Superoxide dismutase and use thereof in prevention or treatment of dry eye

Abstract

The present invention relates to superoxide dismutase and its use in the prevention or treatment of dry eye. More specifically, the present invention relates to a pharmaceutical composition or a treatment method for preventing or treating dry eye. According to the compositions and methods of the present invention, an increase in the secretion amount selected from tear fluid can be achieved; the tear film rupture time is prolonged; minimizing ocular surface damage; goblet cells in the conjunctiva are protected; the number of immune cells and inflammatory cell factors in cornea and conjunctiva is reduced; reduction of oxidative stress in cornea, conjunctiva and lacrimal gland; and a reduction in corneal epithelial cell apoptosis, thereby being effectively used in the prevention or treatment of dry eye.

Description

Technical Field

[0001] The present invention relates to superoxide dismutase and its use in preventing, improving or treating dry eyes. More specifically, the present invention relates to superoxide dismutase, composition or method for preventing, improving or treating dry eyes. Background Art

[0002] Dry eye is an increasingly common major disease, affecting approximately 20% of adults in Korea. In recent years, the concept of dry eye has changed, and dry eye is defined as a multifactorial disease of the tear film and ocular surface, causing discomfort, visual impairment, tear film instability, and ocular surface damage, accompanied by increased tear osmolarity and ocular surface inflammation.

[0003] Although the pathological mechanism of dry eye is not yet clear, various studies have reported that it is related to inflammatory changes in the ocular surface caused by tear hyperosmolarity, and that a variety of inflammatory factors can affect the pathological process of dry eye and ocular discomfort. In the tears and conjunctival epithelium of patients with Sjogren's syndrome, inflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, and IFN-γ, immune activity and adhesion substances in conjunctival epithelial cells such as HLA-DR and ICAM-1, and the expression of cytokines Th-1 or Th-17 related to the inflammatory pathway increase. In addition, increased concentrations and activities of matrix metalloproteinases and increased apoptosis of ocular surface epithelial cells have been confirmed in dry eyes, indicating that dry eye is directly related to inflammation of the ocular surface. Therefore, for the treatment of dry eye, it is important to inhibit ocular surface inflammation caused by tear hyperosmolarity.

[0004] Currently used treatments for dry eyes include environmental adjustment, artificial tears, anti-inflammatory treatments such as steroids or cyclosporine, tear secretion promoters, punctal embolization, serum eye drops, therapeutic contact lenses, surgery, etc., which are selected according to the severity of the disease, but there is no clear treatment method. Among them, the mixed administration of artificial tears and anti-inflammatory eye drops is the most widely used drug treatment method in the treatment of dry eyes. Artificial tears can temporarily relieve the symptoms of dry eyes, but cannot fundamentally treat inflammation of the ocular surface. Among anti-inflammatory preparations, the disadvantage of steroids is that they may induce serious complications such as glaucoma and cataracts, so it is necessary to develop a new dry eye treatment agent.

[0005] In addition, in 2006, Allergan developed a dry eye treatment agent Restasis eye drops using the immunomodulator cyclosporine. It is reported that Restasis eye drops can inhibit the generation and activation of immune cells associated with keratoconjunctivitis sicca and increase tear secretion. However, the above-mentioned preparations exert their efficacy through anti-inflammatory effects, so they need to be used repeatedly for several months to achieve a satisfactory level of efficacy, and the burning sensation as a representative side effect is high, about 17%.

[0006] Therefore, there is a need to develop a preparation that can fundamentally prevent or treat dry eye disease, as opposed to a symptomatic treatment that simply relieves symptoms, and that is safe due to a low incidence of side effects caused by eye drop administration. Summary of the invention

[0007] Technical issues

[0008] The purpose of the present invention is to solve the above problems existing in the prior art.

[0009] One object of the present invention is to provide superoxide dismutase for preventing, improving or treating dry eyes.

[0010] Another object of the present invention is to provide superoxide dismutase derived from Bacillus amyloliques faciens, which ensures oral administration efficacy and safety for preventing, improving or treating dry eyes.

[0011] Another object of the present invention is to provide a superoxide dismutase derived from Bacillus or Bacillus amyloliquefaciens, which is secreted outside the cell and is easy to produce, for use in preventing or treating dry eyes.

[0012] Yet another object of the present invention is to provide a pharmaceutical composition or a treatment method for preventing or treating dry eyes.

[0013] Still another object of the present invention is to provide a food or feed composition for preventing or improving dry eyes.

[0014] Still another object of the present invention is to provide an oral composition comprising superoxide dismutase for preventing, improving or treating dry eyes.

[0015] The object of the present invention is not limited to the above-mentioned object. The object of the present invention will become more apparent from the following description and can be achieved by the means described in the claims and their combination.

[0016] Technical Solution

[0017] Representative embodiments of the present invention for achieving the above-mentioned object are as follows.

[0018] According to one aspect of the present invention, an improved superoxide dismutase (SOD) for preventing, improving or treating dry eyes is provided.

[0019] According to another aspect of the present invention, there is provided a superoxide dismutase for preventing, improving or treating dry eyes, which is derived from generally regarded as safe (GRAS) bacteria, such as Bacillus.

[0020] According to yet another aspect of the present invention, there is provided a superoxide dismutase derived from Bacillus or Bacillus amyloliquefaciens, which is secreted outside the cell to facilitate production, and is used for preventing, improving or treating dry eyes.

[0021] According to yet another aspect of the present invention, there is provided an improved superoxide dismutase, which also exhibits the effect of preventing, improving or treating dry eyes when orally administered.

[0022] According to yet another aspect of the present invention, a pharmaceutical composition for preventing or treating dry eyes is provided, comprising the superoxide dismutase (SOD) as an effective ingredient.

[0023] According to yet another aspect of the present invention, there is provided a food or feed composition for improving or preventing dry eyes, comprising the superoxide dismutase as an effective ingredient.

[0024] According to yet another aspect of the present invention, there is provided an oral composition for preventing, improving or treating dry eyes, comprising the superoxide dismutase as an active ingredient.

[0025] According to yet another aspect of the present invention, a method for preventing, improving or treating dry eyes is provided, comprising the step of administering the superoxide dismutase or the composition to a subject.

[0026] According to yet another aspect of the present invention, there is provided use of the superoxide dismutase in preventing or treating dry eyes.

[0027] According to yet another aspect of the present invention, there is provided use of the superoxide dismutase in preparing a medicament for preventing or treating dry eyes.

[0028] Beneficial Effects

[0029] According to the superoxide dismutase, composition and method of the present invention, at least one effect selected from the group consisting of increase in tear secretion, increase in tear film breakup time, minimization of ocular surface damage, protection of goblet cells in the conjunctiva, reduction in the number of immune cells and / or inflammatory cytokines in the cornea and / or conjunctiva, reduction in oxidative stress in the cornea, conjunctiva and / or lacrimal glands, and reduction in corneal epithelial cell apoptosis can be effectively used for the prevention, improvement or treatment of dry eyes. In particular, it has been confirmed that the superoxide dismutase according to the present invention is also effective for the prevention, improvement or treatment of dry eyes when administered orally.

