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WO2025095595A1 - Superoxyde dismutase et ses utilisations pour la prévention ou le traitement d'une lésion rénale - Google Patents

Superoxyde dismutase et ses utilisations pour la prévention ou le traitement d'une lésion rénale Download PDF

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WO2025095595A1
WO2025095595A1 PCT/KR2024/016851 KR2024016851W WO2025095595A1 WO 2025095595 A1 WO2025095595 A1 WO 2025095595A1 KR 2024016851 W KR2024016851 W KR 2024016851W WO 2025095595 A1 WO2025095595 A1 WO 2025095595A1
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sod
strain
pharmaceutical composition
composition
bacillus
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Korean (ko)
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반재구
김의중
김정현
양지현
조상경
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Biomlogic Inc
Genofocus Inc
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Genofocus Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health

Definitions

  • sequence listing This application includes a sequence listing, which sequence listing has been submitted electronically in XML format and is incorporated by reference herein in its entirety.
  • a copy of the sequence listing, created on October 23, 2024, is named KC24164.xml and is 28.6 kilobytes in size.
  • the present invention relates to superoxide dismutase and uses thereof for preventing, ameliorating or treating renal damage. More particularly, the present invention relates to uses, compositions or methods of Bacillus-derived superoxide dismutase for preventing, ameliorating or treating renal damage.
  • Acute kidney injury a representative example of kidney damage, is defined as the sudden loss of kidney excretory function. This acute kidney injury can occur after a sudden event such as an accident, burn, or trauma.
  • acute kidney injury is one of the common complications in intensive care, and patients with underlying diseases are known to be a particularly high-risk group.
  • Acute kidney injury is fatal, often requiring kidney transplantation, and also contributes to increased length of hospital stay, increased mortality, and increased medical expenses.
  • Acute kidney injury can lead to chronic kidney disease and cardiovascular disease in the long term, so it is essential to prevent acute kidney injury and detect and treat it early.
  • kidney injury has a wide range of causes and severity, making it difficult to approach it as a single disease, and it is difficult to find a treatment method that shows common effects in clinical studies.
  • fluid therapy is mainly used to continuously monitor body fluid status and administer appropriate fluids and diuretics when acute kidney injury occurs.
  • kidney is an organ responsible for maintaining homeostasis, and kidney damage affects almost all systems of the body in various ways.
  • Recent basic and clinical research data have shown that acute kidney injury causes severe systemic inflammation while causing damage to organs far from the kidney, such as the heart, lungs, spleen, brain, liver, and intestines.
  • kidney-gut crosstalk may exist when kidney damage occurs.
  • unlike diabetes, obesity, and inflammatory bowel disease only a few studies have proven the connection between the kidney and the intestines in the field of kidney damage, and further research is needed.
  • Non-patent Document 1 Kellum, J.A., Romagnani, P., Ashuntantang, G. et al. Acute kidney injury. Nat Rev Dis Primers 7, 52 (2021). https://doi.org/10.1038/s41572-021-00284-z
  • Non-patent Document 2 Doi K, Rabb H. Impact of acute kidney injury on distant organ function: recent findings and potential therapeutic targets. Kidney Int 2016; 89: 555-564.
  • Non-patent Document 3 Se won Oh, The Cause and Treatment of Acute Kidney Injury, Korean J Med. 2019; 94(4): 315-321.
  • the present invention aims to solve one or more of the problems of the above-mentioned prior art.
  • the present invention aims to provide a Bacillus species strain, a Bacillus species strain spore and/or a superoxide dismutase (SOD) for preventing or treating kidney damage.
  • SOD superoxide dismutase
  • Another object of the present invention is to provide a composition for preventing, improving or treating kidney damage comprising a Bacillus species strain, a Bacillus species strain spore and/or SOD.
  • Another object of the present invention is to provide a method for preventing, improving or treating kidney damage, comprising the step of administering to a subject a Bacillus species strain, a Bacillus species strain spore and/or SOD.
  • Another object of the present invention is to provide a use of Bacillus species strain, Bacillus species strain spores and/or superoxide dismutase for preventing, ameliorating or treating renal damage.
  • a representative configuration of the present invention to achieve the above purpose is as follows.
  • a pharmaceutical composition for preventing or treating renal damage comprising a Bacillus species strain, a Bacillus species strain spore and/or a polypeptide having superoxide dismutase (SOD) activity as an active ingredient.
  • SOD superoxide dismutase
  • polypeptide having superoxide dismutase (SOD) activity or SOD can be in the form of or be present in the form of a Bacillus strain producing or containing the same or a spore thereof.
  • the polypeptide may be an isolated or purified protein.
  • the polypeptide may be included as a strain lysate, strain culture, strain culture concentrate, strain culture extract, or a dried form thereof.
  • the polypeptide can be Mn-SOD.
  • the polypeptide can be deamidated Mn-SOD.
  • the polypeptide may be derived from a Bacillus species strain.
  • the Bacillus species strain may be a Bacillus velezensis species.
  • the Bacillus species strain may be selected from a Bacillus velezensis strain deposited under accession number KCTC 13222 BP, a Bacillus velezensis strain deposited under accession number KCTC 13227 BP, and a Bacillus velezensis strain deposited under accession number KCTC 15552 BP.
  • the composition may comprise as an active ingredient a polypeptide having superoxide dismutase activity comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
  • the polypeptide may be coated with a coating agent.
  • the coating may include shellac.
  • the composition may be administered orally.
  • the composition may be administered to the subject before, after, or both before and after the onset of renal injury.
  • the composition may be co-administered to a subject prior to, concurrently with, and/or subsequent to administration of another drug that causes or is at risk for causing renal injury.
  • an oral composition for preventing, ameliorating or treating renal damage comprising a superoxide dismutase as disclosed herein as an active ingredient is provided.
  • a food composition for preventing, improving or treating kidney damage comprising superoxide dismutase as disclosed herein as an active ingredient is provided.
  • a feed composition for preventing, improving or treating kidney damage comprising superoxide dismutase as disclosed herein as an active ingredient is provided.
  • a veterinary composition for preventing, improving or treating kidney damage comprising superoxide dismutase as disclosed herein as an active ingredient is provided.
  • a method for preventing or treating kidney damage comprising administering to a subject a polypeptide as disclosed herein.
  • a superoxide dismutase as disclosed herein for the manufacture of a medicament for preventing or treating renal damage.
  • a composition comprising at least one selected from the group consisting of a Bacillus species strain of the present invention, a Bacillus species strain spore, and superoxide dismutase (SOD) exhibits a preventive or therapeutic effect on renal damage (e.g., renal failure, acute renal damage, or chronic renal damage).
  • renal damage e.g., renal failure, acute renal damage, or chronic renal damage.
  • a protective effect on renal damage such as a change in the composition of intestinal microorganisms, a decrease in intestinal epithelial cell death, a decrease in intestinal permeability, a decrease in oxidative stress in the kidney, an alleviation of renal tubular damage, and an improvement in renal function, was observed.
  • the superoxide dismutase according to the present invention can be effectively used to prevent, improve, or treat renal damage.
