Superoxide dismutase and its use for preventing or treating mucositis

Abstract

The present invention relates to a superoxide dismutase and its use for preventing or treating mucositis. The superoxide dismutase according to the present invention can be effectively used to improve or treat the inhibition of proliferation of mucosal epithelial cells, which is a side effect of anticancer drugs or radiation therapy.

Description

[Technical field]

[0001] The present invention relates to the use of superoxide dismutase for the prevention or treatment of mucositis. More specifically, the present invention relates to a superoxide dismutase useful for the prevention or treatment of mucositis, its use, composition or method for the prevention or treatment of mucositis. [Background technology]

[0002] Mucositis is a pathological condition characterized by mild inflammation and, when severe, damage to the mucosa due to the development of ulcers. Mucositis is one of the common side effects following anticancer chemotherapy and radiation therapy, occurring frequently in the oral cavity, as well as in the small intestine, esophagus, stomach, and large intestine. Mucositis occurs in 40% of patients undergoing anticancer chemotherapy, and in head and neck cancer patients, the incidence rate increases to about 90% after chemotherapy and radiation therapy. Currently, several treatment methods have been proposed, but their efficacy is still uncertain (Non-Patent Document 1). In the past, the most major side effects of anticancer therapy were vomiting symptoms and decreased immunity due to decreased bone marrow function, but recently mucositis has been considered one of the serious side effects of treating cancer patients. The epithelial cells of the oral and gastrointestinal mucosa are the fastest growing tissues in the body, and in particular, the small intestine has the fastest replacement cycle, being replaced approximately once every 7 days. Anticancer chemotherapy and radiation therapy may hinder the regeneration of mucosal cells, and nutritional deficiencies in patients may further worsen cell regeneration. It has been suggested that radiation-induced damage to the intestinal mucosa is caused by microvascular damage due to apoptosis of vascular endothelial cells, and it is speculated that vascular endothelial cells and platelets play a role in the pathogenesis of mucositis.

[0003] Mucositis can cause severe pain, increased risk of systemic infection, and reduced nutritional intake in many patients, and in some patients, mucositis can prevent them from receiving adequate doses of anticancer therapy, reducing their motivation to undergo treatment, which can significantly reduce the quality of life of patients. To date, therapeutic agents for oral mucositis are actively being researched, but new methods for preventing or treating oral and gastrointestinal mucositis are still needed. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Oral mucositis: the hidden side of cancer therapy. J Exp Clin Cancer Res 39,210 (2020); https: / / www.ons.org / pep / mucositis Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to solve the problems of the prior art described above.

[0006] Another object of the present invention is to provide a superoxide dismutase for the prevention or treatment of mucositis.

[0007] Another object of the present invention is to provide a Bacillus-derived superoxide dismutase that is effective and safe when orally administered for the prevention or treatment of mucositis.

[0008] Another object of the present invention is to provide a superoxide dismutase derived from Bacillus amyloliquefaciens that is effective and safe when orally administered for the prevention or treatment of mucositis.

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

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

[0011] Another object of the present invention is to provide a food composition for preventing or ameliorating mucositis.

[0012] Another object of the present invention is to provide compositions and methods for the prevention or treatment of mucositis that may occur during anti-cancer therapy.

[0013] 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]

[0014] A representative configuration of the present invention for achieving the above object is as follows.

[0015] According to one embodiment of the present invention, there is provided an improved superoxide dismutase (SOD) that exhibits the effect of preventing, improving or treating mucositis.

[0016] 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 mucositis.

[0017] According to another embodiment of the present invention, there is provided a superoxide dismutase derived from Bacillus amyloliquefaciens, which has ensured efficacy and safety when orally administered for the prevention, amelioration or treatment of mucositis.

[0018] In accordance with another embodiment of the present invention, there is provided a Bacillus-derived or Bacillus amyloliquefaciens-derived superoxide dismutase that is conveniently secreted extracellularly for production for the prevention or treatment of mucositis.

[0019] According to another embodiment of the present invention, there is provided an improved superoxide dismutase that exhibits efficacy in preventing, ameliorating or treating mucositis in subjects undergoing radiation therapy and / or chemotherapy.

[0020] In another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating mucositis, comprising the above superoxide dismutase as an active ingredient.

[0021] According to another embodiment of the present invention, there is provided a food or feed composition for preventing or ameliorating mucositis, which comprises the above-mentioned superoxide dismutase as an active ingredient.

[0022] According to another embodiment of the present invention, there is provided a kit for treating or preventing mucositis, comprising a composition containing the superoxide dismutase as an active ingredient, and instructions including instructions for taking or administering the composition.

[0023] According to another embodiment of the present invention, there is provided a method for preventing, ameliorating or treating mucositis, comprising the step of administering said superoxide dismutase or said composition to a subject.

[0024] According to another embodiment of the present invention, there is provided a use of the superoxide dismutase for the prevention or treatment of mucositis.

[0025] 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 mucositis. Effect of the Invention

[0026] The superoxide dismutase, composition, method and kit according to the present invention can be effectively used for the prevention or treatment of mucositis. In particular, it has been confirmed that the superoxide dismutase according to the present invention is effective for the prevention or treatment of mucositis even at a low dose when orally applied or administered.

[0027] The superoxide dismutase of the present invention is derived from Bacillus or Bacillus amyloliquefaciens, which are bacteria generally regarded as safe (GRAS), thereby ensuring efficacy and safety for oral administration as well as the production advantage of being able to be directly recovered from the supernatant during cultivation.