[0030] The superoxide dismutase according to the present invention is derived from Bacillus or Bacillus amyloliquefaciens, which are generally regarded as safe (GRAS) bacteria, and thus not only ensures oral administration efficacy and safety, but also has a production advantage in that it can be directly recovered from the supernatant during culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the double crossover recombination performed in Example 1.1 is shown.

[0032] Figure 2 The results of confirming the defective gene using PCR in the expression host GFBS220 and the production strain BSBA310 are shown. W indicates the wild-type Bacillus subtilis KCTC 3135, 220 indicates the expression host GFBS220, and 310 indicates the SodA2 production strain BSBA310.

[0033] Figure 3 Shown are the results of a comparison of the amino acid sequences of SodA (Mn-SOD determined by N-terminal sequencing in the culture supernatant of Bacillus amyloliquefaciens strain GF423) and the superoxide dismutase of the present invention (SodA2).

[0034] Figure 4 The expression vector for sodA2 gene overexpression is shown. Among them, rrnB T1T2 represents a transcription terminator; rep(pBR322) represents a replicon from pBR322 that works in E. coli; rep(pUB110) represents a replicon from pUB110 that works in Bacillus subtilis; Kan R represents a kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); and BJ27 promoter represents a strong promoter of Bacillus subtilis (B. subtilis).

[0035] Figure 5 The overall scheme for preparing the production strain BSBA310 is shown.

[0036] Figure 6 The results of measuring tear volume performed in the examples of the present invention are shown.

[0037] Figure 7 The measurement results of tear film break-up time (TBUT) performed in the embodiment of the present invention are shown.

[0038] Figure 8 The results of measuring the corneal fluorescent staining score performed in the embodiment of the present invention are shown.

[0039] Figure 9a and Figure 9b The results of measuring the number of immune cells performed in the examples of the present invention are shown.

[0040] Fig.10a , Fig.10b and Fig.10c The results of active oxygen generation performed in Examples of the present invention are shown.

[0041] Fig.11a and Fig.11b The figures show the results of measuring the number of goblet cells in the conjunctiva performed in the examples of the present invention.

[0042] Fig.12a and Figure 12b The results of measuring cell apoptosis performed in the examples of the present invention are shown. DETAILED DESCRIPTION

[0043] In the detailed description of the invention set forth below, specific embodiments that can implement the invention will be described with reference to specific drawings, but the invention is not limited thereto, and if properly described, the scope of the invention depends on the appended claims including those claimed in the claims and all equivalents. It should be understood that the various embodiments of the invention, although different from each other, are not necessarily mutually exclusive. For example, without departing from the spirit and scope of the invention, the specific shapes, structures and characteristics described herein can be realized by changing from one embodiment to another or by a combination of embodiments. Unless otherwise specified, the terms mentioned when describing the present invention should generally be understood as the usual meaning, and are applicable to both the aspects or embodiments of the invention that define the term, and the same terms used herein. The definitions described below are provided for the purpose of explaining the present invention, and the terms used in the singular will include the plural form where appropriate, and vice versa.

[0044] definition

[0045] The term "about" as used herein refers to the usual error range for each value known to those skilled in the art.

[0046] The term "subject" is used interchangeably with "patient" and refers to a mammal in need of prevention or treatment of dry eye, for example, primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), livestock animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and experimental animals (e.g., rats, guinea pigs, etc.). In one embodiment of the present invention, the subject is a human.

[0047] The term "treatment" includes preventive and / or curative treatment. The preventive and / or curative treatment includes all types of treatments recognized in the art, for example, including administering the pharmaceutical composition of the present invention or SOD to the subject. If administered before clinical symptoms of an undesirable or unpreferred state (e.g., a disease or other undesirable or unpreferred state of the subject), such treatment is preventive treatment (e.g., protecting the subject from developing an undesirable or unpreferred state). In addition, if administered after clinical manifestations of an undesirable or unpreferred state, such treatment is a curative treatment that alleviates, relieves or stabilizes an existing undesirable or unpreferred state or its side effects.

[0048] The meaning of the term "prevention" is well known in the art. When used together with medical conditions such as dry eyes, it means that by administering the pharmaceutical composition or SOD of the present invention, the frequency of medical conditions (e.g., discomfort, visual disturbances, tear film instability, ocular surface damage, increased tear osmotic pressure, ocular surface inflammation, etc.) is reduced or the onset and symptoms are delayed compared to subjects not administered with it.

[0049] The term "administering" refers to providing an active ingredient to a subject for preventive or therapeutic purposes (eg, preventing or treating dry eye).

[0050] Dry eyes

[0051] The present invention is based at least in part on the discovery that administration of superoxide dismutase (SOD), especially oral administration, can effectively prevent or treat dry eyes. Therefore, according to one aspect of the present invention, there is provided a use of SOD in preventing, ameliorating or treating dry eyes.

[0052] The term "dry eye" is used interchangeably with dry eye or dry eye syndrome, and refers to an eye disease characterized by various symptoms (such as eye discomfort, dryness, foreign body sensation, itching, pain, decreased vision, etc.) caused by damage to the ocular surface due to a variety of reasons (such as insufficient tears, excessive evaporation of the tear film, imbalance of tear components, inflammatory changes in tissues involved in the tear secretion process).

[0053] In addition, in the present invention, dry eyes may be caused by other underlying diseases such as Sjogren's syndrome, and may be a complication of blepharitis or intraocular foreign bodies. In addition, in the present invention, dry eyes may be caused by infection or a side effect of medication, and may be a result of exposure to toxins, chemicals or other substances. Therefore, in the present invention, dry eyes may include dry eyes caused by Sjogren's syndrome or dry eyes caused by non-Sjogren's syndrome.

[0054] The SOD according to the present invention can achieve at least one selected from the following: an increase in tear secretion; an increase in tear film breakup time; minimization of ocular surface damage; protection of goblet cells in the conjunctiva; a reduction in the number of immune cells and / or inflammatory cytokines in the cornea and / or conjunctiva; a reduction in oxidative stress in the cornea, conjunctiva and / or lacrimal glands; and a reduction in corneal epithelial cell apoptosis.

[0055] Superoxide dismutase (SOD)

[0056] According to another aspect of the present invention, an improved superoxide dismutase (SOD) for preventing, improving or treating dry eyes is provided.

[0057] Superoxide dismutase (SOD) is an enzyme that can alternately catalyze the ·- ) into general molecular oxygen (O2) and hydrogen peroxide (H2O2). SOD plays an important role in reducing oxidative stress by removing reactive oxygen species. SOD is widely distributed in prokaryotes and eukaryotic cells and is divided into four categories according to different types of metal centers (copper / zinc, nickel, manganese and iron). Manganese-containing SOD [Mn-SOD; manganese-containing superoxide dismutase] is widely present in a variety of bacteria, chloroplasts, mitochondria and cytoplasm of eukaryotic cells. The term "SOD" can be used interchangeably with polypeptides with superoxide dismutase activity.

[0058] In one embodiment, the SOD may be a superoxide dismutase (Mn-SOD) bound to manganese. Specifically, the SOD may be a deamidated Mn-SOD. More specifically, the SOD may be a SOD based on SEQ ID NO: 2, wherein the amino acid residues at positions 73 and 136 are substituted by Asp. More specifically, the SOD may comprise or consist of the amino acid sequence shown in SEQ ID NO: 4.