  • the SOD of the present invention exhibits an effect of preventing or treating acute kidney damage when administered together with a contrast agent that induces acute kidney damage, and therefore can be used as a drug administered concomitantly in an imaging diagnostic examination using a contrast agent to protect against renal damage.
  • the SOD of the present invention is derived from a Bacillus species strain (e.g., a Bacillus velezensis strain) that is generally regarded as safe (GRAS) bacteria, thereby ensuring oral administration efficacy and safety, and also having a process advantage of being able to be directly recovered from the supernatant during culture.
  • GRAS safe
  • Figure 1 shows the procedure for producing a recombinant production strain (BSBA310) expressing SodA2.
  • Figure 2 shows the expression vector for overexpression of the sodA2 gene.
  • rrnBT1T2 represents a transcription terminator
  • rep(pBR322) represents a replicon from pBR322 that operates in E. coli
  • rep(pUB110) is a replicon from pUB110 that operates in B. subtilis
  • KanR represents a kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase)
  • BJ27 promoter represents a strong promoter for B. subtilis.
  • Figures 3a and 3b are schematic diagrams showing the cloning process of the GF427 strain, which has increased SOD activity by replacing the promoter of the GF423 strain.
  • Figure 3a shows the process of producing pUori-cm-amp-10sod using a PCR product obtained from GF423 genomic DNA as a template
  • Figure 3b shows the process of producing pUori-cm-amp-P3-SOD using a PCR product obtained from pUori-cm-amp-10sod as a template.
  • Figure 4a is a graph showing principal coordinates analysis performed on stool samples from the untreated Sham group (Sham), the SOD-BA-administered Sham group (SOD+Sham), the unadministered IRI group (IRI), and the SOD-BA-administered IRI group (SOD+IRI).
  • Figure 5 is a graph showing changes in intestinal permeability according to administration of SOD.
  • Figures 6a and 6b are diagrams showing the degree of intestinal epithelial cell death according to the administration of SOD.
  • Figure 6a is a graph showing the quantification of TUNEL-positive epithelial cells
  • Figures 7a and 7b are diagrams showing changes in the distribution of intestinal immune cells according to the administration of SOD.
  • Figure 7a is a graph showing the quantification of Ly6G positive cells
  • Figures 8a to 8e are drawings showing the kidney improvement effect according to the administration of SOD.
  • Figures 8a and 8b are a graph ( Figure 8a) and a micrograph (Figure 8b) showing the distribution of SOD in the kidney according to the administration of SOD as a staining positivity ratio compared to the total tissue area, respectively.
  • Figures 8c and 8d are a graph ( Figure 8c) and a micrograph ( Figure 8d) showing the distribution of catalase in the kidney according to the administration of SOD as a staining positivity ratio compared to the total tissue area, respectively.
  • Figure 8e shows the oxidative stress relief process of SOD. (All scale bars except for the * mark (1000 ⁇ m) in the drawings are 500 ⁇ m.)
  • Figures 9a to 9d are diagrams showing changes in the distribution of immune cells (changes in the level of infiltration) in renal tissue according to the administration of SOD.
  • Figure 10a is a graph showing the change in blood creatinine concentration according to the administration of SOD (unpaired t-test; *, p ⁇ 0.05; **, p ⁇ 0.05).
  • Figure 10b is a graph showing the change in blood urea nitrogen (BUN) concentration according to the administration of SOD (unpaired t-test; *, p ⁇ 0.05; **, p ⁇ 0.05; #, p ⁇ 0.05).
  • Figure 12 is a schematic diagram for creating a contrast-induced acute kidney injury (CI-AKI) model.
  • CI-AKI contrast-induced acute kidney injury
  • Figures 13a to 13c are diagrams showing the preventive or ameliorating effect on renal damage according to the administration of SOD in a contrast agent-induced acute renal injury model.
  • Figure 13a is a graph showing changes in blood creatinine concentration
  • Figure 13b is a graph showing changes in blood urea nitrogen (BUN) concentration
  • Figure 13c is a graph showing NGAL distribution as a positive ratio relative to the total tissue area.
  • subject is used interchangeably with “subject” or “patient” and can be a mammal in need of prevention, amelioration, or treatment of kidney damage, such as a primate (e.g., human, monkey, chimpanzee, etc.), companion animal (e.g., dog, cat, etc.), livestock animal (e.g., cow, pig, horse, sheep, goat, etc.), and laboratory animal (e.g., rat, mouse, guinea pig, etc.).
  • a primate e.g., human, monkey, chimpanzee, etc.
  • companion animal e.g., dog, cat, etc.
  • livestock animal e.g., cow, pig, horse, sheep, goat, etc.
  • laboratory animal e.g., rat, mouse, guinea pig, etc.
  • the subject is a human.
  • treatment generally means obtaining a desired pharmacological effect and/or physiological effect. Such effect is therapeutic in that it partially or completely cures a disease and/or other unwanted or undesirable condition (e.g., kidney damage, such as renal failure, acute kidney injury or chronic kidney injury). Desirable therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, ameliorating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improved prognosis. Preferably, "treatment” may mean medical intervention for an already manifested disease or disorder.
  • prevention means obtaining the desired prophylactic pharmacological and/or physiological effect with a view to partially or completely preventing a disease or its symptoms.
  • administration means providing an active ingredient to a subject for a preventive or therapeutic purpose (e.g., renal impairment, such as renal failure, acute renal injury, or chronic renal injury).
  • a preventive or therapeutic purpose e.g., renal impairment, such as renal failure, acute renal injury, or chronic renal injury.
  • kidney damage For use in preventing or treating kidney damage
  • the present invention is based at least in part on the surprising discovery that administration of a composition comprising as an active ingredient a polypeptide having superoxide dismutase (SOD) activity is effective in preventing, ameliorating or treating renal damage, such as acute kidney injury.
  • the polypeptide having SOD activity may be included in the composition in the form of a Bacillus species strain expressing SOD, a spore of said Bacillus species strain or free SOD. Accordingly, according to one aspect of the present invention, there is provided a use for preventing or treating renal damage of at least one selected from the group consisting of a Bacillus species strain expressing SOD, a Bacillus species strain spore expressing SOD and superoxide dismutase.
  • a use is provided of a combination of two or more selected from a Bacillus species strain, a Bacillus species strain spore, and SOD for preventing, ameliorating, or treating kidney damage.
  • uses of SOD for preventing or treating kidney damage are provided.
  • Kidney damage refers to a deterioration in kidney function, particularly characterized by a decrease in the ability of the kidney to filter waste products from the blood. Kidney damage may be due to, but is not limited to, trauma, infection, drug toxicity, vascular abnormalities, tumors, aging, surgery, etc. In some embodiments, the kidney damage may be (acute or chronic) renal failure, acute renal injury, or chronic renal damage, such as contrast agent-induced acute renal injury, ischemic acute renal failure, or chronic renal failure or chronic renal damage caused thereby.
  • Bacillus-derived SOD was orally administered during, before, or after induction of renal damage (induction of acute renal failure or contrast agent-induced acute kidney injury due to ischemia-reperfusion injury), the degree of renal damage was significantly improved (see Examples 1 to 5).