[0028] According to a specific embodiment of the present invention, mucositis was induced using experimental animals (rats), and the SOD of the present invention was administered in different concentrations through oral application and oral administration, and the body weight and food intake were measured. As a result, it was confirmed that the body weight and food intake, which were decreased by the induction of mucositis, increased again in the SOD-administered group compared to the SOD-unadministered group. In addition, it was confirmed that the degree of improvement in re-epithelialization and villous atrophy in oral and intestinal mucositis was observed in a SOD concentration-dependent manner, and that the reactive oxygen species (ROS) level in the gastrointestinal tract (e.g., small intestine) and blood was reduced in a SOD concentration-dependent manner. Therefore, the superoxide dismutase according to the present invention can be effectively used to prevent, improve or treat mucositis by reducing the ROS level and re-epithelializing epithelial cells or improving atrophied villous cells, and can be expected to have a weight gain effect by enabling normal nutrient intake. [Brief description of the drawings]

[0029] [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 from 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 amyloliquefaciens strain GF423) and the superoxide dismutase (SodA2) of the present invention. [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) is 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 6a] FIG. 1 shows the results of body weight measurements in an animal model of mucositis. [Figure 6b] FIG. 1 shows the results of measuring food intake in an animal model of mucositis. [Figure 7a] 7 shows the results of evaluation of oral mucositis in an animal model of mucositis, and Fig. 7a shows the results of scoring oral mucositis. [Figure 7b] 7A and 7B show the results of evaluation of oral mucositis in an animal model of mucositis.Figure 7b shows the results of histological evaluation of oral mucositis. [Figure 7c] Fig. 7 shows the results of evaluation of oral mucositis in an animal model of mucositis. Fig. 7c is a graph quantitatively showing the thickness of the re-epithelialized oral mucosal epithelium. [Figure 8a] 8 shows the results of intestinal mucositis evaluation in an animal model of mucositis.Figure 8a shows the results of scoring intestinal mucositis. [Figure 8b] Figure 8b shows the results of histological evaluation of intestinal mucositis in an animal model of mucositis. [Figure 8c] 8 shows the results of evaluating intestinal mucositis in an animal model of mucositis. Fig. 8c is a graph quantitatively showing the length of the intestinal villi. [Figure 9a] FIG. 1 shows the results of evaluation of intestinal ROS. [Figure 9b] FIG. 1 shows the results of evaluation of blood ROS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] The detailed description of the present invention described below will be described with reference to certain drawings of one embodiment in which the present invention can be practiced, but the present invention is not limited thereto, but 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 modified from one embodiment to another, or combined, 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 include the plural where appropriate, and vice versa.

[0031] definition The term "subject" is used interchangeably with "patient" and may refer to a mammal in need of prevention or treatment of mucositis or to undergo radiation therapy and / or chemotherapy, 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.

[0032] 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 a pharmaceutical composition or SOD of the present invention to a subject. If 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, if 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.

[0033] The meaning of the term "prevention" is well known in the art. When used in relation to a medical condition such as mucositis, it means that administration of the pharmaceutical composition or SOD of the present invention reduces the frequency or delays the onset and symptoms of the medical condition (e.g., weight loss, mucositis or ulcers, diarrhea, and high ROS levels in the gastrointestinal tract (e.g., small intestine) or in the blood) compared to a subject not receiving the pharmaceutical composition or SOD.

[0034] The term "administering" means providing an active ingredient to a subject to achieve a prophylactic or therapeutic objective (eg, prevention or treatment of mucositis).

[0035] The term "application" refers to any method of contacting an active ingredient with a biological surface (e.g., skin or mucosa) to achieve a prophylactic or therapeutic purpose in a subject (e.g., prevention or treatment of mucositis), thereby allowing the composition to be absorbed into the skin or mucosa.

[0036] mucositis The present invention is based, at least in part, on the discovery that administration of superoxide dismutase (SOD), particularly oral application or oral administration, is effective in preventing or treating mucositis. Thus, according to one embodiment of the present invention, there is provided a use of SOD for preventing or treating mucositis.

[0037] The term "mucositis" refers to a disease in which inflammation or ulcers develop in the mucous membranes of the oral cavity and digestive tract, from the mouth to the anus.

[0038] In one embodiment, mucositis may occur as a side effect of cancer treatment, including, for example, anti-cancer treatment (e.g., chemotherapy, radiation therapy, or a combination thereof). Chemotherapy and / or radiation therapy conditioning regimens, such as for hematopoietic stem cell transplantation (HSCT), may cause mucositis in many patients. Mucositis may also result from damage to the mucosa due to viral, fungal, or bacterial infections, such as Herpes simplex virus (HSV) or candida, and may also be caused by a variety of diseases or illnesses, such as systemic lupus erythematosus (SLE), neutropenic conditions, such as Behcet's disease (BD), and autoimmune diseases.

[0039] The radiotherapy means a method for preventing or treating a disease using radiation or a medical treatment using radiation, and includes any radiotherapy that may cause mucositis, either alone or in combination with chemotherapy. Additionally, chemotherapy means a method for preventing or treating a disease using a chemical agent or a medical treatment using a chemical agent, and includes any chemotherapy that may cause mucositis, either alone or in combination with radiotherapy.

[0040] In one embodiment, the radiotherapy and chemotherapy may be for the prevention or treatment of cancer, including lung cancer, breast cancer, prostate cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous and intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, female vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic and acute leukemia (e.g. , acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia or chronic lymphocytic leukemia), solid tumors of childhood, lymphocytic lymphoma, bladder cancer, kidney cancer, ureter cancer, renal pelvis cancer, neoplasia of the central nervous system (CNS), primary CNS lymphoma, tumor neovascularization, spinal axonal tumor, brain stem glioma, pituitary adenocarcinoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (e.g., cancer induced by asbestos).

