Superoxide dismutase and its use for preventing or treating diabetic retinopathy or uveitis

Abstract

The present invention relates to superoxide dismutase and its use for the prevention or treatment of diabetic retinopathy or uveitis. The superoxide dismutase according to the present invention can be effectively used for the prevention or treatment of diabetic retinopathy or uveitis, particularly by oral administration.

Description

Technical Field

[0001] The present invention relates to the use of superoxide dismutase for the prevention or treatment of diabetic retinopathy or uveitis. More specifically, the present invention relates to the use, composition or method of superoxide dismutase for the prevention or treatment of diabetic retinopathy or uveitis.

Background Art

[0002] Recently, visual impairment due to various diseases such as age-related retinal nerve decline has been on the increase. The eyeball including the retina is a nerve tissue related to visual function, and once the damage progresses, it is difficult to recover, so special attention is required. Diseases that cause visual impairment include, for example, diabetic retinopathy, uveitis, cataract, age-related macular degeneration, retinal detachment, and the like.

[0003] Diabetic retinopathy is one of the chronic complications of diabetes and one of the major causes of blindness worldwide. Since there is no special pain and the vision deteriorates, the treatment period may be delayed. Diabetic retinopathy is treated by preventing the generation and bleeding of new blood vessels in the eye through laser treatment, which is a common treatment method, and suppressing sudden vision loss. However, when there is diabetic macular edema, it is difficult to improve vision and there are limitations in the application of laser treatment. As a method to complement this, a method of injecting a steroid or a vascular endothelial growth factor inhibitor (for example, dexamethasone) into the vitreous body is used. Although this has the advantage of suppressing vascular endothelial growth factor and improving the chronic inflammatory state of the retina of diabetic retinopathy patients, there is a risk of side effects when used for a long time.

[0004] Uveitis is an inflammatory disease of the uveal tract of the eye. Although it may be a transient onset and recover after treatment, in most cases, the inflammation recurs and persists, and it is often idiopathic with unknown causes. Eventually, it may cause severe visual impairment and even lead to blindness. Uveitis is caused by infectious causes such as viruses, bacteria, fungi, and parasites, as well as non-infectious causes such as autoimmune diseases. Uveitis is a specific endogenous disease in which most causative bacteria are not found, and since abnormalities in the immune system are involved, mainly corticosteroid preparations are administered in the form of eye drops. When acute inflammation occurs, it is treated by instilling a cycloplegic agent together with local steroid eye drops. When the inflammation is severe, systemic steroids are administered, and in the case of chronic diseases, immunosuppressive agents such as cyclosporine are administered.

[0005] Conventional steroid preparations have a risk of side effects when used for a long time, and cycloplegic agents have many drawbacks such as glare and difficulty in seeing nearby characters. Therefore, there is a need to develop a preparation that can prevent or treat eye diseases, particularly diabetic retinopathy and / or uveitis.

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to solve all of the above-mentioned problems.

[0007] One object of the present invention is to provide superoxide dismutase for the prevention, improvement, or treatment of diabetic retinopathy or uveitis.

[0008] Another object of the present invention is to provide superoxide dismutase derived from Bacillus amyloliquefaciens that ensures the efficacy and safety of oral administration for the prevention, improvement, or treatment of diabetic retinopathy or uveitis.

[0009] Another object of the present invention is to provide a Bacillus - derived or Bacillus amyloliquefaciens - derived superoxide dismutase that is conveniently extracellularly secreted for production for the prevention or treatment of diabetic retinopathy or uveitis.

[0010] Another object of the present invention is to provide a pharmaceutical composition or a treatment method for the prevention or treatment of diabetic retinopathy or uveitis.

[0011] Another object of the present invention is to provide a food or feed composition for the prevention or improvement of diabetic retinopathy or uveitis.

[0012] Another object of the present invention is to provide an orally - administered composition containing superoxide dismutase for the prevention, improvement or treatment of diabetic retinopathy or uveitis.

[0013] The object of the present invention is not limited to the above. The object of the present invention will become clearer from the following description and will be realized by the means and their combinations described in the claims.

Means for Solving the Problems

[0014] Typical configurations of the present invention for achieving the above object are as follows.

[0015] According to one embodiment of the present invention, an improved superoxide dismutase (SOD) showing a preventive, ameliorative or therapeutic effect on diabetic retinopathy or uveitis is provided.

[0016] According to another embodiment of the present invention, a generally regarded as safe (GRAS) bacterium - derived superoxide dismutase, for example, a Bacillus - derived superoxide dismutase, for the prevention, amelioration or treatment of diabetic retinopathy or uveitis is provided.

[0017] According to another embodiment of the present invention, there is provided a superoxide dismutase derived from Bacillus or Bacillus amyloliquefaciens that is conveniently extracellularly secreted for production for the prevention, improvement or treatment of diabetic retinopathy or uveitis.

[0018] According to another embodiment of the present invention, there is provided an improved superoxide dismutase that exhibits a preventive, ameliorative or therapeutic effect on diabetic retinopathy or uveitis even when administered orally.

[0019] According to another embodiment of the present invention, there is provided a superoxide dismutase derived from Bacillus amyloliquefaciens that ensures the efficacy and safety of oral administration for the prevention, improvement or treatment of diabetic retinopathy or uveitis.

[0020] According to another embodiment of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of diabetic retinopathy or uveitis, comprising the superoxide dismutase as an active ingredient.

[0021] According to another embodiment of the present invention, there is provided a food or feed composition for the prevention or improvement of diabetic retinopathy or uveitis, comprising the superoxide dismutase as an active ingredient.

[0022] According to another embodiment of the present invention, there is provided an oral administration composition for the prevention, improvement or treatment of diabetic retinopathy or uveitis, comprising the superoxide dismutase as an active ingredient.

[0023] According to another embodiment of the present invention, there is provided a method for the prevention, improvement or treatment of diabetic retinopathy or uveitis, comprising the step of administering the superoxide dismutase or the composition to a subject.

[0024] According to another embodiment of the present invention, there is provided the use of the superoxide dismutase for the prevention, improvement or treatment of diabetic retinopathy or uveitis.

[0025] According to another embodiment of the present invention, there is provided a use for producing a medicament for preventing, improving or treating diabetic retinopathy or uveitis of the superoxide dismutase.

Advantages of the Invention

[0026] The superoxide dismutase, composition, method and kit according to the present invention can be effectively used for the prevention or treatment of diabetic retinopathy or uveitis. In particular, the superoxide dismutase according to the present invention has been confirmed to be effective for the prevention or treatment of diabetic retinopathy or uveitis even when administered orally.

