Superoxide dismutase and use thereof in prevention or treatment of mucositis

Abstract

The present invention relates to superoxide dismutase and its use in the prevention or treatment of mucositis. The superoxide dismutase according to the present invention can be effectively used for improving or treating proliferation inhibition of mucosal epithelial cells which are side effects of anti-cancer drugs or radiotherapy.

Description

Technical Field

[0001] The present invention relates to the use of superoxide dismutase in preventing or treating mucositis. More specifically, the present invention relates to superoxide dismutase useful in preventing or treating mucositis, and its use, composition or method in preventing or treating mucositis. Background Art

[0002] Mucositis is a pathological condition characterized by mucosal damage caused by mild inflammation or even ulceration. Mucositis is one of the common side effects of anticancer chemotherapy and radiotherapy, and it often occurs in the oral cavity, but it is also prone to occur in the small intestine, esophagus, stomach, and large intestine. 40% of patients receiving anticancer chemotherapy will develop mucositis, and the incidence rate of head and neck cancer patients after chemotherapy and radiotherapy increases to about 90%. Several treatments have been proposed, but their efficacy is unclear (Oral mucositis: the hidden side of cancer therapy.); (J Exp Clin Cancer Res 39, 210 (2020); https: / / www.ons.org / pep / mucositis). In the past, the main side effects of anticancer treatment were vomiting symptoms and decreased immunity due to decreased bone marrow function, but recently, mucositis has been recognized as one of the serious side effects of cancer patients during treatment. The epithelial cells of the oral and gastrointestinal mucosa are the fastest growing tissues in the body, especially the small intestine, which has the fastest renewal cycle, about every seven days. Anticancer chemotherapy or radiotherapy interferes with the regeneration of mucosal cells, and malnutrition of patients further hinders cell regeneration. Radiation-induced intestinal mucosal damage is believed to be caused by microvascular damage caused by apoptosis of vascular endothelial cells, and it is speculated that vascular endothelial cells and platelets may play a role in the pathogenesis of mucositis.

[0003] For most patients, mucositis may induce severe pain, increase the risk of systemic infection, and reduce nutritional intake. For some patients, mucositis may lead to the inability to receive appropriate doses of anticancer therapy, resulting in a decrease in the willingness to receive treatment, etc., which seriously reduces the patient's quality of life. Although research on therapeutic agents for oral mucositis is actively ongoing to date, new methods for preventing or treating oral mucositis and gastrointestinal mucositis are still needed. Summary of the invention

[0004] Technical issues

[0005] The purpose of the present invention is to solve the above problems existing in the prior art.

[0006] Another object of the present invention is to provide superoxide dismutase for preventing or treating mucositis.

[0007] Another object of the present invention is to provide a superoxide dismutase derived from Bacillus that ensures oral administration efficacy and safety for preventing or treating mucositis.

[0008] Another object of the present invention is to provide superoxide dismutase derived from Bacillus amyloliques faciens, which ensures oral administration efficacy and safety for preventing or treating mucositis.

[0009] Another object of the present invention is to provide a superoxide dismutase derived from Bacillus or Bacillus amyloliquefaciens, which is secreted outside the cell and is easy to produce, for use in preventing or treating mucositis.

[0010] Still another object of the present invention is to provide a pharmaceutical composition or method for preventing or treating mucositis.

[0011] Still another object of the present invention is to provide a food composition for preventing or improving mucositis.

[0012] Yet another object of the present invention is to provide compositions and methods for preventing or treating mucositis that may occur during anti-cancer treatment.

[0013] The object of the present invention is not limited to the above-mentioned object. The object of the present invention will become more apparent from the following description and can be achieved by the means described in the claims and their combination.

[0014] Technical Solution

[0015] Representative embodiments of the present invention for achieving the above-mentioned object are as follows.

[0016] According to one aspect of the present invention, an improved superoxide dismutase (SOD) is provided, which exhibits the effect of preventing, improving or treating mucositis.

[0017] According to another aspect of the present invention, there is provided a superoxide dismutase for preventing, ameliorating or treating mucositis, wherein the superoxide dismutase is derived from generally regarded as safe (GRAS) bacteria, such as Bacillus.

[0018] According to yet another aspect of the present invention, there is provided a superoxide dismutase derived from Bacillus amyloliques faciens which ensures oral administration efficacy and safety for preventing, improving or treating mucositis.

[0019] According to yet another aspect of the present invention, there is provided a superoxide dismutase derived from Bacillus or Bacillus amyloliquefaciens, which is secreted outside the cell to facilitate production, for use in preventing or treating mucositis.

[0020] According to yet another aspect of the present invention, there is provided an improved superoxide dismutase which exhibits an effect of preventing, improving or treating mucositis in a subject receiving radiotherapy and / or chemotherapy.

[0021] According to yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating mucositis, comprising the superoxide dismutase as an effective ingredient.

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

[0023] According to yet another aspect of the present invention, there is provided a kit for treating or preventing mucositis, comprising: a composition comprising the superoxide dismutase as an active ingredient; and instructions for the composition, including instructions for taking or applying the composition.

[0024] According to yet another aspect of the present invention, a method for preventing, improving or treating mucositis is provided, comprising the following step: administering the superoxide dismutase or the composition to a subject.

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

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

[0027] Beneficial Effects

[0028] The superoxide dismutase, composition, method and kit according to the present invention can be effectively used for preventing or treating mucositis. In particular, it has been confirmed that the superoxide dismutase according to the present invention can effectively prevent or treat mucositis even at a low dose when applied orally orally.

[0029] The superoxide dismutase according to the present invention is derived from Bacillus or Bacillus amyloliquefaciens, which are generally regarded as safe (GRAS) bacteria, and thus not only ensures oral administration efficacy and safety, but also has a production advantage in that it can be directly recovered from the supernatant during culture.

[0030] According to a specific embodiment of the present invention, after mucositis is induced in experimental animals (rats), SOD of the present invention is applied in different concentrations by oral coating and oral administration, and body weight and food intake are measured. The results confirm that the body weight and food intake reduced by mucositis in the SOD administration group are increased again compared to the SOD non-administered group. In addition, it is observed that the degree of improvement of re-epithelialization and villous atrophy is related to the SOD concentration in oral and intestinal mucositis, and it is confirmed that the level of reactive oxygen species (ROS; Reactive oxygen species) in the gastrointestinal tract (e.g., small intestine) and blood is 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 ROS levels and the improvement of epithelial re-epithelialization or atrophic villous cells of epithelial cells, and normal nutrient intake can be achieved, and the effect of weight gain can also be expected. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the double crossover recombination performed in Example 1.1 is shown.

[0032] Figure 2 The results of confirming the defective gene using PCR in the expression host GFBS220 and the production strain BSBA310 are shown. W indicates wild-type B. subtilis KCTC 3135, 220 indicates the expression host GFBS220, and 310 indicates the SodA2 production strain BSBA310.

[0033] Figure 3 Shown are the results of a comparison of the amino acid sequences of SodA (Mn-SOD determined by N-terminal sequencing in the culture supernatant of Bacillus amyloliquefaciens strain GF423) and the superoxide dismutase of the present invention (SodA2).