[0041] The chemotherapy for preventing or treating the cancer may be chemotherapy using an anticancer drug, which may cause mucositis as a side effect. The use of the anticancer drug includes the use of the anticancer drug alone or in combination, and also includes the use of the anticancer drug together with other substances (e.g., drugs other than the anticancer drug, drugs that suppress the side effects of the anticancer drug, etc.) in sequential or reverse order to cause mucositis.

[0042] In one embodiment, the anti-cancer agent capable of causing mucositis may be, but is not limited to, a cytotoxic anti-cancer agent, a targeted anti-cancer agent, an immunological anti-cancer agent, or a combination thereof.

[0043] The cytotoxic anticancer drugs are drugs that attack rapidly differentiating cells, such as nucleoside analogues [e.g., Azacitidine, Capecitabine, Carmofur, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Fluorouracil, Gemcitabine, , Mercaptopurine, Nelarabine, Pentostatin, Tegafur, Tioguanine, etc.], antifolates [e.g. Methotrexate, Pemetrexed, Raltitrexed, etc.], antimetabolites [e.g. Hydroxycarbamide, etc.], Topoisomerase II) Inhibitors [e.g., Irinotecan, Topotecan, etc.], Anthracyclines [e.g., Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, etc.], Podophyllotoxins [e.g., Etoposide, Teniposide, etc.], Taxanes [e.g., Cabazitaxel, Docetaxel] , Paclitaxel, etc.], Vinca alkaloids [e.g. Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, etc.], Alkylating agents [e.g. Bendamustine, Busulfan, Carmustine, Chlorambucil, Chlormethine, thine, Cyclophosphamide, Dacarbazine, Fotemustine, Ifosfamide, Lomustine, Melphalan, Streptozotocin, Temozolomide, etc.], Platinum compounds [e.g. Carboplatin, Cisplatin, Nezaplatin, etc.] aplatin, oxaliplatin, etc.], or other drugs [e.g., altretamine, bleomycin, bortezomib, dactinomycin, estramustine, ixabepilone, mitomycin, procarbazine, etc.], but are not limited to these.

[0044] In addition, a targeted anticancer drug is a drug that targets a specific part of a cancer cell that is different from other cells (e.g., normal cells) and selectively attacks the cancer cell. For example, a monoclonal antibody [e.g., Alemtuzumab, Bevacizumab, Cetuximab, Denosumab, Gemtuzumab ozogamicin, Ibritumomab tucetan] is an anticancer drug.tiuxetan, Ipilimumab, Nivolumab, Ofatumumab, Panitumumab, Pembrolizumab, Pertuzumab, Rituximab, Tositumomab, Trastuzumab, etc.], tyrosine kinase inhibitors [e.g. Afatinib, Aflibercept, etc.] t), Axitinib, Bosutinib, Crizotinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Nilotinib, Pazopanib, Ponatinib, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib nitinib, vandetanib, etc.], mTOR inhibitors [e.g., everolimus, temsirolimus, etc.], retinoids [e.g., alitretinoin, bexarotene, isotretinoin, tamibarotene, tretinoin, etc.], immunomodulators [e.g., lenalidomide, pomalidomide, idomide, thalidomide, etc.), histone deacetylase inhibitors [e.g. panobinostat, romidepsin, valproate, vorinostat, etc.], or other drugs [e.g. anagrelide, arsenictrioxide, asparaginase, BCG vaccine, denileukin difitoxdiftitox, vemurafenib, etc., but are not limited to these.

[0045] In addition, the immune anticancer drug is a drug that attacks cancer cells using the body's immune system, and may be, for example, an immune checkpoint inhibitor [e.g., Atezolizumab, Ipilimumab, Avelumab, Nivolumab, Pembrolizumab, Durvalumab, etc.], an immune cell action enhancer [e.g., blinatumomab, etc.], or other drugs (e.g., Alemtuzumab, Ofatumumab, Elotuzumab, etc.), but is not limited thereto. One drug may be both a targeted anticancer drug and an immune anticancer drug.

[0046] In particular, and in one specific embodiment, the anti-cancer agent capable of inducing mucositis may be, but is not limited to, 5-fluorouracil (5-FU), cyclophosphamide (CPA), docetaxel, doxorubicin, vincristine, prednisone, etoposide, ifosfamide, methotrexate, paclitaxel, gemcitabine, vinorelbine, leucovorin, irinotecan, oxaliplatin, or combinations thereof.

[0047] In one embodiment, the mucositis may be, but is not limited to, oral mucositis (e.g., oral ulcers, recurrent oral ulceration, or pharyngeal mucositis), esophageal mucositis, gastrointestinal mucositis (e.g., intestinal inflammatory disease or intestinal ulcer disease), rectal mucositis, or a combination thereof.

[0048] In one embodiment, mucositis can cause one or more symptoms such as ulcers or inflammation, induction of pain, impaired food intake, diarrhea, weight loss, reduced ROS levels, bleeding, reduced epithelial thickness, erythema, etc. Specifically, mucositis can cause one or more symptoms selected from the group consisting of ulcers or inflammation, induction of pain, impaired food intake, diarrhea, weight loss, reduced ROS levels, bleeding, reduced epithelial thickness, and erythema.

