Superoxide dismutase and use thereof in prevention or treatment of dry macular degeneration

Abstract

The present invention relates to superoxide dismutase and its use in the prevention or treatment of dry age-related macular degeneration. More specifically, the present invention relates to a pharmaceutical composition or a treatment method for preventing or treating dry macular degeneration. According to the composition and the method of the present invention, the composition can be effectively used for preventing or treating dry macular degeneration by reducing or preventing glass membrane wart formation and / or glass membrane wart lesion.

Description

Technical Field

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

[0002] Macular degeneration, also known as age-related macular degeneration (AMD), is a chronic, progressive degenerative disease of the macula that leads to loss of central vision. In adults over 50 years old, AMD is the leading cause of vision loss and irreversible central vision loss. 25 million people worldwide suffer from AMD, and the number of patients with AMD continues to increase rapidly as the elderly population rapidly grows. In addition, the early onset and prevalence of AMD in modern people are increasing due to excessive use of electronic devices such as smartphones and laptops.

[0003] The main causes of macular degeneration are age-related atrophy and decreased function of the retinal pigment epithelium (RPE). The retina plays a key role in visual function, and the retinal pigment epithelium plays an important role in maintaining retinal homeostasis and physiological functions. In addition, age-related abnormal changes in Bruch's membrane and degeneration of the choroid capillaries are also considered to be one of the causes of macular degeneration. Bruch's membrane acts as the basement membrane of the RPE, while the choroid capillaries are located at the outermost part of the sensory neuroretina and provide nutrients and oxygen to the photoreceptor cells that undergo light conversion. It is known that the causes of macular degeneration include excessive reactive oxygen species, mitochondrial dysfunction, inflammation, abnormal lipid metabolism, genetic factors, and environmental factors (such as long-term exposure to smoking and ultraviolet rays), but the exact mechanism is not yet fully understood.

[0004] Macular degeneration can be roughly divided into two types: wet and dry. The dry type accounts for 85% to 90% of diagnosed cases of macular degeneration, and the wet type also begins with the dry type. The main characteristics of dry macular degeneration are yellow deposits of medium size or larger (called drusen) present under the retinal pigment epithelium, pigmentary abnormalities of the retinal pigment epithelium, and deposits called reticular pseudodrusen present under the retina. The formation of drusen is a typical feature of dry macular degeneration, and most medium-sized drusen and large-sized drusen are known to be risk factors for progression to advanced macular degeneration. In addition, the size and density of drusen are correlated with the loss of choriocapillaris.

[0005] Dry macular degeneration usually progresses slowly and may progress to geographic atrophy (GA) in the late stage. Geographic atrophy is a confluent or scattered irreversible degeneration of the retinal pigment epithelium that damages photoreceptors and leads to vision loss when it continues to progress. Geographic atrophy is the main cause of visual impairment with aging, but there are currently no approved treatments or therapeutic drugs that can delay or inhibit the progression of dry macular degeneration.

[0006] As another type of advanced macular degeneration, wet macular degeneration is a condition in which immature blood vessels are generated from the choroid to the retinal side, causing blood or body fluid to leak out. At this time, photoreceptors and nerve cells connected to them are damaged, eventually leading to loss of central vision. Wet macular degeneration progresses faster than dry macular degeneration, and if not treated in time, it may cause blindness within a few months. However, as an anti-angiogenic therapy for preventing neovascularization, a treatment method has been developed that inhibits abnormally generated VEGF by injecting an anti-VEGF agent into the eyeball to terminate the angiogenesis process.

[0007] Therefore, there is a need for a substance and method that can effectively prevent or treat dry macular degeneration. Summary of the invention

[0008] Technical issues

[0009] The purpose of the present invention is to solve the above problems existing in the prior art.

[0010] Another object of the present invention is to provide superoxide dismutase for preventing, improving or treating dry type macular degeneration.

[0011] Yet another object of the present invention is to provide a pharmaceutical composition or a treatment method for preventing or treating dry macular degeneration.

[0012] Still another object of the present invention is to provide a food or feed composition for preventing or improving dry macular degeneration.

[0013] Yet another object of the present invention is to provide a pharmaceutical composition or a therapeutic method for preventing or treating dry macular degeneration by reducing or eliminating drusen formation or drusenoid lesions.

[0014] Still another object of the present invention is to provide a food or feed composition for preventing or improving dry macular degeneration by reducing or eliminating drusen formation or drusen lesions.

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

[0016] Technical Solution

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

[0018] According to one aspect of the present invention, an improved superoxide dismutase (SOD) is provided, which exhibits the effect of preventing, improving or treating dry macular degeneration.

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

[0020] According to yet another aspect of the present invention, there is provided a superoxide dismutase derived from Bacillus amyloliques faciens which ensures oral administration efficacy and safety for preventing, improving or treating dry macular degeneration.

[0021] According to yet another aspect of the present invention, there is provided a superoxide dismutase derived from Bacillus or Bacillus amyloliquefaciens, which is secreted outside the cell to facilitate production, for use in preventing or treating dry macular degeneration.

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

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

[0024] According to yet another aspect of the present invention, there is provided a method for preventing, improving or treating dry macular degeneration, comprising the step of administering the superoxide dismutase to a subject.

[0025] According to yet another aspect of the present invention, there is provided use of the superoxide dismutase in preventing or treating dry macular degeneration.

[0026] According to yet another aspect of the present invention, there is provided use of the superoxide dismutase in preparing a medicament for preventing or treating dry macular degeneration.

[0027] According to yet another aspect of the present invention, there is provided a superoxide dismutase, a polynucleotide sequence encoding the same, an expression vector comprising the polynucleotide sequence, or a cell line transformed with the expression vector, wherein the superoxide dismutase is derived from a Bacillus strain and the amino acid residues at positions 73 and 136 are substituted by Asp based on SEQ ID NO: 2.

[0028] According to yet another aspect of the present invention, a Bacillus strain is provided, comprising the polynucleotide sequence and having a partial gene deletion.

[0029] Beneficial Effects

[0030] The superoxide dismutase, composition and method according to the present invention can be effectively used to prevent, improve or treat dry macular degeneration by reducing or preventing drusen formation and / or drusen lesions. In particular, it has been confirmed that the superoxide dismutase according to the present invention is effective in preventing or treating dry macular degeneration when orally administered.

[0031] The superoxide dismutase according to the present invention is derived from Bacillus or Bacillus amyloliquefaciens, which are generally regarded as safe (GRAS) bacteria, and thus not only ensures oral administration efficacy and safety, but also has a production advantage in that it can be directly recovered from the supernatant during culture.

[0032] By utilizing the polynucleotide sequence encoding the superoxide dismutase according to the present invention, the expression vector comprising the sequence, or the cell line transformed with the expression vector, and the Bacillus strain, an effective ingredient for reducing or preventing drusen formation and / or drusen lesions can be efficiently produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 A schematic diagram of the double crossover recombination performed in Example 1.1 is shown.

[0034] Figure 2 The results of confirming the defective gene using PCR in the expression host GFBS220 and the production strain BSBA310 are shown. W indicates the wild-type Bacillus subtilis KCTC 3135, 220 indicates the expression host GFBS220, and 310 indicates the SodA2 production strain BSBA310.