[0034] Figure 4 The expression vector for sodA2 gene overexpression is shown. Among them, rrnB T1T2 represents a transcription terminator; rep(pBR322) represents a replicon from pBR322 that works in E. coli; rep(pUB110) represents a replicon from pUB110 that works in Bacillus subtilis; Kan Rrepresents the kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); BJ27 promoter represents the strong promoter of Bacillus subtilis (B. subtilis).

[0035] Figure 5 The overall scheme used to prepare the production strain BSBA310 is shown.

[0036] Figure 6a and Figure 6b Shown are the results of measurements of body weight and food intake in an animal model of mucositis.

[0037] Figures 7a to 7c Results of evaluation of oral mucositis in a mucositis animal model are shown. Figure 7a The oral mucositis scoring results are shown. Figure 7b The results of oral mucositis tissue assessment are shown. Figure 7c The thickness of the re-epithelialized oral mucosal epithelium was quantified.

[0038] Figures 8a to 8c Results of evaluation of intestinal mucositis in a mucositis animal model are shown. Figure 8a The intestinal mucositis score results are shown. Figure 8b Results of histological assessment of intestinal mucositis are shown. Figure 8c The length of intestinal villi was quantified.

[0039] Figure 9a and Figure 9b Results of ROS assessment in the intestine and blood are shown. DETAILED DESCRIPTION

[0040] In the detailed description of the invention set forth below, an embodiment that can implement the invention will be described with reference to specific drawings, but the invention is not limited thereto, and if properly described, the scope of the invention depends on the appended claims including those claimed in the claims and all equivalents. It should be understood that the various embodiments of the invention, although different from each other, are not necessarily mutually exclusive. For example, without departing from the spirit and scope of the invention, the specific shapes, structures and characteristics described herein can be realized by changing from one embodiment to another or by a combination of embodiments. Unless otherwise specified, the terms mentioned when describing the present invention should generally be understood as the usual meaning, and are applicable to both the aspects or embodiments of the invention that define the term, and the same terms used herein. The definitions described below are provided for the purpose of explaining the present invention, and the terms used in the singular will include the plural form where appropriate, and vice versa.

[0041] definition

[0042] The term "subject" is used interchangeably with "patient" and refers to a mammal that needs to prevent or treat mucositis, or receive radiotherapy and / or chemotherapy, for example, primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), livestock animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and experimental animals (e.g., rats, guinea pigs, etc.). In one embodiment of the present invention, the subject is a human.

[0043] The term "treatment" includes preventive and / or therapeutic treatments. The preventive and / or therapeutic treatments include all types of treatments recognized in the art, for example, including administering the composition of the present invention or SOD to the subject. If administered before the clinical manifestation of an undesirable or unpreferred state (e.g., a disease or other undesirable or unpreferred state of the subject), such treatment or treatment is a preventive treatment or treatment (e.g., protecting the subject from the progression of the undesirable or unpreferred state of the subject). In addition, if administered after the clinical manifestation of an undesirable or unpreferred state, such treatment or treatment is a therapeutic treatment that alleviates, relieves or stabilizes the existing undesirable or unpreferred state or its side effects.

[0044] The meaning of the term "prevention" is well known in the art. When used in relation to medical symptoms such as mucositis, it means that by administering the pharmaceutical composition or SOD of the present invention, the frequency of medical symptoms (such as weight loss, mucosal inflammation or ulcers, diarrhea, and high levels of ROS in the gastrointestinal tract (such as the small intestine) or blood) is reduced or the onset and symptoms are delayed compared to subjects not administered with it.

[0045] The term "administering" refers to providing an active ingredient to a subject for prophylactic or therapeutic purposes (eg, preventing or treating mucositis).

[0046] The term "application" refers to all methods of contacting the active ingredient with the biological surface of the subject (e.g., skin or mucosa) for preventive or therapeutic purposes (e.g., prevention or treatment of mucositis), thereby achieving the purpose of the composition being absorbed into the skin or mucosa.

[0047] Mucositis

[0048] 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 aspect of the present invention, there is provided a use of SOD in preventing or treating mucositis.

[0049] The term "mucositis" refers to a disease involving inflammation or ulceration of the lining of the oral cavity and digestive tract from the mouth to the anus.

[0050] In one embodiment, mucositis may be caused by side effects of cancer treatments including, for example, anticancer treatments (e.g., chemotherapy, radiotherapy, or combinations thereof). The conditioning therapy for chemotherapy and / or radiotherapy for hematopoietic stem cell transplantation (HSCT) etc. may cause mucositis in most patients. In addition, mucositis may also be caused by mucosal damage caused by infection with viruses, fungi or bacteria (e.g., herpes simplex virus (HSV) or Candida), or by a variety of conditions or diseases such as decreased neutrophil levels and autoimmune diseases such as systemic lupus erythematosus (SLE), Behcet's disease (BD), etc.

[0051] The radiotherapy refers to a method of preventing or treating a disease using radiation, or a medical treatment method using radiation, including any radiotherapy that causes mucositis when performed alone or in combination with chemotherapy. In addition, chemotherapy refers to a method of preventing or treating a disease using chemical drugs, or a medical treatment method using chemical drugs, including any chemotherapy that causes mucositis when performed alone or in combination with radiotherapy.

[0052] In one embodiment, the radiotherapy and chemotherapy can be used to prevent or treat cancer, and the cancer can be selected from lung cancer, breast cancer, prostate cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin and intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine 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 (adrenal cancer), ovarian cancer, uterine 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 (parathyroid cancer), adrenal cancer (adrenal cancer), soft tissue sarcomas, urethral cancer, penile cancer, chronic and acute leukemias (e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia), solid tumors in children, lymphocytic lymphomas, bladder cancer, kidney cancer, ureteral cancer, renal pelvic cancer, neoplasia of the central nervous system (CNS), primary CNS lymphomas, neovascularization in tumors, spinal cord axis tumors, brain stem gliomas, pituitary adenocarcinomas, Kaposi's sarcoma, epidermoid carcinomas, squamous cell carcinomas, T-cell lymphomas, environmentally induced cancers (e.g., cancers caused by asbestos), but are not limited to these.

[0053] The chemotherapy used to prevent or treat the cancer may be chemotherapy using anticancer drugs, which may induce mucositis as a side effect. The use of anticancer drugs includes using anticancer drugs alone or in combination, and may include administering anticancer drugs with other substances (e.g., drugs other than anticancer drugs, drugs that inhibit side effects of anticancer drugs, etc.) simultaneously, sequentially, or in reverse order, thereby inducing mucositis.

[0054] In one embodiment, the anticancer drug that can cause mucositis can be a cytotoxic anticancer drug, a targeted anticancer drug, an immune anticancer drug, or a combination thereof, but is not limited thereto.