[0049] In another embodiment, administration of SOD to a subject in need of prevention, amelioration, or treatment of mucositis can provide effects such as reduced food intake disorders, ulcer recovery, improved re-epithelialization, improved diarrhea symptoms, and improved intestinal villus atrophy in the subject. Specifically, administration of SOD can provide one or more effects selected from reduced food intake disorders, ulcer recovery, improved re-epithelialization, improved diarrhea symptoms, and improved intestinal villus atrophy in the subject. Furthermore, SOD can provide an effect of reducing the level of reactive oxygen species (ROS) in the gastrointestinal tract (e.g., small intestine) or blood of mucositis.

[0050] Superoxide dismutase (SOD) According to another embodiment of the present invention, there is provided an improved superoxide dismutase (SOD) that exhibits the effect of preventing, ameliorating or treating mucositis.

[0051] SOD is a superoxide dismutase (O 2 -) radical of general molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2SODs are enzymes that alternately catalyze the dismutation of reactive oxygen species (ROS) to oxidative stress, and SODs play 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 with superoxide dismutase activity.

[0052] In one embodiment of the present invention, the SOD may be manganese-binding type (Mn-SOD). Specifically, the SOD may be deamidated Mn-SOD. More specifically, the SOD may be one in which the amino acid residues at positions 73 and 136 are substituted with Asp based on SEQ ID NO: 2. Even more specifically, the SOD may comprise or consist of the amino acid sequence shown in SEQ ID NO: 4.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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 may be 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.

[0057] In another embodiment, the SOD may be derived from a recombinant strain comprising an expression vector as shown in FIG. 4. The SOD may also be derived from a recombinant strain produced by a method comprising the method as shown in FIG. 5. The recombinant strain may comprise a nucleotide sequence encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 4. The recombinant strain may also be a Bacillus sp. strain lacking the following genes: AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE, and Xpf. For a detailed process for producing the strain, see Example 1.

[0058] 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.

[0059] In one embodiment, SOD can be isolated or purified from various sources, including natural or recombinant hosts. For example, SOD having activity in preventing or treating mucositis 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 cultured at 25°C to 42°C for 1 to 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).

[0060] 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 recombinantly produced. In particular, the term "substantially free of cellular material" may include 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.

[0061] In another embodiment, it may be desirable to purify SOD by the following purification method, including but not limited to: Cultivating Bacillus amyloliquefaciens strain GF423, centrifuging the resulting culture medium to collect the culture supernatant. The supernatant fraction is pretreated by solid phase extraction, and then isolated and purified by chromatography. In this case, SOD may be purified using various types of chromatography. Preferably, hydrophobic interaction chromatography is used.

[0062] In one embodiment, the SOD may be contained in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof. In this case, the term "strain lysate" refers to a product obtained by culturing a strain and mechanically or chemically disrupting the strain, and may include any product that has been subjected to further processes such as extraction, dilution, concentration, purification, etc. The term "strain culture" refers to the culture liquid itself obtained by culturing a strain or its supernatant. The term "strain culture concentrate" refers to a product that has been purely separated from a strain culture by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or a culture concentrate obtained by ultrafiltration, concentration, etc. The term "strain culture extract" refers to a product extracted from the culture liquid 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.

[0063] According to one embodiment of the present invention, the SOD was administered to a subject in need of prevention or treatment of mucositis, and was confirmed to exhibit one or more of the following effects: reduction in food intake disorder in the stomach, recovery of ulcers, improvement in re-epithelialization, improvement in diarrhea symptoms, and improvement in intestinal villus atrophy. According to another embodiment, the SOD was orally applied or orally administered to a subject in need of prevention or treatment of mucositis, and exhibited the effect of reducing ROS (Reactive oxygen species) levels in the gastrointestinal tract (e.g., small intestine) or blood.

[0064] Dosage The present invention has been accomplished, at least in part, by confirming that the SOD of the present invention is effective in preventing or treating mucositis even when applied or administered orally at a low dose.

[0065] The SOD of the present invention may be administered in an amount of about 50 units / kg to about 2,500 units / kg / day, preferably about 125 units / kg / day to about 1,000 units / kg / day, more preferably about 125 units / kg / day to about 500 units / kg / day or about 250 units / kg / day to about 1,000 units / kg / day, and even more preferably about 250 units / kg / day, about 500 units / kg / day, or about 1,000 units / kg / day, based on the body weight of the subject. In particular, when the SOD of the present invention is orally applied, it can effectively exert a preventive or therapeutic effect on mucositis even if the oral dosage is reduced to half based on the body weight of the subject. For example, when SOD is applied intraorally, it may be applied in an amount of about 25 units / kg / day to about 1,250 units / kg / day, preferably about 125 units / kg / day to about 500 units / kg / day, and more preferably about 250 units / kg / day, about 500 units / kg / day, or about 1,000 units / kg / day.

[0066] Pharmaceutical Compositions According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating mucositis, comprising SOD as an active ingredient.

[0067] 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 mucositis. 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.

[0068] The pharmaceutical or veterinary composition of the present invention may further comprise one or more selected from the group consisting of pharma- ceutically acceptable carriers, excipients and diluents. The pharma- ceutically acceptable carriers, excipients and / or diluents may be those commonly used in the art. Examples of the carriers, excipients or diluents 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.

[0069] 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.

[0070] The pharmaceutical or veterinary compositions of the present invention may be formulated in a preferred 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, liquids, capsules, suspensions, emulsions, injections, plasters, lotions, liniments, limonades, aerosols, extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, patches, etc.

[0071] 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.