[0035] Figure 3Shown 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).

[0036] Figure 4 The expression vector for sodA2 gene overexpression is shown. Among them, rrnB T1T2 represents a transcription terminator; rep(pBR322) represents a replicon from pBR322 that works in E. coli; rep(pUB110) represents a replicon from pUB110 that works in Bacillus subtilis; Kan R represents a kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); and BJ27 promoter represents a strong promoter of Bacillus subtilis (B. subtilis).

[0037] Figure 5 The overall scheme for preparing the production strain BSBA310 is shown.

[0038] Figure 6 Benchmarks for determining clinical outcomes used in embodiments of the present invention are shown.

[0039] Figure 7 The change in the total area of ​​drusen lesions from the baseline according to an embodiment of the present invention is shown. *p<0.05 compared to the area change of the vehicle group.

[0040] Figure 8 The change in size of the largest drusen lesion relative to a baseline is shown according to an embodiment of the present invention.

[0041] Figure 9a and Figure 9b The results of blood biochemistry performed in the examples of the present invention are shown.

[0042] Figures 10a to 10c The results of hematology of blood performed in the examples of the present invention are shown. DETAILED DESCRIPTION

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

[0044] definition

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

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

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

[0048] The meaning of the term "prevention" is well known in the art. When used together with medical conditions such as dry macular degeneration, it means that by administering the pharmaceutical composition or SOD of the present invention, the frequency of medical symptoms (such as blurred vision or vision loss) is reduced or the onset and symptoms are delayed compared to subjects not administered with it.

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

[0050] Dry macular degeneration

[0051] The present invention is based at least in part on the discovery that administration of superoxide dismutase (SOD), particularly oral administration, is effective in preventing or treating dry macular degeneration. Thus, according to one aspect of the present invention, there is provided a use of SOD in preventing or treating dry macular degeneration.

[0052] The term "dry macular degeneration" can be used interchangeably with "dry age-related macular degeneration." It includes early, intermediate and advanced dry macular degeneration.

[0053] The feature of described dry macular degeneration is drusen or drusen lesions.Term " drusen " refers to the waste product formed by the aging thing produced in retina between RPE cell and Bruch's membrane or Bruch's membrane (B) internal deposition along with the growth of age.The formation of drusen is the typical feature (hallmark) of dry type AMD, and the drusen of known multiple medium-sized and large-sized is a risk factor for progressing to late macular degeneration.In addition, there is a correlation between the size and density of drusen and the loss of choroidal capillaries.In addition, term " drusen lesions " refers to the lesions caused by drusen.

[0054] The SOD of the present invention can effectively prevent, improve or treat dry macular degeneration by reducing drusen or drusen-like lesions or preventing their formation.

[0055] The inventors conducted animal experiments using Rhesus monkeys instead of mice as an animal model, which produces drusen with a location and composition similar to that of humans. The reason is that, unlike non-transgenic mice, normal mice cannot be an effective model for dry AMD because there is no evidence that the light-induced oxidative stress model affects the function of the retina (P. Sicard et al., "Protective effect of dietary superoxide dismutase on light-induced retinal oxidative stress in young accelerated senescence mice (SAM)", Invest Ophthalmol Vis Sci 2006; 47: E-Abstract 2089), and considering that drusen, which are characteristic of dry AMD, are not formed (Hema L. Ramkumar et al., "Retinal ultrastructure of a dry age-related macular degeneration (AMD) mouse model", Progress In Retinal And Eye Research 2010; 29(3), 169-190). In addition, as recorded in the literature (Erica L. Fletcher et al., "Using Animal Models to Study Age-Related Macular Degeneration", Optom Vis Sci. 2014; 91(8), 878-886), to date, great apes and primates are the only animals that have drusen with a composition and location similar to that of humans; although some mice develop white lesions as they age, their location and composition are different from those of humans, and therefore they are not an effective model for understanding drusen formation.

[0056] Superoxide dismutase (SOD)

[0057] 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 dry type macular degeneration.

[0058] Superoxide dismutase (SOD) is an enzyme that can alternately catalyze the dismutation of superoxide (O2·-) free radicals into general molecular oxygen (O2) and hydrogen peroxide (H2O2). SOD plays an important role in reducing oxidative stress by removing reactive oxygen species. SOD is widely distributed in prokaryotes and eukaryotic cells and is divided into four categories according to different types of metal centers (copper / zinc, nickel, manganese and iron). Manganese-containing SOD [Mn-SOD; manganese-containing superoxide dismutase] is widely present in a variety of bacteria, chloroplasts, mitochondria and cytoplasm of eukaryotic cells. The term "SOD" can be used interchangeably with polypeptides with superoxide dismutase activity.

[0059] In one embodiment, the SOD may be a superoxide dismutase (Mn-SOD) combined with manganese. Specifically, the SOD may be a deamidated Mn-SOD. More specifically, the SOD may be a SOD based on SEQ ID NO:2 with amino acid residues at positions 73 and 136 substituted by Asp. More specifically, the SOD may comprise or consist of the amino acid sequence shown in SEQ ID NO:4.

[0060] 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 compared sequences are analyzed using an algorithm commonly used in the art, the amino acid sequence shows 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity.

[0061] 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.

[0062] 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.

[0063] In one embodiment, SOD may be derived from a natural or recombinant microorganism. For example, SOD is derived from bacteria. Preferably, SOD is 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 Bacillus amyloliquefaciens GF423 strain (KCTC 13222BP). GF423 strain (KCTC13222BP) was deposited at the Korea Institute of Biotechnology on March 6, 2017. In addition, the SOD may be derived from a strain comprising Figure 4 In addition, the SOD can be derived from a recombinant strain containing the expression vector shown in Figure 5The recombinant strain prepared by the method shown in the method. The recombinant strain may be a Bacillus strain, the Bacillus strain comprises a nucleotide sequence encoding a polypeptide, the polypeptide comprises an amino acid sequence shown in SEQ ID NO: 4, and the AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf genes are deleted in the Bacillus strain. The detailed process of preparing the strain is shown in Example 1.

[0064] 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.

[0065] In one embodiment, SOD can be isolated or purified from a variety of supply sources including natural or recombinant hosts. For example, SOD with the activity of preventing or treating dry macular degeneration 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 medium (pH 6.0 to 7.0) is used to grow the strain at about 25°C to about 42°C for about 1 day to about 4 days. Other suitable culture media for culturing Bacillus amyloliquefaciens GF423 strain include LB (Luria-Bertani) medium, ISP (International Streptomyces Project) medium, NA (nutrient agar) medium, BHI (brain heart infusion agar) medium, SDA (Sabouraud dextrose agar) medium, PDA (potato dextrose agar) medium, NB (nutrient broth) medium. Preferably, LB medium, ISP medium, BHI medium, SDA medium or NB medium can be used. Additionally, SOD can be obtained from other natural or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda-enzymes.org on the World Wide Web).

