Superoxide dismutase and its use for preventing or treating dry macular degeneration

Abstract

The present invention relates to superoxide dismutase and its use for preventing or treating dry age-related macular degeneration. More specifically, the present invention relates to a pharmaceutical composition or a method for preventing or treating dry age-related macular degeneration. The composition and method according to the present invention can be effectively used to prevent or treat dry macular degeneration by reducing or preventing drusen formation and / or drusen lesions.

Description

[Technical field]

[0001] The present invention relates to a superoxide dismutase and its use for the prevention, amelioration or treatment of dry age-related macular degeneration. More specifically, the present invention relates to a superoxide dismutase, composition or method for the prevention, treatment or amelioration of dry age-related macular degeneration. [Background technology]

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

[0003] The main causes of macular degeneration are age-related atrophy and impaired function of the retinal pigment epithelium (RPE). The RPE plays an important role in maintaining the homeostasis and physiological function of the retina, which is the core of visual function. Age-related abnormal changes in Bruch's membrane and degeneration of the choriocapillaris are also thought to contribute to the pathogenesis of macular degeneration. Bruch's membrane acts as the basement membrane of the RPE, while the choriocapillaris is located at the outermost part of the neurosensory retina and provides nutrients and oxygen to the photoreceptor cells where photoconversion occurs. The causes of macular degeneration are said to be excessive reactive oxygen species, mitochondrial dysfunction, inflammation, abnormal lipid metabolism, genetic factors, and environmental factors (e.g., smoking and long-term exposure to ultraviolet light), but the exact mechanism has not been fully elucidated.

[0004] Macular degeneration can be broadly classified as wet or dry. The dry type accounts for 85-90% of cases diagnosed with macular degeneration, and wet types also start as dry types. The main characteristics of dry macular degeneration are medium-sized or larger yellow deposits called drusen that exist under the retinal pigment epithelium layer, pigmentary abnormalities in the retinal pigment epithelium, and deposits called reticular pseudodrusen that exist under the retina. The formation of drusen is a typical feature of dry macular degeneration, and numerous medium-sized and large drusen are known as risk factors for progression to end-stage macular degeneration. The size and density of drusen are also correlated with the loss of choriocapillaris.

[0005] Dry macular degeneration generally progresses slowly and may progress to geographic atrophy (GA) in the end stages. Geographic atrophy is a confluent or scattered irreversible degeneration of the retinal pigment epithelium that damages the photoreceptors and leads to vision loss in advanced stages. Although geographic atrophy is a major cause of age-related vision loss, there are currently no approved treatments or drugs that can slow or inhibit the progression of dry macular degeneration.

[0006] Wet macular degeneration, another type of end-stage macular degeneration, is caused by immature blood vessels that grow from the choroid to the retina, leaking blood or fluid, damaging the photoreceptors and the nerve cells connected to them, and ultimately causing loss of central vision. Wet macular degeneration progresses more quickly than the dry type, and if left untreated, can lead to blindness within a few months. However, a treatment method has been developed to stop the formation of new blood vessels by injecting anti-VEGF preparations into the eyeball to suppress the abnormal production of VEGF, thereby stopping the angiogenesis process. Therefore, there is a need for materials and methods that can effectively prevent or treat dry macular degeneration. Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to solve the above-mentioned problems of the conventional technology. Another object of the present invention is to provide a superoxide dismutase for the prevention, amelioration or treatment of dry macular degeneration. Another object of the present invention is to provide a pharmaceutical composition or a therapeutic method for preventing or treating dry macular degeneration. Another object of the present invention is to provide a food or feed composition for the prevention or amelioration of dry macular degeneration. Another object of the present invention is to provide a pharmaceutical composition or method of treatment for preventing or treating dry macular degeneration by reducing or eliminating drusen formation or drusenoid lesions. Another object of the present invention is to provide a food or feed composition that prevents or ameliorates dry macular degeneration by reducing or eliminating drusen formation or drusen lesions. The object of the present invention is not limited to the above object. The object of the present invention will become clearer from the following description and can be realized by the means and combinations thereof described in the claims. [Means for solving the problem]

[0008] A representative configuration of the present invention for achieving the above object is as follows. According to one embodiment of the present invention, there is provided an improved superoxide dismutase (SOD) that exhibits a preventive, ameliorative or therapeutic effect on dry macular degeneration.

[0009] According to another embodiment of the present invention, there is provided a superoxide dismutase derived from a bacterium generally regarded as safe (GRAS), such as Bacillus, for the prevention, amelioration or treatment of dry macular degeneration.

[0010] According to another embodiment of the present invention, there is provided a superoxide dismutase derived from Bacillus amyloliquefaciens, which is ensured to be effective and safe when orally administered for the prevention, amelioration or treatment of dry macular degeneration.

[0011] According to another embodiment of the present invention, there is provided an extracellularly secreted Bacillus-derived or Bacillus amyloliquefaciens-derived superoxide dismutase which is convenient to produce for the prevention or treatment of dry macular degeneration.

[0012] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating dry macular degeneration, comprising the above-mentioned superoxide dismutase (SOD) as an active ingredient.

[0013] According to another embodiment of the present invention, there is provided a food or feed composition for improving or preventing dry macular degeneration, comprising the above-mentioned superoxide dismutase as an active ingredient.

[0014] According to another embodiment of the present invention, there is provided a method for preventing, ameliorating or treating dry macular degeneration, comprising the step of administering the superoxide dismutase to a subject.

[0015] According to another embodiment of the present invention, there is provided a use of the superoxide dismutase for the prevention or treatment of dry macular degeneration.

[0016] According to another embodiment of the present invention, there is provided a use of the superoxide dismutase for producing a medicament for preventing or treating dry macular degeneration.