[0059] The SOD or polypeptide having SOD activity of the present invention is interpreted as also including amino acid sequences showing substantial identity with respect to the amino acid sequence. The substantial identity means that when the sequence is analyzed and aligned using an algorithm commonly used in the art, the amino acid sequence shows 80% or higher, preferably 90% or higher, more preferably 95% or higher, and most preferably 98% or higher sequence identity.

[0060] In another embodiment, SOD is a modified or engineered polypeptide having SOD enzyme activity, which may include one or more mutations that may or may not affect various aspects (in vivo, in vitro or ex vivo stability, uniformity and / or conformational changes), such as deletion, insertion or substitution of more than one amino acid. In addition, the polypeptide may also include heterologous substances (e.g., tags known in the art including HIS tags, Ha tags, myc tags, GFC and / or Fc domains of antibodies) for purification, detection or increased stability.

[0061] In one embodiment, SOD may be derived from natural or recombinant microorganisms, and may be an enzyme prepared from various supply sources through a process of isolation or purification.

[0062] In one embodiment, SOD may be derived from a natural or recombinant microorganism. For example, SOD may be derived from bacteria. Preferably, SOD may be derived from bacteria that are generally regarded as safe (GRAS) for use in medicines or foods. Specifically, SOD may be derived from a Bacillus strain. More specifically, the SOD may be derived from a Bacillus amyloliquefaciens strain. For example, the SOD may be derived from a Bacillus amyloliquefaciens GF423 strain (KCTC 13222BP). The GF423 strain (KCTC13222BP) was deposited at the Korea Institute of Biotechnology on March 6, 2017. In addition, SOD may be derived from a mixture comprising Figure 4 In addition, the SOD can be derived from a recombinant strain containing the expression vector shown in Figure 5 The recombinant strain prepared by the method shown in the method. The recombinant strain may be a Bacillus strain, the Bacillus strain comprises a nucleotide sequence encoding a polypeptide, the polypeptide comprises an amino acid sequence shown in SEQ ID NO: 4, and the AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf genes are deleted in the Bacillus strain. The detailed process of preparing the strain is shown in Example 1.

[0063] The SOD derived from the strain is an enzyme secreted outside the cell, so when the strain is used to prepare SOD, SOD that is safe for the human body can be produced in large quantities without expensive purification processes (such as column purification), thereby achieving efficient production.

[0064] In one embodiment, SOD can be isolated or purified from a variety of supply sources including natural or recombinant hosts. For example, SOD with the activity of preventing or treating dry eyes can be extracted from the culture supernatant of Bacillus amyloliquefaciens GF423 strain. In short, first, the culture fluid can be obtained by culturing Bacillus amyloliquefaciens GF423 strain in various types of culture media. For example, a composite culture medium (pH 6.0 to 7.0) is used to grow the strain at about 25°C to about 42°C for about 1 day to about 4 days. Other suitable culture media for culturing Bacillus amyloliquefaciens GF423 strains include LB (Luria-Bertani) medium, ISP (International Streptomyces Project) medium, NA (nutrient agar) medium, BHI (brain heart infusion agar) medium, SDA (Sabouraud dextrose agar) medium, PDA (potato dextrose agar) medium, NB (nutrient broth) medium. Preferably, LB medium, ISP medium, BHI medium, SDA medium or NB medium can be used. Additionally, SOD can be obtained from other natural or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda-enzymes.org on the World Wide Web).

[0065] In one embodiment, SOD can be an isolated or purified enzyme. In this case, the isolated or purified SOD, or its biologically active part, is substantially free of cellular material or other contaminating proteins from its source cells or tissues. For example, the purified product can be obtained by single separation from the strain culture by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or a culture concentrate obtained by ultrafiltration, concentration, etc. The sentence "substantially free of cellular material" includes a protein preparation in which the protein is isolated or such protein is separated from the cellular components of cells produced recombinantly. In one embodiment, the sentence "substantially free of cellular material" refers to a protein preparation in which the content of undesirable protein by dry weight is less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5%.

[0066] In one embodiment, SOD can be preferably purified by the following purification method, but is not limited thereto. The culture solution obtained by culturing the Bacillus amyloliquefaciens GF423 strain is centrifuged and the culture supernatant is collected. The supernatant fraction is pretreated by solid-phase extraction, and then isolated and purified by chromatography. At this time, various forms of chromatography can be used to purify SOD. Preferably, hydrophobic interaction chromatography is used.

[0067] In another embodiment, the SOD may be contained in the form of a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dry form thereof. In this case, a "strain lysate" refers to a substance obtained by culturing a strain and pulverizing it mechanically or chemically, and may include substances that have been further subjected to additional processes such as extraction, dilution, concentration, and purification. A "strain culture" may refer to the culture solution itself or its supernatant obtained by culturing a strain. A "strain culture concentrate" refers to a culture concentrate obtained by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or by ultrafiltration, concentration, etc. from a strain culture. A "strain culture extract" refers to a substance extracted from the culture solution or its concentrate, and may include an extract, a dilution or concentrate of the extract, a dry product obtained by drying the extract, or a crude purified product or purified product thereof, and a fraction obtained by dividing it. The dry form may include a freeze-dried form.

[0068] In one embodiment, the SOD of the present invention is orally administered to an individual in need of preventing or treating dry eyes, showing one or more effects selected from the following: an increase in tear secretion; an increase in tear film breakup time; minimization of ocular surface damage; protection of conjunctival goblet cells; a decrease in the number of immune cells and / or inflammatory cytokines in the cornea and / or conjunctiva; a decrease in oxidative stress in the cornea, conjunctiva and / or lacrimal glands; and a decrease in corneal epithelial cell apoptosis.

[0069] Pharmaceutical composition

[0070] According to one aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing dry eyes, comprising superoxide dismutase (SOD) as an effective ingredient.

[0071] In one embodiment, the SOD according to the present invention can be combined with a pharmaceutically acceptable carrier, excipient and / or diluent to form a pharmaceutical composition for preventing or treating dry eye. When applied to animals other than humans, the pharmaceutical composition of the present invention can be used interchangeably with the term "veterinary composition".

[0072] Pharmaceutical composition or veterinary drug composition of the present invention may further comprise one or more selected from pharmaceutically acceptable carrier, excipient and diluent. The pharmaceutically acceptable carrier, excipient and / or diluent may be materials commonly used in the art. As carrier, excipient or diluent, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and silicon dioxide, mineral oil etc. may be enumerated, but are not limited thereto.

[0073] When preparing the formulation, additives such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. can be used for preparation. The additives used for the formulation can be appropriately selected from additives commonly used in the pharmaceutical field.

[0074] In addition, the pharmaceutical composition or veterinary composition of the present invention can be prepared into a preferred dosage form according to the method of use, and in particular, can be formulated by methods well known in the art so that the active ingredient can be provided quickly, continuously or in a delayed release after being applied to a mammal. Specific examples of such dosage forms include tablets, pills, powders, granules, syrups, liquids, capsules, suspensions, emulsions, injection solutions, plasters, lotions, liniments, limonade agents, aerosols, extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, patches, etc.

[0075] Furthermore, the pharmaceutical composition or veterinary composition of the present invention can be prepared into a preferred dosage form by using appropriate methods known in the art or methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, Pennsylvania.