[0055] The cytotoxic anticancer drug is a drug that attacks rapidly differentiating cells, and examples thereof include: nucleoside analogs, such as Azacitidine, Capecitabine, Carmofur, Cladribine, Clofarabine, Cytarabine, Decitabine, Floxuridine, Fludarabine, Fluorouracil, Gemcitabine, Mercaptopurine, Nelarabine, Pentostatin, Tegafur, Tioguanine, etc.; folic acid antagonists, such as Methotrexate, Pemetrexed, Raltitrexed, etc.; antimetabolites, such as hydroxycarbamide, etc.; topoisomerase I inhibitors, such as irinotecan, topotecan, etc.; anthracyclines, such as daunorubicin, doxorubicin, epirubicin, idarubicin, etc.; podophyllotoxin, such as etoposide, teniposide, etc.; taxanes, such as cabazitaxel, docetaxel, paclitaxel, etc.; vinblastine, such as daunorubicin, doxorubicin, epirubicin, idarubicin, etc. alkaloids, such as Vinblastine, Vincristine, Vindesine, Vinflunine, Vinorelbine, etc.;Alkylating agents, such as Bendamustine, Busulfan, Carmustine, Chlorambucil, Chlormethine, Cyclophosphamide, Dacarbazine, Fotemustine, Ifosfamide, Lomustine, Melphalan, Streptozotocin, Temozolomide, etc.; Platinum compounds, such as Carboplatin, Cisplatin, Nedaplatin, Oxaliplatin, etc. in), etc.; or other drugs, such as altretamine, bleomycin, bortezomib, dactinomycin, estramustine, ixabepilone, mitomycin, procarbazine, etc., but not limited thereto. ;

[0056] In addition, targeted anticancer drugs are drugs that selectively attack cancer cells by targeting specific parts of cancer cells that are different from other cells (such as normal cells), and examples include monoclonal antibodies, such as alemtuzumab, bevacizumab, cetuximab, denosumab, gemtuzumab ozogamicin, ibritumomab tiuxetan tiuxetan), ipilimumab, nivolumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, tositumomab, trastuzumab, etc.; tyrosine kinase inhibitors, such as afatinib, aflibercept, acitretinib, Axitinib, Bosutinib, Crizotinib, Erlotinib, Gefitinib, Imatinib, Lapatinib, Nilotinib, Pazopanib, Ponatinib, Regorafenib, Ruxolitinib, Sorafenib, Sunitinib nib), Vandetanib, etc.; mTOR inhibitors, such as Everolimus, Temsirolimus, etc.; retinoids, such as Alitretinoin, Bexarotene, Isotretinoin, Tamibarotene, Tretinoin, etc.; immunomodulators, such as Lenalidomide, Pomalido mide, Thalidomide, etc.; histone deacetylase inhibitors, such as Panobinostat, Ro midepsin, Valproate, Vorinostat, etc.;or other drugs, such as anagrelide, arsenic trioxide, asparaginase, BCG, denileukindiftitox, vemurafenib, etc., but not limited thereto. ;

[0057] In addition, immune anticancer drugs are drugs that use the human immune system to attack cancer cells, and examples include: immune checkpoint inhibitors, such as Atezolizumab, Ipilimumab, Avelumab, Nivolumab, Pembrolizumab, Durvalumab, etc.; immune cell action enhancers, such as blinatu momab, etc.; or other drugs, such as Alemtuzumab, Ofatumumab, Elotuzumab, etc., but not limited to these. A drug can be both a targeted anticancer drug and an immune anticancer drug.

[0058] In addition, in a specific embodiment, the anticancer drug that can cause mucositis can be 5-fluorouracil (5-FU), cyclophosphamide (CPA), docetaxel, doxorubicin, vincristine, prednisone, etoposide, ifosfamide, methotrexate, paclitaxel, gemcitabine, vinorelbine, leucovorin, irinotecan, oxaliplatin, or a combination thereof, but is not limited thereto.

[0059] In one embodiment, mucositis can be oral mucositis (e.g., oral ulcers, recurrent oral ulcers, or pharyngeal mucositis), esophageal mucositis, gastrointestinal mucositis (e.g., intestinal inflammatory disease or intestinal ulcer disease), rectal mucositis, or a combination thereof, but is not limited thereto.

[0060] In one embodiment, mucositis may cause one or more of the following symptoms: ulcers or inflammation, pain, food intake disorders, diarrhea, weight loss, reduced ROS levels, bleeding, reduced epithelial thickness, erythema, etc. Specifically, mucositis may cause one or more symptoms selected from ulcers or inflammation, pain, food intake disorders, diarrhea, weight loss, reduced ROS levels, bleeding, reduced epithelial thickness, and erythema.

[0061] In another embodiment, by applying SOD to a subject who needs to prevent, improve or treat mucositis, the subject can be provided with effects such as reduction of food intake disorder, recovery of ulcer, improvement of re-epithelialization, improvement of diarrhea symptoms and improvement of intestinal villous atrophy. Specifically, the administration of SOD can bring one or more effects selected from reduction of food intake disorder, recovery of ulcer, improvement of re-epithelialization, improvement of diarrhea symptoms and improvement of intestinal villous atrophy to the subject. In addition, SOD can achieve the effect of reducing the level of reactive oxygen species (ROS) in the gastrointestinal tract (e.g., small intestine) or blood of mucositis.

[0062] Superoxide dismutase (SOD)

[0063] According to another aspect of the present invention, there is provided an improved superoxide dismutase (SOD) which exhibits the effect of preventing, improving or treating mucositis.

[0064] Superoxide dismutase (SOD) is an enzyme that can alternately catalyze the release of superoxide free radicals (O2 ·- ) into general molecular oxygen (O2) and hydrogen peroxide (H2O2). SOD plays an important role in reducing oxidative stress by removing reactive oxygen species. SOD is widely distributed in prokaryotes and eukaryotic cells and is divided into four categories according to different types of metal centers (copper / zinc, nickel, manganese and iron). Manganese-containing SOD [Mn-SOD] is widely present in a variety of bacteria, chloroplasts, mitochondria and cytoplasm of eukaryotic cells. The term "SOD" can be used interchangeably with polypeptides with superoxide dismutase activity.

[0065] In one embodiment of the present invention, SOD may be superoxide dismutase (Mn-SOD) combined with manganese. Specifically, SOD may be deamidated Mn-SOD. More specifically, SOD may be a substance in which the amino acid residues at positions 73 and 136 of SEQ ID NO:2 are replaced by Asp. More specifically, it may include the amino acid sequence shown in SEQ ID NO:4, or consist of the amino acid sequence shown in SEQ ID NO:4.

[0066] The SOD or polypeptide having SOD activity of the present invention is interpreted as also including amino acid sequences showing substantial identity with respect to the amino acid sequence. The substantial identity means that when the sequence is analyzed and aligned using an algorithm commonly used in the art, the amino acid sequence shows 80% or higher, preferably 90% or higher, more preferably 95% or higher, and most preferably 98% or higher sequence identity.

[0067] In another embodiment, SOD is a modified or engineered polypeptide having SOD enzyme activity, which may include one or more mutations that may or may not affect various aspects (in vivo, in vitro or ex vivo stability, uniformity and / or conformational changes), such as one or more amino acid deletions, insertions or substitutions. In addition, the polypeptide may also include heterologous substances (e.g., tags known in the art including HIS tags, Ha tags, myc tags, GFC and / or Fc domains of antibodies) for purification, detection or increased stability.

[0068] In one embodiment, SOD may be derived from natural or recombinant microorganisms, and may be an enzyme prepared from various supply sources through a process of isolation or purification.

[0069] In one embodiment, SOD may be derived from a natural or recombinant microorganism. For example, SOD may be derived from bacteria. Preferably, SOD may be derived from bacteria that are generally regarded as safe (GRAS) in medicine or food. Specifically, SOD may be derived from a Bacillus strain. More specifically, the SOD may be derived from a Bacillus amyloliquefaciens strain. For example, the SOD may be derived from a Bacillus amyloliquefaciens GF423 strain (KCTC 13222BP). The GF423 strain (KCTC13222BP) was deposited at the Korea Institute of Life Science on March 6, 2017.