[0017] According to another embodiment of the present invention, there is provided a superoxide dismutase derived from a Bacillus sp. strain in which the amino acid residues 73 and 136 of SEQ ID NO: 2 are substituted with Asp, a polynucleotide sequence encoding the same, an expression vector containing the polynucleotide sequence, or a cell line transformed with the expression vector.

[0018] According to another embodiment of the present invention, there is provided a Bacillus sp. strain comprising the above polynucleotide sequence and in which some genes are deleted. Effect of the Invention

[0019] The superoxide dismutase, compositions and methods according to the present invention can be effectively used to prevent, ameliorate 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 orally administered and is effective in preventing or treating dry macular degeneration.

[0020] The superoxide dismutase of the present invention is derived from Bacillus or Bacillus amyloliquefaciens, which are bacteria generally regarded as safe (GRAS), and not only ensures efficacy and safety for oral administration, but also has the production advantage of being directly recoverable from the supernatant during cultivation.

[0021] The polynucleotide sequence encoding the superoxide dismutase according to the present invention, an expression vector containing said sequence, or a cell line transformed with said expression vector, and a Bacillus species strain can be used to effectively produce an active ingredient for reducing or preventing drusen formation and / or drusen lesions. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows a schematic diagram for the double crossover recombination carried out in Example 1.1. [Diagram 2] The results of confirming the defective gene by PCR in the expression host GFBS220 and the production strain BSBA310 are shown. W indicates the wild-type B. subtilis KCTC 3135, 220 indicates the expression host GFBS220, and 310 indicates the SodA2 production strain BSBA310. [Diagram 3]FIG. 1 is a diagram showing a comparison of the amino acid sequences of SodA (Mn-SOD identified by N-terminal sequencing from the culture supernatant of Bacillus amyloliquesfaciens strain GF423) and the superoxide dismutase of the present invention (SodA2). [Figure 4] The expression vector for overexpression of sodA2 gene is shown, where rrnB T1T2 represents a transcription terminator, rep(pBR322) represents a replicon from pBR322 that functions in E. coli, rep(pUB110) represents a replicon from pUB110 that functions in B. subtilis, KanR represents a kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase), and BJ27 promoter represents a strong promoter for B. subtilis. [Diagram 5] FIG. 1 shows the overall procedure for producing the production strain BSBA310. [Figure 6] FIG. 1 illustrates criteria for clinical outcome measurements used in an embodiment of the present invention. [Figure 7] 1 shows the change from baseline in total drusen lesion area according to an embodiment of the present invention, compared to the area change in the vehicle group, *p<0.05. [Figure 8] 1 shows the change from baseline in maximum drusen lesion size according to an embodiment of the present invention. [Figure 9] FIG. 1 shows blood biochemistry results performed in an embodiment of the present invention. [Figure 10] FIG. 1 shows blood hematology results performed in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The detailed description of the present invention described below will be described with reference to certain drawings of specific embodiments in which the present invention can be practiced, but the present invention is not limited thereto, and is limited only by the appended claims, if properly described, and to the full extent of equivalents thereto. It should be understood that the various embodiments of the present invention are different from each other, but are not necessarily 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 the embodiments, without departing from the spirit and scope of the present invention. The terms presented in describing the present invention should generally be understood as their ordinary meanings, unless otherwise specified, and apply to all the same terms used herein, as well as to the aspects or embodiments of the invention in which the terms are defined. For purposes of interpreting this specification, the following definitions apply, and terms used in the singular form include the plural form where appropriate, and vice versa.

[0024] definition The term "about" as used herein refers to the normal range of error for the respective value known to one of ordinary skill in the art.

[0025] The term "subject" is used interchangeably with "patient" and may refer to a mammal in need of prevention or treatment of dry macular degeneration, such as primates (e.g., humans), pets (e.g., dogs, cats, etc.), livestock animals (e.g., cows, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In one embodiment of the invention, the subject is a human.

[0026] The term "treatment" includes preventive and / or therapeutic treatments. The preventive and / or therapeutic treatments include any type of treatment recognized in the art, including, for example, administering the pharmaceutical composition or SOD of the present invention to a subject. When administered prior to clinical manifestation of an undesirable or unwanted condition (e.g., a disease or other undesirable or unwanted condition in a subject), such treatment or treatment is a preventive treatment or treatment (e.g., protecting the subject from the development of an undesirable or unwanted condition in the subject). On the other hand, when administered after clinical manifestation of an undesirable or unwanted condition, such treatment or treatment is a therapeutic treatment, such as reducing, alleviating or stabilizing an existing undesirable or unwanted condition or its side effects.

[0027] The meaning of the term "prevention" is well known in the art. When used in relation to a medical condition such as dry macular degeneration, it means that administration of a pharmaceutical composition or SOD of the present invention reduces the frequency or delays the onset and symptoms of a medical condition (e.g., blurred vision or vision loss) compared to a subject not receiving the same.

[0028] The term "administering" means providing an active ingredient to a subject to achieve a prophylactic or therapeutic objective (eg, prevention or treatment of dry macular degeneration).

[0029] Dry macular degeneration The present invention is based, at least in part, on the discovery that administration, particularly oral administration, of superoxide dismutase (SOD) is effective in preventing or treating dry macular degeneration. Thus, according to one embodiment of the present invention, there is provided a use of SOD for preventing or treating dry macular degeneration.

[0030] The term "dry macular degeneration" is used interchangeably with "dry age-related macular degeneration," which includes early, intermediate, and advanced stages of dry macular degeneration. The dry macular degeneration is characterized by drusen or drusen lesions. The term "drusen" refers to the product of the accumulation of waste products generated in the retina with aging between RPE cells and Bruch's membrane or inside Bruch's membrane (B). The formation of drusen is a typical hallmark of dry AMD, and numerous medium-sized and large drusen are known as risk factors for progression to end-stage macular degeneration. The size and density of drusen are also correlated with the loss of choriocapillaris. Meanwhile, the term "drusen lesions" refers to lesions caused by drusen.