[0076] In one embodiment, the SOD can be administered orally or parenterally. In the case of oral administration, SOD can be coated with shellac to avoid the influence of gastric acid, but the coating agent is not limited thereto. Examples of coating agents suitable for use in the present invention include shellac, ethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, EUDRAGIT and combinations thereof. When SOD is coated, SOD can be coated in a solution. Specifically, the purified solution and the shellac-containing solution are mixed and then freeze-dried. The freeze-dried sample can be made into a powder and stored at about 4°C until use. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., which can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. in a composite composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin as commonly used simple diluents, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. can also be used.

[0077] In the case of parenteral administration, the administration mode can be selected from sublingual administration, intraocular administration (including intravitreal administration), nasal spray, skin external application, patch, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection.

[0078] In addition, the pharmaceutical composition or veterinary composition of the present invention is administered in a pharmaceutically or veterinary effective amount. The term "pharmaceutically effective amount" or "veterinary effective amount" refers to an amount that is sufficient to treat a disease at a reasonable benefit / risk ratio applicable to pharmaceutical or veterinary treatment, and the effective dose level can be determined based on factors including the patient's or animal's weight, sex, age, health status, severity, activity of the drug, sensitivity to the drug, administration time, route of administration and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0079] The pharmaceutical composition or veterinary composition of the present invention can be used as a single therapeutic agent, or combined with other therapeutic agents and administered sequentially or simultaneously. In addition, the pharmaceutical composition can be administered once or multiple times as needed. Taking all of the above factors into account, it is important to administer the minimum amount that can achieve the maximum effect without side effects, which amount can be easily determined by those skilled in the art.

[0080] Prevention or treatment methods

[0081] According to yet another aspect of the present invention, there is provided a method for treating or preventing dry eye, comprising the following steps: administering the SOD or pharmaceutical composition according to the present invention to a subject suffering from or at risk of dry eye.

[0082] A method for treating or preventing dry eye is also provided, comprising the following steps: administering superoxide dismutase to a subject. The contents of the SOD, dry eye, treatment or prevention are the same as those described in the above-mentioned pharmaceutical composition.

[0083] The term "administration" includes any number of administrations that achieve the desired effect to achieve the therapeutic purpose. The administration may include oral administration. For oral administration, the description in the above-mentioned pharmaceutical composition may be referred to. The route of administration may be oral, parenteral, inhaled, applied externally or topically (e.g., intralesional administration). For example, parenteral administration may include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intraarterial, and intraocular administration. In the case of oral administration, as described above, SOD may be administered in the form of being coated with a coating agent such as shellac.

[0084] The effective dose or effective non-toxic dose of SOD according to the present invention can be determined by routine experiments. For example, the therapeutic activity amount of SOD of the present invention can vary according to factors such as the stage of the disease, the severity of the disease, the age, sex, medical complications and body weight of the object. The dosage and administration therapy of SOD of the present invention can be adjusted to provide the best therapeutic response. For example, several differentiated dosages can be administered every day, every week, every two weeks, every three weeks, every four weeks, etc., and / or the dosage can be reduced or increased in proportion according to the urgency of the treatment situation.

[0085] In one embodiment, the method may include: administering one or more additional preparations for treating or preventing dry eye. The one or more additional preparations and the SOD or pharmaceutical composition may be administered simultaneously, sequentially, or in reverse order. In addition, each component may be administered to the subject by the same or different routes.

[0086] Food composition

[0087] According to yet another aspect of the present invention, a food composition for improving or preventing dry eyes is provided, which comprises superoxide dismutase (SOD) as an effective ingredient. Such food composition includes medical or nutraceutical food compositions.

[0088] The term "medical food" or "health food" refers to food prepared from raw materials or ingredients that may exert beneficial functions on the human body, and is defined by the Ministry of Food and Drug Safety as food that maintains or improves health by maintaining normal functions or activating human physiological functions, but is not limited to this, and its meaning does not exclude any general health food.

[0089] In addition, the food includes various food types, food additives, beverages (for example, functional beverages, natural fruit juices and vegetable beverages), chewing gum, tea, vitamin complexes, health functional foods, other functional foods, etc., but is not limited to these.

[0090] The food can be prepared by conventional methods known in the art. For example, for the purpose of preventing or improving dry eyes, the medical food, health food or health functional food can further include one or more of a carrier, a diluent, an excipient and an additive on the basis of the SOD, and be prepared into a tablet, a pill, a powder, a granule, a powder, a capsule and a liquid dosage form. Specific examples of the carrier, excipient, diluent and additive are well known in the art, and those skilled in the art can prepare it according to the dosage form combined with appropriate ingredients.

[0091] The content of the superoxide dismutase according to the present invention as an active ingredient in the above-mentioned dosage form can be appropriately adjusted according to the form and purpose of use, the patient's condition, the type and severity of symptoms, etc., and can be about 0.001% by weight to about 99.9% by weight, preferably about 0.01% by weight to about 50% by weight, based on the weight of the solid component, but is not limited thereto.

[0092] The dosage of the food of the present invention may vary according to the patient's age, weight, sex, form of administration, health status and degree of disease, and may be divided and administered once or multiple times a day at certain time intervals according to the judgment of a doctor or pharmacist. For example, based on the content of the active ingredient, the daily dose may be about 10 to 1000 mg / kg. The above dosage is an example of an average situation, and the dosage may be higher or lower depending on individual differences. If the daily dose of the health functional food of the present invention is less than the dosage, a significant effect may not be obtained. If it exceeds it, it is not only uneconomical, but also may cause undesirable side effects due to exceeding the range of the commonly used amount.

[0093] Feed composition

[0094] According to yet another aspect of the present invention, a feed composition for preventing or improving dry eyes is provided, which contains the SOD according to the present invention as an effective ingredient. In the present invention, the food composition may include a feed composition. These feed compositions can be prepared in any dosage form commonly used in the field. For example, the feed composition of the present invention may further include: auxiliary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other microbial preparations in the form of live bacteria; cereals, such as crushed or broken wheat, oats, barley, corn, and rice; plant protein feeds, such as materials with rapeseed, soybeans, and sunflower seeds as main ingredients; animal protein feeds, such as blood meal, meat meal, bone meal, and fish meal; sugars and dairy products, such as dry ingredients composed of various milk powders and whey powders; lipids, such as main ingredients such as animal fats and vegetable fats that are liquefied at will by heating; additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventive agents.

[0095] The feed composition of the present invention may be in the form of a powder or liquid preparation, and may contain excipients for feed addition, such as calcium carbonate, wheat bran, zeolite, corn flour or rice bran.

[0096] Polynucleotides, expression vectors and host cells

[0097] According to yet another aspect of the present invention, a polynucleotide encoding the superoxide dismutase polypeptide is provided. Specifically, a nucleic acid encoding the polypeptide is provided, wherein the polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0098] According to yet another aspect of the present invention, an expression vector comprising the nucleic acid is provided. Specifically, the vector may be Figure 4 The expression vector shown.

[0099] According to yet another aspect of the present invention, a host cell comprising the nucleic acid or the expression vector is provided. Specifically, the host cell is a bacterial cell. More specifically, the host cell is a Bacillus strain.

