Superoxide dismutase and application thereof in preventing or treating diabetic retinopathy or uveitis

Abstract

The invention relates to superoxide dismutase and application thereof in preventing or treating diabetic retinopathy or uveitis. The superoxide dismutase according to the present invention can be effectively used for preventing or treating diabetic retinopathy or uveitis, particularly by oral administration.

Description

Technical Field

[0001] The present invention relates to the use of superoxide dismutase in preventing or treating diabetic retinopathy or uveitis. More specifically, the present invention relates to the use, composition or method of superoxide dismutase for preventing or treating diabetic retinopathy or uveitis. Background Art

[0002] In recent years, vision impairment caused by various diseases such as retinal nerve degeneration caused by aging has been on the rise. The eye, including the retina, is a neural tissue related to visual function. Once damaged, it is difficult to recover, so special attention is required. Diseases that cause vision impairment include, for example, diabetic retinopathy, uveitis, cataracts, macular degeneration, and retinal detachment.

[0003] Diabetic retinopathy is one of the chronic complications of diabetes and one of the leading causes of blindness worldwide. Since vision loss is not accompanied by particular pain, treatment may be delayed. For diabetic retinopathy, a common treatment is to prevent the formation of new blood vessels and bleeding in the eye through laser therapy to inhibit rapid vision loss, but in the case of diabetic macular edema, it is difficult to improve vision and the application of laser therapy is limited. To compensate for this, the method used is to inject steroids or vascular endothelial growth factor inhibitors (such as dexamethasone) into the vitreous body. The advantage of this method is that it inhibits vascular endothelial growth factor and improves the chronic inflammatory state of the retina in patients with diabetic retinopathy, but there is a risk of side effects with long-term use.

[0004] Uveitis is an inflammatory disease of the uvea of ​​the eye. It is transient and recoverable after treatment, but the inflammation usually recurs and persists, and is often idiopathic with unknown causes, so it may eventually lead to severe visual impairment and blindness. The causes of uveitis include infectious causes caused by viruses, bacteria, fungi, parasites, etc. and non-infectious causes caused by autoimmune diseases. Uveitis is mostly a specific endogenous disease in which no pathogenic bacteria can be found, and it is related to abnormal immune system. Therefore, adrenocortical hormone preparations are mainly administered in the form of eye drops. If acute inflammation occurs, it is treated by topical eye drops of steroids and eye drops of regulating paralytics. If the inflammation is severe, steroids are administered systemically, or immunosuppressants such as cyclosporine are administered in the case of chronic diseases.

[0005] Conventional steroid preparations have the risk of side effects when used for a long time, while accommodation paralytics may cause glare, difficulty in seeing close text, etc. Therefore, it is desirable to develop a preparation that can prevent or treat eye diseases, especially diabetic retinopathy and / or uveitis. Summary of the invention

[0006] Technical issues

[0007] The purpose of the present invention is to solve all the above technical problems.

[0008] One object of the present invention is to provide superoxide dismutase for preventing, improving or treating diabetic retinopathy or uveitis.

[0009] 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 diabetic retinopathy or uveitis.

[0010] Another object of the present invention is to provide 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 diabetic retinopathy or uveitis.

[0011] Another object of the present invention is to provide a pharmaceutical composition or a therapeutic method for preventing or treating diabetic retinopathy or uveitis.

[0012] Still another object of the present invention is to provide a food or feed composition for preventing or improving diabetic retinopathy or uveitis.

[0013] Still another object of the present invention is to provide an oral composition comprising superoxide dismutase for preventing, improving or treating diabetic retinopathy or uveitis.

[0014] 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.

[0015] Technical Solution

[0016] Representative embodiments of the present invention for achieving the above-mentioned object are as follows.

[0017] According to one aspect of the present invention, an improved superoxide dismutase (SOD) is provided, which exhibits the effect of preventing, improving or treating diabetic retinopathy or uveitis.

[0018] According to another aspect of the present invention, there is provided a superoxide dismutase for preventing, improving or treating diabetic retinopathy or uveitis, wherein the superoxide dismutase is derived from generally regarded as safe (GRAS) bacteria, such as Bacillus.

[0019] 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 and is easy to produce, for use in preventing, improving or treating diabetic retinopathy or uveitis.

[0020] 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 diabetic retinopathy or uveitis when orally administered.

[0021] According to yet another aspect of the present invention, there is provided a superoxide dismutase derived from Bacillus amyloliques faciens which ensures oral administration efficacy and safety for preventing, improving or treating diabetic retinopathy or uveitis.

[0022] According to yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating diabetic retinopathy or uveitis, comprising the superoxide dismutase as an effective ingredient.

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

[0025] According to yet another aspect of the present invention, a method for preventing, improving or treating diabetic retinopathy or uveitis is provided, comprising the following step: 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, improving or treating diabetic retinopathy or uveitis.

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

[0028] Beneficial Effects

[0029] The superoxide dismutase, composition, method and kit according to the present invention can be effectively used for preventing or treating diabetic retinopathy or uveitis. In particular, it has been confirmed that the superoxide dismutase according to the present invention is also effective for preventing or treating diabetic retinopathy or uveitis 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 collected from the supernatant during culture.