[0072] In one embodiment, the composition may be administered through any normal route that can reach the target in the body. For example, the composition may be administered through parenteral routes, including, but not limited to, oral application, oral administration, dermal, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, intrapulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, topical, sublingual, vaginal or rectal routes. For oral administration, the SOD may be coated with shellac for protection from gastric acid, but the coating agent is not limited thereto. Examples of coating agents suitable for use in the present invention include shellac, ethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose 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 solution containing shellac, and then freeze-dried. This freeze-dried sample becomes a powder, and can be stored at about 4°C until use. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be prepared by mixing the composite 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, oral liquids, emulsions, syrups, etc., and various excipients, such as wetting agents, sweeteners, flavorings, preservatives, etc., may be used in addition to water and liquid paraffin, which are commonly used simple diluents.

[0073] The compositions according to the invention may be applied as a liniment or administered as an injection, and may be administered by any device that allows the active ingredient to be delivered to target cells.

[0074] The pharmaceutical or veterinary composition of the present invention is administered in a pharma- ceutical or veterinary effective amount. The term "pharmaceutical effective amount" or "veterinarily effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical or veterinary treatment, and the effective dose level can be determined based on factors including the patient's or animal's body weight, sex, age, health condition, severity, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. Preferably, the pharmaceutical or veterinary composition of the present invention may contain SOD in an amount of about 2 to about 1,500 U / mg, specifically about 5 to about 1,000 U / mg, more specifically about 10 to about 800 U / mg, and even more specifically about 100 to about 500 U / mg, based on the total weight of the composition.

[0075] 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.

[0076] In one embodiment, the compositions of the invention may be administered together with radiation therapy and / or chemotherapy, either sequentially or in reverse order, in particular before, after, or both before and after radiation therapy and / or chemotherapy.

[0077] Treatment or prevention methods According to another embodiment of the invention, there is provided a method for the treatment or prevention of mucositis comprising the step of administering an SOD or composition according to the invention to a subject having or at risk of mucositis.

[0078] "Administration" in the present invention includes any of various administrations that enable prevention or treatment by performing the intended action. With regard to said administration, reference is made to the above description. For example, said method may include administering a therapeutically effective amount of the SOD or composition of the present invention to one or more gastrointestinal regions of a subject having or at risk of developing mucositis. Furthermore, said method may include applying a therapeutically effective amount of the SOD or composition of the present invention to the oral cavity of a subject having or at risk of developing oral mucositis.

[0079] 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 of the present invention may vary depending on factors such as the stage of the disease, the severity of the disease, the age, sex, medical complications, and weight of the subject.The dosage and administration regimen of SOD of the present invention may be adjusted to provide an optimal therapeutic response.For example, several divided doses may be administered daily, weekly, every two weeks, every three weeks, every four weeks, etc., or the dosage may be proportionally reduced or increased depending on the exigencies of the therapeutic situation.

[0080] The method may further include administering one or more other preparations for treating or preventing mucositis. The other preparations include, but are not limited to, any preparation that can improve or ameliorate mucositis, such as a compound, a gene therapy agent, a protein (including an antibody), etc. The one or more other preparations may be administered simultaneously, sequentially, or in reverse order with the SOD or pharmaceutical composition. Also, the individual components may be administered to the subject by the same or different routes.

[0081] However, in one embodiment, the method may include administering the SOD or composition of the invention to the subject before, after, or both before and after the subject undergoes radiation therapy.

[0082] In other embodiments, the methods may include administering the SOD or composition of the invention to the subject before, after, or both before and after the subject undergoes chemotherapy.

[0083] In other embodiments, the methods may include administering an SOD or composition of the invention to a subject undergoing anti-cancer treatment.

[0084] In this case, the SOD or composition of the present invention may be used in combination with chemotherapy (eg, administration of the anticancer agent) and / or radiation therapy, regardless of the order and number of times.

[0085] Food Composition According to another embodiment of the present invention, there is provided a food composition for preventing or improving mucositis, comprising the SOD according to the present invention as an active ingredient. Such a food composition includes a medical or nutraceutical food composition.

[0086] 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.

[0087] 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.

[0088] 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 mucositis. 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.

[0089] 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. %, preferably about 0.01 to about 50 wt. %, based on the solid content weight, but is not limited thereto.

[0090] 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 1,000 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.

[0091] Feed composition According to another embodiment of the present invention, there is provided a feed composition for preventing or improving mucositis, which comprises the SOD according to the present invention as an active ingredient. In the present invention, the food composition may comprise 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 comprise 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.

[0092] 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 calcium carbonate, powdered powder, zeolite, flour, or rice bran).

[0093] kit According to another embodiment of the present invention, there is provided a kit for preventing, ameliorating or treating mucositis, comprising the SOD or composition of the present invention.

[0094] In one embodiment, the kit may further include instructions for use of the SOD or composition of the present invention. The instructions may include instructions for taking or administering the active ingredient. For example, the instructions may include instructions for taking or administering the active ingredient in combination with radiotherapy and / or chemotherapy, or in combination with an anticancer drug. Specifically, the instructions may include instructions regarding the timing, order, number of times, dosage, etc. of administration in combination with radiotherapy and / or chemotherapy, or in combination with an anticancer drug.

[0095] 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. EXAMPLES

[0096] Example 1. Construction of a recombinant strain expressing superoxide dismutase (SodA2) Example 1.1. Preparation 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.

[0097] [Table 1]

[0098] 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 sequences flanking the target gene 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.

[0099] [Table 2]

[0100] [Table 3-1] [Table 3-2]

[0101] 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, which were then subjected to a second pure isolation to select the final recombinant, which was named GFBS220.

[0102] 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.