[0066] 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 sentence "substantially free of cellular material" includes a protein preparation in which the protein is isolated or such protein is separated from the cellular components of a recombinantly produced cell. In one embodiment, the sentence "substantially free of cellular material" refers to a protein preparation in which the content of undesirable protein by dry weight is less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5%.

[0067] 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.

[0068] In another embodiment, the SOD may be contained in a strain lysate, a strain culture solution, a strain culture concentrate, a strain culture extract, or a dry form thereof. In this case, the "strain lysate" may be obtained by culturing the strain and pulverizing it mechanically or chemically. The "strain culture solution" may refer to the culture solution itself or its supernatant obtained by culturing the strain. The "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 the strain culture. The "strain culture extract" refers to a substance extracted from the culture solution or its concentrate, and may include an extract, a dilution or concentrate of the extract, a dry product obtained by drying the extract, or a crude purified product or purified product thereof, and a fraction obtained by dividing it. The dry form may include a freeze-dried form.

[0069] The SOD of the present invention can reduce drusen or drusenic lesions or prevent their formation, and / or, effectively prevent, improve or treat dry macular degeneration. In particular, the SOD of the present invention is useful for preventing, improving or treating dry macular degeneration characterized by drusen or drusenic lesions.

[0070] Pharmaceutical composition

[0071] According to one aspect of the present invention, there is provided a pharmaceutical composition for treating or preventing dry type macular degeneration, comprising superoxide dismutase (SOD) as an effective ingredient.

[0072] The pharmaceutical composition can reduce drusen or drusenic lesions or prevent their formation.

[0073] In one embodiment, the SOD according to the present invention can be combined with a pharmaceutically acceptable carrier, excipient and / or diluent to form a pharmaceutical composition for preventing or treating dry macular degeneration. When applied to animals other than humans, the pharmaceutical composition of the present invention can be used interchangeably with the term "veterinary composition".

[0074] Pharmaceutical composition or veterinary drug composition of the present invention may further comprise pharmaceutically acceptable carrier, excipient and / or diluent. The pharmaceutically acceptable carrier, excipient and / or diluent may be a material 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] In addition, the pharmaceutical composition 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" refers to an amount that is sufficient to treat a disease at a reasonable benefit / risk ratio applicable to pharmaceutical or veterinary treatment, and the effective dosage level can be determined based on factors including the patient's or animal's weight, sex, age, health status, severity, activity of the drug, sensitivity to the drug, administration time, route of administration and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0081] 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.

[0082] Prevention or treatment methods

[0083] According to yet another aspect of the present invention, there is provided a method for treating or preventing dry macular degeneration, comprising the following steps: administering the SOD or pharmaceutical composition according to the present invention to a subject suffering from or at risk of dry macular degeneration.

[0084] A method for treating or preventing dry macular degeneration is also provided, comprising the following steps: administering superoxide dismutase to a subject. The contents of the SOD, dry macular degeneration, treatment or prevention are the same as those described in the above-mentioned pharmaceutical composition.

[0085] The term "administration" includes any number of administrations that achieve the desired effect to achieve the therapeutic purpose. The administration may include oral or parenteral administration. For oral or parenteral administration, the description in the above-mentioned pharmaceutical composition may be referred to. The route of administration may be oral, parenteral, inhaled, applied externally or topically (e.g., intralesional administration). For example, parenteral administration may include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intraarterial, and intraocular administration. In the case of oral administration, as described above, SOD may be administered in the form of being coated with a coating agent such as shellac.

[0086] 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.

[0087] In one embodiment, the method may include: administering one or more additional preparations for treating or preventing dry macular degeneration. The one or more additional preparations and the SOD or pharmaceutical composition may be administered simultaneously, sequentially or in reverse order. In addition, each component may be administered to the subject via the same or different routes.

[0088] Food composition

[0089] According to yet another aspect of the present invention, there is provided a food composition for improving or preventing dry macular degeneration, comprising the SOD according to the present invention as an effective ingredient. Such food composition includes a medical or nutraceutical food composition.

[0090] 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.

[0091] 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.

[0092] The food can be prepared by conventional methods known in the art. For example, for the purpose of preventing or improving mucositis, 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 the dosage form combined with appropriate ingredients.

[0093] 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.

[0094] 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 10 to 1000 mg / kg. The above dosage is an example of an average situation, and the daily dose 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.

[0095] Feed composition

[0096] According to yet another aspect of the present invention, a feed composition for preventing or improving dry macular degeneration is provided, which contains the SOD according to the present invention as an effective ingredient. In the present invention, the food composition may include a feed composition. These feed compositions can be prepared in any dosage form commonly used in the field. For example, the feed composition of the present invention may further include: auxiliary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other microbial preparations in the form of live bacteria; cereals, such as crushed or broken wheat, oats, barley, corn, and rice; plant protein feeds, such as materials with rapeseed, soybeans, and sunflower seeds as main ingredients; animal protein feeds, such as blood meal, meat meal, bone meal, and fish meal; sugars and dairy products, such as dry ingredients composed of various milk powders and whey powders; lipids, such as main ingredients such as animal fats and vegetable fats that are liquefied at will by heating; additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventive agents.

[0097] The feed composition of the present invention may be in the form of a powder or liquid, and may contain feed additive excipients, such as but not limited to calcium carbonate, wheat bran, zeolite, corn flour or rice bran.

[0098] Polynucleotides, expression vectors and host cells

[0099] According to yet another aspect of the present invention, a polynucleotide encoding the superoxide dismutase polypeptide is provided. Specifically, a nucleic acid encoding the polypeptide is provided, wherein the polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 4.

[0100] According to yet another aspect of the present invention, an expression vector comprising the nucleic acid is provided. Specifically, the vector may be Figure 4 The expression vector shown.

[0101] According to yet another aspect of the present invention, a host cell comprising the nucleic acid or the expression vector is provided. Specifically, the host cell is a bacterial cell. More specifically, the host cell is a Bacillus strain.

[0102] According to yet another aspect of the present invention, a Bacillus strain is provided, comprising a polynucleotide sequence encoding a polypeptide, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 4, and the AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf genes are deleted in the Bacillus strain. The detailed process of preparing the strain is shown in Example 1 below.

[0103] Example

[0104] 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.

[0105] [Example 1] Preparation of recombinant strain expressing superoxide dismutase (SodA2)

[0106] [Example 1.1] Preparation of expression host GFBS220

[0107] 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.

[0108]

Table 1

[0109] Gene name Protein name Function AprE Serine alkaline protease (subtilisin E) Extracellular proteases NvE Extracellular neutral metalloproteinases Extracellular proteases Bpr Bacillopeptidase F Extracellular proteases Epr Extracellular serine proteases Extracellular proteases Nb Neutral extracellular proteinase B Extracellular proteases VpR Extracellular serine proteases Extracellular proteases Mpr Extracellular metalloproteinases Extracellular proteases IspA Intracellular serine proteases Intracellular proteases SxR Surfactin synthase Synthesis of Surfactin spoIIAC RNA polymerase sporulation-specific sigma factor (Sigma-F) Spore formation E Putative glycosyltransferase Extracellular polysaccharide XpF RNA polymerase sigma factor Transcription of PBSX prophage genes

[0110] 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.