[0031] The SOD of the present invention can effectively prevent, ameliorate or treat dry macular degeneration by reducing or preventing the formation of drusen or drusen-like lesions.

[0032] The present inventors conducted animal tests using Rhesus monkeys, instead of mice, as an animal model that develops drusen with a location and composition similar to that of humans. The reason is that in the case of general mice, rather than transgenic mice, there is no evidence that the light-induced oxidative stress model affects retinal function (P. Sicard, et. al, "Dietary Superoxide Dismutase Protects Against Light-Induced Retinal Oxidative Stress in Young Senescence Accelerated Mice (SAM)," Invest Ophthalmol Vis Sci 2006;47: E-Abstract 2089) and drusen, which is a characteristic of dry AMD, is not generated (Hema L. Ramkumar, et. al, "Retinal ultrastructure of murine models of dry age-related macular degeneration (AMD)," Progress In Retinal And Eye Research 2010;29(3), 169-190), so they cannot be an effective model of dry AMD. In addition, according to the literature [Erica L. Fletcher, et. al., “Studying Age-Related Macular Degeneration Using Animal Models,” Optom Vis Sci.2014;91(8), 878-886], the only animals that develop drusen with a composition and location similar to that of humans are great apes (primates). Although some mice develop white lesions as they age, the location and composition of these lesions differ from those of humans, so they are not an effective model for understanding the formation of drusen.

[0033] Superoxide dismutase (SOD) According to another embodiment of the present invention, there is provided an improved superoxide dismutase (SOD) that exhibits a preventive, ameliorative or therapeutic effect on dry macular degeneration.

[0034] Superoxide dismutase (SOD) is an enzyme that alternately catalyzes the dismutation of superoxide radicals (O2-) into general molecular oxygen (O2) and hydrogen peroxide (H2O2), and SOD plays an important role in scavenging reactive oxygen species and reducing oxidative stress. SODs are widely distributed in prokaryotic and eukaryotic cells and are classified into four classes according to the different 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 polypeptides that have superoxide dismutase activity.

[0035] In one embodiment, the SOD may be manganese-binding type (Mn-SOD). Specifically, the SOD may be deamidated Mn-SOD. More specifically, the SOD may have amino acid residues at positions 73 and 136 of SEQ ID NO: 2 substituted with Asp. More specifically, the SOD may include or consist of the amino acid sequence shown in SEQ ID NO: 4. The SOD or polypeptide having SOD activity of the present invention is also understood to include an amino acid sequence showing substantial identity to the above amino acid sequence. The term "substantial identity" means an amino acid sequence showing 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity when the aligned sequences are analyzed using an algorithm commonly used in the art.

[0036] In other embodiments, the SOD is a modified or engineered polypeptide having SOD enzymatic activity and may contain one or more mutations, e.g., deletion, insertion, or substitution of one or more amino acids, that may or may not affect various aspects (in vivo, in vitro, or ex vivo stability, homogeneity, and / or morphological changes). Additionally, the polypeptide may further contain a heterologous substance (e.g., tags known in the art, including HIS tag, Ha tag, myc tag, GFC and / or Fc domain of an antibody) for enhancing purification, detection, or stability.

[0037] In one embodiment, the SOD may be an enzyme derived from a natural or recombinant microorganism, or may be produced through a process of being isolated or purified from a variety of sources.

[0038] In one embodiment, the SOD may be derived from a natural or recombinant microorganism. For example, the SOD may be derived from a bacterium. Preferably, the SOD is sourced from a bacterium generally regarded as safe (GRAS) for use in drugs or foods. Specifically, the SOD may be derived from a Bacillus species strain. More specifically, the SOD may be derived from a Bacillus amyloliquefaciens strain. For example, the SOD may be derived from the Bacillus amyloliquefaciens GF423 strain (KCTC 13222 BP). The GF423 strain (KCTC 13222 BP) was deposited at the Korea Institute of Bioscience and Biotechnology on March 6, 2017. The SOD may also be derived from a recombinant strain comprising the expression vector shown in FIG. 4. The SOD may also be derived from a recombinant strain produced by a method comprising the method shown in FIG. 5. The recombinant strain may be a Bacillus sp. strain comprising a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, and deleted in the following genes: AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf. For detailed process for producing the strain, see Example 1.

[0039] Since the SOD derived from the above-mentioned strain is an enzyme secreted extracellularly, when SOD is produced using the above-mentioned strain, it is possible to mass-produce SOD that is safe for humans without going through an expensive purification process (e.g., column purification), thereby enabling efficient production.

[0040] In one embodiment, SOD may be isolated or purified from various sources, including natural or recombinant hosts. For example, SOD having activity in preventing or treating dry macular degeneration can be extracted from the culture supernatant of Bacillus amyloliquefaciens strain GF423. Briefly, Bacillus amyloliquefaciens strain GF423 can be cultured in various types of media to obtain culture media. For example, the strain is grown at about 25°C to about 42°C for about 1 day to about 4 days using a complex medium (pH 6.0 to 7.0). Other suitable media for culturing Bacillus amyloliquefaciens strain GF423 include Luria-Bertani (LB) medium, International Streptomyces Project (ISP) medium, nutrient agar (NA) medium, brain heart infusion agar (BHI) medium, sabouraud dextrose agar (SDA) medium, potato dextrose agar (PDA) medium, nutrient broth (NB) 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).