  • a mouse model of renal damage induced by ischemia-reperfusion injury after administration of Bacillus-derived SOD showed improved intestinal permeability and alleviated intestinal epithelial cell death (improvement of intestinal environment) compared to a mouse model of renal damage not administered with SOD.
  • administration of Bacillus-derived SOD increased the distribution of SOD, an oxidative stress factor, in the kidney, and showed the effect of protecting renal function and alleviating the degree of renal tubular damage (renal protective effect).
  • Bacillus species strain expressing SOD Bacillus species strain expressing SOD, spores of Bacillus species strain expressing SOD and/or composition comprising SOD
  • a composition for preventing, improving or treating kidney damage comprising as an active ingredient at least one selected from the group consisting of a Bacillus species strain expressing SOD, a Bacillus species strain spore expressing SOD, and SOD.
  • the composition may comprise (i) a Bacillus species strain, (ii) a Bacillus species strain spore, (iii) SOD, (iv) a Bacillus species strain and a Bacillus species strain spore, (v) a Bacillus species strain and SOD, (vi) a Bacillus species strain spore and SOD, or (vii) a Bacillus species strain, a Bacillus species strain spore and SOD.
  • spore as used herein may be used interchangeably with "spore”, and Bacillus species strain spores may be obtained by culturing a Bacillus species strain in a suitable medium, inducing sporulation, and then isolating the produced spores.
  • the Bacillus species strain can be sourced from a GRAS bacteria that is generally regarded as safe for use in drugs or foods.
  • Bacillus species strain spores are known to be resistant to proteases and low pH (see Cutting SM. Bacillus probiotics. Food Microbiol . 2011;28:214-220. doi:10.1016/j.fm.2010.03.007; and Wang Y, et al., In vitro assessment of probiotic properties of Bacillus isolated from naturally fermented congee from inner Mongolia of China. World J. Microb. Biot . 2010;26:1369-1377. doi:10.1007/s11274-010-0309-7).
  • Bacillus species strains are GRAS probiotics approved in several countries. Specifically, the Bacillus species strains can be, but are not limited to, B. velezensis, B. amyloliquesfaciens, B. methylotrophicus, B. siamensis, B. subtilis, B. tequilensis, B. atrophaeus, B. mojavensis, or B. vallismortis.
  • the Bacillus species strain spores can be derived from any of the strains described above, but are not limited to.
  • the Bacillus species strain may be a Bacillus velezensis (Bacillus amyloliquefaciens) strain (e.g., GF423 strain, GF424 strain, or GF427 strain).
  • the Bacillus species strain spores may be derived from a Bacillus velezensis (Bacillus amyloliquefaciens) strain (e.g., GF423 strain, GF424 strain, or GF427 strain).
  • the GF423 strain, the GF424 strain, and the GF427 strain were deposited with the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017, March 13, 2017, and August 14, 2023, respectively (Accession No.
  • KCTC 13222 BP Accession No. KCTC 13227 BP, and Accession No. KCTC 15552 BP. Additionally, the characteristics and cultivation method of the GF423 strain are described in Korean Patent No. 1762199, the entire disclosure of which is incorporated herein by reference.
  • the deposited Bacillus species strain was classified and described as Bacillus amyloliquefaciens in a previous application. However, when compared using genome-based classification methods such as DDH and ANI, it was found that the criteria for species level distinction were not satisfied, and thus it was suggested that it should be classified as a microorganism belonging to Bacillus velezensis .
  • Bacillus velezensis in LPSN (List of Prokaryotic names with Standing in Nomenclature; https:/lpsn.dsmz.de/species/bacillus-velezensis) (see literature [Fan, Ben, et al. "Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an "operational group B.amyloliquefaciens" within the B. subtilis species complex.” Frontiers in microbiology 8 (2017): 22.]). Given that Bacillus velezensis and Bacillus amyloliquefaciens are heterotypic synonyms, the two names can be used interchangeably.
  • the Bacillus amyloliquefaciens strains may be used interchangeably with the Bacillus velezensis strains . Accordingly, the Bacillus amyloliquefaciens GF423 strain, the GF424 strain, and the GF427 strain may be understood to be the same strains as the Bacillus velezensis GF423 strain, the GF424 strain, and the GF427 strain, respectively.
  • the process by which the deposited Bacillus species strains were classified as Bacillus velezensis is as follows.
  • the deposited Bacillus species strains were isolated and compared with three highly homologous reference strains through DDH, ANI, and AAI analyses (gene-based classification method) for 16S rRNA genes and whole genomes (see Table 1).
  • the deposited Bacillus species strains through DDH and ANI results were found to be more similar to Bacillus amyloliquefaciens subspecies plantarum than to Bacillus amyloliquefaciens subspecies amyloliquefaciens (the DDH species discrimination criterion is 70% or higher, and the ANI species discrimination criterion is 94% or higher). Meanwhile, Bacillus amyloliquefaciens subsp. plantarum was classified as a microorganism belonging to Bacillus velezensis rather than the same species as Amyloliquefaciens subsp.
  • the Bacillus species strain may be a strain expressing or producing SOD or a mutant strain mutated or recombined to overexpress or overproduce SOD.
  • the Bacillus species strain spore may be a spore of a strain expressing or producing SOD.
  • the Bacillus species strain spore may be derived from a strain mutated or recombined to overexpress or overproduce SOD.
  • the Bacillus species strain may be a naturally isolated strain (e.g., GF423 strain) or may be a mutant strain that has been mutated to overexpress or overproduce SOD (e.g., GF424 or GF427 strains).
  • the Bacillus species strain spores may be derived from another Bacillus species strain that has been recombinantly expressed to express the SOD of a Bacillus velezensis strain. Such recombinant strains are further described below. Sporulation of the Bacillus species strain can be induced using conventional techniques known in the art.
  • Superoxide dismutase is an enzyme that alternately catalyzes the dismutation of superoxide (O 2 ⁇ - ) radicals into ordinary molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ).
  • SOD plays an important role in reducing oxidative stress by removing reactive oxygen species.
  • SODs are widely distributed in prokaryotic and eukaryotic cells, and are classified into four classes according to the various types of metal centers (copper/zinc, nickel, manganese, and iron).
  • Manganese-containing SOD [Mn-SOD] is widely present in the chloroplasts, mitochondria, and cytoplasm of many bacteria or eukaryotic cells.
  • the SOD may be manganese-binding (Mn-SOD).
  • the SOD may be deamidated Mn-SOD.
  • the SOD may comprise or consist of an amino acid sequence represented by SEQ ID NO: 2 (SodA).
  • the SOD may be one in which amino acid residues 74 and 137 of SEQ ID NO: 2 are substituted with Asp.
  • the SOD may comprise or consist of an amino acid sequence represented by SEQ ID NO: 4 (SodA2).
  • the SOD may be a form in which methionine (Met), which is a start codon during protein translation, is deleted.
  • the SOD may be a form in which methionine (Met) of deamidated Mn-SOD is deleted.
  • the SOD may be one in which Met, which is amino acid residue 1 of SEQ ID NO: 2, is deleted.