[0031] According to embodiments of the present invention, SOD of the present invention is orally administered to diabetic mice and normal mice, and the results of retinal vascular leakage assessment, astrocyte immunofluorescence staining assessment, and retinal vascular staining assessment show that the use of SOD can reduce retinal vascular permeability, restore astrocyte foot processes, inhibit pericyte loss, and inhibit the occurrence of acellular capillaries in diabetic mice. In addition, SOD is orally administered to mice that have induced acute and chronic uveitis, and the results of fundus assessment and tissue assessment show that inflammation is improved to mild. Therefore, superoxide dismutase according to the present invention can be effectively used to prevent, improve, or treat diabetic retinopathy or uveitis by reducing retinal vascular leakage and improving inflammatory state. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A schematic diagram of the double crossover recombination performed in Example 1.1 is shown.

[0033] 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 wild-type B. subtilis KCTC 3135, 220 indicates the expression host GFBS220, and 310 indicates the SodA2 production strain BSBA310.

[0034] 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).

[0035] Figure 4The 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 the kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); BJ27 promoter represents the strong promoter of Bacillus subtilis (B. subtilis).

[0036] Figure 5 The overall scheme used to prepare the production strain BSBA310 is shown.

[0037] Figure 6a and Figure 6b The figure shows the qualitative evaluation results of retinal vascular leakage in the 3-week diabetic retinopathy experimental group (scale bar: 500 μm). Figure 6c Quantitative evaluation results are shown.

[0038] Figure 7a and Figure 7b The figure shows the qualitative evaluation results of retinal vascular leakage in the experimental group with diabetic retinopathy for 16 weeks (scale bar: 500 μm). Figure 7c Quantitative evaluation results are shown.

[0039] Figure 8a and Figure 8b The qualitative evaluation results of immunofluorescence staining of astrocytes in the 3-week diabetic retinopathy experimental group are shown. Figure 8c Quantitative evaluation results are shown.

[0040] Figure 9a and Figure 9b The qualitative evaluation results of astrocyte immunofluorescence staining in the 16-week diabetic retinopathy experimental group are shown. Fig.9c Quantitative evaluation results are shown.

[0041] Fig.10a and Fig.10b The figure shows the qualitative evaluation results of retinal blood vessel staining in the 16-week diabetic retinopathy experimental group (scale bar: 100 μm). Fig.10c and Fig.10d Quantitative evaluation results are shown.

[0042] Fig.11a and Fig.11b Shown are the results of clinical scoring for fundus evaluation of uveitis. Fig.11a Fundus evaluation results of the endotoxin-induced uveitis model. Fig.11bFundus evaluation results of the experimental autoimmune uveitis model.

[0043] Fig.12 Four areas of the intraocular retina divided for evaluation of uveitic tissue are shown.

[0044] Figures 13a to 13c Shown are the results of uveitis tissue assessment in the endotoxin-induced uveitis model. Fig.13a The results are for the normal control group (Control) and the normal mouse group administered with SodA2. Fig.13b Results of the endotoxin-induced uveitis model and the model in which SodA2 was administered were shown in Table 1. Fig.13a and Fig.13b The scale bars in the figure are 200 μm on the left and 100 μm on the right. Fig.13c The grade distribution results of the experimental group are shown.

[0045] Fig.14a , Fig.14b and Fig.14c Shown are the results of uveitis tissue assessment in an experimental autoimmune uveitis model. Fig.14a The results for the normal mice group and the normal mice group administered with SodA2 are shown in Table 1. Fig.14b Results of experimental autoimmune uveitis model and SodA2-administered model. Fig.14a and Fig.14b The scale bars in the figure are 200 μm on the left and 100 μm on the right. Fig.14c The grade distribution results of the experimental group are shown. DETAILED DESCRIPTION

[0046] 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.

[0047] definition

[0048] The term "about" as used herein refers to the usual error range for each value known to those skilled in the art.

[0049] The term "subject" is used interchangeably with "patient" and refers to a mammal in need of prevention or treatment of diabetic retinopathy or uveitis, 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.

[0050] The term "treatment" includes preventive and / or therapeutic treatments. The preventive and / or therapeutic treatments include all types of treatments recognized in the art, such as including administering a pharmaceutical composition of the present invention or SOD to a subject. If administered before the clinical manifestation of an undesirable or unpreferred state (e.g., a disease or other undesirable or unpreferred state of a subject), such treatment or treatment is a preventive treatment or treatment (e.g., protecting a subject from the progression of an undesirable or unpreferred state of a subject). In addition, if administered after the clinical manifestation of an undesirable or unpreferred state, such treatment or treatment is a therapeutic treatment that alleviates, relieves, or stabilizes an existing undesirable or unpreferred state or its side effects.

[0051] The meaning of the term "prevention" is well known in the art. When it occurs together with medical conditions such as diabetic retinopathy or uveitis, it means that by administering the pharmaceutical composition or SOD of the present invention, the frequency of the medical condition (discomfort, visual impairment, ocular vascular leakage, ocular surface damage, ocular surface inflammation, etc.) is reduced or the onset and symptoms are delayed compared to subjects not administered with it.

[0052] The term "administering" refers to providing an active ingredient to a subject for preventive or therapeutic purposes (eg, preventing or treating diabetic retinopathy or uveitis).