[0070] In another embodiment, SOD may be derived from a protein comprising Figure 4 In addition, the SOD can be derived from a recombinant strain containing the expression vector shown in Figure 5 The recombinant strain prepared by the method shown in the method. The recombinant strain may include a nucleotide sequence encoding a polypeptide, and the polypeptide includes the amino acid sequence shown in SEQ ID NO:4. In addition, the recombinant strain may be a Bacillus strain in which AprE, NprE, Bpr, Epr, NprB, Vpr, Mpr, IspA, SrfAC, spoIIAc, EpsE and Xpf genes are deleted. The detailed process of preparing the strain is shown in Example 1.

[0071] The SOD derived from the strain is an enzyme secreted outside the cell, so when the strain is used to prepare SOD, SOD that is safe for the human body can be produced in large quantities without expensive purification processes (such as column purification), thereby achieving efficient production.

[0072] In one embodiment, SOD can be isolated or purified from a variety of supply sources including natural or recombinant hosts. For example, SOD with the activity of preventing or treating mucositis can be extracted from the culture supernatant of Bacillus amyloliquefaciens GF423 strain. In short, first, the culture fluid can be obtained by culturing Bacillus amyloliquefaciens GF423 strain in various types of culture medium. For example, a composite culture medium (pH 6.0 to 7.0) is used to grow the strain at 25° C. to 42° C. for 1 to 4 days. Other suitable culture media for culturing Bacillus amyloliquefaciens GF423 strain include LB (Luria-Bertani) culture medium, ISP (International Streptomyces Project) culture medium, NA (nutrient agar) culture medium, BHI (brain heart infusion agar) culture medium, SDA (Sabouraud dextrose agar) culture medium, PDA (potato dextrose agar) culture medium, NB (nutrient broth) culture medium, etc. Preferably, LB culture medium, ISP culture medium, BHI culture medium, SDA culture medium or NB culture medium can be used. Additionally, SOD can be obtained from other natural or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda-enzymes.org on the World Wide Web).

[0073] In one embodiment, the SOD may be an isolated or purified enzyme. In this case, the isolated or purified SOD, or its biologically active portion, is substantially free of cellular material or other contaminating proteins from its source cells or tissues. For example, the purified product may be obtained by single separation from a strain culture by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or a culture concentrate obtained by ultrafiltration, concentration, etc. The phrase "substantially free of cellular material" includes a protein preparation in which the protein is isolated or such protein is separated from the cellular components of a recombinantly produced cell. Specifically, the phrase "substantially free of cellular material" means that the content of undesirable protein by dry weight in the included protein preparation is less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5%.

[0074] In another embodiment, SOD can be preferably purified by the following purification method, but is not limited thereto. The culture solution obtained by culturing Bacillus amyloliquefaciens GF423 strain is centrifuged and the culture supernatant is collected. The supernatant fraction is pretreated by solid-phase extraction and then isolated and purified by chromatography. At this time, various forms of chromatography can be used to purify SOD. Preferably, hydrophobic interaction chromatography is used.

[0075] In one embodiment, the SOD may be contained in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dry form thereof. At this time, "strain lysate" refers to a substance obtained by culturing a strain and pulverizing it mechanically or chemically, and may include a substance that has been further subjected to additional processes such as extraction, dilution, concentration, and purification. "Strain culture" may refer to the culture solution itself or its supernatant obtained by culturing a strain. "Strain culture concentrate" refers to a culture concentrate obtained by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or obtained by ultrafiltration, concentration, etc. from a strain culture. "Strain culture extract" refers to a substance extracted from the culture solution or its concentrate, and may include an extract, a dilution or concentrate of the extract, a dry product obtained by drying the extract, or a crude purified product or purified product thereof, and a fraction obtained by dividing it. The dry form may include a lyophilized form.

[0076] According to one embodiment of the present invention, it is confirmed that by applying the SOD to a subject in need of preventing or treating mucositis, one or more effects of reducing gastrointestinal food intake disorders, healing ulcers, improving re-epithelialization, improving diarrhea symptoms, and improving intestinal villous atrophy can be shown. According to another embodiment, it is confirmed that by orally applying or orally administering the SOD to a subject in need of preventing or treating mucositis, the effect of reducing the level of reactive oxygen species (ROS) in the gastrointestinal tract (e.g., small intestine) or blood can be shown.

[0077] Dosage

[0078] The present invention has been accomplished, at least in part, by recognizing that the SOD of the present invention is effective in preventing or treating mucositis even when orally applied or orally administered at low doses.

[0079] Based on the body weight of the subject, the dosage of the SOD of the present invention may be about 50 to about 2500 units / kg / day, preferably about 125 units / kg / day to about 1000 units / kg / day, more preferably about 125 units / kg / day to about 500 units / kg / day, or about 250 units / kg / day to about 1000 units / kg / day, and further more preferably about 250 units / kg day, about 500 units / kg / day, or about 1,000 units / kg / day. In particular, when the SOD of the present invention is orally applied, the oral dosage based on the body weight of the subject can also be halved to effectively prevent or treat mucositis. For example, when SOD is applied intraorally, the administration dosage may be about 25 units / kg / day to about 1250 units / kg / day, preferably about 125 units / kg / day to about 500 units / kg / day, more preferably about 250 units / kg / day, about 500 units / kg / day, or about 1000 units / kg / day.

[0080] Pharmaceutical composition

[0081] According to yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating mucositis, comprising superoxide dismutase as an effective ingredient.

[0082] In one embodiment, the SOD according to the present invention can be combined with a pharmaceutically acceptable carrier, excipient and / or diluent to form a pharmaceutical composition for preventing or treating mucositis. When applied to animals other than humans, the pharmaceutical composition of the present invention can be used interchangeably with the term "veterinary composition".

[0083] Pharmaceutical composition or veterinary drug composition of the present invention may further comprise one or more selected from pharmaceutically acceptable carrier, excipient and diluent. The pharmaceutically acceptable carrier, excipient and / or diluent may be materials commonly used in the art. As carrier, excipient or diluent, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and silicon dioxide, mineral oil etc. may be enumerated, but are not limited thereto.

[0084] When preparing the formulation, additives such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. can be used for preparation. The additives used for the formulation can be appropriately selected from additives commonly used in the pharmaceutical field.

[0085] In addition, the pharmaceutical composition or veterinary composition of the present invention can be prepared into a preferred dosage form according to the method of use, and in particular, can be formulated by methods well known in the art so that the active ingredient can be provided quickly, continuously or in a delayed release after being applied to a mammal. Specific examples of such dosage forms include tablets, pills, powders, granules, syrups, liquids, capsules, suspensions, emulsions, injection solutions, plasters, lotions, liniments, limonade agents, aerosols, extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, patches, etc.

[0086] Furthermore, the pharmaceutical composition or veterinary composition of the present invention can be prepared into a preferred dosage form by using appropriate methods known in the art or methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton, Pennsylvania.