[0111]

Table 2

[0112]

[0113]

Table 3

[0114]

[0115]

[0116] After the vector prepared in this way is introduced into the Bacillus subtilis cells, the transformant is screened at 37°C (non-replication temperature) using a culture medium containing chloramphenicol (single cross homologous recombination). The screened transformant is cultivated at 30°C (permitting replication temperature) using LB culture medium without antibiotics (second crossover), and then cultivated at 39°C using LB agar culture medium without antibiotics. From the generated bacterium colonies, strains sensitive to chloramphenicol are screened (plasmid solidification). PCR is performed on the screened bacterium colonies to obtain double cross-over recombinants, and a second single separation is performed to screen out the final recombinant, named as GFBS220.

[0117] After amplification using the primers shown in Table 4 and PCR, the defective gene site of GFBS220 was confirmed by agarose gel electrophoresis. The results of confirming the defective gene by PCR in the expression host GFBS220 are as follows: Figure 2 shown.

[0118]

Table 4

[0119]

[0120]

[0121] [Example 1.2] Preparation of expression vector pBE1-sodA2

[0122] 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).

[0123] 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.

[0124]

Table 5

[0125]

[0126] 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.

[0127] [Example 1.3] Preparation of production strain BSBA310

[0128] The expression vector pBE1-sodA2 was transformed into Bacillus subtilis GFBS220. Strains were selected from the transformants and pure clones were established by two single colony isolation procedures in LB2 medium. The selected strain was named BSBA310 and stored at -80°C using the glycerol stock method. The results of confirming the defective gene using PCR in the production strain BSBA310 are as follows: Figure 2 In addition, Figure 5 Summary The overall process for preparing the production strain BSBA310 is shown.

[0129] [Example 2] Superoxide dismutase (SodA2) was isolated and purified from the production strain BSBA310.

[0130] [Example 2.1] Cultivation of production strain BSBA310

[0131] 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.

[0132] [Example 2.2] Isolation and purification of SodA2

[0133] The cell culture fluid obtained in Example 2.1 was centrifuged at 4°C and 3,578×g for 20 minutes and the supernatant was collected, and then concentrated 10 times by ultrafiltration (Ultrafiltration, hereinafter referred to as UF, MWCO 10,000). 390g of ammonium sulfate was added to each 1L of concentrated cell culture, stirred for 20 minutes, centrifuged and the supernatant was collected. The obtained supernatant was purified using a phenyl agarose HP column. During the purification process, 50mM potassium phosphate pH7.0 with a concentration of 2M ammonium sulfate was used to balance the column, and then the supernatant obtained by adding ammonium sulfate was passed through the column, and 50mM potassium phosphate pH7.0 containing 1.6M ammonium sulfate was used to recover SodA2 attached to the column. The purified solution purified by the column was collected, and the high concentration of salts formed during the purification process was removed by ultrafiltration and concentrated. After filtering the concentrated solution with a sterilizing filter, it was freeze-dried. The activity of SODA2 was analyzed using a SOD assay kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits 50% of superoxide free radicals. The activity of dried SodA2 enzyme is 1000 to 1200 U / mg.

[0134] [Example 3] Preparation of SodA2 for animal testing

[0135] Preparation of administration substances

[0136] Shellac (EXCELACS co., LTD., Bangkok) was dissolved in 100% ethanol to a concentration of 3%, and prepared for filtration using a 0.2 um sterile filter. The shellac solution dissolved in ethanol was diluted to 1 / 20 with sterilized 1xPBS to prepare a shellac solution.

[0137] Freeze-dried purified SodA2 was dissolved at 20 mg / ml and prepared sterile using a 0.2 um filter.

[0138] Mix the prepared shellac solution and dissolved SOD in a ratio of 1:1 and stir. Then, continue stirring for 10 minutes.

[0139] The thoroughly stirred material was freeze-dried, thereby finally preparing freeze-dried shellac-coated SodA2.

[0140] Dextrin was added to freeze-dried shellac-coated SodA2 at a ratio of 1:9-12 (shellac-coated SodA2:dextrin), and the final activity was determined to be 90-110 U / mg.

[0141] [Example 4] Animal test

[0142] In this example, SodA2 prepared in Example 3 was orally administered to rhesus monkeys suffering from idiopathic moderate dry age-related macular degeneration (AMD) to evaluate its safety and efficacy.

[0143] The abbreviations used in this example are defined as follows:

[0144]

[0145]

[0146] [Example 4.1] Preparation of test substances and control substances The vehicle administered to SodA2 and the control group had the characteristics and conditions shown below.

[0147]

Table 6

[0148]

[0149] [Example 4.2] Preparation of experimental animals

[0150] The rhesus monkeys used in this example were obtained and used after screening according to the following criteria.

[0151]

Table 7

[0152]

[0153]

[0154] Inclusion criteria

[0155] Monkeys with any of the following characteristics were diagnosed with moderate dry AMD in at least one eye, where the diagnosis was made using the AREDS classification for drusen size and area in fundus photographs (see Table 8 for AREDS classification for grading digital fundus photographs).

[0156] - Presence of soft drusen and progression of drusen size or area within 2 years;

[0157] - Multiple drusen including at least one large drusen (Grade 4, drusen greater than 116 μm in diameter);

[0158] - Total lesion area of ​​drusenic lesions of at least grade 4 (grade 4, drusen area of ​​1-2, 0.089 mm 2 above).

[0159]

Table 8

[0160]

[0161] Exclusion criteria

[0162] - Evidence or history of neovascular AMD or diabetic retinopathy in either eye;

[0163] - Severe heart failure (New York Heart Association class III and IV) or left ventricular ejection fraction less than 35% on all examinations;

[0164] - Severe liver disease, with ALT levels elevated to more than three times the upper limit of normal;

[0165] - Severe renal disease with any of the following symptoms: (1) Dialysis or eGFR less than 20 ml / min / 1.73 m 2 ; (2) Urine protein / urine creatinine is 1 g / g or more; (3) Serum creatinine or albumin / urine creatinine is 600 mg / g or more;

[0166] - Confounding eye diseases such as aphakia; optic atrophy (pallor of the optic nerve head); all eye diseases that the investigators believe may be confounding factors in this study.

[0167] The prepared monkeys were housed under the following conditions. All procedures in this experiment complied with animal welfare regulations and were performed in a laboratory approved for experimental animal welfare.

[0168] Environmental conditions

[0169] The monkeys were housed in a facility with a 12-hour light / dark cycle and controlled temperature (18°C to 26°C) and humidity (40% to 70%).

[0170] Food intake

[0171] Each monkey was fed 350 g of Primed diet per day. Food intake was quantitatively assessed daily.

[0172] water

[0173] All monkeys had free access to purified tap water.

[0174] [Example 4.3] Experimental design

[0175] [Example 4.3.1] Group allocation

[0176] A total of 8 rhesus monkeys diagnosed with idiopathic moderate dry AMD within the past 2 years were enrolled for the pre-study phase (4 weeks) of baseline imaging examination. Among the 8 monkeys, 2 monkeys had retinal drusen area that met grade 6 (area greater than or equal to 1 / 2DA and less than 1DA), and 6 monkeys met grades 4 (area greater than or equal to I-2 and less than O-2) to 5 (area greater than or equal to O-2 and less than 1 / 2DA); the monkeys were randomly divided into two groups, each including 4 monkeys.