[0053] Diabetic retinopathy and uveitis

[0054] The present invention is based at least in part on the discovery that administration of superoxide dismutase (SOD), especially oral administration, is effective in preventing or treating diabetic retinopathy or uveitis. Thus, according to one aspect of the present invention, there is provided the use of SOD in preventing or treating diabetic retinopathy or uveitis.

[0055] In one embodiment, diabetic retinopathy or uveitis may present with one or more symptoms selected from the group consisting of vision impairment, ocular vascular leakage, or ocular inflammation.

[0056] The term "diabetic retinopathy" refers to the eye complication of decreased vision caused by persistent high blood sugar levels in diabetes that leads to damage to capillaries. Diabetic retinopathy does not have any symptoms in the early stages, but as the macula is affected, vision loss occurs. Diabetic retinopathy is a disease that develops or progresses with a variety of pathological features, including microaneurysms, venous dilatation, retinal hemorrhage, retinal infarction, macular edema, neovascularization, vitreous hemorrhage, and tractional membranes.

[0057] In one embodiment, SOD can bring about one or more effects selected from the following: reducing vascular permeability in diabetic retinopathy, restoring astrocyte foot processes, inhibiting pericyte loss, and inhibiting the formation of non-cellular capillaries.

[0058] The term "uveitis" refers to inflammation of the inside of the eye, particularly inflammation of the middle layer of the eye (uvea). More specifically, uveitis includes anterior uveitis, which is inflammation of the anterior half of the uveal system, including inflammation of the iris (iritis), and inflammation of the iris and ciliary body (cyclitis); intermediate uveitis, which is inflammation of the hyaline membrane; and posterior uveitis, which is inflammation of the part of the uveal system behind the lens of the eye, including inflammation of the choroid (choroiditis), and inflammation of the choroid and retina (chorioretinitis), and also includes panuveitis involving the entire uveal system.

[0059] In one embodiment, uveitis may include, but is not limited to, one or more selected from infectious causes and non-infectious causes. Specifically, the infectious causes of uveitis may include one or more selected from bacteria, viruses, fungi and parasites, and the non-infectious causes may include one or more selected from autoimmune diseases, tumors, trauma, surgery and systemic diseases.

[0060] In one embodiment, the SOD can show the following effects on uveitis: it has an improvement or relief effect in one or more fundus assessment indicators selected from focal lesions, linear lesions, chorioretinopathy, confluent lesions, vasculitis, vitreitis, vitreous hemorrhage, optic disc edema and retinal detachment; and / or, it has an improvement or relief effect in one or more tissue assessment indicators selected from inflammatory cell infiltration, retinal wrinkles, intraretinal / subretinal hemorrhage, intraretinal / subretinal exudate and retinal damage.

[0061] Superoxide dismutase (SOD)

[0062] According to another aspect of the present invention, there is provided an improved superoxide dismutase (SOD) which exhibits the effect of preventing, improving or treating diabetic retinopathy or uveitis.

[0063] 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] 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.

[0064] In one embodiment, SOD may be superoxide dismutase (Mn-SOD) combined with manganese. Specifically, SOD may be deamidated Mn-SOD. More specifically, SOD may be a substance in which amino acid residues at positions 73 and 136 of SEQ ID NO:2 are replaced by Asp. More specifically, it may comprise the amino acid sequence shown in SEQ ID NO:4, or consist of the amino acid sequence shown in SEQ ID NO:4.

[0065] 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.

[0066] 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 one or more amino acid deletions, insertions or substitutions. 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.

[0067] 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.

[0068] 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 comprising a nucleotide sequence encoding a polypeptide, the polypeptide comprising the amino acid sequence shown in SEQ ID NO:4, and wherein AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf genes are deleted. The detailed process of preparing the strain is shown in Example 1.

[0069] 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.

[0070] 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 diabetic retinopathy or uveitis 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 medium. For example, a composite culture medium (pH 6.0 to 7.0) is used to grow the strain at 25° C. to 42° C. for 1 to 4 days. Other suitable culture media for culturing Bacillus amyloliquefaciens GF423 strain include LB (Luria-Bertani) culture medium, ISP (International Streptomyces Project) culture medium, NA (nutrient agar) culture medium, BHI (brain heart infusion agar) culture medium, SDA (Sabouraud dextrose agar) culture medium, PDA (potato dextrose agar) culture medium, NB (nutrient broth) culture medium, etc. Preferably, LB culture medium, ISP culture medium, BHI culture medium, SDA culture medium or NB culture 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).

[0071] In one embodiment, the SOD may be an isolated or purified enzyme. In this case, the isolated or purified SOD, or its biologically active portion, is substantially free of cellular material or other contaminating proteins from its source cells or tissues. For example, the purified product may be obtained by single separation from a strain culture by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or a culture concentrate obtained by ultrafiltration, concentration, etc. The phrase "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 a recombinantly produced cell. Specifically, the phrase "substantially free of cellular material" means that the content of undesirable protein by dry weight in the included protein preparation is less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5%.

[0072] In another embodiment, SOD can be preferably purified by the following purification method, but is not limited thereto. The culture solution obtained by culturing 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.