[0027] Since the superoxide dismutase according to the present invention is derived from Bacillus or Bacillus amyloliquefaciens, which are bacteria generally regarded as safe (GRAS), not only the efficacy and safety of oral administration are ensured, but also the production advantage that it can be directly recovered from the supernatant during culture is obtained.

[0028] According to the examples of the present invention, the SOD of the present invention was orally administered to diabetic-induced mice and normal mice, and as a result of evaluating retinal vascular leakage, astrocyte immunofluorescence staining, and retinal vascular staining, it was confirmed that there were effects of reducing retinal vascular permeability, restoring astrocyte foot processes, suppressing pericyte loss, and suppressing acellular capillary formation in SOD-administered diabetic-induced mice. In addition, as a result of orally administering SOD to mice induced with acute and chronic uveitis and performing fundus evaluation and tissue evaluation, it was confirmed that the inflammatory findings were improved to a lower grade. Therefore, the superoxide dismutase according to the present invention can be effectively used for preventing, improving or treating diabetic retinopathy or uveitis through reducing retinal vascular leakage and improving the inflammatory state.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0030] The detailed description of the present invention to be described below will be described with reference to specific drawings for specific embodiments in which the present invention can be implemented. However, the present invention is not limited thereto, and if appropriately described, it is limited only by the appended claims, and is limited to all ranges equivalent to those described in the claims. It should be understood that the various embodiments of the present invention are different from each other but need not be mutually exclusive. For example, the specific shapes, structures, and characteristics described herein can be changed from one embodiment to another or implemented in combination with embodiments without departing from the spirit and scope of the present invention. The terms presented when describing the present invention should be understood in their ordinary meaning in general, unless otherwise specified, and apply not only to the embodiments or implementations of the invention in which the terms are defined, but also to the same terms used herein. For the purpose of interpreting this specification, the following definitions apply, and terms used in the singular include the plural where appropriate, and vice versa.

[0031] Definition As used herein, the term "about" refers to the normal error range of each value known to those skilled in the art.

[0032] The term "subject" is used interchangeably with "patient" and refers to a mammal in need of prevention or treatment of diabetic retinopathy or uveitis, such as a primate (e.g., human), a pet (e.g., dog, cat, etc.), a livestock animal (e.g., cow, pig, horse, sheep, goat, etc.), and a laboratory animal (e.g., rat, mouse, guinea pig, etc.). In one embodiment of the present invention, the subject is a human.

[0033] The term "treatment" includes prophylactic and / or therapeutic measures. The prophylactic and / or therapeutic measures include any kind of measures recognized in the art, for example, administering the pharmaceutical composition or SOD of the present invention to a subject. If administered prior to the clinical symptoms of an undesired or unwanted condition (e.g., a disease or other undesired or unwanted condition in a subject), such a measure or treatment is a prophylactic measure or treatment (e.g., protecting the subject from the progression of an undesired or unwanted condition in the subject). On the other hand, if administered after the clinical symptoms of an undesired or unwanted condition, such a measure or treatment is a therapeutic measure such as reducing, alleviating or stabilizing the existing undesired or unwanted condition or its side effects.

[0034] The meaning of the term "prevention" is well known in the art. When used in relation to a medical condition such as diabetic retinopathy or uveitis, administering the pharmaceutical composition or SOD of the present invention means reducing the frequency of a medical condition (such as discomfort, visual impairment, ocular vascular leakage, damage to the ocular surface, inflammation of the ocular surface, etc.) or delaying the onset and symptoms as compared to a subject not receiving the administration.

[0035] The term "administration" means providing an active ingredient to a subject to achieve a prophylactic or therapeutic purpose (e.g., prevention or treatment of diabetic retinopathy or uveitis).

[0036] Diabetic retinopathy and uveitis The present invention is based, at least in part, on the discovery that administration of superoxide dismutase (SOD), particularly oral administration, is effective for the prevention or treatment of diabetic retinopathy or uveitis. Accordingly, according to one embodiment of the present invention, there is provided the use of SOD for the prevention or treatment of diabetic retinopathy or uveitis.

[0037] In one embodiment, diabetic retinopathy or uveitis can exhibit one or more symptoms selected from the group including visual impairment, ocular vascular leakage, or ocular inflammation.

[0038] The term "diabetic retinopathy" refers to an eye complication in which persistent hyperglycemia due to diabetes causes damage to capillaries and results in vision loss. Diabetic retinopathy is asymptomatic in its early stages but exhibits vision loss due to the invasion of the macula. Diabetic retinopathy is a disease that develops or progresses with various pathological features such as microaneurysms, venous dilation, retinal hemorrhage, retinal infarction, macular edema, neovascularization, vitreous hemorrhage, and traction membranes.

[0039] In one embodiment, SOD can bring about one or more effects selected from a decrease in vascular permeability in diabetic retinopathy, restoration of astrocytic end-feet, suppression of pericytes loss, and suppression of acellular capillary formation.

[0040] The term "uveitis" refers to inflammation of the interior of the eye, particularly the uvea, the middle layer of the eye. More specifically, uveitis includes anterior uveitis, which is inflammation of the anterior segment of the uveal tract, including inflammation of the iris (iritis) and inflammation of the iris and ciliary body (cyclitis); intermediate uveitis, which is inflammation in the vitreous; posterior uveitis, which is inflammation of a part of the uveal tract behind the lens of the eye, including inflammation of the choroid (choroiditis) and inflammation of the choroid and retina (choroidoretinitis); and panuveitis, which is uveitis that affects the entire uveal tract.

[0041] In one embodiment, uveitis may include, but is not limited to, one or more selected from the group including 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.

[0042] In one embodiment, the SOD can exhibit an improvement or remission effect in one or more fundus evaluation indicators selected from uveitis focal lesions, linear lesions, choroidal lesions, confluent lesions, vasculitis, vitritis, vitreous hemorrhage, papilledema, and retinal detachment, an improvement or remission effect in one or more tissue evaluation indicators selected from inflammatory cell infiltration, retinal folds, intraretinal and subretinal hemorrhages, intraretinal and subretinal exudates, and retinal damage, and both of the above improvement or remission effects.

[0043] Superoxide dismutase (SOD) According to another embodiment of the present invention, an improved superoxide dismutase (SOD) is provided that exhibits a preventive, ameliorating, or therapeutic effect on diabetic retinopathy or uveitis.