[0177] The baseline characteristics of the monkeys are shown in Table 9, and the animal groups and administration are shown in Table 10.

[0178]

Table 9

[0179]

[0180]

[0181]

Table 10

[0182]

[0183] [Example 4.3.2] Administration

[0184] SodA2 and vehicle were filled into dried kiwi fruit slices. Vehicle and SodA2 were orally administered to each monkey in the vehicle group and SodA2 group once a day for 4 months. SodA2 and vehicle were prepared before daily oral administration.

[0185] [Example 4.3.3] Test process

[0186] Observation of retinal drusen

[0187] On the 30th day (D30), 60th day (D60), 90th day (D90) and 120th day (D120) after administration, the size and area of ​​retinal drusen of the test animals (including the change of retinal drusen size and area relative to the baseline) were measured by fundus photography (FP). Each baseline has been measured in the previous study stage.

[0188] Prior to image acquisition, monkeys were anesthetized with an intramuscular injection of a 1:1 mixture of ketamine and xylazine (8 mg / kg ketamine). Two drops of tropicamide and phenylephrine eye drops were placed in each eye to dilate the pupil. After anesthesia, the monkeys were placed in a dark room until the pupil diameter was greater than 6 mm. A self-retaining eyelid speculum was placed on the eye.

[0189] FP images were acquired using a retinal camera (KOWA VX-20) according to the following procedure. The monkey's forehead was kept against the forehead support. The operating distance was adjusted and the focus was performed before taking the image. Confirmation was made whether the central image of the macula was obtained. During the examination, the monkey was managed according to the relevant standard operating procedures (SOP). The size and area of ​​drusen were measured by two experienced image assessors using Image J (Version 1.52, Wayne Rasband National Institutes of Health, USA). The Age-Related Eye Disease Study 2 (AREDS2) system was used to grade drusen lesions and AMD (Table 11). The rating involved a modified grid template derived from the ETDRS (Early Treatment Diabetic Retinopathy Study), using a series of scaled circles to assess the maximum drusen size and pigment abnormalities and the total area associated with drusen. All measurements included the number of drusen lesions, the total area of ​​lesions, and the size of the largest lesion.

[0190]

Table 11

[0191]

[0192] Observation in the cage

[0193] Each monkey was observed and physically examined once daily for changes in behavior, autonomic activity, psychological performance, body color, excreta, and eye appearance.

[0194] Food intake

[0195] Food intake was assessed qualitatively and once daily during the study period.

[0196] weight

[0197] Monkeys were weighed using a scale after a 14-hour overnight fast during the pre-study period (baseline) and on Days 7, 14, 21, 30, 37, 45, 52, 60, 67, 75, 82, 90, 97, 105, 112, and 120 after administration.

[0198] Biochemical tests of blood

[0199] Equipment: Roche Cobas6000 Analyzer Series C501

[0200] Sample type: plasma

[0201] Frequency: Baseline and 14, 30, 45, 60, 75, 90, 105, 120 days after administration

[0202] MDA and C3 were measured only at three time points (day 90, day 105 and day 120). The parameters of blood biochemistry examination are shown in Table 12.

[0203]

Table 12

[0204]

[0205]

[0206] Hematological tests of the blood

[0207] Equipment: Siemens ADVIA 212i / SYSMEX XN1000

[0208] Sample type: whole blood

[0209] Frequency: Baseline and 14, 30, 45, 60, 75, 90, 105, 120 days after administration

[0210] The parameters of blood hematological examination are shown in Table 13.

[0211]

Table 13

[0212]

[0213]

[0214] Data analysis

[0215] Data analysis included total area of ​​drusenic lesions, size of largest drusenic lesion, number of drusenic lesions, body weight, blood biochemistry, and hematology.

[0216] Microsoft Office Excel 2013 and SPSS Statistics 23.0 were used for data processing and statistical analysis. The total area of ​​drusenic lesions, the size of the largest drusenic lesion, and the amount of drusenic lesions were analyzed by paired sample t-test. Differences were considered statistically significant at p-value < 0.05.

[0217] Criteria for clinical outcome measures are found in Figure 6 .

[0218] [Example 4.4] Results

[0219] [Example 4.4.1] Total area of ​​drusen lesions

[0220] The total area of ​​drusenic lesions is shown in Table 14, and the changes in area are shown in Tables 15 and Figure 7 shown.

[0221] In the 7 eyes of the vehicle group, the total area of ​​drusen lesions increased significantly at 30, 60, 90, and 120 days after application compared with the baseline (p<0.05). This indicates that dry AMD in the vehicle group occurred significantly after 4 months of vehicle application.

[0222] In the 7 eyes of the 2.5 mg / kg SodA2 group, the total area of ​​drusen lesions did not change significantly at 30, 60, 90, and 120 days after administration compared with the baseline, and the change in the total area of ​​drusen lesions in the 2.5 mg / kg SodA2 group was significantly lower than that in the vehicle group (p<0.05). This suggests that SodA2 slows the progression of dry AMD.

[0223]

Table 14

[0224]

[0225]

Table 15

[0226]

[0227]

[0228] [Example 4.4.2] Size of the largest drusen lesion

[0229] The size of the largest drusen lesion is shown in Table 16, and the area change is shown in Tables 17 and Figure 8 shown.

[0230] In the seven eyes in the vehicle group, the size of the largest drusen lesion increased at days 90 and 120, but was not statistically significant compared with baseline. This suggests that the progression of dry AMD in the vehicle group was significantly advanced after 4 months of vehicle administration.

[0231] In the 7 eyes of the 2.5 mg / kg SodA2 group, the size of the largest drusen lesion did not change significantly after administration compared with baseline. This suggests that 2.5 mg / kg SodA2 slowed the progression of dry AMD.

[0232]

Table 16

[0233]

[0234]

Table 17

[0235]

[0236] [Example 4.4.3] Observation in cage

[0237] No obvious abnormalities were found in the behavior, autonomic nervous system activity, skin, hair, excrement and eyes of the monkeys in each group before and after administration. No toxicity-related issues occurred in this study.

[0238] [Example 4.4.4] Body weight

[0239] The body weight of the monkeys in each group did not change significantly during the administration period. The relevant results are shown in Table 18, and the body weight is in kg (mean ± SD).

[0240]

Table 18

[0241]

[0242]

[0243] [Example 4.4.5] Blood biochemistry

[0244] During the administration period, no significant changes were observed in the blood biochemistry of the monkeys in each group. The relevant results are shown in Figure 9 (expressed as mean ± SD).

[0245] [Example 4.4.6] Blood Hematology

[0246] During the administration period, no significant changes were observed in the hematology of the blood of the monkeys in each group. The relevant results are shown in Figure 10 (expressed as mean ± SD).

[0247] [Example 4.4.7] Conclusion

[0248] In the 7 eyes of the vehicle group, the total area of ​​drusenic lesions increased significantly at 30, 60, 90, and 120 days after administration compared with baseline (p<0.05); the size of the largest drusenic lesion increased at 90 and 120 days, but was not statistically significant compared with baseline; this indicates that drusenic lesions occurred in monkeys in the vehicle group after 4 months of vehicle administration.