[0100] According to yet another aspect of the present invention, a Bacillus strain is provided, comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4, and the AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf genes are deleted in the Bacillus strain. The detailed process of preparing the strain is shown in Example 1 below.

[0101] Example

[0102] Hereinafter, the present invention will be described in more detail in conjunction with the following examples. The following examples are provided for a thorough understanding of the present invention and are not intended to and should not be construed as limiting the scope of the present invention in any way.

[0103] [Example 1] Preparation of recombinant strain expressing superoxide dismutase (SodA2)

[0104] [Example 1.1] Preparation of expression host GFBS220

[0105] The expression host GFBS220 was prepared using Bacillus subtilis KCTC 3135. Bacillus subtilis KCTC 3135 was obtained from the Bioresource Center (KCTC) of the Korea Institute of Biotechnology. In order to facilitate the subsequent steps, the genes shown in Table 1 below were removed from the genome of Bacillus subtilis KCTC 3135.

[0106]

Table 1

[0107]

[0108]

[0109] Double cross-over recombination is used to remove genes from the genome. Figure 1 Specifically, DNA fragments (up frag. and down frag.) with homology to the DNA base sequences on both sides of the gene to be operated were cloned into the vector pUCori-ts-cm with a temperature-sensitive replication origin. The PCR conditions and primers used for cloning are shown in Tables 2 and 3, respectively.

[0110]

Table 2

[0111]

[0112]

Table 3

[0113]

[0114]

[0115] After the vector prepared in this way is introduced into the Bacillus subtilis cells, the transformant is screened at 37°C (non-replication temperature) using a culture medium containing chloramphenicol (single cross homologous recombination). The screened transformant is cultivated at 30°C (permitting replication temperature) using LB culture medium without antibiotics (second crossover), and then cultivated at 39°C using LB agar culture medium without antibiotics. From the generated bacterium colonies, strains sensitive to chloramphenicol are screened (plasmid solidification). PCR is performed on the screened bacterium colonies to obtain double cross-over recombinants, and a second single separation is performed to screen out the final recombinant, named as GFBS220.

[0116] After amplification using the primers shown in Table 4 and PCR, the defective gene site of GFBS220 was confirmed by agarose gel electrophoresis. The results of confirming the defective gene by PCR in the expression host GFBS220 are as follows: Figure 2 shown.

[0117]

Table 4

[0118]

[0119] [Example 1.2] Preparation of expression vector pBE1-sodA2

[0120] Mn-SOD was confirmed in the culture supernatant of Bacillus amyloliquefaciens GF423 strain by N-terminal sequencing (see Kang et al., 2018). The GF423 strain was deposited in the Korea Biotechnology Research Institute on March 6, 2017 (with the deposit number KCTC 13222BP). The gene of Mn-SOD was named sodA according to the nomenclature of bacterial SOD, and its amino acid sequence is as follows Figure 3 and SEQ ID NO: 2 (its nucleotide sequence is shown in SEQ ID NO: 1). The LC-UV-MS / MS peptide map of the enzyme preparation yielded 100% amino acid sequence coverage, but deamidation was observed at residues Asn73 and Asn136 with abundances of 73% and 17%, respectively. Therefore, in order to improve the homogeneity of the purified enzyme, the two Asn residues were replaced with Asp. This variant sodA was named SodA2 and its amino acid sequence is shown in Figure 3 and SEQ ID NO:4 (its nucleotide sequence is shown in SEQ ID NO:3).

[0121] The sodA2 gene was amplified using repeat extension PCR using four primers (Table 5) and the Bacillus amyloliquefaciens GF423 genome as template. The nucleotides used to replace Asn with Asp are underlined.

[0122]

Table 5

[0123]

[0124] The amplified DNA fragment containing the sodA2 gene and the plasmid pBE1 linearized by NdeI and HindIII treatment were assembled in vitro by the SLIC method (see Jeong et al., 2012), and the resulting construct was transformed into Escherichia coli C2984H. Correct clones were screened by restriction enzyme mapping and confirmed by nucleotide sequencing. The expression vector pBE1-sodA2 generated in this way is as follows Figure 4 shown.

[0125] [Example 1.3] Preparation of production strain BSBA310

[0126] The expression vector pBE1-sodA2 was transformed into Bacillus subtilis GFBS220. Strains were selected from the transformants and pure clones were established by two single colony isolation procedures in LB2 medium. The selected strain was named BSBA310 and stored at -80°C using the glycerol stock method. The results of confirming the defective gene using PCR in the production strain BSBA310 are as follows: Figure 2 In addition, Figure 5 Summary The overall process for preparing the production strain BSBA310 is shown.

[0127] [Example 2] Isolation and purification of superoxide dismutase (SodA2) from production strain BSBA310

[0128] [Example 2.1] Cultivation of production strain BSBA310

[0129] In order to culture the production strain BSBA310 obtained in Example 1, a single colony formed in LB agar medium (LB (Luria-Bertani) agar; tryptophan 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L) was inoculated into 30 ml of LB medium and cultured at 37° C. for 12 hours. The seed culture was again inoculated into 3 L of LB medium containing 1 mM manganese sulfate (MnSO4) and cultured at 37° C. for 20 hours.

[0130] [Example 2.2] Isolation and purification of SodA2

[0131] The cell culture fluid obtained in Example 2.1 was centrifuged at 4°C and 3,578×g for 20 minutes and the supernatant was collected, and then concentrated 10 times by ultrafiltration (Ultrafiltration, hereinafter referred to as UF, MWCO 10,000). 390g of ammonium sulfate was added to each 1L of concentrated cell culture, stirred for 20 minutes, centrifuged and the supernatant was collected. The obtained supernatant was purified using a phenyl agarose HP column. During the purification process, 50mM potassium phosphate pH7.0 with a concentration of 2M ammonium sulfate was used to balance the column, and then the supernatant obtained by adding ammonium sulfate was passed through the column, and 50mM potassium phosphate pH7.0 containing 1.6M ammonium sulfate was used to recover SodA2 attached to the column. The purified solution purified by the column was collected, and the high concentration of salts formed during the purification process was removed by ultrafiltration and concentrated. After filtering the concentrated solution with a sterilizing filter, it was freeze-dried. The activity of SODA2 was analyzed using a SOD assay kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits 50% of superoxide free radicals. The activity of dried SodA2 enzyme is 1000 to 1200 U / mg.

[0132] [Example 3] Preparation of administration material

[0133] Shellac (EXCELACS co., LTD., Bangkok) was dissolved to 3% ethanol concentration and sterilized using a 0.2um sterile filter. The shellac dissolved in ethanol was diluted with PBS. Freeze-dried SodA2 was dissolved to 20 mg / ml, and the shellac solution and SOD solution were mixed at a ratio of 1:1 and then freeze-dried. Dextrin was mixed into freeze-dried shellac-coated SodA2 at a ratio of 1:9 to 12 (shellac-coated SodA2:dextrin) to prepare the material for animal testing. The SOD activity of the final material was made to reach 90 to 110 U / mg.