[0044] Superoxide dismutase is an enzyme that alternately catalyzes the dismutation of superoxide (O2−) radicals to ordinary molecular oxygen (O2) and hydrogen peroxide (H2O2). SOD plays an important role in removing reactive oxygen species and reducing oxidative stress. SOD is widely distributed in prokaryotic and eukaryotic cells and is classified into four classes according to various types of metal centers (copper / zinc, nickel, manganese, and iron). Manganese-containing SOD (Mn-SOD) is widely present in various bacteria, chloroplasts, mitochondria, and cytoplasm of eukaryotic cells. The term "SOD" can be used interchangeably with a polypeptide having superoxide dismutase activity.

[0045] In one embodiment, the SOD may be a manganese-bound type (Mn-SOD). Specifically, the SOD may be deamidated Mn-SOD. More specifically, the SOD may have the 73rd and 136th amino acid residues replaced with Asp based on SEQ ID NO: 2. Even more specifically, it may include or be constituted by the amino acid sequence shown in SEQ ID NO: 4.

[0046] The SOD or polypeptide having SOD activity of the present invention is construed to also include an amino acid sequence that exhibits substantial identity to the above amino acid sequence. The substantial identity means an amino acid sequence that exhibits a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more when analyzing the sequences aligned using algorithms commonly used in the art.

[0047] In other embodiments, the SOD is a modified or engineered polypeptide having SOD enzyme activity and may include one or more mutations that affect or do not affect various aspects (in vivo, in vitro, or ex vivo stability, homogeneity, and / or morphological changes), such as deletions, insertions, or substitutions of one or more amino acids. Further, the polypeptide may further include heterologous substances (e.g., tags known in the art including HIS tag, Ha tag, myc tag, GFC and / or the Fc domain of an antibody) for enhancing purification, detection, or stability.

[0048] In one embodiment, the SOD may be derived from natural or recombinant microorganisms and may be an enzyme produced through a process of isolation or purification from various sources.

[0049] In one embodiment, the SOD may be derived from natural or recombinant microorganisms. For example, the SOD may be derived from bacteria. Preferably, the SOD may be supplied from bacteria generally regarded as safe (GRAS) for use in drugs or foods. Specifically, the SOD may be derived from Bacillus species strains. More specifically, the SOD may be derived from Bacillus amyloliquefaciens strains. For example, the SOD may be derived from Bacillus amyloliquefaciens GF423 strain (KCTC 13222 BP). The GF423 strain (KCTC 13222 BP) was deposited with the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017. Also, the SOD may be derived from a recombinant strain containing the expression vector shown in FIG. 4. Also, the SOD may be derived from a recombinant strain produced by a method including the method shown in FIG. 5. The recombinant strain may be a Bacillus species strain containing a nucleotide sequence encoding a polypeptide containing the amino acid sequence of SEQ ID NO: 4 and lacking the following genes: AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf. For the detailed process for the production of the strain, refer to Example 1.

[0050] Since the SOD derived from the strain is an enzyme secreted extracellularly, when producing SOD using the strain, it is possible to mass-produce SOD with ensured safety for humans without undergoing an expensive purification process (e.g., column purification), so efficient production is possible.

[0051] In one embodiment, SOD can be isolated or purified from various sources including natural or recombinant hosts. For example, SOD having the activity of preventing or treating diabetic retinopathy or uveitis can be extracted from the culture supernatant of Bacillus amyloliquefaciens strain GF423. Briefly, first, Bacillus amyloliquefaciens strain GF423 can be cultured in various types of media to obtain a culture solution. For example, using a complex medium (pH 6.0 - 7.0), the strain can be cultured at 25°C - 42°C for 1 day - 4 days. Other suitable media for culturing Bacillus amyloliquefaciens strain GF423 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, etc. Preferably, LB medium, ISP medium, BHI medium, SDA medium, or NB medium may be used. Furthermore, SOD can be supplied from other natural or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda - enzymes.org on the World Wide Web).

[0052] In one embodiment, the SOD may be an isolated or purified enzyme. At this time, the isolated or purified SOD or its biologically active portion is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it is derived. For example, the purified product may be one that is purely separated 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 products of proteins that are isolated or from which such proteins are separated from the cellular components of the cells in which they are recombinantly produced. Specifically, the phrase "substantially free of cellular material" may include protein products in which the undesired protein is less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% based on dry weight.

[0053] In other embodiments, the SOD may desirably be purified by, but is not limited to, the following purification methods. The culture solution obtained by culturing the Bacillus amyloliquefaciens strain GF423 is centrifuged to collect the culture supernatant. The supernatant fraction is pretreated by solid-phase extraction and then isolated and purified by chromatography. At this time, the SOD may be purified using various modes of chromatography. Preferably, hydrophobic interaction chromatography is used.

[0054] In other embodiments, 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 dried form thereof. At this time, the "strain lysate" means a product obtained by culturing a strain and mechanically or chemically disrupting it, and may include all products that have undergone further processes such as extraction, dilution, concentration, and purification. The "strain culture" means the culture solution itself obtained by culturing a strain or its supernatant. The "strain culture concentrate" refers to a product purely separated from the strain culture by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or a culture concentrate obtained by ultrafiltration, concentration, etc. The "strain culture extract" means a product extracted from the culture solution or its concentrate, and may include an extract, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, or its crude or purified product, or a fraction obtained by fractionating this. The dried form may include a freeze-dried form.

[0055] In one embodiment, the SOD is orally administered to a subject in need of prevention or treatment of diabetic retinopathy, and it has been confirmed to exhibit one or more effects among a decrease in vascular permeability, recovery of astrocytic foot processes, suppression of pericytes loss, and suppression of acellular capillary formation.

[0056] In other embodiments, the SOD is orally administered to a subject in need of prevention or treatment of uveitis, and it has shown an improvement or alleviation effect in one or more fundus evaluation indicators selected from focal lesions, linear lesions, chorioretinal lesions, confluent lesions, vasculitis, vitritis, vitreous hemorrhage, papilledema, and retinal detachment, and an improvement or alleviation effect in one or more tissue evaluation indicators selected from inflammatory cell infiltration, retinal folds, intraretinal and subretinal hemorrhages, intraretinal and subretinal exudates, and retinal damage, or both of the above improvement or alleviation effects.

[0057] Pharmaceutical composition According to another embodiment of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of diabetic retinopathy or uveitis containing SOD as an active ingredient.

