytotoxicity test:

[0132] Cultured human corneal epithelial cells (HCE-T) were digested with 0.25% trypsin solution to prepare a cell suspension of 5000 cells / mL, which was then seeded into 96-well plates (100 μL / well). After culturing for 24 h, the supernatant was discarded, and different concentrations of recombinant type III humanized collagen (0.2 mg / ml, 0.4 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1 mg / ml) were prepared. Positive control wells (1% DMSO solution) and negative control wells (DMEM medium) were also set up, with 6 replicates for each concentration. The wells were surrounded by D-PBS and cultured at 37℃ and 5% CO2 for 24 h. After discarding the supernatant, CCK8 and DMEM medium were added and cultured for 2 h. The absorbance (OD) at 450 nm was measured using a microplate reader. The cell viability (%) was calculated as follows: (OD value of sample wells - OD value of blank wells) / (OD value of negative control wells - OD value of blank wells) × 100.

[0133] The cytotoxicity test results are shown in Figure 1. According to the CCK-8 assay, a higher OD value indicates higher activity. Compared to the positive and negative control groups, with increasing sample concentration, the activity of human corneal epithelial cells in the wells containing recombinant type III humanized collagen gradually increased. Recombinant type III humanized collagen can reduce corneal epithelial cell damage and promote corneal epithelial cell repair.

[0134] (2) Cell adhesion experiment:

[0135] Recombinant type III humanized collagen was diluted to 1.00 mg / ml, 0.5 mg / ml, 0.1 mg / ml, 0.05 mg / ml, and 0.01 mg / ml using D-PBS. Positive control (1 mg / ml bovine type I collagen) and negative control (D-PBS) were added, with 100 μL per well and 5 replicates per group. The mixture was incubated overnight at 4°C. The supernatant was discarded, and 100 μL of 1% BSA (inactivated at 56°C for 30 min) was added. The mixture was incubated at 37°C for 1 h. The supernatant was discarded, and the sample was washed three times with D-PBS. 1×10⁻⁶ BSA was added to each well. 6 HCE-T cells in good condition, resuspended in D-PBS, were incubated at 37°C for 2 hours. The cells were washed twice with D-PBS. The absorbance at OD492 nm was measured using an LDH detection kit (Roche, 04744926001). The cell adhesion rate was calculated based on the values ​​of the blank control. The formula is as follows: Cell adhesion rate = (Test wells - Blank wells) / (Positive wells - Blank wells) × 100%.

[0136] The results of the cell adhesion experiment are shown in Figure 2. Different concentrations of recombinant human type III collagen exhibited better adhesion activity compared to both the positive and negative controls. Compared to the positive control, the cell adhesion activity was significantly different at concentrations of 1.00 mg / ml, 0.5 mg / ml, 0.1 mg / ml, and 0.01 mg / ml (P < 0.001, P < 0.001, P < 0.01, P < 0.05), while there was no significant difference in cell adhesion activity at the 0.05 mg / ml group (P > 0.05). Recombinant human type III collagen significantly improved cell adhesion activity, and the concentration of recombinant human type III collagen was determined to be 0.5 mg / ml (0.05%) in subsequent experiments. Cell adhesion is fundamental to maintaining tissue structure. Recombinant type III humanized collagen provides cells with a high-quality external environment in a short period of time, helping cells adhere to the wall and exhibiting excellent cell adhesion activity.

[0137] (3) Cell scratch assay:

[0138] Experimental groups: The experiment was divided into a PBS group, a commercially available group (commercially available type III collagen group, purchased from Jilin Guoda Biotechnology Co., Ltd.), and a 0.5 mg / ml recombinant type III humanized collagen group. 0.4% serum culture medium served as a negative control (NC), and 10 ng / ml epidermal growth factor (EGF) served as a positive control (PC).

[0139] Draw lines on the back of the 12-well plate with a marker to facilitate subsequent positioning and photography. Add 1 mL of 1×10⁻⁶ solution to each well of the 12-well plate. 6 HCE-T cell suspension was incubated at / mL for 24h in an incubator. The supernatant was discarded, and 1mL of 0.4% serum culture medium was added for starvation treatment for 12h. After the cells reached confluence, vertical scratches were marked using a 200μL yellow pipette tip. The supernatant was discarded, and the cells were washed three times with PBS. 1mL of each sample was added to each group, and cell healing was observed at 0, 12, and 24h. The scratch healing area was estimated using ImageJ software.