  • the SOD may comprise or consist of an amino acid sequence represented by SEQ ID NO: 5 (Met-deleted SodA).
  • SOD may be one in which Met, the 1st amino acid residue of SEQ ID NO: 4, is deleted. More preferably, SOD may comprise or consist of an amino acid sequence represented by SEQ ID NO: 6 (Met-deleted SodA2).
  • the SOD or SOD activity-having polypeptide of the present invention is interpreted to also include an amino acid sequence showing substantial identity to the above amino acid sequence.
  • the substantial identity means an amino acid sequence showing a sequence identity of 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more, when analyzing aligned sequences using an algorithm commonly used in the art.
  • the SOD is a modified or engineered polypeptide having SOD enzymatic activity, which may or may not include one or more mutations, such as deletions, insertions or substitutions of one or more amino acids, that may or may not affect various aspects (e.g., in vivo, in vitro or ex vivo stability, uniformity, and/or conformational changes).
  • the polypeptide may further include a heterologous material (e.g., a tag known in the art including a HIS tag, an HA tag, a myc tag, a GFC and/or an Fc domain of an antibody) for purification, detection, in vivo delivery or increased stability.
  • the SOD of the present invention can be derived from various sources, including natural, mutant or recombinant microorganisms.
  • the SOD can be derived from bacteria.
  • the SOD can be derived from a bacterium generally regarded as safe (GRAS) for use in drugs or foods, such as a Bacillus sp. strain or a mutant or recombinant thereof.
  • GRAS bacterium generally regarded as safe
  • the SOD can be obtained from a Bacillus velezensis strain (e.g., a GF423 strain, a GF424 strain or a GF427 strain) or a culture supernatant thereof.
  • the Bacillus velezensis GF424 strain is a strain obtained by mutagenizing the Bacillus velezensis GF423 strain by UV irradiation to improve the expression of the sod gene.
  • the Bacillus velezensis GF427 strain is a strain obtained by replacing the promoter sequence of the Bacillus velezensis GF423 strain with a base sequence having a stronger promoter performance in order to improve the expression of the sod gene.
  • the SOD enzyme (SodA) derived from the Bacillus velezensis GF423, GF424 or GF427 strain is Mn-SOD and may comprise or consist of the amino acid sequence of SEQ ID NO: 2.
  • SOD may be a recombinant polypeptide.
  • SOD may be a deamidated SOD in which amino acid residues 74 and 137 of SEQ ID NO: 2 are substituted with Asp (SodA2), and this may comprise or consist of the amino acid sequence of SEQ ID NO: 4.
  • SodA2 Asp
  • SOD can be a polypeptide in which the start codon methionine (Met) is deleted.
  • SOD SOD in which amino acid residue 1 is deleted based on SEQ ID NO: 2 or SEQ ID NO: 4, and it can include or consist of an amino acid sequence of SEQ ID NO: 5 or 6.
  • the sequences of SEQ ID NOs: 1 to 6 are as shown in Table 2.
  • the SOD may be derived from another recombinant strain (e.g., a Bacillus subtilis strain) containing the SOD expression gene of a Bacillus velezensis strain.
  • the recombinant strain may be produced by recombinant technology using a conventional protein production strain known in the art.
  • the Bacillus subtilis strain (which is the parent strain of the recombinant strain) may be KCTC 3135, and the KCTC 3135 strain may be provided by the Korea Research Institute of Bioscience and Biotechnology (KCTC).
  • the recombinant strain may have one or more of the genes shown in Table 3 below deleted to facilitate the post-process.
  • a recombinant strain can be produced through the process illustrated in FIG. 1.
  • the recombinant strain can include the expression vector illustrated in FIG. 2.
  • sodA and sodA2 represent genes encoding SOD. Since the SOD derived from the strain is an enzyme secreted outside the cell, when SOD is produced using the strain, SOD can be produced in large quantities without an expensive purification process (e.g., column purification), and thus efficient production is possible.
  • SOD can be obtained by culturing a natural, mutant, or recombinant microorganism in various culture media.
  • SOD can be isolated from the culture supernatant of Bacillus velezensis GF423 strain, GF424 strain, or GF427 strain.
  • a culture solution can be obtained by first culturing a Bacillus velezensis strain in various types of media. For example, the strain is grown at about 25° C. to about 42° C. for about 1 to about 4 days using a complex medium (pH 6.0 to 7.0).
  • LB medium 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, and the like.
  • ISP International Streptomyces Project
  • NA nutrient agar
  • BHI brain heart infusion agar
  • SDA sabouraud dextrose agar
  • PDA potato dextrose agar
  • NB medium nutrient broth
  • SOD can be sourced from other natural, mutant or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda-enzymes.org on the World Wide Web).
  • the SOD can be isolated or purified from a culture of a natural, mutant or recombinant strain.
  • 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.
  • the purified product can be purified from a culture of the strain by ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or can be a culture concentrate obtained by ultrafiltration, concentration, etc.
  • substantially free of cellular material includes preparations of the protein where the protein is separated from cellular components of the cells from which the protein is isolated or recombinantly produced. In some embodiments, the phrase “substantially free of cellular material” includes preparations of protein having less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% by dry weight of unwanted protein.
  • SOD may be purified preferably by the following purification methods, but is not limited thereto.
  • the culture solution obtained by previously culturing the Bacillus velezensis strain is centrifuged to collect the culture supernatant.
  • the supernatant fraction is pretreated by solid phase extraction, and then isolated and purified by chromatography.
  • SOD can be purified using various chromatography methods. Preferably, hydrophobic interaction chromatography is used.
  • SOD may be included in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof.
  • strain lysate means a product obtained by culturing a strain and mechanically or chemically disrupting the strain, and may include all products obtained therefrom by additional processes such as extraction, dilution, concentration, and purification.
  • strain culture may mean a culture solution obtained by culturing a strain or a supernatant thereof.
  • strain culture concentrate refers to a product purified from a strain culture by ultrafiltration, ammonium sulfate treatment, column purification, concentration, or the like, or a culture concentrate obtained by ultrafiltration, concentration, or the like.
  • strain culture extract means a product extracted from the culture solution or a concentrate thereof, and may include an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or a preparation or purified product thereof, and a fraction obtained by fractionating the extract.
  • the dried form may include a freeze-dried form.
  • the SOD may comprise cellular material from the cell or tissue source from which it is derived, such as extracellular vesicles.
  • the cellular material comprising the SOD may be cultured from various sources, including natural, mutant or recombinant hosts, using conventional techniques known in the art, as described above, and separated from the culture solution by filtration, concentration, or the like.
  • a method of preventing, ameliorating, or treating renal impairment comprising administering to a subject a composition comprising a SOD as disclosed herein.
  • the method may comprise administering to a subject having or at risk of developing renal impairment a composition comprising a Bacillus species strain, a Bacillus species strain spore, and/or a SOD as an active ingredient, in a therapeutically or nutritionally effective amount.
  • the renal impairment is as described herein.