[0058] In one embodiment, the SOD according to the present invention can form a pharmaceutical composition for preventing or treating diabetic retinopathy or uveitis in combination with a pharmaceutically acceptable carrier, excipient, and / or diluent. When the pharmaceutical composition of the present invention is applied to animals other than humans, it may be used interchangeably with the term veterinary composition.

[0059] The pharmaceutical or veterinary composition of the present invention may further contain one or more selected from the group consisting of a pharmaceutically acceptable carrier, excipient, and diluent. The pharmaceutically acceptable carrier, excipient, and / or diluent may be those commonly used in the art. Examples of the carrier, excipient, or diluent include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and silicon dioxide, and mineral oil and the like.

[0060] When formulating, additives such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used for preparation. Appropriate additives for the formulation may be selected from those commonly used in the pharmaceutical field.

[0061] In addition, the pharmaceutical or veterinary composition of the present invention can be formulated into a preferred form according to the method of use, and in particular, in order to provide rapid, sustained or delayed release of the active ingredient after administration to mammals, it can be formulated by adopting methods known in the art. Specific examples of such dosage forms include tablets, pills, powders, granules, syrups, solutions, capsules, suspensions, emulsions, injection solutions, plasters, lotions, liniments, lemonades, aerosols, extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, patches, and the like.

[0062] Furthermore, the pharmaceutical or veterinary composition of the present invention can be suitably formulated using appropriate methods known in the art or the methods disclosed in Remington's Pharmaceutical Science (latest edition, Mack Publishing Company, Easton PA).

[0063] In one embodiment, the SOD may be administered orally or parenterally. In the case of oral administration, the SOD may be coated with shellac for protection from gastric acid, but the coating agent is not limited thereto. Examples of coating agents suitable for use in the present invention include shellac, ethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, zein, Eudragit, and combinations thereof. When the SOD is coated, the SOD may be coated in solution. Specifically, a purified solution and a shellac-containing solution are mixed and then lyophilized. This lyophilized sample becomes a powder and can be stored at about 4 °C until use. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., in the composite composition. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and various excipients, such as wetting agents, sweetening agents, flavoring agents, preservatives, etc., may be used in addition to water and liquid paraffin, which are commonly used simple diluents.

[0064] In the case of parenteral administration, injection methods such as sublingual administration, intraocular administration including intravitreal administration, intranasal spray, topical skin application, patch, intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection may be selected.

[0065] In addition, the pharmaceutical or veterinary composition of the present invention is administered in a pharmaceutically or veterinarily effective amount. The term "pharmaceutically effective amount" or "veterinarily effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical or veterinary treatment. The effective dosage level can be determined based on factors including the weight, sex, age, health status, severity, drug activity, sensitivity to the drug, administration time, administration route and excretion rate of the patient or animal, treatment duration, factors including drugs used simultaneously, and other factors well known in the medical field. Preferably, the pharmaceutical or veterinary composition of the present invention may contain SOD in an amount of about 2 to about 1,500 U / mg, specifically about 5 to about 1,000 U / mg, more specifically about 10 to about 800 U / mg, and even more specifically about 100 to about 500 U / mg, based on the total weight of the composition.

[0066] The pharmaceutical or veterinary composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, either sequentially or simultaneously. Further, the pharmaceutical composition may be administered once or multiple times as needed. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects considering all the above factors, and such an amount can be easily determined by those skilled in the art.

[0067] Treatment or prevention method According to another embodiment of the present invention, there is provided a method for the treatment or prevention of diabetic retinopathy or uveitis, comprising the step of administering the SOD or pharmaceutical composition according to the present invention to a subject having or at risk of diabetic retinopathy or uveitis.

[0068] "Administration" in the present invention includes any of a variety of administrations that enable treatment by performing the intended action. The route of administration may be oral, parenteral, inhalation, topical or local administration (e.g., intralesional administration). For example, parenteral administration may include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intraarterial, and intravitreal administration. In the case of oral administration, as described above, SOD may be administered in a form coated with a coating agent such as shellac.

[0069] The effective amount or effective non-toxic amount of SOD according to the present invention can be determined by ordinary experiments. For example, the therapeutic active amount of SOD of the present invention may vary depending on factors such as the stage of the disease, the severity of the disease, the age, sex, medical complications, and body weight of the subject. The dosage and administration therapy of SOD of the present invention may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, weekly, every two weeks, every three weeks, every four weeks, etc., or the dosage may be proportionally decreased or increased according to the urgency of the treatment situation.

[0070] In one embodiment, the method may further include administering one or more other formulations for treating or preventing diabetic retinopathy or uveitis. The other formulations include, but are not limited to, any formulations that can improve or ameliorate diabetic retinopathy or uveitis, such as compounds, gene therapy agents, proteins (including antibodies). The one or more other formulations may be administered simultaneously with, sequentially or in reverse order to the SOD or pharmaceutical composition. Also, the individual components may be administered to the subject by the same or different routes.

[0071] Food composition According to another embodiment of the present invention, there is provided a food composition for improving or preventing diabetic retinopathy or uveitis containing SOD according to the present invention as an active ingredient. Such food compositions include medical or nutraceutical food compositions.

[0072] The term "medical food" or "nutraceutical food" refers to food products manufactured from raw materials or ingredients that may have beneficial functions for the human body, and are defined by the Food and Drug Administration as foods that maintain normal functions or activate the physiological functions of the human body to maintain or improve health. However, it is not limited to this, and does not exclude any ordinary health food in this sense.

[0073] In addition, the said foods include, but are not limited to, various foodstuffs, food additives, beverages (such as functional beverages, natural fruit juices and vegetable beverages), gums, teas, vitamin complexes, health functional foods, and other functional foods.

[0074] The said foods may be manufactured by ordinary methods known in the art. For example, for the purpose of preventing or improving diabetic retinopathy or uveitis, the said medical food, nutraceutical food or health functional food may further contain one or more of a carrier, a diluent, an excipient and an additive in addition to the said SOD, and may be formulated into one selected from the group consisting of tablet, pill, acid, granule, powder, capsule and liquid dosage forms. Specific examples of the said carrier, excipient, diluent and additive are well known in the art, and those skilled in the art may manufacture by appropriately combining suitable components according to the dosage form.

[0075] The content of superoxide dismutase according to the present invention as the active ingredient in the above-mentioned dosage forms may be appropriately adjusted according to the usage form and purpose, the patient's condition, the type and severity of the symptoms, etc., and may be about 0.001 to about 99.9% by weight, preferably about 0.01 to about 50% by weight on a solid content weight basis, but is not limited thereto.