[0134] [Example 4] Confirmation of the effect of treating or preventing dry eyes

[0135] In this example, in an experimental dry eye (EDE) induced C57BL / 6 mouse model, an attempt was made to analyze the effects of oral SodA2 at concentrations of 2.5 mg / kg, 5 mg / kg, and 10 mg / kg on tear secretion, tear film breakup time, ocular surface damage, conjunctival goblet cells, inflammatory cytokines on the ocular surface, CD4+T immune cells, oxidative stress and reactive oxygen species, and corneal epithelial cell apoptosis.

[0136] [Example 4.1] Preparation of experimental dry eye (EDE) animal model

[0137] To induce experimental dry eye, 7 to 8 week old C57BL / 6 mice (obtained from DAMOOL SCIENCE) were placed in a state of being exposed to ventilation for 18 hours a day and subcutaneously injected with 0.5 mg / 0.2 ml of scopolamine hydrochloride 3 times a day. Humidity was maintained at 30% and temperature was maintained at 25° C. According to the concentration of the oral drug administered, the C57BL / 6 mice were divided into 7 groups, as shown in Table 6.

[0138]

Table 6

[0139]

[0140] [Example 4.2] Drug administration and test procedures

[0141] After experimental dry eye was induced in the experimental mice, the treatment group was orally administered with the drug prepared in Example 3, 2 μL per time, once a day (administered at 13:30 every day). At the baseline, 5th day and 10th day, the tear secretion and tear film breakup time were measured for both eyes of all mice, and the corneal fluorescence staining score was examined. Then, the mice were sacrificed and tissue analysis was performed. There were 6 animals in each group.

[0142] Determination of tear volume

[0143] A cotton thread soaked in phenol red was placed in contact with the lateral canthal conjunctival sac of each mouse for 20 seconds, and its length was measured using SMZ microscopy and then substituted into the given formula to convert it into volume (see reference: Villareal AL, Farley W, Pflugfelder SC; Effect of topical ophthalmic epinastine and olopatadine on tear volume in mice; Eye Contact Lens 2006; 32: 272-6).

[0144] Determination of tear film breakup time

[0145] In order to evaluate the tear film stability, which is known to be the most important clinical factor for dry eyes, the tear film breakup time was measured. After instilling 1 μl of 1% fluorescent dye solution into the lower conjunctival sac of the mouse, the mouse was made to blink about 3 times, and then the eye was gently opened. The time (seconds) required for the first defect to appear in the dyed tear film was measured using a slit lamp microscope equipped with a cobalt blue filter. This was repeated 3 times and the average value was taken.

[0146] Determination of corneal fluorescent staining score

[0147] After instilling 1 μl of 1% fluorescent staining solution and rinsing with saline, the cornea of ​​the mouse was observed under a slit lamp microscope to score the degree of epithelial damage (degree of fluorescent staining). For this purpose, the cornea was divided into four areas, and each area was evaluated with a score of 0 to 4 as shown in Table 7, and then added together, with a total score of 0 to 16 (see literature: Pauly A, Brignole-Baudouin F, Labbe A, Liang H, Warnet JM, Baudouin C; New tools for the evaluation of toxic ocular surface changes in the rat; Invest Ophthalmol Vis Sci. 2007; 48: 5473-83).

[0148]

Table 7

[0149] 0 none 1 Mild punctate staining with fewer than 30 spots 2 Punctate staining with fewer than 30 spots but not diffuse 3 Severe diffuse staining but no positive plaques 4 Severe diffuse staining with positive fluorescein plaques

[0150] Determination of malondialdehyde (MDA) by ELISA

[0151] Cornea, conjunctiva and lacrimal gland were removed from both eyes (6 eyes) of 3 animals in each group for tissue analysis. After homogenization of the samples in PBS, the lysate was centrifuged at 10000xg for 10 minutes at 4°C. Then, the supernatant was removed and the total amount of protein was determined using the Bradford assay. MDA was detected using an ELISA (enzyme-linked immunosorbent assay) kit (manufacturer: Quanti kine, R&D Systems, Minneapolis, MN) containing capture and detection antibodies according to the product manual. Using ELISA, the MDA values ​​of the cornea, conjunctiva and lacrimal gland were measured and recorded separately.

[0152] Measuring inflammation using the Luminex multiplex immunobeadassay Cytokines

[0153] Conjunctival tissues were removed from six eyes of three animals in each group and immersed in a lysis buffer containing protease inhibitors for 30 min before centrifugation and storage at −80° C. The concentrations of IFN-γ, IL-1β, and TNF-α in the cornea and conjunctiva were then analyzed using the Luminex multiplex staining bead assay.

[0154] Flow cytometry was used to measure the number of immune cells and reactive oxygen species (DCF-DA)

[0155] The cornea, conjunctiva and lacrimal gland were removed, cut with scissors, and then shaken at 37°C for 60 minutes with 0.5 mg / ml collagenase type D (manufacturer: Roche Applied Science, Indianapolis, Indiana, USA). After the tissue was pulverized, it was passed through a cell strainer and then centrifuged. Flow cytometry was performed using fluorescein-conjugated anti-CD4 antibody and phycoerythrin-conjugated anti-IFN-γ antibody to count CD4+IFN-γ+T cells, and the number of DCF-DA cells was counted using the CM-H2DCFDA kit (manufacturer: Thermo Fisher Scientific, Waltham, MA, USA).

[0156] Histological analysis

[0157] The eye and accessory tissues were removed from one eye of one animal in each group, fixed with 4% paraformaldehyde and embedded in paraffin, and then two sections of the tissue were stained with PAS. In each section, the density of goblet cells in the conjunctiva was calculated using a microscope and image analysis software.

[0158] Immunofluorescent staining

[0159] The eye and accessory tissues were removed from one eye of one animal in each group, fixed with 4% paraformaldehyde and embedded in paraffin, and then two sections of the tissue were stained with TUNEL. In each section, the degree of apoptosis of the conjunctiva was confirmed using a microscope (Leica TCS SP5AOBS laser scanning confocal microscope (Zeiss GmbH)) and image analysis software (NISElements version 4.1; Nikon, Melville, NY, USA).

[0160] Statistical analysis

[0161] SPSS 22.0 was used for analysis, and the results of continuous variables were expressed as mean ± SD. Normality was confirmed by Shapiro-Wilk test. Comparisons between groups were performed by one-way analysis of variance (ANOVA) followed by Fisher's LSD post hoc test. P values ​​less than 0.05 were considered statistically significant.

[0162] [Example 4.3] Results

[0163] Tear secretion

[0164] In terms of tear secretion, before drug administration, the UT group was 0.046±0.004μL, the EDE group was 0.023±0.004μL, the vehicle group was 0.024±0.003μL, the 0.05% CsA (cyclosporine A) group was 0.023±0.003μL, the 2.5mg / kg oral SodA2 group was 0.024±0.003μL, the 5mg / kg oral SodA2 group was 0.022±0.004μL, and the 10mg / kg oral SodA2 group was 0.023±0.004μL.

[0165] On the 5th day of administration, the UT group was 0.045±0.006μL, the EDE group was 0.02±0.003μL, the vehicle group was 0.021±0.003μL, the 0.05% CsA group was 0.026±0.004μL, the 2.5mg / kg oral SodA2 group was 0.026±0.005μL, the 5mg / kg oral Sod A2 group was 0.026±0.005μL, and the 10mg / kg oral SodA2 group was 0.026±0.005μL.