  • the method can comprise administering to the subject a Bacillus species strain, a Bacillus species strain spore, or a SOD alone as disclosed herein. In other embodiments, the method can comprise administering to the subject a combination of one or more selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and a SOD as disclosed herein.
  • the administration can be oral or parenteral, and is preferably oral. In one embodiment of the present invention, it was confirmed that SOD can effectively prevent or treat kidney damage even when administered orally to the subject.
  • the effective amount or effective non-toxic amount of a composition according to the present invention can be determined by routine experimentation.
  • the therapeutically active amount of a composition comprising the Bacillus species strain, the Bacillus species strain spore, and/or the SOD of the present invention can 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, and the ability of the species strain spore to express SOD to induce the desired response in the subject.
  • the dosage and administration regimen of the composition of the present invention can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily, weekly, every two weeks, every three weeks, every four weeks, etc., and/or the dosage can be proportionally reduced or increased as the exigencies of the therapeutic situation dictate.
  • Bacillus species strain or Bacillus species strain spores can be included in the composition at 10 4 to 10 12 , 10 4 to 10 10 , 10 4 to 10 9 , 10 5 to 10 12 , 10 5 to 10 11 , 10 5 to 10 10 or 10 5 to 10 9 cfu per dose.
  • composition of the present invention may be administered simultaneously with, or before or after, the administration of one or more other agents that cause or are likely to cause renal damage, for the purpose of preventing, improving, or treating renal damage.
  • other agents include, but are not limited to, any agent that is administered for the purpose of improving or ameliorating other diseases, such as contrast agents, antibiotics, immunosuppressants, drugs for heart disease, and cancer chemotherapy drugs, and that has the potential to cause unintended renal damage.
  • compositions comprising one or more selected from the group consisting of Bacillus species strains, Bacillus species strain spores, and/or SOD disclosed herein are administered together with another agent, they may be administered simultaneously, sequentially, or in reverse order, and may be administered before the administration of the other agent.
  • Each component may be administered to the subject at a different time from when the other components are administered.
  • each administration may be given non-simultaneously (e.g., separately or sequentially) at multiple intervals over a given period of time.
  • the individual components may be administered to the subject by the same or different routes. The routes by which administration may be administered are detailed in the pharmaceutical composition.
  • the Bacillus species strain, the Bacillus species strain spore, and/or the polypeptide having SOD activity of the present invention can be administered simultaneously, sequentially, or in reverse order.
  • a composition comprising the following components (i) to (iv) can be administered simultaneously, sequentially, or in reverse order to exhibit a preventive or therapeutic effect on renal damage: (i) the Bacillus species strain and the Bacillus species strain spore, (ii) the Bacillus species strain and SOD, (iii) the Bacillus species strain spore and SOD, or (iv) the Bacillus species strain, the Bacillus species strain spore, and SOD.
  • the Bacillus species strain spore and the SOD can be administered simultaneously, sequentially, or in reverse order to exhibit a preventive or therapeutic effect on renal damage.
  • compositions comprising:
  • a pharmaceutical or veterinary composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and SOD as an active ingredient.
  • the pharmaceutical composition of the present invention may be used interchangeably with the term veterinary composition when applied to animals other than humans.
  • Examples of the carriers, excipients or diluents include, but are not limited to, mineral oils such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and silicon dioxide.
  • mineral oils such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl
  • additives such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants.
  • the additives for the above formulation can be appropriately selected from those commonly used in the pharmaceutical field.
  • the SOD may be coated with shellac for protection from gastric acid, but the coating agent is not limited thereto.
  • coating agents suitable for use in the present invention include shellac, ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof.
  • the SOD may be coated in solution. Specifically, the purified solution and the shellac-containing solution are mixed, and then lyophilized. The lyophilized sample may be powdered and stored at about 4° C. until use.
  • the SOD may be an oral SOD having enhanced stability in gastric acid through the shellac coating.
  • the SOD may be a shellac-coated SOD in which amino acid residue 1, Met, is deleted based on SEQ ID NO: 4. More preferably, the SOD may be a shellac-coated SOD comprising or consisting of an amino acid sequence represented by SEQ ID NO: 6.
  • the pharmaceutical or veterinary composition of the present invention is administered in a pharmaceutically or veterinarily effective amount.
  • pharmaceutically effective amount or “veterinarily effective amount” means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical or veterinary treatment, and the effective dosage level can be determined based on factors including the body weight, sex, age, health status, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.
  • the pharmaceutical or veterinary composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously.
  • the pharmaceutical composition may be administered singly or in multiple doses as needed. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and such an amount can be easily determined by a person skilled in the art.
  • the composition may have various forms suitable for human administration known in the art, including liquid form, solid form, gel form, powder form, paste form, and the like.
  • a food composition comprising at least one selected from the group consisting of a Bacillus species strain, a Bacillus species strain spore, and SOD as described above.
  • the food composition comprises a medical or nutraceutical food composition. Accordingly, the food composition can be provided for use in preventing or improving kidney damage as described herein.
  • medical food or “nutraceutical food” refers to a food manufactured with raw materials or ingredients that have the potential to have a beneficial function for the human body, and that maintains or improves health by maintaining normal functions or activating physiological functions of the human body, and is regulated by the Ministry of Food and Drug Safety, but is not limited thereto, and does not exclude any conventional health food from its meaning.
  • food includes, but is not limited to, various food products, food additives, beverages (e.g., functional beverages, natural fruit juices and vegetable beverages), gum, tea, vitamin complexes, health functional foods, and other functional foods.
  • beverages e.g., functional beverages, natural fruit juices and vegetable beverages
  • gum e.g., tea, vitamin complexes, health functional foods, and other functional foods.
  • Foods can be prepared by conventional methods known in the art.
  • medical foods, nutraceutical foods or health functional foods can be formulated as one selected from the group consisting of tablets, pills, powders, granules, powders, capsules and liquid formulations by additionally including one or more of carriers, diluents, excipients and additives in addition to the SOD for the purpose of preventing or improving kidney damage.
  • carriers, diluents, excipients and additives are well known in the art, and a person skilled in the art can prepare them by combining appropriate ingredients according to the formulation.
  • the content of the Bacillus species strain, strain spores and/or SOD according to the present invention as an effective ingredient in the formulation described above can be appropriately adjusted depending on the form and purpose of use, patient condition, type and severity of symptoms, etc., and may be 0.001 to 99.9 wt%, preferably 0.01 to 50 wt%, based on the solid weight, but is not limited thereto.
  • the dosage of the food of the present invention may vary depending on the patient's age, weight, sex, dosage form, health condition, and disease severity, and may be administered once a day or several times in divided doses at regular intervals depending on the judgment of a doctor or pharmacist.
  • 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, a significant effect may not be obtained, and if it exceeds it, it is not only uneconomical, but also goes beyond the range of commercial dosage, so undesirable side effects may occur.
  • a feed composition comprising at least one selected from the group consisting of Bacillus species strains, Bacillus species strain spores, and SOD disclosed herein is provided.
  • the feed composition of the present invention can be prepared in any formulation commonly used in the art.