[0073] In one embodiment, the SOD may be contained in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dry form thereof. At this time, "strain lysate" refers to a substance obtained by culturing a strain and pulverizing it mechanically or chemically, and may include a substance that has been further subjected to additional processes such as extraction, dilution, concentration, and purification. "Strain culture" may refer to the culture solution itself or its supernatant obtained by culturing a strain. "Strain culture concentrate" refers to a culture concentrate obtained by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or obtained by ultrafiltration, concentration, etc. from a strain culture. "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 lyophilized form.

[0074] In one embodiment, by orally administering the SOD to a subject in need of preventing or treating diabetic retinopathy, one or more effects of reducing vascular permeability, restoring astrocyte foot processes, inhibiting pericyte loss, and inhibiting non-cellular capillary angiogenesis can be achieved.

[0075] In another embodiment, by orally administering the SOD to a subject in need of preventing or treating uveitis, the following effects can be achieved: an improvement or alleviation effect in one or more fundus assessment indicators selected from focal lesions, linear lesions, chorioretinopathy, confluent lesions, vasculitis, vitreitis, vitreous hemorrhage, optic disc edema and retinal detachment; and / or, an improvement or alleviation effect in one or more tissue assessment indicators selected from inflammatory cell infiltration, retinal wrinkles, intraretinal / subretinal hemorrhage, intraretinal / subretinal exudates and retinal damage.

[0076] Pharmaceutical composition

[0077] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating diabetic retinopathy or uveitis, comprising SOD as an effective ingredient.

[0078] 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 diabetic retinopathy or uveitis. When applied to animals other than humans, the pharmaceutical composition of the present invention can be used interchangeably with the term "veterinary composition".

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] In addition, the pharmaceutical composition of the present invention or veterinary composition is applied with pharmaceutically or veterinary effective amount. The term "pharmaceutically effective amount" or "veterinary effective amount" refers to the amount of a disease that is sufficient to treat a reasonable benefit / risk ratio applicable to pharmaceutical or veterinary treatment, and the effective dose level can be determined according to factors including the body weight, sex, age, health status, severity, activity of the drug, sensitivity to the drug, administration time, route of administration and discharge ratio, treatment period, and other factors well-known in the medical field including the patient or animal's body weight, sex, age, health status, severity, drug activity, drug sensitivity, administration time, route of administration and discharge ratio, treatment period, and drugs used simultaneously. Preferably, based on the gross weight of the composition, the content of SOD in the pharmaceutical composition of the present invention or veterinary composition can be about 2 to about 1500U / mg, specifically about 5 to about 1000U / mg, more specifically about 10 to about 800U / mg, and further more specifically about 100 to about 500U / mg.

[0086] 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.

[0087] Treatment or prevention methods

[0088] According to yet another aspect of the present invention, a method for treating or preventing diabetic retinopathy or uveitis is provided, comprising the following steps: administering the SOD or pharmaceutical composition according to the present invention to a subject suffering from, or at risk of, diabetic retinopathy or uveitis.

[0089] In the present invention, "administration" includes any variety of administrations that achieve the desired effect to achieve the therapeutic purpose. The administration route may be oral, parenteral, inhalation, external application or topical administration (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 can be administered in the form of being coated with a coating agent such as shellac.

[0090] 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 separate dosages can be applied 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.

[0091] In one embodiment, the method may include: administering one or more additional preparations for treating or preventing diabetic retinopathy or uveitis. The additional preparations include but are not limited to any preparations that can alleviate or improve diabetic retinopathy or uveitis, such as compounds, gene therapy agents, proteins (including antibodies), etc. The one or more additional preparations can be administered simultaneously, sequentially, or in reverse order with the SOD or pharmaceutical composition. In addition, each component can be administered to the subject by the same or different routes.

[0092] Food composition

[0093] According to yet another aspect of the present invention, a food composition for improving or preventing diabetic retinopathy or uveitis is provided, which comprises the SOD according to the present invention as an effective ingredient. Such food composition includes medical or nutraceutical food compositions.

[0094] 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.

[0095] 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.

[0096] The food can be prepared by conventional methods known in the art. For example, for the purpose of preventing or improving diabetic retinopathy or uveitis, 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. The 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 its dosage form combination of appropriate ingredients.

[0097] 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.

[0098] 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.

[0099] Feed composition

[0100] According to yet another aspect of the present invention, a feed composition for preventing or improving diabetic retinopathy or uveitis is provided, which comprises 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 comprise: 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 feed, such as materials with rapeseed, soybeans, and sunflower seeds as main ingredients; animal protein feed, such as blood meal, meat meal, bone meal, and fish meal; sugar 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.

[0101] The feed composition of the present invention may be in the form of a powder or liquid preparation, and may contain an excipient for feed addition (calcium carbonate, wheat shortbread, zeolite, corn flour, rice bran, etc.).

[0102] Box

[0103] The composition of the present invention can be provided in the form of a kit. For example, the composition of the present invention (e.g., a modified polypeptide, a pharmaceutical composition, a food composition, a feed composition, a co-administered composition comprising at least one additional agent, or a combination thereof) packaged in a suitable container can be included, and a guide for its administration can also be included. In addition, the kit can also include components such as an administration tool, which is packaged in another container.

[0104] Example

[0105] 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.