[0166] On the 10th day of administration, the UT group was 0.043±0.005μL, the EDE group was 0.017±0.003μL, the vehicle group was 0.018±0.003μL, the 0.05% CsA group was 0.027±0.005μL, the 2.5mg / kg oral SodA2 group was 0.027±0.005μL, the 5mg / kg oral Sod A2 group was 0.03±0.005μL, and the 10mg / kg oral SodA2 group was 0.028±0.004μL.

[0167] On the 5th and 10th days after administration, the 0.05% CsA group, the 2.5 mg / kg oral SodA2 group, the 5 mg / kg oral SodA2 group, and the 10 mg / kg oral SodA2 group all showed a significant increase in tear secretion compared to the EDE group and the vehicle group. In particular, the 5 mg / kg oral SodA2 group showed a significant increase that was superior to the 0.05% CsA group. The tear secretion measurement results as described above are shown in Figure 6 .

[0168] Tear film breakup time

[0169] In terms of tear film breakup time, before drug administration, it was 2±0.45 seconds in the UT group, 1.43±0.19 seconds in the EDE group, 1.43±0.23 seconds in the vehicle group, 1.46±0.13 seconds in the 0.05% CsA group, 1.44±0.15 seconds in the 2.5 mg / kg oral SodA2 group, 1.42±0.16 seconds in the 5 mg / kg oral SodA2 group, and 1.41±0.25 seconds in the 10 mg / kg oral SodA2 group.

[0170] On the 5th day of administration, the UT group was 2.21±0.4 seconds, the EDE group was 1.02±0.2 seconds, the vehicle group was 1.06±0.23 seconds, the 0.05% Cs A group was 1.3±0.2 seconds, the 2.5 mg / kg oral SodA2 group was 1.23±0.2 seconds, the 5 mg / kg oral SodA2 group was 1.31±0.3 seconds, and the 10 mg / kg oral SodA2 group was 1.28±0.3 seconds.

[0171] On the 10th day of administration, the UT group was 2.1±0.4 seconds, the EDE group was 0.91±0.2 seconds, the vehicle group was 0.93±0.2 seconds, the 0.05% CsA group was 1.39±0.2 seconds, the 2.5 mg / kg oral SodA2 group was 1.21±0.3 seconds, the 5 mg / kg oral SodA2 group was 1.28±0.3 seconds, and the 10 mg / kg oral SodA2 group was 1.3±0.3 seconds.

[0172] On the 5th and 10th days of administration, the 0.05% CsA group, the 2.5 mg / kg oral SodA2 group, the 5 mg / kg oral SodA2 group, and the 10 mg / kg oral SodA2 group all showed a significant increase in tear breakup time compared to the EDE group and the vehicle group. In particular, the tear breakup time of the 5 mg / kg oral SodA2 group and the 10 mg / kg oral SodA2 group increased to be similar to that of the 0.05% CsA group. The tear breakup time measurement results as described above are shown in Figure 7 .

[0173] Corneal fluorescence staining score

[0174] In terms of corneal fluorescence staining scores, before drug administration, the UT group was 1.8±1.1 points, the EDE group was 9.5±1.8 points, the vehicle group was 9.1±2 points, the 0.05% CsA group was 9.1±2 points, the 2.5 mg / kg oral SodA2 group was 9.2±1.7 points, the 5 mg / kg oral SodA2 group was 9.4±1.7 points, and the 10 mg / kg oral SodA2 group was 9.3±1.7 points.

[0175] On the 5th day of administration, the UT group was 2.9±1.4 points, the EDE group was 12.1±2.1 points, the vehicle group was 11.7±2.2 points, the 0.05% CsA group was 10.4±2.5 points, the 2.5 mg / kg oral SodA2 group was 10.4±1.9 points, the 5 mg / kg oral SodA2 group was 9.9±2.5 points, and the 10 mg / kg oral SodA2 group was 10.1±2.9 points.

[0176] On the 10th day of administration, the UT group was 3.6±1.4 points, the EDE group was 14.1±1.8 points, the vehicle group was 13.5±1.6 points, the 0.05% CsA group was 8.9±2 points, the 2.5 mg / kg oral SodA2 group was 8.8±2 points, the 5 mg / kg oral SodA2 group was 7.8±1.9 points, and the 10 mg / kg oral SodA2 group was 8.2±1.9 points.

[0177] On the 5th and 10th days after administration, the 0.05% CsA group, the 2.5 mg / kg oral SodA2 group, the 5 mg / kg oral SodA2 group, and the 10 mg / kg oral SodA2 group all showed a significant decrease in corneal fluorescence staining scores compared to the EDE group and the vehicle group. In particular, the 5 mg / kg oral SodA2 group showed a significant decrease that was superior to the 0.05% CsA group. The corneal fluorescence staining score measurement results as described above are shown in Figure 8 .

[0178] Malondialdehyde (MDA) concentration

[0179] The results of the determination of MDA concentrations in the cornea, conjunctiva and lacrimal gland are shown in Table 8. The unit is pmol / ml.

[0180]

Table 8

[0181]

[0182] As shown in Table 8 above, both the CsA group and the oral SodA2 treatment group showed a decrease in the MDA concentration in the cornea, conjunctiva and lacrimal gland compared to the EDE group and the vehicle group. In particular, the 10 mg / kg oral SodA2 group showed an efficacy similar to (cornea, lacrimal gland) or better than (conjunctiva) the 0.05% CsA group.

[0183] Inflammatory cytokines

[0184] The results of the determination of inflammatory cytokines in the cornea and conjunctiva are shown in Table 9. The unit is pg / ml.

[0185]

Table 9

[0186]

[0187] As shown in Table 9 above, the CsA group and the oral SodA2 treatment group showed a tendency to decrease the concentrations of IFN-γ, IL-1β, and TNF-α in the cornea and conjunctiva compared with the EDE and vehicle groups. In particular, all doses of oral SodA2 showed a trend of reducing inflammatory cytokines better than CsA.

[0188] The number of immune cells (CD4+IFN-γT cells) was determined by flow cytometry.

[0189] In terms of the ratio of CD4+IFN-γ+T cells in the cornea, the UT group was 16.53±1.65%, the EDE group was 48.39±1.34%, the vehicle group was 47.02±0.04%, the 0.05% CsA group was 26.05±0.18%, the 2.5 mg / kg oral SodA2 group was 21.18±0.15%, the 5 mg / kg oral SodA2 group was 23.87±0.17%, and the 10 mg / kg oral SodA2 group was 30.24±1.33%.

[0190] At the same time, the ratio of CD4+IFN-γ+T cells in the conjunctiva was 17.40±3.43% in the UT group, 46.63±1.27% in the EDE group, 43.65±2.68% in the vehicle group, 22.67±0.47% in the 0.05% CsA group, 21.00±0.63% in the 2.5 mg / kg oral SodA2 group, 22.87±2.47% in the 5 mg / kg oral SodA2 group, and 25.83±0.27% in the 10 mg / kg oral SodA2 group.