  • the feed composition of the present invention can further comprise auxiliary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes, and other microbial preparations in the form of live cells; grains such as pulverized or crushed wheat, oats, barley, corn, and rice; plant-based protein feeds such as those mainly composed of rapeseed, soybeans, and sunflower; animal-based protein feeds such as blood meal, meat meal, bone meal, and fish meal; dry ingredients composed of sugars and dairy products such as various types of milk powder and whey powder; lipids such as animal fats and plant-based fats optionally liquefied by heating; and additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventive agents.
  • auxiliary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes, and other microbial preparations in the form of live cells
  • grains such as pul
  • the feed composition of the present invention may be in the form of a powder or liquid formulation, and may include excipients for feed addition (calcium carbonate, malt powder, zeolite, corn powder, rice bran, etc.).
  • the Bacillus velezensis ( Bacillus amyloliquefaciens ) strain expressing SOD used in this example was a Bacillus velezensis strain deposited at the Korea Culture and Technology Center (KCTC) (Accession No.: KCTC 13222BP, Deposit Date: March 6, 2017) (hereinafter referred to as "GF423 strain”), a Bacillus velezensis strain deposited at the Korea Culture and Technology Center (KCTC) (Accession No.: KCTC 13227BP, Deposit Date: March 13, 2017) (hereinafter referred to as "GF424 strain”), or a Bacillus velezensis strain deposited at the Korea Culture and Technology Center (KCTC) (Accession No.: KCTC 15552BP, Deposit Date: August 14, 2023) (hereinafter referred to as "GF427 strain”).
  • Spores of the strain were prepared according to the following method.
  • a single colony of the Bacillus velezensis strain was inoculated into 1 mL of LB in a 14 mL tube and cultured at 37°C and 200 rpm for 12 h.
  • 1 mL of the culture was transferred to 50 mL of LB medium in a 500 mL flask and cultured at 37°C and 200 rpm for 12 h.
  • 20 mL of the culture medium was transferred to 1 L of SYP or DSM in a 2.5 L baffled flask.
  • the inoculated culture was cultured at 37°C and 200 rpm for 24 to 120 h.
  • SYP medium contains 1.5% soy tone, 0.5% yeast extract, 0.5% K2HPO4, 0.1% MnSO4 , 0.1% MgSO4 , 10 mM FeSO4 , 0.04% (NH4) 2SO4 , 0.04% ( NH4 ) 2PO4 , 0.1% CaCl2 and 2 % glucose
  • DSM medium contains 8 g /L bacto nutrient broth, 1 g/L KCl, 0.25 g/L MgSO4 , 0.16415 g /L Ca( NO3 ) 2 , 0.9521 mg/L MnCl2 and 0.152 mg FeSO4.
  • MnSO 4 , MgSO 4 , FeSO 4 , (NH 4 ) 2 SO 4 , (NH 4 ) 2 PO 4 and CaCl 2 added to the above medium were dissolved in ddH 2 O before use and added.
  • lysozyme 0.5 g/L was added to the culture broth and incubated at 37°C and 200 rpm for 1 hour to remove any remaining vegetative cells.
  • the crude spores were collected by centrifugation at 6000 rpm for 10 minutes. The collected crude spores were purified by washing twice with water, washing with 0.02% SDS, washing twice with water again, and then suspending in PBS solution. The spore suspension was stored at -20°C.
  • the number of spores was determined by spreading the diluted spore solution on an LB agar plate and counting the colonies. Spores were dissolved in phosphate-buffered saline (PBS) according to experimental conditions and prepared in a volume of 100 ⁇ l.
  • PBS phosphate-buffered saline
  • the Bacillus velezensis GF427 strain expressing SOD was prepared from the GF423 strain by replacing the promoter sequence of the GF423 sodA gene with a base sequence having a stronger promoter performance to increase SOD activity according to the following method.
  • the promoter sequences of the original GF423 strain and the mutant GF427 strain are as follows: GF423 promoter sequence (5'-TTGATTACCACGCTTTCTTTT-GTTACATT-3') (SEQ ID NO: 7) and GF427 promoter sequence (5'-TTGACTTT-ACGCTTTCTTATAGGTTATAAT-3') (SEQ ID NO: 8).
  • PCR was performed using GF423 genomic DNA as a template and the primers SOD up F and Psodmut R to prepare a PCR product
  • a PCR product was prepared using GF423 genomic DNA as a template and the primers Psodmut F and SOD dw R to prepare a PCR product.
  • the PCR product was cloned into pUori-cm-amp-tsrepA digested with BamHI using the LIC method, to produce pUori-cm-amp-10sod (Fig. 3a).
  • PCR was performed using pUori-cm-amp-10sod as a template and the SOD PF and SOD P3R primers to prepare the PCR products, and the PCR products were prepared using the same template and the SOD P3F and SOD PR primers to prepare the PCR products.
  • the PCR products were cloned into pUori-cm-amp-10sod digested with HindIII and ClaI using the LIC method, to produce pUori-cm-amp-P3-SOD to be used for replacement (Fig. 3b).
  • the vector was produced using E. coli C2984H, and the base sequences of the primers used, PCR conditions, and LIC reaction solution compositions are shown in Tables 4, 5, and 6, respectively.
  • PCR step temperature hour First transformation 95°C 30 seconds 30 cycles 95°C 30 seconds 50°C 30 seconds 72°C 1 min/kb Final height 72°C 10 minutes
  • the DNA polymerase used for PCR was Taq DNA polymerase (TAKARA, JAPAN).
  • GF423 strain was inoculated into LB-Sor medium (1x LB, 0.5 M sorbitol) and cultured at 37°C, 200 rpm. When OD was 0.8, glycine was added to a final concentration of 10 mg/ml, and cultured for additional 1.5 h. After culture, cells were placed on ice, cooled for 15 min, and centrifuged at 4000 rpm, 4°C, and 10 min.
  • the harvested cells were washed three times with electrotransfection buffer (containing 0.5 M sorbitol, 0.5 M mannitol, and 10% glycerol), and the cells were resuspended in 1/50 of the culture volume using the same buffer and used for electroporation.
  • electrotransfection buffer containing 0.5 M sorbitol, 0.5 M mannitol, and 10% glycerol
  • the mixture was placed in a 1 mm gap electroporation cuvette that had been cooled and reacted on ice for 3 min.
  • PCR was performed using primers Cm CF and SOD mid R to obtain colonies in which single crossover recombination had occurred.
  • the obtained colonies were inoculated back into LB broth and cultured at 28°C for 20 hours, then 1/10 5 was diluted and plated on LB solid medium and cultured.
  • the confirmed colonies were stamped on both antibiotic solid medium and general solid medium to confirm colonies from which the plasmid had been removed.
  • PCR conditions were set so that only colonies that successfully underwent engineering were confirmed to show a band using primers containing the mutation site, and colonies that successfully underwent mutation were selected.
  • the primers used were SOD DC F2, -10 SOD R pair, SOD DC F2, SOD P2 R pair.
  • the selected colonies were subjected to PCR using 424 conf F and 424 conf R primers, and the PCR product was gel purified.
  • the sample was sequenced to confirm the correct substitution and named 'GF427'.