[0249] In the 7 eyes of the 2.5 mg / kg SodA2 group, the total area of ​​drusen lesions did not change significantly after administration compared with the baseline; the change in the total area of ​​drusen lesions was significantly lower than that in the vehicle group (p<0.05); the size of the largest drusen lesion did not change significantly after administration compared with the baseline, but showed a trend of decrease. This suggests that 2.5 mg / kg SodA2 slowed the progression of drusen lesions.

[0250] At the same time, no significant changes were found in the body weight, blood biochemistry and hematology of the monkeys in each group during the 4-month administration period. No toxicity-related issues occurred in this study. In summary, oral administration of SodA2 can delay the progression of drusen lesions in dry AMD without toxicity-related issues. <110> Biologic Co., Ltd. Geno Fox Co., Ltd. <120> Superoxide dismutase and its use in preventing or treating dry macular degeneration <130> FC22010 / PCT <150> 10-2021-0185912 <151> 2021-12-23 <160> 72 <170> KoPatentIn 3.0 <210> 1 <211> 609 <212> DNA <213> Bacillus amyloliquefaciens <400> 1 atggcttaca aacttccaga attgccttac gctttatgatg ctttagaacc tcatatcgat 60 aaggaaacga tgacgattca ccatacgaag caccataaca catacgtgac aaacctcaac 120 aaagcgatcg aaggatctgc gcttgcagag aaatctgtag atgagcttgt tgctgatttg 180 aacgcagtgc cggaggacat ccgcacggca gtccgcaaca atggcggcgg acatgcaaac 240 cactctttat tctggactct tttatctccg aacggcggag gcgaaccgac tggtgagctt 300 gctgaagaga tcaaaagcac gttcggaagc ttcgatcaat ttaaagaaaa attcgccgca 360 gcagctgcag gccgtttcgg ttcaggctgg gcttggctcg ttgtaaacaa cggcaaactt 420 gaaattacaa gcacgccaaa ccaagattca ccgctttcag aaggtaaaac acctgttctc 480 ggtcttgatg tttgggagca tgcgtactac ctgaactacc aaaaccgccg tcctgattac 540 atttcagctt tctggaatgt tgtgaactgg gatgaagttg cccgtcttta cagcgaagca 600 aaataatga 609 <210> 2 <211> 201 <212> PRT <213> Bacillus amyloliquefaciens <400> 2 Met Ala Tyr Lys Leu Pro Glu Leu Pro Tyr Ala Tyr Asp Ala Leu Glu 1 5 10 15 Pro His Ile Asp Lys Glu Thr Met Thr Ile His His Thr Lys His His 20 25 30 Asn Thr Tyr Val Thr Asn Leu Asn Lys Ala Ile Glu Gly Ser Ala Leu 35 40 45 Ala Glu Lys Ser Val Asp Glu Leu Val Ala Asp Leu Asn Ala Val Pro 50 55 60 Glu Asp Ile Arg Thr Ala Val Arg Asn Asn Gly Gly Gly His Ala Asn 65 70 75 80 His Ser Leu Phe Trp Thr Leu Leu Ser Pro Asn Gly Gly Gly Glu Pro 85 90 95 Thr Gly Glu Leu Ala Glu Glu Ile Lys Ser Thr Phe Gly Ser Phe Asp 100 105 110 Gln Phe Lys Glu Lys Phe Ala Ala Ala Ala Ala Gly Arg Phe Gly Ser 115 120 125 Gly Trp Ala Trp Leu Val Val Asn Asn Gly Lys Leu Glu Ile Thr Ser 130 135 140 Thr Pro Asn Gln Asp Ser Pro Leu Ser Glu Gly Lys Thr Pro Val Leu 145 150 155 160 Gly Leu Asp Val Trp Glu His Ala Tyr Tyr Leu Asn Tyr Gln Asn Arg 165 170 175 Arg Pro Asp Tyr Ile Ser Ala Phe Trp Asn Val Val Asn Trp Asp Glu 180 185 190 Val Ala Arg Leu Tyr Ser Glu Ala Lys 195 200 <210> 3 <211> 609 <212> DNA <213> Bacillus amyloliquefaciens <400> 3 atggcttaca aacttccaga attgccttac gcttatgatg ctttagaacc tcatatcgat 60 aaggaaacga tgacgattca ccatacgaag caccataaca catacgtgac aaacctcaac 120 aaagcgatcg aaggatctgc gcttgcagag aaatctgtag atgagcttgt tgctgatttg 180 aacgcagtgc cggaggacat ccgcacggca gtccgcaacg atggcggcgg acatgcaaac 240 cactctttat tctggactct tttatctccg aacggcggag gcgaaccgac tggtgagctt 300 gctgaagaga tcaaaagcac gttcggaagc ttcgatcaat ttaaagaaaa attcgccgca 360 gcagctgcag gccgtttcgg ttcaggctgg gcttggctcg ttgtaaacga cggcaaactt 420 gaaattacaa gcacgccaaa ccaagattca ccgctttcag aaggtaaaac acctgttctc 480 ggtcttgatg tttgggagca tgcgtactac ctgaactacc aaaaccgccg tcctgattac 540 atttcagctt tctggaatgt tgtgaactgg gatgaagttg cccgtcttta cagcgaagca 600 aaataatga 609 <210> 4 <211> 201 <212> PRT <213> Bacillus amyloliquefaciens <400> 4 Met Ala Tyr Lys Leu Pro Glu Leu Pro Tyr Ala Tyr Asp Ala Leu Glu 1 5 10 15 Pro His Ile Asp Lys Glu Thr Met Thr Ile His His Thr Lys His His 20 25 30 Asn Thr Tyr Val Thr Asn Leu Asn Lys Ala Ile Glu Gly Ser Ala Leu 35 40 45 Ala Glu Lys Ser Val Asp Glu Leu Val Ala Asp Leu Asn Ala Val Pro 50 55 60 Glu Asp Ile Arg Thr Ala Val Arg Asn Asp Gly Gly Gly His Ala Asn 65 70 75 80 His Ser Leu Phe Trp Thr Leu Leu Ser Pro Asn Gly Gly Gly Glu Pro 85 90 95 Thr Gly Glu Leu Ala Glu Glu Ile Lys Ser Thr Phe Gly Ser Phe Asp 100 105 110 Gln Phe Lys Glu Lys Phe Ala Ala Ala Ala Ala Gly Arg Phe Gly Ser 115 120 125 Gly Trp Ala Trp Leu Val Val Asn Asp Gly Lys Leu Glu Ile Thr Ser 130 135 140 Thr Pro Asn Gln Asp Ser Pro Leu Ser Glu Gly Lys Thr Pro Val Leu 145 150 155 160 Gly Leu Asp Val Trp Glu His Ala Tyr Tyr Leu Asn Tyr Gln Asn Arg 165 170 175 Arg Pro Asp Tyr Ile Ser Ala Phe Trp Asn Val Val Asn Trp Asp Glu 180 185 190 Val Ala Arg White Tyr Ser Glu Ala Lys 195 200 <210> 5 <211> 37 <212> DNA <213> The snowstorm <220> <223> AprE_frgament 1_forward primer <400> 5 ctacggggtc tgacgcagat atcgtaaccg aagaacg <210> 6 <211> 28 <212> DNA <213> The snowstorm <220> <223> AprE_frgament 1_reverse primer <400> 6 28. tgttgcagac atgttgctga acgccatc <210> 7 <211> 28 <212> DNA <213> The snowstorm <220> <223> AprE_frgament 2_forward primer <400> 7 caacatgtct gcaacatc ttggaac <210> 8 <211> 38 <212> DNA <213> Artificial sequence <220> <223> AprE_frgament 2_reverse <400> 8 gataagctgt caaaccagag attggatagg ctatcaag 38 <210> 9 <211> 38 <212> DNA <213> Artificial sequence <220> <223> NprE_frgament 1_forward primer <400> 9 ctacggggtc tgacgcaggg cattactgca ccataatc 38 <210> 10 <211> 28 <212> DNA <213> Artificial sequence <220> <223> NprE_frgament 1_reverse primer <400> 10 gaggtacgcc ttcagcagcc tgaacacc 28 <210> 11 <211> 28 <212> DNA <213> Artificial sequence <220> <223> NprE_frgament 2_forward primer <400> 11 gctgaaggcg tacctcacgc cttcttcc 28 <210> 12 <211> 38 <212> DNA <213> Artificial sequence <220> <223> NprE_frgament 2_reverse primer <400> 12 gataagctgt caaaccagtt gtcagatttg catccttc 38 <210> 13 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Bpr_frgament 1_forward primer <400> 13 ctacggggtc tgacgcagag caatttccag cccgcttc 38 <210> 14 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Bpr_frgament 1_reverse primer <400> 14 gtatgatgag gcatgaacag caatcccg 28 <210> 15 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Bpr_frgament 2_forward primer <400> 15 tcatgcctca tcatactcaa caagggtg 28 <210> 16 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Bpr_frgament 2_reverse primer <400> 16 gataagctgt caaaccagtt cccagccgga tgttcaac 38 <210> 17 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Epr_frgament 1_forward primer <400> 17 ctacggggtc tgacgcaggc tttctgccag ccgatagg 38 <210> 18 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Epr_frgament 1_reverse primer <400> 18 tgtgtcaaag ccgttatgct tggctccg 28 <210> 19 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Epr_frgament 2_forward primer <400> 19 taacggcttt gacacagcac aaactgcc 28 <210> 20 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Epr_frgament 2_reverse primer <400> 20 gataagctgt caaaccaggt ctcccataca tgaacgac 38 <210> twenty one <211> 38 <212> DNA <213> Artificial sequence <220> <223> NprB_frgament 1_forward primer <400> twenty