[0087] In one embodiment, the composition can be applied by any conventional route that can reach the target in the organism. For example, it can be applied by oral coating, oral use, non-oral administration routes including percutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, intrapulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, intragastrointestinal, topical, sublingual, intravaginal or rectal routes, but is not limited thereto. In the case of oral use, SOD can be coated with shellac to avoid the influence of gastric acid, but the coating agent is not limited thereto. Examples of coating agents suitable for use in the present invention include shellac, ethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, EUDRAGIT and combinations thereof. When SOD is coated, SOD can be coated in a solution. Specifically, the purified solution and the shellac-containing solution are mixed and then freeze-dried. The freeze-dried sample can be made into a powder and stored at about 4°C until use. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. Such solid preparations can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. in a composite composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to water and liquid paraffin as commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. can also be used.

[0088] The composition according to the present invention can be applied in the form of a coating agent or administered in the form of an injection. In addition, the composition according to the present invention can be administered by any device that can deliver the active ingredient to the target cell.

[0089] In addition, the pharmaceutical composition of the present invention or veterinary composition is applied with pharmaceutically or veterinary effective amount. The term "pharmaceutically effective amount" or "veterinary effective amount" refers to the amount of a disease that is sufficient to treat a reasonable benefit / risk ratio applicable to pharmaceutical or veterinary treatment, and the effective dose level can be determined according to factors including the body weight, sex, age, health status, severity, activity of the drug, sensitivity to the drug, administration time, route of administration and discharge ratio, treatment period, and other factors well-known in the medical field including the patient or animal's body weight, sex, age, health status, severity, drug activity, drug sensitivity, administration time, route of administration and discharge ratio, treatment period, and drugs used simultaneously. Preferably, based on the gross weight of the composition, the content of SOD in the pharmaceutical composition of the present invention or veterinary composition can be about 2 to about 1500U / mg, specifically about 5 to about 1000U / mg, more specifically about 10 to about 800U / mg, and further more specifically about 100 to about 500U / mg.

[0090] The pharmaceutical composition or veterinary composition of the present invention can be used as a single therapeutic agent, or combined with other therapeutic agents and administered sequentially or simultaneously. In addition, the pharmaceutical composition can be administered once or multiple times as needed. Taking all of the above factors into account, it is important to administer the minimum amount that can achieve the maximum effect without side effects, which amount can be easily determined by those skilled in the art.

[0091] In one embodiment, the composition of the present invention can be administered simultaneously, sequentially or in reverse order with radiotherapy and / or chemotherapy. Specifically, the composition of the present invention can be administered before, after, or before and after the implementation of radiotherapy and / or chemotherapy.

[0092] Treatment or prevention methods

[0093] According to yet another aspect of the present invention, there is provided a method for treating or preventing mucositis, comprising the step of administering the SOD or composition according to the present invention to a subject suffering from or at risk of mucositis.

[0094] In the present invention, "administration" includes any variety of administrations that achieve the desired effect to achieve the purpose of prevention or treatment. The route of administration can refer to the above. For example, the method may include: administering the SOD or composition of the present invention in a therapeutically effective amount to one or more digestive tract areas of a subject suffering from mucositis or at risk of mucositis. In addition, the method may include: applying the SOD or composition of the present invention in a therapeutically effective amount to the oral cavity of a subject suffering from oral mucositis or at risk of this.

[0095] The effective dose or effective non-toxic dose of SOD according to the present invention can be determined by routine experiments. For example, the therapeutic activity amount of SOD of the present invention can vary according to factors such as the stage of the disease, the severity of the disease, the age, sex, medical complications and body weight of the object. The dosage and administration therapy of SOD of the present invention can be adjusted to provide the best therapeutic response. For example, several separate dosages can be applied every day, every week, every two weeks, every three weeks, every four weeks, etc., and / or the dosage can be reduced or increased in proportion according to the urgency of the treatment situation.

[0096] 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 preparations capable of alleviating or improving mucositis, such as compounds, gene therapy agents, proteins (including antibodies), etc. The one or more other preparations may be administered simultaneously, sequentially, or in reverse order with the SOD or pharmaceutical composition. In addition, the individual components may be administered to the subject by the same or different routes.

[0097] In addition, in one embodiment, the method may include administering the SOD or composition of the present invention to the subject before, after, or both before and after the subject receives radiation therapy.

[0098] In another embodiment, the method may include administering the SOD or composition of the present invention to the subject before, after, or both before and after the subject receives chemotherapy.

[0099] In yet another embodiment, the method may include: administering the SOD or composition of the present invention to a subject receiving anti-cancer treatment.

[0100] At this time, the SOD or composition of the present invention may be used in combination with chemotherapy (eg, administration of the above-mentioned anticancer drugs) and / or radiotherapy, and the order and number of times thereof are not limited.

[0101] Food composition

[0102] According to yet another aspect of the present invention, there is provided a food composition for preventing or improving mucositis, which comprises the SOD according to the present invention as an effective ingredient. Such food composition includes a medical or nutraceutical food composition.

[0103] The term "medical food" or "health food" refers to food prepared from raw materials or ingredients that may exert beneficial functions on the human body, and is defined by the Ministry of Food and Drug Safety as food that maintains or improves health by maintaining normal functions or activating human physiological functions, but is not limited to this, and its meaning does not exclude any general health food.

[0104] In addition, the food includes various food types, food additives, beverages (for example, functional beverages, natural fruit juices and vegetable beverages), chewing gum, tea, vitamin complexes, health functional foods, other functional foods, etc., but is not limited to these.

[0105] The food can be prepared by conventional methods known in the art. For example, for the purpose of preventing or improving mucositis, the medical food, health food or health functional food can further include one or more of a carrier, a diluent, an excipient and an additive on the basis of the SOD, and be prepared into a tablet, a pill, a powder, a granule, a powder, a capsule and a liquid dosage form. The specific examples of the carrier, excipient, diluent and additive are well known in the art, and those skilled in the art can prepare it according to the dosage form combined with appropriate ingredients.

[0106] The content of the superoxide dismutase according to the present invention as an active ingredient in the above-mentioned dosage form can be appropriately adjusted according to the form and purpose of use, the patient's condition, the type and severity of symptoms, etc., and can be about 0.001% by weight to about 99.9% by weight, preferably about 0.01% by weight to about 50% by weight, based on the weight of the solid component, but is not limited thereto.

[0107] The dosage of the food of the present invention may vary according to the patient's age, weight, sex, form of administration, health status and degree of disease, and may be divided and administered once or multiple times a day at certain time intervals according to the judgment of a doctor or pharmacist. For example, based on the content of the active ingredient, the daily dose may be about 10 to about 1000 mg / kg. The above dosage is an example of an average situation, and the daily dose may be higher or lower depending on individual differences. If the daily dose of the health functional food of the present invention is less than the dosage, a significant effect may not be obtained. If it exceeds it, it is not only uneconomical, but also may cause undesirable side effects due to exceeding the range of the commonly used amount.

[0108] Feed composition

[0109] According to yet another aspect of the present invention, a feed composition for preventing or improving mucositis is provided, which contains the SOD according to the present invention as an effective ingredient. In the present invention, the food composition may include a feed composition. These feed compositions can be prepared in any dosage form commonly used in this field. For example, the feed composition of the present invention may further contain: auxiliary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other microbial preparations in the form of live bacteria; cereals, such as crushed or broken wheat, oats, barley, corn, and rice; plant protein feeds, such as materials with rapeseed, soybeans, and sunflower seeds as main ingredients; animal protein feeds, such as blood meal, meat meal, bone meal, and fish meal; sugars and dairy products, such as dry ingredients composed of various milk powders and whey powders; lipids, such as main ingredients such as animal fats and vegetable fats that are liquefied at will by heating; additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventive agents.