  • This strain was deposited with the Korea Research Institute of Bioscience and Biotechnology on August 14, 2023 (KCTC 15552 BP).
  • KCTC 15552 BP Korea Research Institute of Bioscience and Biotechnology on August 14, 2023
  • SOD expression amount of the GF427 strain it was compared with the SOD expression amount of the GF424 strain, which is a form with improved SOD activity.
  • the SOD activity of the culture supernatant was measured as follows.
  • Glass SOD was prepared by extracting and purifying a culture solution of Bacillus velezensis strain using the following method.
  • LB agar medium (LB (Luria-Bertani) agar; tryptophan 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L) was inoculated into 30 ml of LB medium and cultured at 37°C for 12 hours. This seed culture was then inoculated into 3 L of LB medium containing 1 mM manganese sulfate (MnSO4) and cultured at 37°C for 20 hours. The cell culture obtained in this way was centrifuged at 3,578 ⁇ g for 20 minutes at 4°C, and the supernatant was collected and concentrated 10-fold using ultrafiltration (UF, MWCO 10,000).
  • LB Lia-Bertani
  • the purified solution purified through the column was collected and concentrated while removing the high-concentration salts formed during the purification process through ultrafiltration.
  • the concentrated solution was filtered through a sterilizing filter and then lyophilized.
  • the activity of SodA2 was analyzed using a SOD assay kit (Cayman Chemical, Michigan, USA).
  • One unit of SOD activity is defined as the amount of enzyme that inhibits 50% of superoxide radicals.
  • GF103 Metal-deleted SodA2 of SEQ ID NO: 6
  • Shellac EXCELACS co., LTD., Bangkok
  • the shellac solution dissolved in ethanol was diluted 1/20 times with sterile 1x PBS to prepare a shellac solution.
  • Lyophilized purified SodA2 was dissolved to 20 mg/ml and sterilized using a 0.2 ⁇ m pore size filter.
  • the prepared shellac solution and the dissolved SOD were mixed in a 1:1 ratio with stirring. Stirring was then continued for 10 minutes.
  • shellac-coated SodA2 that was finally freeze-dried was prepared.
  • Dextrin was added to freeze-dried shellac-coated SodA2 at a ratio of 1:9 to 12 (shellac-coated SodA2: dextrin), and the final activity was measured to be 90 to 110 U/mg.
  • Example 1 Preparation of an ischemia-reperfusion acute renal failure animal model and administration of SOD
  • mice Six-week-old male C57BL/6 mice were purchased from Orient Bio and raised in a specific pathogen free 1 facility with free access to water and feed. The mice were randomly assigned to the "untreated Sham group (Sham)", "untreated IRI group (IRI)", “SOD-BA-administered Sham group (SOD+Sham)", and "SOD-BA-administered IRI group (SOD+IRI)".
  • the IRI group is a group in which ischemia-reperfusion injury is induced to cause acute renal failure
  • the "untreated IRI group” is a group in which ischemia-reperfusion injury is induced in mice that were not administered SOD-BA
  • the "SOD-BA-administered IRI group” is a group in which ischemia-reperfusion injury is induced 24 hours after SOD-BA administration.
  • the Sham group was a group in which ischemia-reperfusion injury was not induced. Similar to the IRI group, dorsal incision was performed after intraperitoneal anesthesia, but renal blood vessel ligation was not performed. Depending on whether SOD-BA was administered before surgery, they were referred to as the “non-administered Sham group” and the “SOD-BA-administered Sham group.”
  • IRI bilateral ischemic reperfusion injury
  • Ischemia-reperfusion injury was induced by ligating the renal vessels on both sides through a dorsal incision under intraperitoneal anesthesia, blocking blood flow for 24 to 25 minutes, and then reperfusion. The mice were monitored on a heating pad until they woke up after surgery. This study was approved by the Institutional Review Board of the Animal Research Center, Korea University College of Medicine (IRB Number: KOREA-2020-0061).
  • the preparation of the drug substance (SOD-BA) in Manufacturing Example 4 was orally administered once per mouse at a dose of 20 U one day before vascular ligation.
  • At least two fecal pellets per mouse were collected, stored at -70°C, and analyzed in batches. 16S rRNA pyrosequencing analysis was used for quantitative microbiome analysis.
  • the untreated Sham group (Sham), the SOD-BA-administered Sham group (SOD+Sham), the unadministered IRI group (IRI), and the SOD-BA-administered IRI group (SOD+IRI) showed different microbiome-gut microbiota compositions in the principal coordinate analysis (PCoA) (Fig. 4a).
  • the gut microbiota diversity index remained similar to that in the unadministered IRI group (IRI), but the composition ratio of Bacteroidaceae increased.
  • the composition ratio of Bacteroidaceae showed a greater difference than that between the unadministered Sham group (Sham) and the SOD-BA-administered Sham group (SOD+sham) (Fig. 4b).
  • Example 2.2 Intestinal permeability analysis
  • FITC-dextran assay was performed.
  • fluorescent dextran Fluorescein isothiocyanate-conjugated dextran (FITC-dextran); catalog number FD4; Sigma-Aldrich, St. Louis, MO, USA
  • PBS 100 mg/mL
  • Fluorescence was extracted from the blood 4 hours later.
  • Apoptosis of colonic epithelial cells was quantified by counting TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling)-positive epithelial cells in 5–8 high-power fields ( ⁇ 200, high power field, HPF). Images were digitized and analyzed using a slide scanner, an automatic image capture system (Axio Scan Z1, Zeiss Korea, Seoul).
  • Example 2.4 Evaluation of intestinal inflammatory cell infiltration
  • Colonic tissues were stained with F4/80 monoclonal antibody (1:100; mch-497-GA; Bio-Rad Laboratories, Hercules, CA, USA) and Ly6G monoclonal antibody (1:200; 14-59-85; eBioscience) to assess macrophage/neutrophil infiltration levels.
  • the neutrophil marker (Ly6G) was significantly increased in the untreated IRI group and decreased in the SOD-BA-administered IRI group compared to the unadministered IRI group ( Figures 7a and 7b).
  • SOD and catalase are oxidative stress factors that play an important role in improving oxidative stress up to the final metabolic product, water.
  • the improvement of renal oxidative stress in acute renal failure was evaluated by measuring the distribution of SOD and catalase in the kidney.
  • SOD and catalase were stained with SOD monoclonal antibody (1:500; MA1-105, ThermoFisher) and catalase monoclonal antibody (1:500, PA5-29183, ThermoFisher), respectively, and the distribution was evaluated comparatively as the staining positivity ratio (%) relative to the total tissue area at low magnification.
  • Renal tissues were stained with F4/80 monoclonal antibody (1:100; mch-497-GA; Bio-Rad Laboratories, Hercules, CA, USA) and Ly6G monoclonal antibody (1:200; 14-59-85; eBioscience) to assess macrophage/neutrophil infiltration levels.
  • BUN blood urea nitrogen
  • ATN score improvement in the renal tubular damage score
  • Example 4.1 Blood urea nitrogen and creatinine tests
  • Creatinine and urea nitrogen are normally filtered and excreted by the kidneys, so they exist in small amounts in the blood. However, when there is a problem with kidney function, they are not filtered normally, so their concentration in the blood increases and they are used as factors for evaluating kidney function.