one ctacggggtc tgacgcagag taatcttgta ggaggctg 38 <210> twenty two <211> 28 <212> DNA <213> Artificial sequence <220> <223> NprB_frgament 1_reverse primer <400> twenty two tgtgactgtc atattcaccg tcgtgtgg 28 <210> twenty three <211> 29 <212> DNA <213> Artificial sequence <220> <223> NprB_frgament 2_forward primer <400> twenty three tgaatatgac agtcacacag tattccgcc 29 <210> twenty four <211> 38 <212> DNA <213> Artificial sequence <220> <223> NprB_frgament 2_reverse primer <400> twenty four gataagctgt caaaccagat taggatacgg tctggctg 38 <210> 25 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Vpr_frgament 1_forward primer <400> 25 ctacggggtc tgacgcagat gtacgctgag ccgaatag 38 <210> 26 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Vpr_frgament 1_reverse primer <400> 26 ggaatcacat ttgcggagat acggcgtc 28 <210> 27 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Vpr_frgament 2_forward primer <400> 27 ccgcaaatgt gattccggca catcaaac 28 <210> 28 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Vpr_frgament 2_reverse primer <400> 28 gataagctgt caaaccagac cgttttccga atctgacc 38 <210> 29 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Mpr_frgament 1_forward primer <400> 29 ctacggggtc tgacgcagcg ttatcccgaa tgcccgtg 38 <210> 30 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Mpr_frgament 1_reverse primer <400> 30 ggctttgttc ctttagtgtc aaccaccc 28 <210> 31 <211> 28 <212> DNA <213> Artificial sequence <220> <223> Mpr_frgament 2_forward primer <400> 31 ctaaaggaac aaagccgatt cgctccgc 28 <210> 32 <211> 38 <212> DNA <213> Artificial sequence <220> <223> Mpr_frgament 2_reverse primer <400> 32 gataagctgt caaaccagaa attgactgct ccacgctg 38 <210> 33 <211> 38 <212> DNA <213> Artificial sequence <220> <223> SrfAC_frgament 1_forward primer <400> 33 ctacggggtc tgacgcagga tatgtattac ctatcgcc 38 <210> 34 <211> 29 <212> DNA <213> Artificial sequence <220> <223> SrfAC_frgament 1_reverse primer <400> 34 gccccttcat ccagttcact gcttccttg 29 <210> 35 <211> 33 <212> DNA <213> Artificial sequence <220> <223> SrfAC_frgament 2_forward primer <400> 35 ggaagcagtg aactggatga aggggcttcg ctg 33 <210> 36 <211> 38 <212> DNA <213> Artificial sequence <220> <223> SrfAC_frgament 2_reverse primer <400> 36 gataagctgt caaaccagaa cgcttcgaca tcactttc 38 <210> 37 <211> 38 <212> DNA <213> Artificial sequence <220> <223> spoIIA_frgament 1_forward primer <400> 37 ctacggggtc tgacgcagag cggacgatgg gagactgg 38 <210> 38 <211> 28 <212> DNA <213> Artificial sequence <220> <223> spoIIA_frgament 1_reverse primer <400> 38 gccacctcat gcagccgatc tggaagag 28 <210> 39 <211> 28 <212> DNA <213> Artificial sequence <220> <223> spoIIA_frgament 2_forward primer <400> 39 ggctgcatga ggtggctgag cggctcgg 28 <210> 40 <211> 38 <212> DNA <213> Artificial sequence <220> <223> spoIIA_frgament 2_reverse primer <400> 40 gataagctgt caaaccagct cattgttttg gtgagctg 38 <210> 41 <211> 38 <212> DNA <213> Artificial sequence <220> <223> IspA_frgament 1_forward primer <400> 41 ctacggggtc tgacgcagtg ctgcttggca ttcatttc 38 <210> 42 <211> 29 <212> DNA <213> Artificial sequence <220> <223> IspA_frgament 1_reverse primer <400> 42 gccattgaag caatgcctcc gtttgaatc 29 <210> 43 <211> 33 <212> DNA <213> Artificial sequence <220> <223> IspA_frgament 2_forward primer <400> 43 acggaggcat tgcttcaatg gctgcccctc atg 33 <210> 44 <211> 38 <212> DNA <213> Artificial sequence <220> <223> IspA_frgament 2_reverse primer <400> 44 gataagctgt caaaccagtc aatgctctcg tcatattc 38 <210> 45 <211> 38 <212> DNA <213> Artificial sequence <220> <223> EpsE_frgament 1_forward primer <400> 45 ctacggggtc tgacgcagca agcaaatggg ctgggagg 38 <210> 46 <211> 28 <212> DNA <213> Artificial sequence <220> <223> EpsE_frgament 1_reverse primer <400> 46 gacaagccat gctttctgcc aaagtgcg 28 <210> 47 <211> 28 <212> DNA <213> Artificial sequence <220> <223> EpsE_frgament 2_forward primer <400> 47 gaaagcatgg cttgtcaagc atgaatag 28 <210> 48 <211> 38 <212> DNA <213> Artificial sequence <220> <223> EpsE_frgament 2_reverse primer <400> 48 gataagctgt caaaccagtg ttgtatacat tcagcagc 38 <210> 49 <211> 29 <212> DNA <213> Artificial sequence <220> <223> Xpf_frgament 1_forward primer <400> 49 gatcctctac ggttcatcca tgtccgaac 29 <210> 50 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Xpf_frgament 1_reverse primer <400> 50 tctatgatcc tcctcatttt tg 22 <210> 51 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Xpf_frgament 2_forward primer <400> 51 gaggaggatc atagaaagct tgtcatacgt ttgcc 35 <210> 52 <211> 33 <212> DNA <213> Artificial sequence <220> <223> Xpf_frgament 2_reverse primer <400> 52 gagttttcgt tcggatcctc cgctttttgt ttg 33 <210> 53 <211> 20 <212> DNA <213> Artificial sequence <220> <223> AprE_PCR primer_forward <400> 53 caaggcttga aataacgttg 20 <210> 54 <211> 20 <212> DNA <213> Artificial sequence <220> <223> AprE_PCR primer_reverse <400> 54 ttcgctgatt acaacattgg 20 <210> 55 <211> 20 <212> DNA <213> Artificial sequence <220> <223> NprE_PCR primer_forward <400> 55 ggaacagatg atagctcatc 20 <210> 56 <211> 20 <212> DNA <213> Artificial sequence <220> <223> NprE_PCR primer_reverse <400> 56 acactccgag aaggctgaag 20 <210> 57 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Vpr_PCR primer_forward <400> 57 ctgcggcctg cacagccgcc 20 <210> 58 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Vpr_PCR primer_reverse <400> 58 cgctgaatga cggtggtaag 20 <210> 59 <211> 20 <212> DNA <213> Artificial sequence <220> <223> NprB_PCR primer_forward <400> 59 agtaatcttg taggaggctg 20 <210> 60 <211> 20 <212> DNA <213> Artificial sequence <220> <223> NprB_PCR primer_reverse <400> 60 cgccaatgaa gtgaaggagg 20 <210> 61 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Mpr_PCR primer_forward <400> 61 ggttgaggcg attgcgacgg 20 <210> 62 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Mpr_PCR primer_reverse <400> 62 ccattctgca aaatcagctc 20 <210> 63 <211> 20 <212> DNA <213> Artificial sequence <220> <223> spoIIAC_PCR primer_forward <400> 63 aagatatgaa gaaagccggg 20 <210> 64 <211> 20 <212> DNA <213> Artificial sequence <220> <223> spoIIAC_PCR primer_reverse <400> 64 acagccgctt cataagcctg 20 <210> 65 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ThrC_PCR primer_forward <400> 65 tcaagctgtc atgtacggag 20 <210> 66 <211> 20 <212> DNA <213> Artificial sequence <220> <223> ThrC_PCR primer_reverse <400> 66 tccgatacga atggctgtcg 20 <210> 67 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> SOD_forward primer <400> 67 ggaggaatta caatggctta caa 23 <210> 68 <211> 27 <212> DNA <213> Artificial sequence <220> <223> D74_reverse primer <400> 68 tgcatgtccg ccgccatcgt tgcggac 27 <210> 69 <211> 27 <212> DNA <213> Artificial sequence <220> <223> D74_forward primer <400> 69 cagtccgcaa cgatggcggc ggacatg 27 <210> 70 <211> 28 <212> DNA <213> Artificial sequence <220> <223> D137_reverse primer <400> 70 ttcaagtttg ccgtcgttta caacgagc 28 <210> 71 <211> 25 <212> DNA <213> Artificial sequence <220> <223> D137_forward primer <400> 71 ctcgttgtaa acgacggcaa acttg 25 <210> 72 <211> 26 <212> DNA <213> Artificial sequence <220> <223> SOD_reverse primer <400> 72 caaaacagaa gcttcattattttgct 26