[0076] The dosage of the food of the present invention varies depending on the patient's age, weight, gender, dosage form, health condition, and degree of the disease, and may be administered once or several times a day at regular time intervals according to the judgment of a doctor or a pharmacist. For example, the daily dosage may be 10 to 1,000 mg / kg based on the active ingredient content. The above dosage is an example of an average case, and the dosage may be higher or lower due to individual differences. If the daily dosage of the health functional food of the present invention is less than the above dosage, it may not be possible to obtain a significant effect. In more cases, not only is it uneconomical, but it may also exceed the normal dosage range, resulting in possible undesirable side effects.

[0077] Feed composition According to another embodiment of the present invention, there is provided a feed composition for preventing or improving diabetic retinopathy or uveitis containing SOD according to the present invention as an active ingredient. In the present invention, the food composition may include a feed composition. Such a feed composition may be manufactured in any dosage form commonly used in the industry. For example, the feed composition of the present invention includes auxiliary agent components such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and microbial preparations in other viable forms; grains such as ground or crushed wheat, oats, barley, corn, and rice; vegetable protein feeds mainly composed of rape, soybeans, and sunflowers; animal protein feeds such as blood meal, meat meal, bone meal, and fish meal; sugars and dairy products such as dry components composed of various powdered milks and whey powder; lipids such as animal fats and vegetable fats arbitrarily liquefied by heating, etc. as main components; and may further include additives such as nutritional supplements, digestion and absorption improvers, growth promoters, and disease preventives.

[0078] The feed composition of the present invention may be in a powder or liquid dosage form and may contain excipients for feed addition (such as calcium carbonate, fine powder, zeolite, starch, or rice bran).

[0079] Kit The composition of the present invention may 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 combination administration composition containing at least one or more additional formulations, or a combination thereof) may be packaged in a suitable container and may further include instructions for these uses. Furthermore, the kit may include components such as administration tools packaged in separate containers.

[0080] Hereinafter, the present invention will be described in more detail with reference to the following examples. The following examples are presented to assist in the understanding of the present invention and are not intended to limit its scope in any way and should not be construed as limiting.

Examples

[0081] Example 1. Production of a recombinant strain expressing superoxide dismutase (SodA2) Example 1.1. Production of the expression host GFBS220 The expression host GFBS220 was produced using Bacillus subtilis KCTC 3135. B. subtilis KCTC 3135 was obtained from the Korea Research Institute of Bioscience and Biotechnology's Biological Resource Center (KCTC). To facilitate the subsequent steps, the genes shown in Table 1 below were removed from the genome of B. subtilis KCTC 3135.

[0082]

Table 1

[0083] The removal of the gene was performed using double cross-over recombination on the genome (Figure 1). Specifically, DNA fragments (up frag. and down frag.) having homology with the DNA base sequences flanking the gene to be manipulated were cloned into the vector pUCori-ts-cm having a temperature-sensitive replication origin. The PCR conditions and primers used for cloning are shown in Table 2 and Table 3, respectively.

[0084]

Table 2

[0085]

Table 3-1

Table 3-2

[0086] After introducing the vector thus produced into Bacillus subtilis cells, transformants (single cross-homologous recombination) were selected using a chloramphenicol-containing medium at 37°C (non-replicating temperature). The selected transformants were cultured (second cross) in LB medium without antibiotics at 30°C (permissive replication temperature), and then cultured on LB agar medium without antibiotics at 39°C. Chloramphenicol-sensitive strains (plasmid curing) were selected from the resulting colonies. PCR was performed on the selected colonies to secure double cross-over recombinants, followed by secondary purification and selection of the final recombinants, which were named GFBS220.

[0087] The deleted gene region of GFBS220 was amplified using the primers shown in Table 4 and PCR, and then confirmed using agarose gel electrophoresis. The results of confirming the deleted gene using PCR in the expression host GFBS220 are shown in Figure 2.

[0088]

Table 4

[0089] Example 1.2. Preparation of the expression vector pBE1-sodA2 Mn-SOD was identified by N-terminal sequencing (reference [Kang et al., 2018]) from the culture supernatant of Bacillus amyloliquesfaciens strain GF423. The GF423 strain was deposited at the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017, respectively (accession number KCTC 13222 BP). The gene of the Mn-SOD was named sodA according to the nomenclature of bacterial SOD, and its amino acid sequence is shown in Figure 3 and SEQ ID NO: 2 (its nucleotide sequence is shown in SEQ ID NO: 1). LC-UV-MS / MS peptide mapping of the enzyme product generated 100% amino acid sequence coverage, but deamidation was observed at the Asn73 and Asn136 residues with abundance ratios of 73% and 17%, respectively. Therefore, in order to improve the homogeneity of the purified enzyme, both Asn residues were replaced with Asp. The amino acid sequence of this mutant sodA named SodA2 is shown in Figure 3 and SEQ ID NO: 4 (its nucleotide sequence is shown in SEQ ID NO: 3).

[0090] The sodA2 gene was amplified using overlap extension PCR with four primers (Table 5) and the Bacillus amyloliquefaciens GF423 genome as a template. The nucleotides for the substitution of Asn with Asp are underlined.

[0091]

Table 5

[0092] 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 (reference [Jeong et al., 2012]), and then the obtained product was transformed into E. coli C2984H. Accurate clones were screened by restriction enzyme mapping and confirmed by nucleotide sequencing. The expression vector pBE1-sodA2 thus generated is shown in Figure 4.

[0093] Example 1.3. Production of the production strain BSBA310 The expression vector pBE1-sodA2 was transformed into B. subtilis GFBS220. Strains were selected from the transformants, and pure clones were established by a single colony isolation procedure twice in LB2 medium. The selected strain was named BSBA310 and stored at -80 °C by the glycerol stock method. The results of confirming the defective gene from this production strain BSBA310 using PCR are shown in Figure 2. Also, the entire procedure for producing the production strain BSBA310 is summarized in Figure 5.

[0094] Example 2. Isolation and purification of superoxide dismutase (SodA2) from the production strain BSBA310 Example 2.1. Cultivation of the production strain BSBA310 For the culture of the production strain BSBA310 obtained in Example 1, a single colony formed on an 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. This seed culture was inoculated again into 3 L of LB medium containing 1 mM manganese sulfate (MnSO4) and cultured at 37 °C for 20 hours.