[0140] The results of the cell scratch assay are shown in Figure 3. Using an inverted microscope, it was observed that from 0 h to 24 h, the cell migration rate of the recombinant type III humanized collagen group was superior to the other four groups, and by 24 h, the cells had essentially covered the entire field of view. Calculations of the average cell migration rate at 12 h and 24 h using software confirmed that the recombinant type III humanized collagen group had the largest migration area, thus demonstrating that recombinant type III humanized collagen can enhance the migration ability of corneal epithelial cells and promote corneal damage repair.

[0141] Example 2: rhCol III eye drops enhance tear film stability

[0142] Preparation of rhCol III group: A sterile environment was prepared in the early stages of the experiment. A laminar flow hood was sterilized with UV light, and all necessary equipment was sterilized in an autoclave at 121°C for 15 minutes. In the laminar flow hood, 0.1 parts sodium hyaluronate, 0.85 parts sodium chloride, 0.02 parts anhydrous disodium hydrogen phosphate, and 0.01 parts sodium dihydrogen phosphate monohydrate were weighed according to the specified ratio and dissolved in the prescribed amount of water (the amount of water should ensure a final total of 100 parts of eye drops). The solution was placed in a sterilized container and thoroughly stirred until homogeneous. The solution was then sterilized at 121°C for 15 minutes to obtain the matrix. After cooling, the pH of the matrix was measured and should be between 6 and 8. 0.05% recombinant type III humanized collagen, filtered through a membrane, was added. The commercially available type III collagen group was prepared in the same manner.

[0143] Several healthy Japanese white rabbits were randomly divided into an experimental group (recombinant type III humanized collagen group), a commercially available group (commercially available type III collagen group, purchased from Jilin Guoda Biotechnology Co., Ltd.), a model group (intervention with buffered saline solution), and a normal group (no intervention).

[0144] All Japanese White rabbits were placed in the animal facility for one week for acclimatization, with free access to water. The rabbits were then restrained, and 2 mL / kg was injected via the marginal ear vein. -1 General anesthesia was administered with chloral hydrate, followed by local anesthesia with 2 drops of promecaine hydrochloride eye drops placed in the surgical area. After the anesthesia took effect, the tip of the third eyelid was lifted with forceps, and part of the tissue was removed. Tobramycin eye drops were then instilled 2 drops three times a day for 4 days. After the wound healed, 3 rabbits were randomly selected as the normal control group (hereinafter referred to as the normal control group). The remaining groups received 0.2% benzalkonium chloride eye drops, 2 drops three times a day for 7 consecutive days. Seven days after modeling, increased ocular surface secretions, eyelid redness and swelling, severe conjunctival congestion, and corneal opacity were observed, ensuring the successful establishment of the inflammation model. The experimental group, the commercially available group, and the model group were treated with eye drops, 2 drops three times a day for 7 and 14 days respectively.

[0145] Tear film stability was assessed by aspirating rabbit tears from the lower eyelid conjunctival sac using a disposable micropipette onto a glass slide, spreading them evenly, and allowing them to dry. Tear film stability was determined based on the morphology of tear crystals (graded from regular, intact, and dense fern-like crystals to irregular, amorphous, or even no crystals, classified as grades I-IV; grades I and II represent normal tear crystals, while grades III and IV represent abnormal crystals). Mucin in normal tears can form a branching fern-like pattern.

[0146] The experimental results are shown in Figure 4. On day 0 of treatment, except for the normal group, only a small number of irregular crystals were observed in other groups, mainly grade III and IV. On day 7 of treatment, the recombinant type III humanized collagen eye drops group showed more regular crystal shapes and increased numbers compared to the model group. On day 14 of treatment, complete, large fern-like crystals were observed in the recombinant type III humanized collagen eye drops group. This demonstrates that after using recombinant type III humanized collagen eye drops, the tear film crystal morphology is closer to normal (grades I and II), indicating that it can replenish tear film components, alleviate tear hyperosmolarity, and enhance tear film stability.