[0041] In one embodiment, the SOD may be an isolated or purified enzyme, where the isolated or purified SOD or biologically active portion thereof 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 isolated in pure form from a culture of a strain by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or may be a culture retentate obtained by ultrafiltration, concentration, etc. The term "substantially free of cellular material" includes preparations of a protein in which such a protein has been separated from cellular components of the cells from which the protein is isolated or from which the protein is recombinantly produced. In one embodiment, the term "substantially free of cellular material" includes preparations of a protein having less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of undesired protein by dry weight.

[0042] In another embodiment, it may be desirable to purify SOD by the following purification method, including but not limited to the following: Bacillus amyloliquefaciens strain GF423 is cultured, and the resulting culture broth is centrifuged to collect the culture supernatant. The supernatant fraction is pretreated by solid phase extraction, and then separated and purified by chromatography. In this case, SOD may be purified using various types of chromatography. Preferably, hydrophobic interaction chromatography is used.

[0043] In another embodiment, the SOD may be contained in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof. In this case, the "strain lysate" may be obtained by culturing a strain and mechanically or chemically disrupting the strain. The "strain culture" refers to the culture itself obtained by culturing a strain or its supernatant. The "strain culture concentrate" refers to a pure product 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" refers to a product extracted from the culture or the concentrate, and may include an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, a crude product or purified product thereof, or a fraction obtained by fractionating the same. The dried form may include a freeze-dried form.

[0044] The SOD of the present invention can reduce or prevent the formation of drusen or drusen lesions and / or effectively prevent, ameliorate or treat dry macular degeneration. In particular, the SOD of the present invention is useful for preventing, ameliorating or treating dry macular degeneration characterized by drusen or drusen lesions.

[0045] Pharmaceutical Compositions According to one embodiment of the present invention, there is provided a pharmaceutical composition for treating or preventing dry macular degeneration, comprising superoxide dismutase (SOD) as an active ingredient. The pharmaceutical composition may reduce or prevent the formation of drusen or drusen lesions.

[0046] In one embodiment, the SOD according to the present invention can be combined with a pharma- ceutically acceptable carrier, excipient and / or diluent to form a pharmaceutical composition for the prevention or treatment of dry macular degeneration. 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.

[0047] The pharmaceutical or veterinary compositions of the present invention may further comprise a pharma- ceutically acceptable carrier, excipient and / or diluent. The pharma- ceutically acceptable carrier, excipient and / or diluent may be one 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, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils such as silicon dioxide.

[0048] When the formulation is made, additives such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, etc. may be used for preparation. The additives for the formulation may be appropriately selected from those commonly used in the pharmaceutical field.

[0049] Furthermore, the pharmaceutical or veterinary compositions of the present invention may be formulated in any suitable form depending on the method of use, and may be formulated by employing methods known in the art to provide quick, sustained or delayed release of the active ingredient after administration to a mammal, in particular. Specific examples of such dosage forms include tablets, pills, acids, granules, syrups, solutions, capsules, suspensions, emulsions, injections, plasters, lotions, liniments, limeades, aerosols, extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, patches, etc.

[0050] Moreover, the pharmaceutical or veterinary compositions of the present invention may be suitably formulated using any suitable method known in the art or as disclosed in Remington's Pharmaceutical Sciences (latest edition), Mack Publishing Company, Easton Pa.

[0051] In one embodiment, the SOD may be administered orally or parenterally. For oral administration, the SOD may be coated with shellac to protect it from stomach 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 methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof. When the SOD is coated, the SOD may be coated in solution. Specifically, the purified solution is mixed with a shellac-containing solution and then freeze-dried. The freeze-dried sample becomes a powder and can be stored at about 4°C until use. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid formulations may be prepared by mixing the complex composition with at least one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquids for internal use, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. may be used.

[0052] For parenteral administration, the following injection methods may be selected: sublingual administration, intraocular administration including intravitreal administration, intranasal spray, topical application on the skin, patch, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0053] Also, the pharmaceutical or veterinary compositions of the present invention are administered in a pharma- ceutical or veterinarily effective amount. The term "pharmaceutical effective amount" or "veterinarily effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical or veterinary treatment, and the effective dose level can be determined based on factors including the patient's or animal's weight, sex, age, health condition, severity, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical arts.

[0054] The pharmaceutical or veterinary compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, either sequentially or simultaneously. The pharmaceutical compositions may be administered in single or multiple doses as needed. It is important to administer an amount that provides maximum efficacy at a minimum dose without side effects, taking into account all of the above factors, and such an amount can be readily determined by one of skill in the art.

[0055] Preventive or therapeutic methods According to another embodiment of the present invention, there is provided a method for treating or preventing dry macular degeneration comprising administering an SOD or a pharmaceutical composition according to the present invention to a subject having or at risk of having dry macular degeneration.

[0056] Also provided is a method for treating or preventing dry macular degeneration, comprising administering superoxide dismutase to a subject. The details regarding SOD, dry macular degeneration, and treatment or prevention are as described above for the pharmaceutical composition.

[0057] The term "administration" includes any of various administrations that enable treatment by performing a desired action. The administration may include oral or parenteral administration. For oral or parenteral administration, see the description of the pharmaceutical composition above. The administration route 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 intraocular administration. In the case of oral administration, SOD may be administered in a form coated with a coating agent such as shellac, as described above.

[0058] The effective amount or effective non-toxic amount of SOD according to the present invention can be determined by routine experimentation. For example, the therapeutically active amount of SOD according to 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 weight of the subject. The dosage and administration regimen of SOD according to 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 reduced or increased depending on the exigencies of the therapeutic situation.