[0106] [Example 1] Preparation of recombinant strain expressing superoxide dismutase (SodA2)

[0107] [Example 1.1] Preparation of expression host GFBS220

[0108] 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.

[0109]

Table 1

[0110]

[0111]

[0112] 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.

[0113]

Table 2

[0114]

[0115]

Table 3

[0116]

[0117]

[0118] After the carrier prepared in this way is introduced into the subtilis cell, the substratum containing chloramphenicol is used to screen transformant (single cross homologous recombination) at 37 ℃ (non-replication temperature). The transformant screened is cultivated (second crossover) using LB substratum without antibiotics at 30 ℃ (permitting replication temperature), and then cultivated using LB agar medium without antibiotics at 39 ℃. From the bacterium colony generated, strains sensitive to chloramphenicol are screened (plasmid solidification). PCR is performed on the bacterium colony screened to obtain double cross-over recombinant (double cross-over rec ombinant), and single separation is performed for the second time to screen out final recombinant, named after GFBS220.

[0119] The defective gene site of GFBS220 was confirmed by agarose gel electrophoresis after amplification using the primers and PCR shown in Table 4. The results of confirming the defective gene by PCR in the expression host GFBS220 are as follows: Figure 2 shown.

[0120]

Table 4

[0121]

[0122] [Example 1.2] Preparation of expression vector pBE1-sodA2

[0123] Mn-SOD was confirmed in the culture supernatant of Bacillus amyloliquefaciens strain GF423 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 bacterial SOD nomenclature, 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).

[0124] 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.

[0125]

Table 5

[0126]

[0127]

[0128] 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.

[0129] [Example 1.3] Preparation of production strain BSBA310

[0130] 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 storage 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.

[0131] [Example 2] Isolation and purification of superoxide dismutase (SodA2) from production strain BSBA310

[0132] [Example 2.1] Cultivation of production strain BSBA310

[0133] 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.

[0134] [Example 2.2] Isolation and purification of SodA2

[0135] 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 analysis kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits superoxide free radicals by 50%. The activity of dried SodA2 enzyme is 1000 to 1200 U / mg.

[0136] [Example 3] Preparation of administration material

[0137] Shellac (EXCELACS co., LTD., Bangkok) was dissolved in ethanol to a concentration of 3% and sterilized using a 0.2um sterile filter. The shellac dissolved in ethanol was diluted with PBS. The freeze-dried SodA2 was dissolved to 20 mg / mL, and the shellac solution and SOD solution were mixed in a 1:1 ratio and then freeze-dried. The freeze-dried shellac-coated SodA2 and dextrin were mixed in a ratio of 1:9 to 12 (shellac-coated SodA2: dextrin) to prepare the substance used in the animal test. The SOD activity of the final substance reached 90 to 110 U / mg.

[0138] [Example 4] Construction of diabetic retinopathy model

[0139] 120 6-week-old male C57BL / 6 mice (purchased from Koatech) were purchased and maintained under a 12-hour light / dark cycle to allow mice to freely access food and water. Mice induced with diabetes (hereinafter referred to as "DM") were obtained by intraperitoneal injection of streptozotocin (Streptozotocin, hereinafter referred to as STZ) solution (10mM citrate buffer (pH 4.5), 180mg / kg), and blood glucose levels were confirmed after 3 days of injection. Only mice with blood glucose levels of 300mg / dl or higher were selected as diabetic mice. According to the concentration of the oral drug administered and whether diabetes was induced, C57BL / 6 mice were divided into 10 groups as shown in Table 6, and divided into 3-week experimental groups (60) and 16-week experimental groups (60) according to the SodA2 administration period for experiments. For the SodA2 administration group, SodA2 was administered every day from the streptozotocin induction stage regardless of whether diabetes was present.

[0140]

Table 6

[0141]

[0142] [Example 5] Construction of uveitis model

[0143] One hundred and twenty 6-week-old male C57BL / 6 mice (purchased from Koatech) were purchased and maintained under a 12-hour light / dark cycle with free access to food and water. Of the total 120 animals, 60 were used in the endotoxin-induced uveitis model experimental group and 60 were used in the experimental autoimmune uveitis model experimental group. All animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology's statement for the use of animals in ophthalmic and vision research and approved by the Institutional Animal Care and Use Committee of Seoul National University and Seoul National University Hospital.

[0144] [Example 5.1] Construction of endotoxin-induced uveitis model and administration of SodA2

[0145] In order to construct the endotoxin-induced uveitis (EIU) model, SodA2 was orally administered at concentrations of 1, 5, 10, and 20 units for 7 days (once a day, 200ul), and then 1ng of the drug was injected into the vitreous body using LPS (lipopolysaccharide, L2880-10MG), an outer membrane component of Gram-negative bacteria, to induce acute uveitis. The mouse model used in the acute uveitis experiment is shown in Table 7.