[0147] Examples 1 and 2 demonstrate that recombinant type III humanized collagen has the ability to promote corneal epithelial renewal and repair. Damage to the conjunctiva / cornea / eyelid triggers an immune response, releasing a series of inflammatory factors such as interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and matrix metalloproteinases (MMPs). Example 3 uses cellular experiments to examine the effect of recombinant type III humanized collagen on these inflammatory factors, determining whether it can inhibit and treat ocular inflammation at its site.

[0148] Example 3: Cellular assay of rhCol III inhibiting inflammation

[0149] Enzyme-linked immunosorbent assay (ELISA):

[0150] Experimental groups: normal control group (DMEM / F12 medium), inflammation group (LPS 1μM), experimental group (0.05% recombinant type III humanized collagen), and commercially available group (0.05% commercially available type III collagen, purchased from Jilin Guoda Biotechnology Co., Ltd.). Experimental procedure: HCE-T cells in the logarithmic growth phase were cultured in complete medium at a concentration of 1.5 × 10⁻⁶ cells / year. 5Seed 1 mL of cell / mL solution into each well of a 6-well plate. After cell adhesion, add 700 μL of the corresponding group solution and continue culturing for 24 h. Collect the cell supernatant. Remove the ELISA kit from the refrigerator and allow it to equilibrate at room temperature for about 30 minutes to ensure the reagents are stable. Dilute the capture antibody to 5 μg / mL with coating buffer. Add 100-200 μL of the diluted capture antibody to each well of the ELISA plate. Seal the plate with sealing film and place it in a 4°C refrigerator overnight to allow the antibody to fully adsorb onto the well walls. After overnight coating, remove the plate and discard the liquid in the wells. Add 200-300 μL of wash PBS buffer to the wells. Let stand for a moment, then discard the wash buffer. Repeat the washing step 3-5 times to ensure thorough washing. Automated washing can be performed using a plate washer to ensure consistency. Add blocking buffer (PBS buffer containing 1%-5% BSA) to each well of the washed ELISA plate, 200-300 μL per well. Place the plate in a microplate reader and select a suitable wavelength (e.g., 450 nm) for absorbance measurement. The microplate reader will measure the absorbance value in each well. Detect the levels of inflammatory factors TNF-α, IL-1β, MMP-1, and MMP-9.

[0151] The lower the levels of these four inflammatory factors, the milder the inflammatory response. The experimental results are shown in Table 1. The levels of the four inflammatory factors were measured. Compared with the commercially available group, the recombinant type III humanized collagen experimental group showed inhibitory effects on all four inflammatory factors, with the most significant inhibitory effect on MMP-9, demonstrating that recombinant type III humanized collagen has an anti-inflammatory effect.

[0152] Table 1. Results of enzyme-linked immunosorbent assay (ELISA) for detecting inflammatory factor levels.

[0153] Example 3 illustrates that recombinant type III humanized collagen is effective against all these inflammatory factors, with a significant inhibitory effect on MMP-9. Furthermore, all four factors, especially MMP-9, are significantly elevated in eyelid / cornea / conjunctival inflammation. Example 4 further investigates the efficacy of recombinant type III humanized collagen in an animal model of eyelid / cornea / conjunctival inflammation.

[0154] Example 4: Animal experiments on the inhibitory effect of rhCol III on inflammation

[0155] (1) General view photograph:

[0156] Several healthy Japanese white rabbits were randomly divided into an experimental group (0.05% recombinant type III humanized collagen), a commercially available group (0.05% commercially available type III collagen, purchased from Jilin Guoda Biotechnology Co., Ltd.), a model group (intervention with buffered saline solution), and a normal group (no intervention).