Claims

1. A pharmaceutical composition for treating or preventing dry type macular degeneration, comprising superoxide dismutase as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein The composition reduces drusen or drusenous lesions, or prevents their formation.

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

4. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is bound to manganese.

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

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

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

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

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

4.

10. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is administered orally or parenterally.

11. A veterinary composition for treating or preventing dry type macular degeneration, comprising superoxide dismutase as an active ingredient.

12. A food composition for improving or preventing dry macular degeneration, comprising superoxide dismutase as an effective ingredient.

13. The food composition according to claim 12, wherein The superoxide dismutase is an isolated or purified enzyme.

14. The food composition according to claim 12, wherein The superoxide dismutase is included in the form of a strain culture, a strain culture concentrate, or a dry form thereof.

15. The food composition according to claim 12, wherein The superoxide dismutase is bound to manganese.

16. The food composition according to claim 12, wherein The superoxide dismutase is deamidated Mn-SOD.

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

18. The food composition according to claim 12, wherein The superoxide dismutase is derived from Bacillus amyloliquefaciens.

19. The food composition according to claim 12, wherein The superoxide dismutase is a superoxide dismutase based on SEQ ID NO: 2, wherein the amino acid residues at positions 73 and 136 are substituted by Asp.

20. A feed composition for improving or preventing dry macular degeneration, comprising superoxide dismutase as an effective ingredient.

21. A method for treating or preventing dry macular degeneration, comprising the step of administering the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 10 to a subject.

22. The method according to claim 21, wherein: The superoxide dismutase is administered to reduce drusen or drusenic lesions in a subject, or prevents their formation.

23. The method according to claim 21, wherein: The superoxide dismutase is administered orally or parenterally.

24. A superoxide dismutase derived from a Bacillus strain, wherein the amino acid residues at positions 73 and 136 are substituted with Asp based on SEQ ID NO:

2.

25. The superoxide dismutase according to claim 24, which consists of the amino acid sequence shown in SEQ ID NO:

4.

26. A polynucleotide encoding the superoxide dismutase polypeptide according to claim 24 or 25.

27. A Bacillus strain comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence based on SEQ ID NO: 2 with amino acid residues at positions 73 and 136 substituted by Asp, In addition, the AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, Eps E and Xpf genes are deleted in the Bacillus strain.

28. A recombinant strain comprising the expression vector as shown in Figure 4.

29. Use of the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 10 in preventing or treating dry macular degeneration.

30. Use of the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 10 in the preparation of a medicament for preventing or treating dry macular degeneration.

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