[0059] In one embodiment, the method may include administering one or more additional agents for treating or preventing dry macular degeneration. The one or more additional agents may be administered simultaneously, sequentially, or in reverse order with the SOD or pharmaceutical composition. Additionally, the individual components may be administered to a subject by the same route or by different routes.

[0060] Food Composition According to another embodiment 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 active ingredient. Such a food composition includes a medical or nutraceutical food composition.

[0061] The term "medical food" or "nutraceutical food" is defined by the Ministry of Food and Drug Safety as a food made from ingredients or components that may have beneficial functions on the human body and that maintains or improves health by maintaining normal functions or activating physiological functions of the human body, but is not limited thereto and does not exclude any ordinary health food from its meaning.

[0062] In addition, the food products include, but are not limited to, various foods, food additives, beverages (e.g., functional beverages, natural fruit juice and vegetable beverages), gums, teas, vitamin complexes, health functional foods, and other functional foods.

[0063] The food may be prepared by a conventional method known in the art. For example, the medical food, nutraceutical food, or health functional food may further contain one or more of carriers, diluents, excipients, and additives in addition to the SOD, and may be formulated into one selected from the group consisting of tablets, pills, acidulants, granules, powders, capsules, and liquid dosage forms, for the purpose of preventing or improving dry macular degeneration. Specific examples of the carriers, excipients, diluents, and additives are well known in the art, and those skilled in the art may prepare them by combining appropriate ingredients according to the dosage form.

[0064] The content of the superoxide dismutase according to the present invention as an active ingredient in the above-mentioned dosage form may be appropriately adjusted depending on the form and purpose of use, the condition of the patient, the type and severity of symptoms, etc., and may be about 0.001 to about 99.9 wt% based on the solid content weight, preferably about 0.01 to about 50 wt%, but is not limited thereto.

[0065] The dosage of the food of the present invention varies depending on the age, weight, sex, dosage form, health condition and severity of the disease of the patient, and may be administered once or several times a day at regular intervals at the discretion of a doctor or pharmacist. For example, the daily dosage may be 10 to 1000 mg / kg based on the content of the active ingredient. The above dosage is an example of an average case, and the dosage may be higher or lower depending on 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 meaningful effect, and if it is more than the above dosage, it is not only uneconomical but also exceeds the range of the normal dosage, and undesirable side effects may occur.

[0066] Feed composition According to another embodiment of the present invention, there is provided a feed composition for preventing or improving dry macular degeneration, which contains the 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 prepared in any dosage form commonly used in the art. For example, the feed composition of the present invention may further include supplementary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other live microbial preparations; grains such as crushed or shredded wheat, oats, barley, corn, and rice; vegetable protein feeds such as those based on rapeseed, soybeans, and sunflower; animal protein feeds such as blood meal, meat meal, bone meal, and fish meal; dry ingredients consisting of sugar and dairy products such as various types of milk powder and whey powder; lipids such as animal fats and vegetable fats optionally liquefied by heating; and additives such as nutritional supplements, digestive and absorption improvers, growth promoters, and disease preventive agents.

[0067] The feed composition of the present invention may be in the form of a powder or liquid formulation and may contain a feed additive excipient, such as, but not limited to, calcium carbonate, powdered powder, zeolite, ball powder, or rice bran.

[0068] Polynucleotides, expression vectors and host cells In accordance with another embodiment of the present invention, there is provided a polynucleotide encoding the superoxide dismutase polypeptide, specifically a nucleic acid encoding a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO:4. According to another embodiment of the present invention, there is provided an expression vector comprising the nucleic acid. Specifically, the vector may be the expression vector shown in FIG. According to another embodiment of the present invention, there is provided a host cell comprising said nucleic acid or said expression vector. In particular, said host cell is a bacterial cell. More particularly, said host cell is a strain of Bacillus sp.

[0069] According to another embodiment of the present invention, there is provided a Bacillus sp. strain comprising a polynucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4, and deleted in the following genes: AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf. For detailed process for producing said strain, see Example 1 below.

[0070] The present invention will now be described in more detail with reference to the following examples, which are presented to aid in the understanding of the present invention and are not intended, and should not be construed as, limiting its scope in any manner.

[0071] Example 1. Preparation of recombinant strain expressing superoxide dismutase (SodA2) Example 1.1. Construction of expression host GFBS220 The expression host GFBS220 was produced using Bacillus subtilis KCTC 3135. B. subtilis KCTC 3135 was provided by the Korea Comprehensive Biotechnology and Technology Center (KCTC), Korea Institute of Bioscience and Biotechnology. To facilitate downstream processing, the genes shown in Table 1 below were deleted from the genome of B. subtilis KCTC 3135.

[0072] [Table 1]

[0073] The gene was removed by double cross-over recombination on the genome (Figure 1). Specifically, DNA fragments (up and down frags) that are homologous to the DNA base sequences on both sides of the gene to be manipulated were cloned into the vector pUCori-ts-cm, which has a temperature-sensitive replication origin. The PCR conditions and primers used for cloning are shown in Tables 2 and 3, respectively.

[0074] [Table 2]

[0075] [Table 3]

[0076] The vector thus prepared was introduced into B. subtilis cells, and transformants (single crossover homologous recombination) were selected using a chloramphenicol-containing medium at 37°C (non-replicative temperature). The selected transformants were cultured in LB medium without antibiotics at 30°C (permissive temperature for replication) (second crossover), and then cultured in LB agar medium without antibiotics at 39°C. The resulting colonies were screened for chloramphenicol-sensitive strains (plasmid curing). PCR was performed on the selected colonies to obtain double crossover recombinants, and a second pure isolation was performed to select the final recombinant, which was named GFBS220.

[0077] The deleted gene site of GFBS220 was amplified using PCR with the primers shown in Table 4, 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.