[0157] All Japanese White rabbits were placed in the animal facility for one week for acclimatization, with free access to water. The rabbits were then restrained, and 2 mL / kg was injected via the marginal ear vein. -1 General anesthesia was administered with chloral hydrate, followed by local anesthesia with 2 drops of promecaine hydrochloride eye drops placed in the surgical area. After the anesthesia took effect, the tip of the third eyelid was lifted with forceps, and part of the tissue was removed. Tobramycin eye drops were then instilled 2 drops three times daily for 4 days. After the wound healed, 3 rabbits were randomly selected as the normal control group (hereinafter referred to as the normal control group). The remaining groups received 0.2% benzalkonium chloride eye drops, 2 drops three times daily for 7 consecutive days. Seven days after modeling, increased ocular surface secretions, eyelid redness and swelling, severe conjunctival congestion, and corneal opacity were observed, confirming the successful establishment of the inflammation model.

[0158] The experimental group, the commercially available group, and the model group were all treated with a solution, 2 drops each time, 3 times a day, for 7 and 14 consecutive days, and the changes in the rabbits' eyes were observed.

[0159] The experimental results are shown in Figure 5. On day 7 of treatment, the experimental group showed reduced secretions and alleviated symptoms of eye congestion and edema compared to the model group. On day 14 of treatment, neither the experimental nor the commercially available group showed any secretions. Regarding the degree of corneal opacity and the extent of eyelid and conjunctival edema, the experimental group showed better recovery than the commercially available group. This demonstrates that recombinant type III humanized collagen has a good effect in treating ocular inflammation.

[0160] (2) Observation of corneal / conjunctival pathological sections:

[0161] After treatment, rabbits were euthanized, and their eyeballs were fixed in a fixative (such as 4% paraformaldehyde) for 48 hours. After routine dehydration, clearing, paraffin embedding, and embedding, 4-6 μm thick sections were prepared. Hematoxylin-eosin (HE) staining was performed, and changes in corneal / conjunctival tissue structure were observed under a light microscope.

[0162] The experimental results are shown in Figure 6. Pathological examination of the sections revealed that, for corneal tissue, normal corneal epithelium consisted of 2-3 layers of squamous epithelial cells, arranged neatly and uniformly. On day 0 of treatment, except for the normal group, the corneal epithelium in other groups was partially or completely detached, and the corneal endothelium was edematous and detached from the posterior border. On day 14 of treatment, the experimental group showed a more intact corneal epithelium than the model group and the commercially available group, with neatly arranged cells and relatively normal morphology; no edema or detachment of the corneal endothelium was observed. For conjunctival tissue, normal conjunctival tissue was tightly connected, and the columnar epithelium was regular in morphology and neatly arranged. On day 0 of treatment, the interstitial spaces in the experimental group, model group, and commercially available group were widened, and the stroma was loose. On day 14 of treatment, the cells in the experimental group were more neatly arranged than those in the model group and the commercially available group. This demonstrates that the experimental group has a good repair effect on the damage to the cellular tissues of the cornea / conjunctiva, and that recombinant type III humanized collagen has a therapeutic effect on the keratitis / conjunctivitis model.

[0163] From a cellular perspective, recombinant type III humanized collagen can significantly enhance the proliferation and differentiation of corneal epithelial cells and stromal cells, accelerate the renewal and repair of epithelial cells, and thus cover damaged areas more quickly. Furthermore, through tear film experiments, recombinant type III humanized collagen can significantly improve the effect of dry eyes. Its moisturizing effect can further provide an external environment for the proliferation and repair of damaged cells. In addition, recombinant type III humanized collagen can reduce the release of inflammatory mediators and inhibit the infiltration and activation of inflammatory cells, thereby alleviating the inflammatory response of the eyelids / cornea / conjunctiva, which is conducive to the repair of eye tissues. The repaired eye tissues can better perform their physiological functions.

Claims

1. Use of a recombinant humanized collagen type III in the manufacture of a product for the prevention and / or treatment of ocular inflammation; wherein, The amino acid sequence of the recombinant type III humanized collagen contains n repeats of the sequence shown in SEQ ID NO.1, where n is an integer greater than or equal to 1, and when n is an integer greater than or equal to 2, the repeat sequences are directly connected. Preferably, the amino acid sequence of the recombinant type III humanized collagen includes any one of the following (a) to (d): (a) A sequence as shown in SEQ ID NO.3; (b) A sequence having 90%, 92%, 95%, 96%, 97%, 98% or 99% identity with the sequence shown in SEQ ID NO.3, and retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3; (c) A sequence in which one or more amino acid residues are added, substituted or deleted in the sequence shown in SEQ ID NO.3, and which retains the cell adhesion effect of the sequence shown in SEQ ID NO.3; (d) An amino acid sequence encoded by a nucleotide sequence hybridizing with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.3 under stringent conditions, and the amino acid sequence retaining the cell adhesion effect of the sequence shown in SEQ ID NO.3, wherein the stringent conditions are moderately stringent, medium-high stringent, high stringent, or very high stringent.