[0110] The feed composition of the present invention may be in the form of a powder or liquid preparation, and may contain an excipient for feed addition (calcium carbonate, wheat shortbread, zeolite, corn flour, rice bran, etc.).

[0111] Box

[0112] According to yet another aspect of the present invention, a kit for preventing, improving or treating mucositis is provided, comprising the SOD or composition of the present invention.

[0113] In one embodiment, the kit may further include instructions for use of the SOD or composition of the present invention. The instructions may include guidelines for taking or applying the active ingredient. For example, the instructions may include guidelines for taking or applying when used in combination with radiotherapy and / or chemotherapy, or when used in combination with anticancer drugs. Specifically, the instructions may include guidelines for the time, sequence, number of times, dosage, etc. of application when used in combination with radiotherapy and / or chemotherapy, or when used in combination with anticancer drugs.

[0114] Example

[0115] Hereinafter, the present invention will be described in more detail in conjunction with the following examples. The following examples are provided for a thorough understanding of the present invention and are not intended to and should not be construed as limiting the scope of the present invention in any way.

[0116] [Example 1] Preparation of recombinant strain expressing superoxide dismutase (SodA2)

[0117] [Example 1.1] Preparation of expression host GFBS220

[0118] The expression host GFBS220 was prepared using Bacillus subtilis KCTC 3135. Bacillus subtilis KCTC 3135 was obtained from the Bioresource Center (KCTC) of the Korea Institute of Biotechnology. In order to facilitate the subsequent steps, the genes shown in Table 1 below were removed from the genome of Bacillus subtilis KCTC 3135.

[0119]

Table 1

[0120]

[0121]

[0122] Double cross-over recombination is used to remove genes from the genome. Figure 1 Specifically, DNA fragments (up frag. and down frag.) with homology to the DNA base sequences on both sides of the gene to be operated were cloned into the vector pUCori-ts-cm with a temperature-sensitive replication origin. The PCR conditions and primers used for cloning are shown in Tables 2 and 3, respectively.

[0123]

Table 2

[0124]

[0125]

Table 3

[0126]

[0127]

[0128]

[0129] After the carrier prepared in this way is introduced into the subtilis cell, the substratum containing chloramphenicol is used to screen transformant (single cross homologous recombination) at 37 ℃ (non-replication temperature). The transformant screened is cultivated (second crossover) using LB substratum without antibiotics at 30 ℃ (permitting replication temperature), and then cultivated using LB agar medium without antibiotics at 39 ℃. From the bacterium colony generated, strains sensitive to chloramphenicol are screened (plasmid solidification). PCR is performed on the bacterium colony screened to obtain double cross-over recombinant (double cross-over rec ombinant), and single separation is performed for the second time to screen out final recombinant, named after GFBS220.

[0130] The defective gene site of GFBS220 was confirmed by agarose gel electrophoresis after amplification using the primers and PCR shown in Table 4. The results of confirming the defective gene by PCR in the expression host GFBS220 are as follows: Figure 2 shown.

[0131]

Table 4

[0132]

[0133] [Example 1.2] Preparation of expression vector pBE1-sodA2

[0134] Mn-SOD was confirmed in the culture supernatant of Bacillus amyloliquefaciens strain GF423 by N-terminal sequencing (see Kang et al., 2018). The GF423 strain was deposited in the Korea Biotechnology Research Institute on March 6, 2017 (with the deposit number KCTC 13222BP). The gene of Mn-SOD was named sodA according to the bacterial SOD nomenclature, and its amino acid sequence is as follows Figure 3 and SEQ ID NO: 2 (its nucleotide sequence is shown in SEQ ID NO: 1). The LC-UV-MS / MS peptide map of the enzyme preparation yielded 100% amino acid sequence coverage, but deamidation was observed at residues Asn73 and Asn136 with abundances of 73% and 17%, respectively. Therefore, in order to improve the homogeneity of the purified enzyme, the two Asn residues were replaced with Asp. This variant sodA was named SodA2 and its amino acid sequence is shown in Figure 3 and SEQ ID NO:4 (its nucleotide sequence is shown in SEQ ID NO:3).

[0135] The sodA2 gene was amplified using repeat extension PCR using four primers (Table 5) and the Bacillus amyloliquefaciens GF423 genome as template. The nucleotides used to replace Asn with Asp are underlined.

[0136]

Table 5

[0137]

[0138] The amplified DNA fragment containing the sodA2 gene and the plasmid pBE1 linearized by NdeI and HindIII treatment were assembled in vitro by the SLIC method (see Jeong et al., 2012), and the resulting construct was transformed into Escherichia coli C2984H. Correct clones were screened by restriction enzyme mapping and confirmed by nucleotide sequencing. The expression vector pBE1-sodA2 generated in this way is as follows Figure 4 shown.

[0139] [Example 1.3] Preparation of production strain BSBA310

[0140] The expression vector pBE1-sodA2 was transformed into Bacillus subtilis GFBS220. Strains were selected from the transformants and pure clones were established by two single colony isolation procedures in LB2 medium. The selected strain was named BSBA310 and stored at -80°C using the glycerol stock storage method. The results of confirming the defective gene using PCR in the production strain BSBA310 are as follows: Figure 2 In addition, Figure 5 Summary The overall process for preparing the production strain BSBA310 is shown.

[0141] [Example 2] Isolation and purification of superoxide dismutase (SodA2) from production strain BSBA310

[0142] [Example 2.1] Cultivation of production strain BSBA310

[0143] In order to culture the production strain BSBA310 obtained in Example 1, a single colony formed in LB agar medium (LB (Luria-Bertani) agar; tryptophan 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L) was inoculated into 30 ml of LB medium and cultured at 37° C. for 12 hours. The seed culture was again inoculated into 3 L of LB medium containing 1 mM manganese sulfate (MnSO4) and cultured at 37° C. for 20 hours.

[0144] [Example 2.2] Isolation and purification of SodA2

[0145] The cell culture fluid obtained in Example 2.1 was centrifuged at 4°C and 3,578×g for 20 minutes, and after collecting the supernatant, it was concentrated 10 times by ultrafiltration (Ultrafiltration, hereinafter referred to as UF, MWCO 10,000). 390g of ammonium sulfate was added to each 1L of concentrated cell culture, stirred for 20 minutes, centrifuged and the supernatant was collected. The obtained supernatant was purified using a phenyl agarose HP column. During the purification process, 50mM potassium phosphate pH7.0 with a concentration of 2M ammonium sulfate was used to balance the column, and then the supernatant obtained by adding ammonium sulfate was passed through the column, and 50mM potassium phosphate pH7.0 containing 1.6M ammonium sulfate was used to recover SodA2 attached to the column. The purified solution purified by the column was collected, and the high concentration of salts formed during the purification process was removed by ultrafiltration and concentrated. After filtering the concentrated solution with a sterilizing filter, it was freeze-dried. The activity of SodA2 was analyzed using a SOD analysis kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits superoxide radicals by 50%. The activity of dried SodA2 enzyme is 1000 to 1,500 U / mg.