[0078] [Table 4]

[0079] Example 1.2. Construction of expression vector pBE1-sodA2 Mn-SOD was identified from the culture supernatant of Bacillus amyloliquesfaciens strain GF423 by N-terminal sequencing (Kang et al., 2018). The GF423 strain was deposited at the Korea Center for Biotechnology and Technology (KCTC) on March 6, 2017 (Accession No. KCTC 13222 BP). The gene for Mn-SOD was named sodA according to the nomenclature of bacterial SOD, and its amino acid sequence is shown in FIG. 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 preparation produced 100% amino acid sequence coverage, but deamidation was observed at Asn73 and Asn136 residues with abundances of 73% and 17%, respectively. Therefore, to improve the homogeneity of the purified enzyme, both Asn residues were replaced with Asp. The amino acid sequence of this mutant sodA, designated SodA2, is shown in FIG. 3 and SEQ ID NO:4 (its nucleotide sequence is shown in SEQ ID NO:3).

[0080] The sodA2 gene was amplified using overlap extension PCR with four primers (Table 5) and the B. amyloliquefaciens GF423 genome as template. Nucleotides for Asn to Asp substitution are underlined.

[0081] [Table 5]

[0082] The amplified DNA fragment containing the sodA2 gene and plasmid pBE1 linearized by NdeI and HindIII treatment were assembled in vitro by the SLIC method (reference [Jeong et al. 2012]), and then the resulting construct was transformed into E. coli C2984H. Correct clones were screened by restriction enzyme mapping and confirmed by nucleotide sequencing. The expression vector thus generated, pBE1-sodA2, is shown in Figure 4.

[0083] Example 1.3. Construction of production strain BSBA310 The expression vector pBE1-sodA2 was transformed into B. subtilis GFBS220. A strain was selected from the transformants, and a pure clone was established by two single colony isolation procedures in LB2 medium. The selected strain was named BSBA310 and stored at -80°C in glycerol stock method. The defective gene was confirmed from this production strain BSBA310 using PCR, and the result is shown in Figure 2. Also, the whole procedure for producing the production strain BSBA310 is summarized in Figure 5.

[0084] Example 2. Isolation and purification of superoxide dismutase (SodA2) from the production strain BSBA310 Example 2.1. Cultivation of the production strain BSBA310 To cultivate the production strain BSBA310 obtained in Example 1, a single colony formed on 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 again inoculated into 3 L of LB medium containing 1 mM manganese sulfate (MnSO4) and cultured at 37° C. for 20 hours.

[0085] Example 2.2. Isolation and purification of SodA2 The cell culture 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 times using ultrafiltration (UF, MWCO 10,000). 390 g of ammonium sulfate was added per 1 L of concentrated cell culture, stirred for 20 minutes, and centrifuged to collect the supernatant. The collected supernatant was purified using a phenyl sepharose HP column. The purification process was performed by equilibrating the column using 50 mM potassium phosphate pH 7.0 with a concentration of 2 M ammonium sulfate, then adding ammonium sulfate to pass the resulting supernatant through the column, and recovering the SodA2 attached to the column using 50 mM potassium phosphate pH 7.0 containing 1.6 M ammonium sulfate. The purified solution purified through the column was collected, and the high concentration salt formed during the purification process was removed through ultrafiltration, followed by concentration. The concentrated solution was filtered through a sterilization filter and then freeze-dried. 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 50% of superoxide radicals. The activity of the dried SodA2 enzyme was 1000-1200 U / mg.

[0086] Example 3. Preparation of SodA2 for animal experiments Production of the administered substance Shellac (EXCELACS Co., Ltd., Bangkok) was dissolved in 100% ethanol to a concentration of 3%, and a 0.2 um sterile filter was used to prepare the filter. The shellac dissolved in ethanol was diluted 1 / 20 with sterilized 1x PBS to prepare a shellac solution. Lyophilized tablets of SodA2 were dissolved at 20 mg / ml and prepared as sterile using a 0.2 um size filter. The prepared shellac solution and the dissolved SOD were mixed in a 1:1 ratio with stirring, and then stirring was continued for 10 minutes. The well-mixed material was freeze-dried, thus preparing the final freeze-dried shellac-coated SodA2.

[0087] Lyophilized shellac-coated SodA2 was mixed with dextrin at a ratio of 1:9-12 (shellac-coated SodA2:dextrin) and the final activity was 90-110 U / mg through activity measurement.

[0088] Example 4. Animal experiments In this example, SodA2 prepared in Example 3 was orally administered to rhesus monkeys with idiopathic moderate dry age-related macular degeneration (AMD), and its safety and efficacy were evaluated. The abbreviations used in this example are defined as follows:

[0089] [Table 5-1]

[0090] Example 4.1. Preparation of Test and Control Articles The vehicle administered to the SodA2 and control groups has the following characteristics and conditions:

[0091] [Table 6]

[0092] Example 4.2. Preparation of Experimental Animals The rhesus monkeys used in this example were selected and supplied according to the following criteria.

[0093] [Table 7]

[0094] Inclusion criteria Monkeys with moderate dry AMD in at least one eye, diagnosed using the AREDS classification of size and area of ​​retinal drusen on fundus photographs (see AREDS Classification in Table 8 for grading of digital fundus photographs), characterized by any of the following features: - Demonstrating soft drusen and progression of drusen size or area within 2 years; -Multiple drusen, including at least one large drusen (grade 4, drusen diameter >116 μm); - Total drusen lesion area of ​​at least grade 4 (grade 4, drusen area I-2 0.089 mm 2 That's all.