[0103] [Table 4]

[0104] 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).

[0105] 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.

[0106] [Table 5]

[0107] The amplified DNA fragment containing sodA2 gene and plasmid pBE1 linearized by NdeI and HindIII digestion 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 pBE1-sodA2 thus generated is shown in Figure 4.

[0108] Example 1.3. Preparation 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 a glycerol stock method. The defective gene was confirmed from this production strain BSBA310 using PCR, and the results are shown in Figure 2. Also, the entire procedure for producing the production strain BSBA310 is summarized in Figure 5.

[0109] 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 cultivated at 37°C for 12 hours. This seed culture was again diluted with 1 mM manganese sulfate (MnSO 4 The strain was inoculated into 3 L of LB medium containing 100% ethanol and cultured at 37° C. for 20 hours.

[0110] Example 2.2. Isolation and purification of SodA2 The cell culture obtained in Example 2.1 was centrifuged at 4°C and 3,578xg for 20 minutes to collect the supernatant, which was then concentrated 10-fold using ultrafiltration (UF, MWCO 10,000). 390g of ammonium sulfate was added per 1L 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 carried out by equilibrating the column using 50mM potassium phosphate pH 7.0 with a concentration of 2M ammonium sulfate, then adding ammonium sulfate to pass the resulting supernatant through the column, and recovering the SodA2 attached to the column using 50mM potassium phosphate pH 7.0 containing 1.6M 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 1,000-1,500 U / mg.

[0111] Example 3. Preparation of dosage materials For the oral intragastric administration group, shellac (EXCELACS co., LTD., Bangkok) was dissolved in ethanol to a concentration of 3% and sterilized using a 0.2um sterile filter. The shellac dissolved in ethanol was diluted with PBS. Freeze-dried SodA2 was dissolved at 20mg / mL, and the shellac solution and SOD solution were mixed in a 1:1 ratio and freeze-dried. The freeze-dried shellac-coated SodA2 was mixed with dextrin in a ratio of 1:9-12 (shellac-coated SodA2:dextrin) to produce the substance used in the animal test. The produced SOD activity was adjusted to 90-110U / mg. For the intragastric administration group, the final dilution was 10U / mg.

[0112] For the oral application group, SodA2 was suspended in 2% methylcellulose (Sigma) gel and used. The SOD activity in the oral application group was adjusted to 100 U / mg.

[0113] Example 4. Preparation of mucositis model and administration of SodA2 To prepare the mucositis model, 6-week-old Sprague Dawley rats (purchased from Orient Bio) were purchased, purified for one week, and then randomly divided into eight groups. The eight groups were as follows: G1 (control group), G2 (disease group), oral application groups (G3, G4, G5), and oral intragastric administration groups (G6, G7, G8). See Table 6 for the test groups, administration concentrations, and animal numbers for the mucositis model. Mucositis was induced by intraperitoneally administering 5-fluorouracil (purchased from Sigma, 100 mg / kg) twice on the first and third days, and anesthetizing with ketamine (purchased from Yuhan Corporation, 40 mg / kg) on ​​the fifth day. The left oral mucosa was scratched with an 18-gauge needle to induce chronic irritation that occurs during chewing movements in patients undergoing anticancer treatment, inducing mechanical ulcer lesions. The control group, G1, underwent a sham operation.

[0114] [Table 6]

[0115] For the oral application group, the test substance was suspended and prepared using 2% methylcellulose gel as a vehicle, and 100 ul was applied using a micropipette to ensure that the entire drug was applied to the oral mucosa. For the intragastric administration group, the test substance was orally administered at a volume of 10 mL / kg using PBS as a vehicle. The administration period was 10 days, from the completion of mechanical surgery to induce oral mucositis until the ulcers in the normal group were sutured, and the drug was administered once a day at around 10:00 a.m. G1 (control group) and G2 (disease group) were administered only the vehicle.

[0116] Example 5. Measurement of body weight and food intake To evaluate the energy absorption disorder due to the induction of oral and gastrointestinal mucositis, body weight and food intake were measured at 2-3 day intervals. The body weight was measured before feeding the cage the previous day, and the amount of food remaining the next day was measured to calculate the intake, which was then divided by the number of individuals in the cage to calculate the average food intake.

[0117] In the normal group G1, body weight and food intake steadily increased for 10 days. On the other hand, in the cases of G2 to G8 in which mucositis was induced, food intake was significantly decreased compared to G1, and body weight increased for a while in the early stages, but then showed a continuous decrease. On the other hand, in the drug-treated groups (G3 to G8), body weight and food intake increased compared to G2, and in particular, body weight increased statistically significantly in the medium and high concentration SodA2 treated groups G5, G7, and G8, and food intake increased statistically significantly in G5 and G8, which had high concentrations of SodA2. Graphs of body weight and food intake of the test groups are shown in Figures 6a and 6b.

[0118] Example 6. Scoring and histological evaluation of oral mucositis Scoring of oral mucositis The ulcer sites were observed at 3-4 day intervals immediately after mechanical ulceration, and oral mucositis was scored according to the criteria in Table 7. The scoring criteria are based on the literature (Araujo et al. PLoS One 2015, 10: e0116799).

[0119] [Table 7]

[0120] For oral mucositis, the degree of re-epithelialization of the ulcer site was analyzed for 10 days after mechanical ulcer induction. In the normal G1 group, the ulcer site recovered quickly, and re-epithelialization was completed by the 10th day, with only minor traces of the wound site being observed. On the other hand, in the G2 group, 5-FU inhibited epithelial cell differentiation at the ulcer site, with little re-epithelialization occurring, and in some individuals, the ulcer site appeared to become larger due to continuous chewing stimulation. In the drug-administered groups, re-epithelialization was promoted in a concentration-dependent manner, and the ulcer site was sutured (Figure 7a).