[0146] [Example 3] Preparation of administration material

[0147] For the intragastric administration group administered orally, shellac (EXCELACS co., LTD., Bangkok) was dissolved in ethanol to a concentration of 3% and sterilized using a 0.2um sterile filter. The shellac dissolved in ethanol was diluted with PBS. The freeze-dried SodA2 was dissolved to 20 mg / mL, and the shellac solution and the SOD solution were mixed at a ratio of 1:1 and then freeze-dried. The freeze-dried shellac-coated SodA2 and dextrin were mixed at a ratio of 1:9 to 12 (shellac-coated SodA2: dextrin) to prepare the substance used in the animal experiment. The prepared SOD activity reached 90 to 110 U / mg. For the intragastric administration group, it was finally diluted to 10 U / mg for use.

[0148] For the oral application group, SodA2 was suspended in 2% methylcellulose (Sigma) gel and used as a standby. The SOD activity of the oral application group was adjusted to 100 U / mg.

[0149] [Example 4] Preparation of mucositis model and administration of SodA2

[0150] In order to construct a mucositis model, 6-week-old Sprague Dawley rats (purchased from Orient Bio) were purchased, allowed to adapt for one week, and then randomly divided into eight groups. The eight groups are as follows: G1 (control group), G2 (disease group), oral coating group (G3, G4, G5) and intragastric administration group (G6, G7, G8) by oral administration. The test groups, administration concentrations and number of animals of the mucositis model are shown in Table 6. The induction method of mucositis is: 5-fluorouracil (Sigma, 100 mg / kg) is administered intraperitoneally twice on the 1st and 3rd days, and ketamine (Yuhan, 40 mg / kg) is used for anesthesia on the 5th day. Then, in order to create chronic irritation (chronic irritation) that occurs when patients receiving anticancer treatment perform chewing movements, a wound is made on the left oral mucosa with an 18-gauge needle to induce mechanical ulcer lesions. Sham operation is performed on G1 as a control group.

[0151]

Table 6

[0152]

[0153]

[0154] For the oral application group, 2% methylcellulose gel was used as a vehicle to suspend the test substance, and then 100ul of the drug was applied to the oral mucosa with a micropipette. For the intragastric administration group, PBS was used as a vehicle and the test substance was orally administered at a dose of 10mL / kg. After the mechanical surgery to induce oral mucositis was completed, the 10 days of ulcer healing in the normal group were used as the application cycle, and it was applied once every day at about 10 am. G1 (control group) and G2 (disease group) were only administered with a vehicle.

[0155] [Example 5] Measurement of body weight and food intake

[0156] To evaluate the energy absorption disorder caused by oral and gastrointestinal mucositis, body weight and feed intake were measured every 2 to 3 days. For the measurement of feed intake, the weight was measured before the feed was supplied to the cage on the previous day, and then the amount of feed remaining was measured on the next day to calculate the feed intake, and the average feed intake was calculated by dividing by the number of animals in the cage.

[0157] In the normal group G1, body weight and feed intake continued to increase steadily over 10 days. In contrast, in G2 to G8 where mucositis was induced, feed intake was significantly lower than that in G1, and body weight showed a continuous downward trend after an initial short-term increase. In addition, in the drug administration groups (G3 to G8), increases in body weight and feed intake were observed compared to G2. In particular, in the medium / high concentration administration groups of SodA2 (G5, G7, and G8), body weight increased statistically significantly, and in the high concentration administration groups of SodA2 (G5 and G8), feed intake increased statistically significantly. The body weight and feed intake graphs of the experimental groups are shown in the figure below. Figure 6a and 6b shown.

[0158] [Example 6] Scoring and tissue evaluation of oral mucositis

[0159] Scoring of oral mucositis

[0160] Immediately after induction of mechanical ulcer lesions, the ulcer sites were observed every 3 to 4 days, and oral mucositis was scored according to the criteria in Table 7. The scoring criteria were based on the literature [Araujo et al., PLoS One 2015, 10: e0116799].

[0161]

Table 7

[0162]

[0163]

[0164] For oral mucositis, the degree of re-epithelialization of the ulcer site was analyzed during the 10 days after the induction of mechanical ulcers. In the normal group G1, the ulcer site recovered quickly, and by the 10th day, re-epithelialization was complete, and only slight wound marks were visible. On the contrary, in the G2 group, the differentiation of epithelial cells in the ulcer site caused by 5-FU was inhibited, and re-epithelialization almost did not occur, and in some individuals, the ulcer site became larger due to continuous chewing stimulation. In the drug administration group, it was confirmed that re-epithelialization was promoted in a concentration-dependent manner, thereby healing the ulcer site ( Figure 7a ).

[0165] Tissue Evaluation of Oral Mucositis

[0166] Tissues containing oral mucosa were collected, fixed in 10% formalin, and then subjected to conventional tissue processing to prepare H&E (hematoxylin-eosin) stained slides. Afterwards, the lesions caused by mucositis were examined microscopically, the degree of re-epithelialization of the mucositis site was evaluated, and the thickness of the oral mucosal epithelium was analyzed.

[0167] In G1, re-epithelialization was complete in all sites where ulcerative mucositis was induced, and slightly raised tissue due to proliferation of granulation tissue was observed in the submucosal area. Although there were still some individuals with mild inflammation, most of them had healed and showed normal structure ( Figure 7b In G2, re-epithelialization did not occur at all in all sites where ulcerative mucositis was induced, and calcification, necrosis, and proliferation of granulation tissue (inflammatory cell infiltration) were observed. In G3 and G6 (SodA2 low concentration administration groups), there was no significant difference from the G2 group, and severe ulcerative mucositis sites were observed. On the contrary, in G4, G5, G7, and G8 (SodA2 medium and high concentration administration groups), re-epithelialization occurred and the ulcer size decreased, and inflammation and necrosis in the submucosal site also gradually decreased ( Figure 7b ). Referring to the quantitative analysis results of the degree of re-epithelialization, similar to the results of the oral mucositis score chart described above, the degree of re-epithelialization was significantly higher in the normal group G1, while almost no re-epithelialization occurred in G2. In the drug administration groups, re-epithelialization was promoted in a concentration-dependent manner, and the re-epithelialization in the medium- and high-concentration administration groups (G4, G5, G7, and G8) of SodA2 was statistically significantly increased compared with G2 ( Figure 7c ).

[0168] [Example 7] Scoring and tissue evaluation of intestinal mucositis

[0169] Scoring of intestinal mucositis

[0170] During the 10-day test period, the perianal area and feces were visually observed once a day, and the degree of diarrhea was evaluated according to the scoring method shown in Table 8 below.

[0171]

Table 8

[0172]

[0173]

[0174] In G1, diarrhea did not occur during the 10-day test period, but in the 5-FU administration groups (G2 to G8), diarrhea occurred from the start of drug administration, and severe wet diarrhea continued during the 5-day period of drug administration, and the diarrhea gradually improved during the following 5 days. In particular, in the medium- and high-concentration SodA2 administration groups (G4, G5, G7, and G8), the diarrhea scores were statistically significantly lower than those in G2 ( Figure 8a ).