2. The use according to claim 1, characterized in that, The ocular inflammation includes any one or more of the following: blepharitis, keratitis, conjunctivitis, scleritis, meibomianitis, dacryocystitis, optic neuritis, and uveitis; Preferably, the eye inflammation is caused by wearing contact lenses.

3. The use according to claim 1 or 2, characterized in that, The ocular inflammation is an ocular surface inflammation, which includes any one or more of blepharitis, keratitis, and conjunctivitis; Preferably, the ocular surface inflammation is blepharitis, keratitis, or conjunctivitis; More preferably, the blepharitis has symptoms including eyelid defects, the keratitis has symptoms including corneal ulcers, and the conjunctivitis has symptoms including conjunctival scarring.

4. The use according to any one of claims 1 to 3, characterized in that, The prevention and / or treatment of ocular inflammation includes any one or more of the following: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more cells among eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues in the eyelids, cornea and / or conjunctiva and enhancing tear film stability.

5. The use according to claim 4, characterized in that, The inflammatory mediators include any one or more of MMP-1, MMP-9, TNF-α, and IL-1β; Preferably, the inflammatory mediators include MMP-1, MMP-9, TNF-α, and IL-1β; More preferably, the inflammatory mediator is a combination of MMP-1, MMP-9, TNF-α and IL-1β.

6. The use according to claim 4 or 5, characterized in that, The enhancement of tear film stability includes any one or more of increasing the mucin content in the tear fluid and reducing the osmotic pressure of the tear fluid.

7. A composition comprising the following components in weight percentage: 0.001%-0.1% recombinant type III humanized collagen, 0.5%-1.5% osmotic pressure regulator, 0.01%-0.5% buffer salt, 0.02%-1% sodium hyaluronate, and the balance being water; Preferably, the composition is an eye drop.

8. Use of the composition according to claim 7 in the preparation of products for the prevention and / or treatment of ocular inflammation and / or dry eyes.

9. The use according to claim 8, characterized in that, The ocular inflammation includes any one or more of the following: blepharitis, keratitis, conjunctivitis, scleritis, meibomianitis, dacryocystitis, optic neuritis, and uveitis; Preferably, the eye inflammation and / or dryness are caused by wearing contact lenses.

10. The use according to claim 8 or 9, characterized in that, The ocular inflammation is an ocular surface inflammation, which includes any one or more of blepharitis, keratitis, and conjunctivitis; Preferably, the ocular surface inflammation is blepharitis, keratitis, or conjunctivitis; More preferably, the blepharitis has symptoms including eyelid defects, the keratitis has symptoms including corneal ulcers, and the conjunctivitis has symptoms including conjunctival scarring.

11. The use according to any one of claims 8 to 10, characterized in that, The prevention and / or treatment of ocular inflammation and / or dry eyes includes any one or more of the following: reducing the content of ocular inflammatory mediators, promoting the proliferation, migration and / or adhesion of any one or more cells among eyelid epithelial cells, corneal epithelial cells and conjunctival epithelial cells, maintaining the normal structural morphology of any one or more tissues in the eyelids, cornea and / or conjunctiva and enhancing tear film stability.

12. The use according to claim 11, characterized in that, The inflammatory mediators include any one or more of MMP-1, MMP-9, TNF-α, and IL-1β; Preferably, the inflammatory mediators include MMP-1, MMP-9, TNF-α, and IL-1β; More preferably, the inflammatory mediator is a combination of MMP-1, MMP-9, TNF-α and IL-1β.

13. The use according to claim 11 or 12, characterized in that, The enhancement of tear film stability includes any one or more of increasing the mucin content in the tear fluid and reducing the osmotic pressure of the tear fluid.

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