[0121] Histological evaluation of oral mucositis Tissues including the oral mucosa were collected and fixed in 10% formalin, then processed through standard tissue processing to prepare H&E (hematoxylin and eosin) stained slides. The lesions caused by mucositis were then examined under a microscope to evaluate the degree of re-epithelialization of the mucositis area and analyze the thickness of the oral mucosal epithelium.

[0122] In the case of G1, all of the sites where ulcerative mucositis was induced had completed re-epithelialization, and mild swelling of the tissue due to proliferation of granulation tissue was observed in the submucosal area. Although some individuals had mild inflammation, most of the sites had completed healing and showed normal structure (lower left side of Figure 7b). In the case of G2, no re-epithelialization occurred in all of the sites where ulcerative mucositis was induced, and calcification, necrosis, proliferation of granulation tissue (infiltration of inflammatory cells), etc. were observed. In the cases of G3 and G6 (low concentration SodA2 administration groups), there was no significant difference from the G2 group, and severe ulcerative mucositis sites were observed. On the other hand, in the cases of G4, G5, G7, and G8 (medium and high concentration SodA2 administration groups), re-epithelialization occurred, the size of the ulcer was reduced, and inflammation and necrosis in the submucosal area also gradually decreased (Figure 7b). Quantitative analysis of the degree of re-epithelialization showed that, similar to the appearance of the oral mucositis scoring graph, the normal group G1 showed a significantly high degree of re-epithelialization, while G2 showed almost no re-epithelialization. In the drug-treated groups, re-epithelialization was promoted in a concentration-dependent manner, and in the high-concentration SodA2 groups G4, G5, G7, and G8, re-epithelialization was statistically significantly increased compared to G2 (Figure 7c).

[0123] Example 7. Intestinal mucositis scoring and histological evaluation Scoring of intestinal mucositis During the 10-day test period, the anal area and feces were visually observed once a day, and the severity of diarrhea was evaluated using the scoring method shown in Table 8 below.

[0124] [Table 8]

[0125] In the case of G1, no diarrhea occurred during the 10-day test period, but in the 5-FU-treated groups (G2 to G8), diarrhea was observed from the start of drug administration, and severe wet diarrhea was maintained for 5 days after drug administration, after which diarrhea gradually improved over the next 5 days. In particular, in the medium and high concentration SodA2 treated groups G4, G5, G7, and G8, diarrhea scores were statistically significantly reduced compared to G2 (Figure 8a).

[0126] Histological evaluation of intestinal mucositis Tissues containing the intestinal mucosa were collected and fixed in 10% formalin, then standard tissue processing procedures were used to prepare slides stained with H&E (hematoxylin and eosin).The lesions caused by mucositis were then examined under a microscope to evaluate changes in the intestinal villi at the mucositis sites, and the length of the intestinal villi was analyzed.

[0127] In the case of G1, normal small intestinal anatomical histological structures such as villi and crypts were observed. On the other hand, in the case of G2, severe atrophy of villus was characteristically observed, and it was found that diarrhea and weight loss occurred due to malabsorption. In the drug-treated groups (G3 to G8), the improvement of villus atrophy was clearly observed in a concentration-dependent manner, and the infiltration of inflammatory cells was mild and did not show any difference between the groups (Figure 8b). Quantitative analysis of the length of the intestinal villi also showed that the atrophy of the villus occurred in G2 compared to G1, and the length of the villus was reduced. In the drug-treated groups (G3 to G8), the improvement of the atrophy of the villus was significantly observed in a concentration-dependent manner compared to G2. In particular, in the cases of G5, G7, and G8, the length of the intestinal villi was found to increase significantly (Figure 8c).

[0128] Example 8. Evaluation of ROS in oral and intestinal mucositis Assessment of ROS in the small intestine After autopsy, intestinal tissue was collected, 100 mg of intestinal tissue was washed twice with PBS, and 1 mL of ice-cold 50 mM TBS (pH 7.4) containing a protease inhibitor mixture was added, followed by disruption of the tissue using a bead homogenizer, centrifugation at 12,000 rpm for 10 min at 4°C, and the supernatant was saved. The tissue homogenate was diluted with ice-cold lock buffer (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 2.0 mM CaCl2, 10 mM D-glucose and 5 mM HEPES, pH 7.4) to a final concentration of 5 mg tissue / mL. Locke's buffer (pH 7.4), 0.2 mL of the homogenate, and 10 μL of DCFH-DA (purchased from Sigma, 5 μM) were then mixed, and the resulting reaction mixture was incubated for 45 min. DCF fluorescence was measured using a spectrofluorometer (Excitation: 484 nm, Emission: 530 nm). The blank was corrected for the background fluorescence of the homogenate without DCFH-DA.

[0129] Analysis of ROS levels in small intestinal tissues using DCFH-DA showed that ROS levels in G2 were more than twice as high as those in G1. In the drug-administered groups, both the oral application group (G3-G5) and the intragastric administration group (G6-G8) showed a concentration-dependent decrease compared to G2 (Figure 9a). In other words, administration of SodA2 in 5-FU-induced mucositis was confirmed to have a concentration-dependent effect of decreasing ROS levels in both the oral application group and the intragastric administration group.