[0175] Tissue evaluation of intestinal mucositis

[0176] The tissue containing the intestinal mucosa was collected, fixed in 10% formalin, and then subjected to conventional tissue processing to prepare H&E (hematoxylin-eosin) stained slides. Afterwards, the lesions caused by mucositis were examined microscopically, the changes in the intestinal villi at the mucositis site were evaluated, and the length of the intestinal villi was analyzed.

[0177] In G1, normal anatomical histological structures of the small intestine such as villi and crypts were confirmed. In contrast, in G2, severe villous atrophy was characteristically observed, and it was known that the malabsorption caused by this would lead to diarrhea and weight loss. In the drug administration groups (G3 to G8), villous atrophy was significantly improved in a concentration-dependent manner, and inflammatory cell infiltration was mild and did not differ between groups ( Figure 8b ). Referring to the results of quantitative analysis of intestinal villus length, the villus length decreased in G2 due to villus atrophy compared with G1. In the drug administration groups (G3 to G8), villus atrophy was significantly improved in a concentration-dependent manner compared with G2. In particular, in G5, G7, and G8, the length of intestinal villi was statistically significantly increased ( Figure 8c ).

[0178] [Example 8] ROS evaluation of oral and intestinal mucositis

[0179] Assessment of ROS in the small intestine

[0180] After collecting intestinal tissue by dissection, 100 mg of intestinal tissue was washed twice with PBS, 1 mL of ice-cold 50 mM TBS (pH 7.4) containing a proteolytic enzyme inhibitor cocktail was added, and then the tissue was broken using a bead homogenizer, and the lysate was centrifuged at 12,000 rpm for 10 minutes at 4°C, and the supernatant was saved. The tissue lysate was diluted with ice-cold Locke's 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. Next, Locke's buffer (pH 7.4), 0.2 mL of the lysate and 10 μL of DCFH-DA (purchased from Sigma, 5 μM) were mixed, 1 mL of the reaction mixture was incubated for 45 minutes, and the fluorescence of DCF was measured using a fluorescence spectrophotometer (excitation: 484 nm, emission: 530 nm). The blank was corrected by the background fluorescence of the lysate without the addition of DCFH-DA.

[0181] The results of DCFH-DA analysis of ROS levels in small intestinal tissue showed that the ROS level in G2 increased more than two times compared with G1. In the drug administration group, a concentration-dependent decrease was observed in both the oral application group (G3 to G5) and the intragastric administration group (G6 to G8) compared with G2 ( Figure 9a ). That is, for 5-FU-induced mucositis, SodA2 showed a concentration-dependent effect of reducing the ROS level in both the oral application group and the intragastric administration group.

[0182] Assessment of ROS in blood

[0183] Luminol-amplified chemiluminescence signal was used to measure the ROS level in the blood. 40 μL of plasma was dispensed into a 96-well plate, and the baseline value was measured using a chemiluminescence spectrometer. Next, 200 μL of luminol (purchased from Sigma, 200 mM, pH 7.4) was added to the 96-well plate, incubated for 10 minutes, and then the luminescence value was measured every 1 minute and corrected with the baseline value.

[0184] The results of analyzing the ROS level in the blood by luminol luminescence showed that the luminol luminescence in G2 increased by about 0.5 times compared with G1. In the drug administration group, a concentration-dependent decrease was observed in both the oral application group (G3 to G5) and the intragastric administration group (G6 to G8) compared with G2. In particular, after 6 minutes of adding luminol, there was a statistically significant decrease in G5, G7 and G8, after 7 minutes, there was a statistically significant decrease in G5, G7 and G8, after 8 to 9 minutes, there was a statistically significant decrease in G5 and G8, and after 10 minutes, there was a statistically significant decrease in G8 ( Figure 9b ).

[0185] [Example 9] Statistical analysis

[0186] All numerical data were statistically analyzed using GraphPad Prism statistical program (Ver.8.0). First, Levene's test was performed to evaluate the homogeneity of variance, and then one-way ANOVA test was performed to confirm the significance between the experimental groups. If the results of one-way ANOVA confirmed the significance between the experimental groups, Tukey's post hoc test was performed based on whether the variance was homogeneous. All data are expressed as mean ± SEM, and the confidence interval was set at 95%.

[0187] [Example 10] Conclusion and discussion

[0188] After inducing oral mucositis and intestinal mucositis by intraperitoneal administration of the anticancer drug 5-FU, SodA2 was administered by oral application and intragastric (oral) administration for 10 days. The results showed that the efficacy was that the re-epithelialization of the oral mucosal ulcer site was promoted, and diarrhea and intestinal villous atrophy were improved in a concentration-dependent manner. In addition, it was confirmed that the symptoms were improved by reducing the level of ROS in the intestinal tissue and blood of mucositis. There was no significant difference in the case of different drug administration routes.

[0189] In summary, SodA2 (SOD enzyme derived from Bacillus) can be an effective substance for inhibiting and treating oral and intestinal mucositis caused by the side effects of anticancer drugs.

Claims

1. A pharmaceutical composition for treating or preventing mucositis, comprising superoxide dismutase derived from Bacillus as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein The mucositis is induced by chemotherapy, radiation therapy, or a combination thereof.

3. The pharmaceutical composition according to claim 1, wherein The mucositis includes oral mucositis, esophageal mucositis, gastrointestinal mucositis, rectal mucositis or a combination thereof.

4. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase has one or more effects selected from the group consisting of reduction of food intake disorder, recovery of ulcer, improvement of re-epithelialization, improvement of diarrheal symptoms, improvement of intestinal villus atrophy, and reduction of active oxygen levels in the gastrointestinal tract or blood.

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

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

7. The pharmaceutical composition according to claim 1, wherein The composition is administered to a subject receiving anti-cancer therapy.

8. The pharmaceutical composition according to claim 6, wherein The anticancer drugs used in the chemotherapy include one or more selected from cytotoxic anticancer drugs, targeted anticancer drugs, immune anticancer drugs or a combination thereof.

9. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is a manganese-containing superoxide dismutase derived from Bacillus.

10. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase comprises the amino acid sequence shown in SEQ ID NO:

4.

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

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

13. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is contained in the form of strain lysate, strain culture, strain culture concentrate, strain culture extract or a dry form thereof.

14. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is administered by oral application or oral administration.

15. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is administered at a dosage of about 50 units / kg to about 2500 units / kg / day based on the subject's body weight.

16. The pharmaceutical composition according to claim 1, wherein The superoxide dismutase is administered at a dosage of about 125 units / kg / day to about 1000 units / kg / day based on the subject's body weight.

17. A veterinary composition for treating or preventing mucositis, comprising superoxide dismutase derived from Bacillus as an active ingredient.

18. A food composition for treating or preventing mucositis, comprising superoxide dismutase derived from Bacillus as an effective ingredient.

19. A kit for treating or preventing mucositis, comprising: A composition according to any one of claims 1 to 18; and an instruction book comprising instructions for taking or applying the composition.

20. The kit of claim 19, wherein: The instructions include content guiding the combined use with radiotherapy and / or chemotherapy, or the combined use with anticancer drugs.

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

22. Use of the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 18 in preventing or treating mucositis.

23. Use of the pharmaceutical composition or superoxide dismutase according to any one of claims 1 to 18 in the preparation of a medicament for preventing or treating mucositis.

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