[0146]

Table 7

[0147]

[0148] [Example 5.2] Construction of experimental autoimmune uveitis model and administration of SodA2

[0149] The T cell-mediated chronic uveitis induction process for constructing the experimental autoimmune uveitis (hereinafter referred to as "EAU") model is as follows. 30 mg of human IRBP 1-20 (Peptron, GPTHLFQPSLVLDMAKVLLD 95%) was dissolved in PBS and incubated at 26°C for 30 minutes. Next, Mycobacterium tuberculosis H37 Ra (BD, 231141) and complete Freund's adjuvant (CFA; Sigma, F5881) were mixed and incubated at 26°C for 30 minutes, and then mixed with human IRBP 1-20 Mixed in a ratio of 1:1. A total of 200 μl of the mixed reagent was subcutaneously injected into the back of the neck and hind limbs of C57BL / 6 mice, and then 200 μl of pertussis toxin (Sigma, P7208) of Bordetella pertussis was injected intraperitoneally to induce chronic uveitis. Next, Sod A2 drug was orally administered at concentrations of 1, 5, 10, and 20 units for 21 days (once a day, 200 ul). The mouse model used in the chronic uveitis experiment is shown in Table 8.

[0150]

Table 8

[0151]

[0152] [Example 6] Confirmation of effects in diabetic retinopathy model

[0153] [Example 6.1] Assessment of retinal vascular leakage

[0154] In order to confirm the degree of vascular leakage in the 3-week experimental group and the 16-week experimental group after the end of the SodA2 administration period, the mice were anesthetized, and Evans blue (20 mg / ml, 150 ul, dissolved in PBS) was injected intravenously and the eyeballs were removed after perfusion for 1 hour. All eyeball removal processes were carried out in accordance with the statement of the Association for Research in Vision and Ophthalmology on the use of animals in ophthalmic and vision research. In order to qualitatively analyze retinal vascular leakage, the removed eyeballs were fixed with 4% paraformaldehyde (PFA), flatmounted in 1xPBS and placed on a slide, and then the entire retina was photographed at x40 times by a fluorescence microscope (Eclipse 90i, Nikon, Tokyo, Japan). In addition, in order to quantitatively analyze retinal vascular leakage, the entire retina was photographed with red fluorescence, and the image was adjusted to the corresponding color threshold using the ImageJ (1.47v, NIH, Bethesda, Maryland, USA) program and based on the automatic ISODATA algorithm. The red area was measured and expressed as the degree of retinal vascular leakage (%) and compared with the non-diabetic normal control group.

[0155] 3-week administration group

[0156] The results of qualitative analysis using a microscope showed that no vascular leakage was observed in the normal control group without SodA2 administration and the normal mouse group administered SodA2 (Sod A2 concentration was 1, 5, 10, and 20 units), and there was no change in normal retinal vascular permeability regardless of SodA2 administration ( Figure 6a In the diabetic control group, 3 weeks after diabetes induction, the contrast in the retinal tissue was enhanced due to increased vascular permeability ( Figure 6b In the diabetic group administered with SodA2, a significant decrease in retinal vascular permeability was observed compared with the control group ( Figure 6b 2 to 5 in the figure).

[0157] The quantitative analysis results of retinal vascular leakage were consistent with the qualitative evaluation results. No retinal vascular leakage was found in the normal control group without SodA2 administration and the normal mouse group with SodA2 administration, and the diabetic control group had higher retinal vascular leakage. In the 3-week diabetes-induced experimental group administered with SodA2, retinal vascular permeability showed a decreasing trend compared with the diabetic control group, especially in the diabetic group administered with 5 units of SodA2, which showed a statistically significant decrease. Quantitative evaluation of retinal vascular leakage in the 3-week experimental group Figure 6c shown.

[0158] 16-week administration group

[0159] The results of qualitative analysis by microscopy showed that no vascular leakage was observed in the normal control group without SodA2 administration and the normal mouse group with SodA2 administration ( Figure 7a In some normal mouse groups administered with 10 and 20 units of SodA2, retinal vascular permeability increased compared with the non-diabetic control group, but the increase was not significant. In the diabetic control group, after 16 weeks of diabetes induction, the contrast in the retinal tissue was enhanced due to increased vascular permeability ( Figure 7b In the diabetic group administered with SodA2, retinal vascular permeability was reduced compared with the control group ( Figure 7b The vascular permeability was significantly reduced, especially in the diabetic group administered with 5 units of SodA2.

[0160] The quantitative analysis results of retinal vascular leakage were similar to the qualitative evaluation results. No retinal vascular leakage was observed in the normal control group without SodA2 administration and the normal mouse group administered with SodA2 (the retinal vascular permeability increased in the normal mouse group administered with 20 units of SodA2, but it was not significant), and the retinal vascular permeability was higher in the diabetic control group. In the 16-week diabetic induced administration group administered with SodA2, the retinal vascular permeability showed a decreasing trend compared with the diabetic control group, especially in the diabetic group administered with 5 units of SodA2, which showed a statistically significant decrease. Quantitative evaluation of retinal vascular leakage in the 16-week experimental group Figure 7c shown.

[0161] [Example 6.2] Evaluation of astrocyte immunofluorescence staining

[0162] After the retina was isolated from the eyeball fixed in 4% paraformaldehyde, anti-GFAP antibody (1:100; Invitrogen, 53-9392-82) and isolectin B4-594 (1:100; Invitrogen, I21412) were added to Perm / Block solution (PBS solution containing 0.3% Triton-X and 0.2% BS A) and incubated overnight at 4°C. The next day, the retina was washed with PBS and then mounted with fluorescent aqueous mounting medium (Sigma-Aldrich). To confirm the distribution of astrocyte foot processes outside the pericytes surrounding the retinal vascular network (the better the distribution of astrocyte foot processes, the better the vascular permeability is suppressed), the retina was stained and photographed using a confocal microscope (Leica TCS STED, Leica Microsystems, Wetzlar, Germany). The middle part of the retina was photographed at 400 times magnification, and the astrocytes in each group were quantified using the ImageJ (1.47v, NIH, Bethesda, MD, USA) program.