[0095] [Table 8]

[0096] Exclusion criteria - Evidence or history of neovascular AMD or diabetic retinopathy [either eye]; -Severe heart failure (New York Heart Association grades III and IV) or left ventricular ejection fraction <35% on all tests; - Severe liver disease, with ALT levels increased more than 3 times the upper limit of normal. - Severe renal disease with any of the following signs: (1) dialysis or eGFR <20ml / min / 1.73m2, (2) urinary protein / urinary creatinine ≥1g / g, (3) blood creatinine or albumin / urinary creatinine ≥600mg / g. - Disabling eye disease such as aphakia; optic atrophy (pallor of the optic disc); any eye disease that the investigator believes may be a disabling factor in this study.

[0097] The prepared monkeys were kept under the following conditions: The entire course of this experiment was carried out in a laboratory approved for experimental animal welfare, in compliance with animal welfare regulations.

[0098] environmental conditions Monkeys were housed in a temperature-controlled (18°C–26°C) and humidity-controlled (40%–70%) facility with a 12-h light / dark cycle. Food intake Each monkey was fed 350 g of PriMed Diet daily, and food intake was quantitatively assessed daily. water All monkeys had free access to purified tap water.

[0099] Example 4.3. Study Design Example 4.3.1. Group assignment A total of eight rhesus monkeys with idiopathic moderate dry AMD confirmed within the last 2 years were enrolled during the pre-study period (4 weeks) for baseline imaging examination. Of the eight monkeys, two had retinal drusen areas that met grade 6 (area ≥1 / 2 DA but <1 DA), and six met grade 4 (area ≥I-2 but <O-2) or grade 5 (area ≥O-2 but <1 / 2 DA), and the monkeys were randomly divided into two groups, each containing four monkeys. Baseline characteristics of the monkeys are shown in Table 9, and animal groupings and dosing are shown in Table 10.

[0100] [Table 9]

[0101] [Table 10]

[0102] Example 4.3.2. Administration Dried kiwi slices were loaded with SodA2 and vehicle. Monkeys in the vehicle and SodA2 groups were orally administered vehicle and SodA2 once a day for 4 months, respectively. SodA2 and vehicle were prepared before daily oral administration.

[0103] Example 4.3.3. Test Procedure Observation of retinal drusen On days 30 (D30), 60 (D60), 90 (D90), and 120 (D120) after dosing, retinal drusen size and area (including changes from baseline in retinal drusen size and area) of the test animals were measured by fundus photography (FP). Each baseline was measured during the pre-study period. Prior to image acquisition, monkeys were anesthetized with an intramuscular injection of a ketamine:xylazine mixture (1:1.8 mg / kg ketamine). After anesthesia, two drops of tropicamide phenylephrine eye drops were instilled into each eye to dilate the pupils. After anesthesia, monkeys were placed in a dark room until pupil diameter was greater than 6 mm. A self-retaining eye dilator was placed over the eye.

[0104] FP images were acquired using a retinal camera (KOWA VX-20) as follows: The monkey's forehead was held tilted onto the forehead support. The working distance was adjusted and the focus was adjusted before taking the image. It was confirmed that a central macular image was obtained. During the examination, the monkeys were managed according to the relevant standard operating procedures (SOPs). 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). Drusen lesions and AMD were graded using the Age-Related Eye Disease Study 2 (AREDS2) system (Table 12). Grading included a modified grid template modified from the Early Treatment Diabetic Retinopathy Study (ETDRS), which uses a series of graduated circles to assess the largest drusen size and the total area associated with pigmentary abnormalities and drusen. All measurements included the number of drusen lesions, the total area of ​​lesions, and the size of the largest lesion.

[0105] [Table 11]

[0106] Cage Observation Each monkey was observed and physically examined once daily for changes in behavior, autonomic activity, mental performance, body coloration, excretion, and eye appearance. Food intake Food intake was assessed qualitatively and was assessed once daily for the duration of the study. body weight After a 14-h overnight fast, monkeys were weighed using a scale during the prestudy period (baseline) and on days 7, 14, 21, 30, 37, 45, 52, 60, 67, 75, 82, 90, 97, 105, 112, and 120 after dosing.

[0107] Blood Chemistry Test Instrument: Roche Cobas6000 Analyzer Series C501 Sample type: Plasma Frequency: Baseline and days 14, 30, 45, 60, 75, 90, 105, and 120 after dosing. MDA and C3 were measured at only three time points: days 90, 105, and 120. Blood chemistry parameters are shown in Table 12.

[0108] [Table 12]

[0109] Blood Hematology Tests Equipment:Siemens ADVIA 212i / SYSMEX XN1000 Sample type: Whole blood Frequency: Baseline and days 14, 30, 45, 60, 75, 90, 105, and 120 after dosing. Blood hematology parameters are shown in Table 13.

[0110] [Table 13]

[0111] Data analysis Data analysis will include total drusen lesion area, largest drusen lesion size, drusen lesion volume, body weight, blood biochemistry, blood hematology, and the like. Microsoft Office Excel 2013 and SPSS Statistics 23.0 were used for data processing and statistical analysis. The total area of ​​drusen lesions, the size of the largest drusen lesion, and the volume of drusen lesions were analyzed by paired-sample T-test. Differences were considered statistically significant when the p-value was <0.05. For criteria for clinical outcome measurements see FIG.

[0112] Example 4.4. Results Example 4.4.1. Total area of ​​drusen lesions The total area of ​​drusen lesions is shown in Table 14, and the change in area is shown in Table 15 and FIG. In seven eyes in the vehicle group, the total area of ​​drusen lesions significantly increased at 30, 60, 90, and 120 days after treatment compared with baseline (p<0.05), suggesting that dry AMD in the vehicle group developed significantly after 4 months of vehicle treatment.

[0113] In seven eyes in the 2.5 mg / kg SodA2 group, the total area of ​​drusen lesions did not change significantly compared to baseline at 30, 60, 90, and 120 days after treatment, 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), suggesting that SodA2 slowed the progression of dry AMD.