[0130] Assessment of blood ROS Blood ROS levels were measured using luminol-amplified chemiluminescence signal. After dispensing 40 μL of plasma into a 96-well plate, the baseline value was measured using a chemiluminescence spectrum analyzer. Then, 200 μL of luminol (purchased from Sigma, 200 mM, pH 7.4) was added to the 96-well plate, followed by incubation for 10 min. Luminescence values ​​were measured at 1-min intervals and corrected to the baseline value.

[0131] Analysis of blood ROS levels by luminol luminescence showed that luminol luminescence in G2 was approximately 0.5 times higher than that in G1. In the drug-administered groups, both the oral administration (G3-G5) and intragastric administration (G6-G8) groups showed a concentration-dependent decrease compared to G2. In particular, G5, G7, and G8 showed a statistically significant decrease 6 minutes after luminol addition, G5, G7, and G8 at 7 minutes, G5 and G8 at 8-9 minutes, and G8 at 10 minutes (Figure 9b).

[0132] Example 9. Statistical Analysis All numerical data were statistically analyzed using the GraphPad Prism statistical program (Ver. 8.0). First, Levene's test was performed to assess homogeneity of variance, followed by one-way ANOVA test to confirm the significance between each test group. If the significance between the test groups was confirmed as a result of the one-way ANOVA, Tukey's post-hoc test was performed depending on the presence or absence of homogeneity of variance. All data were presented as Mean ± SEM, and confidence intervals were set at 95%.

[0133] Example 10. Conclusions and Observations After inducing oral and intestinal mucositis simultaneously by intraperitoneal administration of 5-FU anticancer drug, SodA2 was applied to the oral cavity and administered intragastrically (orally) for 10 days. As a result, it was confirmed that SodA2 has the effect of promoting re-epithelialization of ulcerated areas of the oral mucosa, improving diarrhea and atrophy of intestinal villi in a concentration-dependent manner. It was also confirmed that it has the effect of reducing ROS levels in intestinal tissue and blood where mucositis has occurred, improving symptoms. It was confirmed that there was no significant difference depending on the route of drug administration.

[0134] In conclusion, it has been confirmed that SodA2 (Bacillus-derived SOD enzyme) is a useful material as an inhibitor and therapeutic agent for oral and intestinal mucositis caused by side effects of anticancer drugs.

Claims

1. A pharmaceutical composition for treating or preventing mucositis, comprising a Bacillus-derived superoxide dismutase (SOD) as an active ingredient.

2. The pharmaceutical composition of claim 1 , wherein the mucositis is caused by chemotherapy, radiation therapy, or a combination thereof.

3. 2. The pharmaceutical composition of claim 1, wherein the mucositis comprises oral mucositis, esophageal mucositis, gastrointestinal mucositis, rectal mucositis, or a combination thereof.

4. 2. The pharmaceutical composition of claim 1, wherein the SOD provides one or more effects selected from the following: reduction in food intake disorders, ulcer healing, improvement in re-epithelialization, improvement in diarrhea symptoms, improvement in intestinal villus atrophy, and reduction in reactive oxygen species (ROS) levels in the gastrointestinal tract or blood.

5. The pharmaceutical composition of claim 1 , wherein the composition is administered to a subject before the subject undergoes radiation therapy, after the subject undergoes radiation therapy, or both before and after the subject undergoes radiation therapy.

6. The pharmaceutical composition of claim 1 , wherein the composition is administered to a subject before the subject undergoes chemotherapy, after the subject undergoes chemotherapy, or both before and after the subject undergoes chemotherapy.

7. The pharmaceutical composition of claim 1 , wherein the composition is administered to a subject undergoing anti-cancer treatment.

8. The pharmaceutical composition according to claim 6, wherein the anti-cancer agent used in the chemotherapy comprises one or more selected from a cytotoxic anti-cancer agent, a targeted anti-cancer agent, an immune anti-cancer agent, or a combination thereof.

9. The pharmaceutical composition according to claim 1 , wherein the SOD is Mn-SOD derived from Bacillus.

10. The pharmaceutical composition of claim 1 , wherein the SOD comprises the amino acid sequence shown in SEQ ID NO:

4.

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

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

13. 2. The pharmaceutical composition of claim 1, wherein the SOD is in the form of a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract or a dried form thereof.

14. The pharmaceutical composition according to claim 1 , wherein the SOD is applied to the oral cavity or administered orally.

15. 2. The pharmaceutical composition according to claim 1, wherein the SOD is for administration or application in an amount of about 50 units / kg to about 2,500 units / kg / day based on the body weight of the subject.

16. 10. The pharmaceutical composition of claim 1, wherein the SOD is for administration or application in an amount of about 125 units / kg / day to about 1,000 units / kg / day based on the subject's body weight.

17. A veterinary composition for the treatment or prevention of mucositis, comprising a Bacillus-derived superoxide dismutase as an active ingredient.

18. A food composition for treating or preventing mucositis, comprising a Bacillus-derived superoxide dismutase as an active ingredient.

19. 20. A kit for treating or preventing mucositis comprising a composition according to any one of claims 1 to 18 and instructions including directions for taking or administering said composition.

20. The kit of claim 19 , wherein the instructions include instructions for use in combination with radiation therapy and / or chemotherapy, or in combination with an anti-cancer drug.

21. 20. A method for preventing or treating mucositis comprising administering to a subject a composition or superoxide dismutase according to any one of claims 1 to 18.

22. 20. Use of a composition or superoxide dismutase according to any one of claims 1 to 18 for the prevention or treatment of mucositis.

23. 20. Use of a composition or superoxide dismutase according to any one of claims 1 to 18 for the manufacture of a medicament for the prevention or treatment of mucositis.

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