[0095] Example 2.2. Isolation and purification of SodA2 The cell culture solution obtained in Example 2.1 was centrifuged at 4°C and 3,578 x g for 20 minutes to collect the supernatant, which was then concentrated 10-fold using ultrafiltration (UF, MWCO 10,000). 390 g of ammonium sulfate per liter of the concentrated cell culture solution was added, stirred for 20 minutes, and centrifuged to collect the supernatant. The collected supernatant was purified using a phenyl sepharose HP column. The purification process involved equilibrating the column with 50 mM potassium phosphate pH 7.0 at a 2M ammonium sulfate concentration, then passing the supernatant obtained by adding ammonium sulfate through the column, and recovering the SodA2 attached to the column using 50 mM potassium phosphate pH 7.0 containing 1.6M ammonium sulfate. The purified solutions purified through the column were collected, and the high-concentration salts formed during the purification process were removed through ultrafiltration and concentrated. The concentrated solution was filtered through a sterilizing filter and then lyophilized. The activity of SodA2 was analyzed using an SOD assay kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits superoxide radicals by 50%. The activity of the dried SodA2 enzyme was 1,000 - 1,200 U / mg.

[0096] Example 3. Production of the administered substance Shellac (EXCELACS co., LTD., Bangkok) was dissolved to an ethanol concentration of 3% and sterilized using a 0.2 µm sterile filter. The shellac dissolved in ethanol was diluted with PBS. The lyophilized SodA2 was dissolved at 20 mg / mL, and the shellac solution and the SOD solution were mixed at a 1:1 ratio and then lyophilized. Dextrin was used to mix with the lyophilized shellac-coated SodA2 at a ratio of 1:9 - 12 (shellac-coated SodA2: dextrin) to produce the substance used in the animal test. The SOD activity of the final substance was adjusted to 90 - 110 U / mg.

[0097] Example 4. Preparation of a diabetic retinopathy model 120 six-week-old male C57BL / 6 mice (source: Coretech) were purchased, maintained on a 12-hour light / dark cycle, and allowed free access to food and water. Diabetic (diabetes mellitus, hereinafter referred to as "DM") mice were induced by intraperitoneal injection of streptozotocin (hereinafter referred to as "STZ") solution (10 mM citrate buffer (pH 4.5), 180 mg / kg), and blood glucose levels were checked 3 days after injection. Only mice with blood glucose levels of 300 mg / dl or higher were selected as diabetic mice. C57BL / 6 mice were classified into 10 groups as shown in Table 6 according to the concentration of the oral drug to be administered and the presence or absence of diabetes induction, and experiments were conducted by dividing them into a 3-week experimental group (60 mice) and a 16-week experimental group (60 mice) according to the SodA2 administration period. In the case of the SodA2 administration group, SodA2 was administered daily starting from the streptozotocin induction step regardless of the presence or absence of diabetes.

[0098]

Table 6

[0099] Example 5. Preparation of a uveitis model 120 six-week-old male C57BL / 6 mice (source: Coretech) were purchased, maintained on a 12-hour light / dark cycle, and allowed free access to food and water. Of the total 120 mice, 60 were used in the endotoxin-induced uveitis model experimental group and 60 were used in the experimental autoimmune uveitis model. All animal experiments were conducted in accordance with the statement of the Association for Research in Vision and Ophthalmology for the use of animals in vision and ophthalmic research, and were approved by the Institutional Animal Care and Use Committee of Seoul National University and Seoul National University Hospital.

[0100] Example 5.1. Preparation of an endotoxin-induced uveitis model and administration of SodA2 To establish an endotoxin-induced uveitis (EIU) model, after orally administering 1, 5, 10, and 20 units of the SodA2 drug once a day (200 μl) for 7 days according to the concentration, 1 ng of LPS (Lipopolysaccharide, L2880 - 10MG), which is an outer membrane component of Gram-negative bacteria, was injected into the vitreous body to acutely induce it. Refer to Table 7 for the mouse model used in the acute uveitis experiment.

[0101]

Table 7

[0102] Example 5.2. Preparation of an experimental autoimmune uveitis model and administration of SodA2 The induction process of T cell-mediated chronic uveitis to establish an experimental autoimmune uveitis (EAU) model follows the following process. Human IRBP 1-20 (Peptron, GPTHLFQPSLVLDMAKVLLD 95%) (Peptron) 30 mg 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 in a 1:1 ratio. A total of 200 μl of the mixed reagent was subcutaneously injected into two parts, behind the neck and the hind legs of C57BL / 6 mice, and then 200 μl of pertussis toxin (sigma, P7208) of Bordetella pertussis was intraperitoneally injected to chronically induce uveitis. Thereafter, 1, 5, 10, and 20 units of the SodA2 drug were orally administered once a day (200 μl) for 21 days. Refer to Table 8 for the mouse model used in the chronic uveitis experiment.

[0103]

Table 8

[0104] Example 6. Example of efficacy confirmation in a diabetic retinopathy model 6.1. Evaluation of retinal vascular leakage After the end of the SodA2 administration period, to confirm the degree of vascular leakage in the 3-week experimental group and the 16-week experimental group, the mice were anesthetized, and then Evans blue (20 mg / ml, 150 μl, dissolved in PBS) was intravenously injected. After perfusion for 1 hour, the eyeballs were enucleated. All enucleation procedures were performed in accordance with the statement of the Association for Research in Vision and Ophthalmology regarding the use of animals in ophthalmology and vision research. For the qualitative analysis of retinal vascular leakage, the enucleated eyeballs were fixed with 4% paraformaldehyde (PFA), then flat-mounted with 1xPBS, placed on slides, and then the entire retina was photographed at a magnification of x40 using a fluorescence microscope (Eclipse 90i, Nikon, Tokyo, Japan). Also, for the quantitative analysis of retinal vascular leakage, the entire retina was photographed with red fluorescence. After the images were adjusted according to the color threshold based on the automatic ISODATA algorithm using the ImageJ (1.47v, NIH, Bethesda, MD, USA) program, the red area was measured and expressed as the degree (%) of retinal vascular leakage, and compared with the non-diabetic normal control group.

[0105] 3-week administration group As a result of microscopic qualitative analysis, no vascular leakage was observed in either the normal control group without SodA2 administration or the normal mouse groups administered with SodA2 (SodA2 concentrations of 1, 5, 10, 20 units), confirming that there was no change in the vascular permeability of the normal retina regardless of the administration of SodA2 (Figure 6a). In the diabetic control group, after 3 weeks of diabetes induction, enhanced contrast in the retinal tissue due to increased vascular permeability was confirmed (the first photo in Figure 6b). On the other hand, in the diabetic group administered with SodA2, a significant decrease in retinal vascular permeability was observed compared with the control group (the second to fifth photos in Figure 6b).