[0114] [Table 14]

[0115] [Table 15]

[0116] Example 4.4.2. Size of the Largest Drusen Lesion The size of the largest drusen lesion is shown in Table 16, and the area change is shown in Table 17 and FIG. In seven eyes in the vehicle group, the size of the largest drusen lesion increased at days 90 and 120, but this did not reach statistical significance compared with baseline, suggesting that the progression of dry AMD in the vehicle group was evident after 4 months of vehicle treatment.

[0117] In seven eyes in the 2.5 mg / kg SodA2 group, the size of the largest drusen lesion did not change significantly after treatment compared with baseline, suggesting that 2.5 mg / kg SodA2 slowed the progression of dry AMD.

[0118] [Table 16]

[0119] [Table 17]

[0120] Example 4.4.3. Cage Observation No significant abnormalities were observed in the behavior, autonomic nerve activity, skin, hair, excretion, or eyes of the monkeys in each group before and after administration. No toxicity problems were found in this study.

[0121] Example 4.4.4. Body weight No obvious changes were observed in the body weight of the monkeys in each group during the treatment period. The relevant results are shown in Table 18, and the body weights are in kg (mean ± SD).

[0122] [Table 18]

[0123] Example 4.4.5. Blood Chemistry No obvious changes were observed in the blood biochemistry of the monkeys in each group during the treatment period. The relevant results are shown in Figure 9 (expressed as mean ± SD).

[0124] Example 4.4.6. Hematology Hematology No obvious changes were observed in the blood hematology of the monkeys in each group during the treatment period. The relevant results are shown in Figure 10 (expressed as mean ± SD).

[0125] Example 4.4.7. Conclusion In seven eyes in the vehicle group, the total area of ​​drusen lesions was significantly increased (p<0.05) compared to baseline at 30, 60, 90, and 120 days after treatment, and the size of the largest drusen lesion was increased at 90 and 120 days, but did not reach statistical significance compared to baseline, suggesting that drusen lesions developed in monkeys in the vehicle group that received vehicle for 4 months.

[0126] In seven eyes in the 2.5 mg / kg SodA2 group, the total area of ​​drusen lesions did not change significantly after treatment compared to baseline, the change in the total area of ​​drusen lesions was significantly lower than in the vehicle group (p<0.05), and the size of the largest drusen lesion did not change significantly after treatment compared to baseline but showed a decreasing trend, suggesting that 2.5 mg / kg SodA2 delayed the progression of drusen lesions. Meanwhile, no obvious changes were observed in the body weight, blood biochemistry, or hematology of the monkeys in each group during the 4-month treatment period. No toxicity-related problems were found in this study. In conclusion, oral administration of SodA2 can slow the progression of drusen lesions in dry AMD without toxicity-related problems.

Claims

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

2. The pharmaceutical composition of claim 1 , wherein the composition reduces or prevents the formation of drusen or drusen lesions.

3. The pharmaceutical composition of claim 1 , wherein the SOD is an isolated or purified enzyme.

4. The pharmaceutical composition of claim 1 , wherein the SOD binds manganese.

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

6. The pharmaceutical composition of claim 1 , wherein the SOD is derived from a Bacillus species strain.

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

8. The pharmaceutical composition according to claim 1, wherein the SOD has amino acid residues at positions 73 and 136 substituted with Asp based on SEQ ID NO:

2.

9. The pharmaceutical composition of claim 1 , wherein the SOD has an amino acid sequence as set forth in SEQ ID NO:

4.

10. The pharmaceutical composition of claim 1 , wherein the SOD is administered orally or parenterally.

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

12. A food composition for improving or preventing dry macular degeneration, comprising superoxide dismutase (SOD) as an active ingredient.

13. The food composition of claim 12 , wherein the SOD is an isolated or purified enzyme.

14. 13. The food composition of claim 12, wherein the SOD is comprised in a strain culture, a strain culture concentrate, or a dried form thereof.

15. The food composition of claim 12 , wherein the SOD binds manganese.

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

17. The food composition of claim 12 , wherein the SOD is derived from a Bacillus species strain.

18. The food composition of claim 12 , wherein the SOD is derived from Bacillus amyloliquefaciens.

19. The food composition according to claim 12, wherein the SOD has amino acid residues at positions 73 and 136 of SEQ ID NO: 2 substituted with Asp.

20. A feed composition for improving or preventing dry macular degeneration, comprising superoxide dismutase (SOD) as an active ingredient.

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

22. 22. The method of claim 21, wherein administration of the SOD reduces or prevents the formation of drusen or drusen lesions in the subject.

23. 22. The method of claim 21, wherein the SOD is administered orally or parenterally.

24. A superoxide dismutase derived from a Bacillus species strain, in which the amino acid residues at positions 73 and 136 of SEQ ID NO:2 are substituted with Asp.

25. 25. The superoxide dismutase of claim 24, consisting of the amino acid sequence of SEQ ID NO:

4.

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

27. A Bacillus sp. strain comprising a nucleotide sequence encoding a polypeptide comprising an amino acid sequence in which the 73rd and 136th amino acid residues of SEQ ID NO:2 are substituted with Asp, and in which the following genes are deleted: AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE, and Xpf.

28. A recombinant strain containing the expression vector shown in FIG.

29. 11. Use of the pharmaceutical composition or superoxide dismutase (SOD) according to any one of claims 1 to 10 for preventing or treating dry macular degeneration.

30. 11. Use of the pharmaceutical composition or superoxide dismutase (SOD) according to any one of claims 1 to 10 for the manufacture of a medicament for the prevention or treatment of dry macular degeneration.

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