[0163] 3-week administration group

[0164] The results of qualitative analysis using a microscope showed that in the normal control group without SodA2 administration and the normal mouse group administered with SodA2 (Sod A2 concentrations of 1, 5, 10, and 20 units), the distribution of astrocyte foot processes did not change regardless of SodA2 administration ( Figure 8a In the diabetic control group, after 3 weeks of diabetes induction, the foot processes of astrocytes decreased compared with the normal control group ( Figure 8b In the diabetic group administered with SodA2, the distribution of astrocyte foot processes was observed to increase significantly and recover compared with the control group ( Figure 8b 2 to 5 in the figure).

[0165] The results of quantitative analysis of astrocyte immunofluorescence staining were consistent with the results of qualitative evaluation. No changes were observed in the distribution of astrocyte foot processes in the normal control group without SodA2 administration and the normal mouse group administered with SodA2. In the diabetic control group, astrocyte foot processes decreased after 3 weeks of diabetes induction. Compared with the diabetic control group that showed a decrease in astrocyte foot processes, the distribution of astrocyte foot processes increased in the 3-week diabetes-induced experimental group administered with SodA2. Quantitative evaluation of astrocyte immunofluorescence staining in the 3-week experimental group Figure 8c shown.

[0166] 16-week administration group

[0167] Referring to the results of qualitative analysis by microscopy, unlike the normal control group to which SodA2 was not administered, the distribution of astrocyte foot processes was reduced in some normal mouse groups to which SodA2 was administered, but the change was not significant ( Figure 9a In the diabetic control group, after 16 weeks of diabetes induction, the foot processes of astrocytes were significantly reduced compared with the normal control group ( Figure 9b In the diabetic group administered with SodA2, the distribution of astrocyte foot processes was observed to increase significantly and recover compared with the control group ( Figure 9b 2 to 5 in the figure).

[0168] The quantitative analysis results of astrocyte immunofluorescence staining showed that the results were consistent with the qualitative analysis. After 16 weeks of diabetes induction, the distribution of astrocyte foot processes in the SodA2-administered diabetes 16-week experimental group was significantly increased compared with the diabetic control group, where astrocyte foot processes were reduced. Quantitative evaluation of astrocyte immunofluorescence staining in the 16-week experimental group was as follows Fig.9c shown.

[0169] [Example 6.3] Evaluation of retinal vascular staining

[0170] After isolating the retina from the eyeball fixed in 4% paraformaldehyde, the retina was placed in a 24-well plate and washed four times with PBS. Next, a 3% trypsin solution (Difco 1:250, 0.1M Tris (pH 7.8)) was added to the plate containing the retina and incubated at 37°C for 2 hours. After the reaction was completed, the trypsin was carefully removed, washed with water, transferred to a slide, and non-vascular tissue was carefully removed with water. After drying the slide that only retained the blood vessels, H&E staining was performed.

[0171] Diabetic retinopathy may lead to the development of acellular capillaries with loss of perivascular pericytes, as confirmed by retinal vascular staining.

[0172] The results of qualitative analysis by microscope showed that no loss of pericytes (indicated by arrows) and the occurrence of non-cellular capillaries (indicated by triangles) were observed in the normal control group without SodA2 administration and the normal mouse group administered SodA2 for 16 weeks ( Fig.10a In the diabetic control group, after 16 weeks of diabetes induction, there was an increase in pericyte loss and an increase in acellular capillary formation ( Fig.10bIn the diabetic group administered with SodA2, however, inhibition of pericyte loss and inhibition of non-cellular capillary angiogenesis were observed ( Fig.10b 2 to 5 in the figure).

[0173] Further quantitative analysis results showed that the values ​​of pericytes and non-cellular capillaries did not change in the normal control group without SodA2 administration and the normal mouse group with SodA2 administration ( Fig.10c and Fig.10d 1 to 5 columns from the left in the figure). After 16 weeks of diabetes induction, the pericyte count decreased in the diabetic control group (increased pericyte loss), but the pericyte count increased significantly in the group administered SodA2 during the 16-week period ( Fig.10c In addition, after 16 weeks of diabetes induction, in the diabetic group administered with SodA2, especially in the 5-unit administration group, although statistical significance was not ensured, the occurrence of non-cellular capillaries decreased ( Fig.10d ).

[0174] [Example 7] Experimental evaluation of ocular diseases in uveitis model

[0175] [Example 7.1] Fundus assessment in uveitis

[0176] For the endotoxin-induced uveitis (EIU) model, fundus examination was performed 24 hours after LPS injection (induction acute), and for the experimental autoimmune uveitis (EAU) model, fundus examination was performed on days 14, 16, 18, and 21 after induction of uveitis (induction chronic). In the fundus assessment of uveitis, the score ranged from 0 points for no inflammation in the eye to 4 points based on bleeding or eye status. The scoring criteria are shown in Table 9.