[0106] As a result of the quantitative analysis of retinal vascular leakage, similar to the qualitative evaluation results, there was no retinal vascular leakage in both the normal control group without SodA2 administration and the normal mouse group with SodA2 administration, and high retinal vascular leakage was confirmed in the diabetic control group. In the 3-week experimental group of diabetic induction with SodA2 administration, a tendency of decreased retinal vascular permeability was observed compared to the diabetic control group, and a statistically significant decrease was particularly observed in the diabetic group administered with 5 units of SodA2. The quantitative evaluation of retinal vascular leakage in the 3-week experimental group is shown in Fig. 6c.

[0107] 16-week administration group As a result of the qualitative analysis by microscope, no vascular leakage was observed in both the normal control group without SodA2 administration and the normal mouse group with SodA2 administration (Fig. 7a). In some of the normal mouse groups administered with 10 and 20 units of SodA2, a tendency of increased retinal vascular permeability was observed compared to the non-diabetic control group, but it was not a significant increase. In the diabetic control group, after 16 weeks of diabetes induction, enhanced contrast in the retinal tissue due to increased vascular permeability was observed (the first photo in Fig. 7b). On the other hand, in the diabetic group with SodA2 administration, the retinal vascular permeability decreased compared to the control group (the second to fifth photos in Fig. 7b), and particularly, the vascular permeability significantly decreased in the diabetic group administered with 5 units of SodA2.

[0108] As a result of the quantitative analysis of retinal vascular leakage, 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 with SodA2 administration (although there was a tendency of increased retinal vascular permeability in the normal mouse group administered with 20 units of SodA2, it was not significant), and high retinal vascular permeability was shown in the diabetic control group. On the other hand, in the 16-week administration group of diabetic induction with SodA2 administration, a tendency of decreased retinal vascular permeability was observed compared to the diabetic control group, and a statistically significant decrease was particularly observed in the diabetic group administered with 5 units of SodA2. The quantitative evaluation of retinal vascular leakage in the 16-week experimental group is shown in Fig. 7c.

[0109] Example 6.2. Evaluation by immunofluorescence staining of astrocytes Retinas were isolated from 4% paraformaldehyde-fixed eyeballs and incubated overnight at 4°C with anti-GFAP antibody (1:100; Invitrogen, 53-9392-82) and isolectin B4-594 (1:100; Invitrogen, I21412) added to Perm / Block solution (PBS solution supplemented with 0.3% Triton-X and 0.2% BSA). The next day, the retinas were washed with PBS and then mounted with fluoromount aqueous mounting medium (Sigma-Aldrich). To confirm the distribution of astrocyte foot processes outside pericytes surrounding the retinal vascular network (it is interpreted that the better the distribution of astrocyte foot processes, the better the suppression of vascular permeability), after staining the retinas, images were taken with a confocal microscope (Leica TCS STED, Leica Microsystems, Wetzlar, Germany), and the central part of the retinas was photographed at a magnification of x400. Subsequently, astrocytes were quantified separately using the ImageJ (1.47v, NIH, Bethesda, MD, USA) program.

[0110] 3-week administration group As a result of microscopic qualitative analysis, in both the non-administered normal control group and the normal mouse groups administered with SodA2 (SodA2 concentrations of 1, 5, 10, and 20 units), there were no changes in the distribution of astrocyte foot processes regardless of SodA2 administration (Figure 8a). In the diabetic control group, a decrease in astrocyte foot processes was confirmed 3 weeks after diabetes induction compared to the normal control group (the first photograph in Figure 8b). On the other hand, in the diabetic group administered with SodA2, a significant increase and recovery in the distribution of astrocyte foot processes were observed compared to the control group (the second to fifth photographs in Figure 8b).

[0111] As a result of the quantitative analysis by immunofluorescence staining of astrocytes, similar to the qualitative evaluation results, no changes were observed in the distribution of astrocytic end-feet in both the normal control group without SodA2 administration and the normal mouse group with SodA2 administration. In the diabetic control group, the astrocytic end-feet decreased 3 weeks after diabetes induction. Compared with the diabetic control group showing a decrease in astrocytic end-feet, the distribution of astrocytic end-feet increased in the experimental group with diabetes induced for 3 weeks and administered with SodA2. The quantitative evaluation of the immunofluorescence staining of astrocytes in the 3-week experimental group is shown in Fig. 8c.

[0112] 16-week administration group As a result of the qualitative analysis by microscope, unlike the normal control group without SodA2 administration, a decrease in the distribution of astrocytic end-feet was observed in some of the normal mice administered with SodA2, but it was not a significant change (Fig. 9a). In the diabetic control group, a significant decrease in astrocytic end-feet was confirmed 16 weeks after diabetes induction compared with the normal control group (the first photo in Fig. 9b). Compared with the control group, in the diabetic group administered with SodA2, an obvious increase and recovery in the distribution of astrocytic end-feet were observed (the second to fifth photos in Fig. 9b).

[0113] As a result of the quantitative analysis by immunofluorescence staining of astrocytes, it showed the same pattern as the qualitative analysis results. 16 weeks after diabetes induction, compared with the diabetic control group in which astrocytic end-feet decreased, the distribution of astrocytic end-feet showed a significant increase in the experimental group with diabetes induced for 16 weeks and administered with SodA2. The quantitative evaluation of the immunofluorescence staining of astrocytes in the 16-week experimental group is shown in Fig. 9c.

[0114] Example 6.3. Staining evaluation of retinal blood vessels After separating the retina from the eyeball fixed with 4% paraformaldehyde, the retina was placed in a 24-well plate and washed 4 times with PBS. Then, 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 the non-vascular tissue was carefully removed with water. After drying the slide with only blood vessels remaining, H&E staining was performed.

[0115] The development of acellular capillaries accompanied by pericyte loss around blood vessels may appear due to diabetic retinopathy, and this was confirmed by retinal vascular staining.

[0116] As a result of microscopic qualitative analysis, in the 16-week non-administered SodA2 normal control group and the SodA2-administered normal mouse group, loss of pericytes (indicated by arrows) and the development of acellular capillaries (indicated by arrowheads) were not observed (Figure 10a). In the diabetic control group, after 16 weeks of diabetes induction, the loss of pericytes increased and the development of acellular capillaries increased (the first photograph in Figure 10b). On the other hand, in the diabetic group administered with SodA2, suppression of pericyte loss and suppression of acellular capillary development were observed (the second to fifth photographs in Figure 10b).