[0191] Compared with the EDE group and the vehicle group, the CsA group and the oral SodA2 group showed a significant decrease in the ratio of CD4+IFN-γ+T cells in the cornea and conjunctiva. In particular, the 2.5 mg / kg oral SodA2 group showed a significantly lower effect than the 0.05% CsA group in the ratio of CD4+IFN-γ+T cells in the cornea. The results of the analysis of the number of CD4+IFN-γ+T cells in the cornea and conjunctiva as described above are shown in Figure 9a and 9b .

[0192] Measurement of Reactive Oxygen Species

[0193] Regarding the reactive oxygen species produced in the cornea due to dry eyes, the UT group was 33.93±24.62%, the EDE group was 87.43±49.83%, the vehicle group was 76.25±49.54%, the 0.05% CsA group was 50.27±11.27%, the 2.5 mg / kg oral SodA2 group was 33.57±26.51%, the 5 mg / kg oral SodA2 group was 54.53±13.53%, and the 10 mg / kg oral SodA2 group was 57.38±23.43%.

[0194] In the conjunctiva, the UT group was 42.25±23.23%, the EDE group was 97.69±54.19%, the vehicle group was 95.48±54.54%, the 0.05% CsA group was 58.16±5.54%, the 2.5 mg / kg oral SodA2 group was 55.13±7.41%, the 5 mg / kg oral SodA2 group was 57.63±8.16%, and the 10 mg / kg oral SodA2 group was 53.29±1.19%.

[0195] In the lacrimal gland, the UT group was 36.88±26.91%, the EDE group was 100.90±80.95%, the vehicle group was 114.26±65.19%, the 0.05% CsA group was 58.66±11.11%, the 2.5 mg / kg oral SodA2 group was 59.05±10.77%, the 5 mg / kg oral SodA2 group was 56.03±6.37%, and the 10 mg / kg oral SodA2 group was 49.92±4.19%.

[0196] In the cornea, conjunctiva and lacrimal gland, the SodA2 treatment group showed similar or better reactive oxygen species reduction effects than the CsA treatment group. In particular, in the cornea, the 2.5 mg / kg oral SodA2 group showed a better effect of reducing reactive oxygen species than the CsA group. The results of reactive oxygen species generation in the cornea, conjunctiva and lacrimal gland as described above are shown in Fig.10a , 10b and 10c.

[0197] Determination of the number of goblet cells in the conjunctiva

[0198] The number of goblet cells in the conjunctiva was 52.1±9.6 cells / 100μm in the UT group, 14.1±3.6 cells / 100μm in the EDE group, and 14.6±2.4 cells / 100μm in the vehicle group. The number of goblet cells in the 0.05% CsA group was 25.2±3.4 cells / 100μm, the number of cells in the 2.5mg / kg oral Sod A2 group was 27.9±6.9 cells / 100μm, the number of cells in the 5mg / kg oral Sod A2 group was 35.8±10.2 cells / 100μm, and the number of cells in the 10mg / kg oral Sod A2 group was 32.2±3.9 cells / 100μm. It can be seen that all treatment groups showed a significant increase in the number of goblet cells. In particular, the 5mg / kg oral Sod A2 group showed a significantly better increase in the number of goblet cells than the 0.05% CsA group. The results of the above-mentioned determination of the number of goblet cells in the conjunctiva are shown in Fig.11a (PAS staining image) and Fig.11b (goblet cell count).

[0199] Determination of cell apoptosis

[0200] The number of necrotic cells in the cornea was 1.6±0.5 cells / 100μm in the UT group, 14.8±3.3 cells / 100μm in the EDE group, and 12.1±3.0 cells / 100μm in the vehicle group, which showed a significant increase due to dry eye stimulation. In contrast, the number of necrotic cells in the 0.05% CsA group was 6.1±2.1 cells / 100μm, the number of cells in the 2.5mg / kg oral SodA2 group was 6.2±1.1 cells / 100μm, the number of cells in the 5mg / kg oral SodA2 group was 4.0±1.3 cells / 100μm, and the number of cells in the 10mg / kg oral SodA2 group was 4.6±1.3 cells / 100μm. It can be seen that compared with the EDE group and the vehicle group, the CsA and oral SodA2 treatment groups showed a significant decrease in the number of apoptotic cells in the cornea. The results of the apoptosis assay as described above are shown in Fig.12a (TUNEL staining images) and Figure 12b (number of apoptosis-positive cells).

[0201] [Example 4.4] Conclusion

[0202] Based on the above results, it can be seen that for mice with induced dry eyes, oral administration of SodA2 can improve the tear secretion volume, tear film breakup time and corneal epithelial damage on the ocular surface. In addition, inflammatory T cells and cytokines in the cornea and conjunctiva showed a downward trend. It can also be shown that the oxidative stress of the cornea, conjunctiva and lacrimal glands is reduced, the number of goblet cells in the conjunctiva is increased, and the apoptosis of corneal epithelial cells is reduced. In addition, in terms of corneal fluorescence staining scores, corneal inflammatory T cells, and goblet cell density in the conjunctiva, oral SodA2 can show similar or better efficacy than 0.05% cyclosporin. The newly developed oral SodA2 can minimize damage to the ocular surface and reduce inflammation, so it is expected to be effectively used in the future to treat patients with dry eyes.

Claims

1. A pharmaceutical composition for treating or preventing dry eyes, comprising superoxide dismutase as an effective ingredient.

2. The pharmaceutical composition according to claim 1, wherein The composition exhibits at least one selected from an increase in tear secretion; an increase in tear film breakup time; minimization of ocular surface damage; protection of goblet cells in the conjunctiva; a reduction in the number of immune cells or inflammatory cytokines in the cornea or conjunctiva; a reduction in oxidative stress in the cornea, conjunctiva or lacrimal glands; and a reduction in corneal epithelial cell apoptosis.

3. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is an isolated or purified enzyme.

4. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is contained in the form of strain lysate, strain culture, strain culture concentrate, strain culture extract, or a dry form thereof.

5. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is Mn-SOD.

6. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is a deamidated manganese-containing superoxide dismutase.

7. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is derived from a strain of Bacillus.

8. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is derived from Bacillus amyloliquesfaciens GF423 strain (KCTC 13222BP).

9. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is a superoxide dismutase based on SEQ ID NO: 2, wherein the amino acid residues at positions 73 and 136 are substituted by Asp.

10. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase has an amino acid sequence shown in SEQ ID NO:

4.

11. The pharmaceutical composition according to claim 1, wherein The dry eye is dry eye caused by Sjögren's syndrome or non-Sjögren's syndrome.

12. The pharmaceutical composition according to claim 1, wherein The composition is for oral administration.

13. The pharmaceutical composition according to claim 12, wherein The superoxide dismutase in the composition is coated with a coating agent.

14. The pharmaceutical composition according to claim 13, wherein The coating agent comprises shellac.

15. A veterinary composition for treating or preventing dry eyes, comprising superoxide dismutase as an active ingredient.

16. A food composition for improving or preventing dry eyes, comprising superoxide dismutase as an effective ingredient.

17. A feed composition for improving or preventing dry eyes, comprising superoxide dismutase as an effective ingredient.

18. A method for preventing or treating dry eyes, comprising the step of administering the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 14 to a subject.

19. Use of the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 14 for preventing or treating dry eyes.

20. Use of the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 14 in the preparation of a medicament for preventing or treating dry eyes.

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