[0177]

Table 9

[0178]

[0179] When evaluated according to the above evaluation criteria, in the EIU model (acute induction group), the SodA2-administered group showed a significant reduction effect compared with the non-administered group ( Fig.11a ).

[0180] In the EAU model (chronic induction group), the group administered with SodA2 showed a reduced effect on the fundus assessment score of uveitis compared with the group not administered with SodA2 ( Fig.11b ). In particular, inflammation was most improved in the 5-unit SodA2 administration group.

[0181] [Example 7.2] H&E staining and tissue evaluation

[0182] To evaluate uveitis tissue, the enucleated eyeballs were fixed in 4% paraformaldehyde and made into paraffin blocks. The paraffin blocks were cut into 4 μm thick sections and then stained with H&E (hematoxylin & eosin). For information on the criteria for evaluating uveitis tissue, see Table 10 and Fig.12 .

[0183]

Table 10

[0184]

[0185]

[0186] EIU model (acute induction group)

[0187] No histological abnormalities were found in the normal control group without SodA2 administration and the normal mouse group with SodA2 administration (SodA2 concentration was 1, 5, 10, and 20 units). Fig.13a In the EIU control group induced by LPS injection, severe inflammation in the vitreous cavity and retinal tissue was observed ( Fig.13b In the EIU mouse group administered with SodA2, reduced inflammation in the vitreous cavity and reduced inflammatory cell infiltration in the retinal tissue were observed compared with the EIU control group ( Fig.13b 2nd to 5th photos in the figure).

[0188] The results of histological grade evaluation showed that in the EIU mouse group administered with SodA2, histological inflammation was improved to a low grade ( Fig.13c ).

[0189] EAU model (chronic induction group)

[0190] No histological abnormalities were found in the normal control group without SodA2 administration and the normal mouse group with SodA2 administration (SodA2 concentration was 1, 5, 10, and 20 units). Fig.14a In the control group of EIU with autoimmune uveitis, inflammatory cell infiltration in the retinal tissue and the resulting retinal changes were observed ( Fig.14b In the EAU mouse group administered with SodA2, the infiltration of inflammatory cells in the retinal tissue and the retinal changes caused by it were significantly reduced compared with the EAU control group, especially in the 5-unit and 10-unit groups ( Fig.14b 2nd to 4th photos in the figure).

[0191] Similar to the tissue evaluation, the results of the histological grade evaluation showed that the histological inflammation in the EAU mouse group administered with SodA2 was improved to a low grade compared with the EAU control group, especially in the EAU mouse groups administered with 5 units and 10 units of SodA2 ( Fig.14c ).

[0192] Statistical processing

[0193] One-way ANOVA and Tukey's multiple comparisons test were used for statistical analysis, and GraphPad Prism 7.0 was used for statistical processing.

Claims

1. A pharmaceutical composition for treating or preventing diabetic retinopathy or uveitis, comprising superoxide dismutase as an effective ingredient.

2. The pharmaceutical composition according to claim 1, wherein The diabetic retinopathy or uveitis includes one or more symptoms selected from the group consisting of vision impairment, ocular vascular leakage, and ocular inflammation.

3. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase has one or more effects selected from the group consisting of reducing vascular permeability, restoring astrocyte foot processes, inhibiting pericyte loss, and inhibiting the occurrence of non-cellular capillaries on diabetic retinopathy.

4. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase has the following effects on uveitis: There is an improvement or alleviation effect in one or more fundus assessment indicators selected from focal lesions, linear lesions, chorioretinopathy, confluent lesions, vasculitis, vitreous hemorrhage, optic disc edema and retinal detachment; and / or The invention has an improvement or alleviation effect in one or more tissue assessment indicators selected from the group consisting of inflammatory cell infiltration, retinal wrinkles, intraretinal / subretinal hemorrhage, intraretinal / subretinal exudate and retinal damage.

5. The pharmaceutical composition according to claim 1, wherein The uveitis may be caused by infectious or non-infectious causes.

6. The pharmaceutical composition according to claim 5, wherein The infectious causes of uveitis include one or more selected from bacteria, viruses, fungi and parasites.

7. The pharmaceutical composition according to claim 5, wherein The non-infectious causes of uveitis include one or more selected from autoimmune diseases, tumors, trauma, surgery and systemic diseases.

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

9. 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.

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

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

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

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

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

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

4.

16. The pharmaceutical composition according to claim 1, wherein The composition is an oral administration composition.

17. The pharmaceutical composition according to claim 16, wherein The superoxide dismutase is coated with a coating agent.

18. A veterinary composition for treating or preventing diabetic retinopathy or uveitis, comprising superoxide dismutase as an active ingredient.

19. A food composition for improving or preventing diabetic retinopathy or uveitis, comprising superoxide dismutase as an effective ingredient.

20. A feed composition for improving or preventing diabetic retinopathy or uveitis, comprising superoxide dismutase as an effective ingredient.

21. A method for treating or preventing diabetic retinopathy or uveitis, comprising the step of administering the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 17 to a subject.

22. Use of the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 17 in preventing or treating diabetic retinopathy or uveitis.

23. Use of the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 17 in the preparation of a medicament for preventing or treating diabetic retinopathy or uveitis.

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