[0117] As a result of further quantitative analysis, in both the non-administered SodA2 normal control group and the SodA2-administered normal mouse group, no changes were observed in the pericyte numerical value and the acellular capillary numerical value (the first to fifth columns from the left in Figures 10c and 10d). After 16 weeks of diabetes induction, a decrease in the pericyte numerical value (an increase in pericyte loss) was confirmed in the diabetic control group, but the level of pericytes significantly increased in the group administered with SodA2 for 16 weeks (the sixth to tenth columns from the left in Figure 10c). Furthermore, 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 development of acellular capillaries showed a tendency to decrease (Figure 10d).

[0118] Example 7. Experimental evaluation of eye diseases in a uveitis model Example 7.1. Fundus evaluation of uveitis For the endotoxin-induced uveitis (EIU) model, fundus examination was performed 24 hours after LPS injection (acute induction), and for the experimental autoimmune uveitis (EAU) model, fundus examination was performed 14, 16, 18, and 21 days after uveitis induction (chronic induction). The fundus evaluation of uveitis was scored from 0 points when there was no intraocular inflammation to 4 points according to the criteria of bleeding and eye conditions. Refer to Table 9 for the scoring criteria.

[0119]

Table 9

[0120] When evaluated according to the above evaluation criteria, in the case of the EIU model (acute induction group), administration of SodA2 showed a significant decreasing effect compared with the non - administration group (Figure 11a).

[0121] In the case of the EAU model (chronic induction group), administration of SodA2 showed a decreasing effect on the fundus evaluation scoring of uveitis compared with the non - administration group (Figure 11b). In particular, the inflammation was most improved in the 5 - unit SodA2 administration group.

[0122] Example 7.2. H&E staining and tissue evaluation After fixing the enucleated eyes removed for uveitis tissue evaluation in 4% paraformaldehyde, paraffin blocks were prepared. After preparing the paraffin blocks into sections with a thickness of 4 μm, H&E (hematoxylin & eosin) staining was performed. Refer to Table 10 and Figure 12 for information on the uveitis tissue evaluation criteria.

[0123]

Table 10

[0124] EIU model (acute induction group) No histological abnormal findings were observed in both the normal control group without SodA2 administration and the normal mouse group administered with SodA2 (SodA2 concentrations of 1, 5, 10, and 20 units) (Figure 13a). In the EIU control group induced by LPS injection, severe vitreous inflammation and retinal tissue inflammation were observed (the first photo in Figure 13b). On the other hand, in the SodA2 - administered EIU mouse group, a decrease in vitreous inflammation and a decrease in the infiltration of inflammatory cells in the retinal tissue were observed compared with the EIU control group (the second to fifth photos in Figure 13b).

[0125] As a result of histological grading, it was confirmed that in the SodA2-administered EIU mouse group, histological inflammation was improved to a lower grade compared to the EIU control group (Figure 13c).

[0126] EAU model (chronic induction group) No histological abnormalities were observed in either the normal control group without SodA2 administration or the normal mouse group administered with SodA2 (SodA2 concentrations of 1, 5, 10, and 20 units) (Figure 14a). In the case of the EIU control group in which uveitis was induced by autoimmunity, inflammatory cell infiltration in the retinal tissue and accompanying retinal changes were observed (the first photograph in Figure 14b). On the other hand, in the SodA2-administered EAU mouse group, a significant decrease in inflammatory cell infiltration in the retinal tissue and accompanying retinal changes was observed compared to the EAU control group, and particularly significant decreases were observed at 5 units and 10 units (the second to fourth photographs in Figure 14b).

[0127] As a result of histological grading, similar to the tissue evaluation, it was confirmed that in the SodA2-administered EAU mouse group, histological inflammatory findings were improved to a lower grade compared to the EAU control group, and it was particularly confirmed that the groups of EAU mice administered with 5 units and 10 units of SodA2 were improved to a lower grade (Figure 14c).

[0128] Statistical analysis Statistics were performed using one-way ANOVA and Tukey's multiple comparisons test. The statistical processing program used was GraphPad Prism 7.0.

Claims

1. A pharmaceutical composition for the treatment or prevention of diabetic retinopathy or uveitis, comprising superoxide dismutase (SOD) as an active ingredient.

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

3. The pharmaceutical composition according to claim 1, wherein the SOD brings about one or more effects selected from reduction of vascular permeability in diabetic retinopathy, recovery of astrocyte foot processes, suppression of pericyte loss, and suppression of acellular capillary development.

4. The SOD according to claim 1, which improves or alleviates one or more fundus evaluation indicators selected from focal lesions, linear lesions, choroidoretinal lesions, confluent lesions, vasculitis, vitritis, vitreous hemorrhage, optic disc edema, and retinal detachment in uveitis, or improves or alleviates one or more tissue evaluation indicators selected from inflammatory cell infiltration, retinal folds, intraretinal and subretinal hemorrhages, intraretinal and subretinal exudates, and retinal damage in uveitis, or The pharmaceutical composition according to claim 1, which brings about both improvement or alleviation of the fundus evaluation indicators and improvement or alleviation of the tissue evaluation indicators.

5. The pharmaceutical composition according to claim 1, wherein the uveitis is caused by an infectious or non-infectious cause.

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

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

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

9. The pharmaceutical composition according to claim 1, wherein the SOD is contained in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract or a dried form thereof.

10. The pharmaceutical composition according to claim 1, wherein the SOD is Mn-SOD.

11. The pharmaceutical composition according to claim 1, wherein the SOD is deamidated Mn-SOD.

12. The pharmaceutical composition according to claim 1, wherein the SOD is derived from a Bacillus species strain.

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

14. The pharmaceutical composition according to claim 1, wherein the 73rd and 136th amino acid residues are substituted with Asp based on SEQ ID NO:

2.

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

4.

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

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

18. A veterinary composition for the treatment or prevention of diabetic retinopathy or uveitis, comprising superoxide dismutase (SOD) as an active ingredient.

19. A food composition for improving or preventing diabetic retinopathy or uveitis, comprising superoxide dismutase (SOD) as an active ingredient.

20. A feed composition for improving or preventing diabetic retinopathy or uveitis, comprising superoxide dismutase (SOD) as an active ingredient.

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

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

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

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