  • Plasma creatinine and urea nitrogen were measured using Beckman AU® 5821 Beckman (Beckman Coulter, USA). After intraperitoneal anesthesia, tissues were removed, and liver and colon tissues were fixed with 4% paraformaldehyde and paraffin. Kidney tissues were evaluated using a standard light microscope.
  • the extent of tubular damage was assessed in a dark-blind manner in five random fields ( ⁇ 100) throughout the renal tissue using periodic acid-Schiff stain (PAS). The extent of damage was assessed semiquantitatively and classified into grade 0, 0–25% (grade 1), 25–50% (grade 2), 50–75% (grade 3), and 75–100% (grade 4) according to the level of visible necrosis.
  • Example 5.1 Construction of a contrast agent-induced acute renal injury model
  • CI-AKI contrast-induced acute kidney injury
  • mice were randomly assigned to the “Vehicle-administered CI-AKI group (PBS+CI-AKI)” and the “SOD-BA-administered CI-AKI group (SOD+CI-AKI)” groups, with 5 to 7 mice per group.
  • the prepared administration material (SOD-BA) in Manufacturing Example 4 was orally administered once or twice per mouse at a dose of 20 U before contrast injection, and the dehydration state was maintained for 48 hours.
  • the CI-AKI group administered vehicle was orally administered PBS in the same amount as SOD.

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Abstract

La présente invention porte sur une utilisation d'une superoxyde dismutase dérivée de bacille pour prévenir ou traiter une lésion rénale. Plus particulièrement, la présente invention porte sur une composition pharmaceutique ou une méthode thérapeutique de prévention ou de traitement d'une lésion rénale. La composition et la méthode selon la présente invention présentent au moins un effet choisi dans le groupe constitué par un changement de la composition microbienne intestinale, une réduction de la mort des cellules épithéliales intestinales, une réduction de la perméabilité intestinale, une réduction du stress oxydatif dans le rein, un soulagement d'une lésion tubulaire et une amélioration de la fonction rénale, et peuvent ainsi être efficacement utilisées pour prévenir ou traiter une lésion rénale.
PCT/KR2024/016851 2023-10-30 2024-10-30 Superoxyde dismutase et ses utilisations pour la prévention ou le traitement d'une lésion rénale Pending WO2025095595A1 (fr)

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KR10-2023-0147284 2023-10-30
KR20230147284 2023-10-30
KR20230147282 2023-10-30
KR10-2023-0147282 2023-10-30
KR10-2024-0075277 2024-06-10
KR20240075277 2024-06-10

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018164468A1 (fr) * 2017-03-09 2018-09-13 Genofocus, Inc. Souche de bacillus amyloliquefaciens gf423, et composition ayant des activités anti-oxydantes et anti-inflammatoires ou de prévention ou de traitement de l'hyperlipidémie, y compris un polypeptide produit par celle-ci
KR20200027624A (ko) * 2018-09-04 2020-03-13 주식회사 제노포커스 염증성 장질환 예방 또는 치료용 약학적 조성물
KR20230017372A (ko) * 2021-07-21 2023-02-03 주식회사 제노포커스 수퍼옥시드 디스뮤타제 및/또는 바실러스 균주 포자를 포함하는 조성물 및 이의 구강 건강 개선용 용도
KR20230099603A (ko) * 2021-12-27 2023-07-04 주식회사 제노포커스 수퍼옥시드 디스뮤타제 및 이의 건성안의 예방 또는 치료용 용도

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018164468A1 (fr) * 2017-03-09 2018-09-13 Genofocus, Inc. Souche de bacillus amyloliquefaciens gf423, et composition ayant des activités anti-oxydantes et anti-inflammatoires ou de prévention ou de traitement de l'hyperlipidémie, y compris un polypeptide produit par celle-ci
KR20200027624A (ko) * 2018-09-04 2020-03-13 주식회사 제노포커스 염증성 장질환 예방 또는 치료용 약학적 조성물
KR20230017372A (ko) * 2021-07-21 2023-02-03 주식회사 제노포커스 수퍼옥시드 디스뮤타제 및/또는 바실러스 균주 포자를 포함하는 조성물 및 이의 구강 건강 개선용 용도
KR20230099603A (ko) * 2021-12-27 2023-07-04 주식회사 제노포커스 수퍼옥시드 디스뮤타제 및 이의 건성안의 예방 또는 치료용 용도

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
HOU XINYUE, SHI JIANXIANG, ZHANG JIE, WANG ZHIGANG, ZHANG SEN, LI RUIFENG, JIANG WEI, HUANG TINGTING, GUO JIANCHENG, SHANG WENJUN: "Treatment of Acute Kidney Injury Using a Dual Enzyme Embedded Zeolitic Imidazolate Frameworks Cascade That Catalyzes In Vivo Reactive Oxygen Species Scavenging", FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, FRONTIERS RESEARCH FOUNDATION, CH, vol. 9, CH , XP093310057, ISSN: 2296-4185, DOI: 10.3389/fbioe.2021.800428 *
ISMY JUFITRIANI, SYUKRI MAIMUN, EMRIL DESSY R., SEKARWANA NANAN, ISMY JUFRIADY, SUHANDA RACHMAD: "The Effect of Superoxide Dismutase on Inhibition of Acute Kidney Injury Induced by Sepsis Based on Kidney Tissue Histology and Murine Sepsis Score", THE SCIENTIFIC WORLD JOURNAL, HINDAWI LIMITED, vol. 2021, 16 December 2021 (2021-12-16), pages 1 - 7, XP093310054, ISSN: 2356-6140, DOI: 10.1155/2021/1827296 *
ISMY JUFITRIANI, SYUKRI MAIMUN, EMRIL DESSY R., SEKARWANA NANAN, ISMY JUFRIADY: "Superoxide Dismutase Reduces Creatinine and NGAL by Restoring Oxidative Balance during Sepsis", EMERGING SCIENCE JOURNAL, vol. 6, no. 2, pages 286 - 294, XP093310055, ISSN: 2610-9182, DOI: 10.28991/ESJ-2022-06-02-06 *
KANDIL ASLI: "The Effects of a Probiotic (Bacillus clausii) in Acute Kidney Injury in a Rat Model of LPS-Induced Endotoxemia", EUROPEAN JOURNAL OF BIOLOGY, vol. 80, no. 1, 16 June 2021 (2021-06-16), pages 48 - 53, XP093310061, ISSN: 2602-2575, DOI: 10.26650/EurJBiol.2021.931755 *
KANG, JI-EUN; KIM, HYUN-DO; PARK, SOO-YOUNG; PAN, JAE-GU; KIM, JEONG HYUN; YUM, DO-YOUNG: "Dietary supplementation with a bacillus superoxide dismutase protects against gamma-radiation-induced oxidative stress and ameliorates dextran sulphate sodium-induced ulcerative colitis in mice", JOURNAL OF CROHN'S AND COLITIS, ELSEVIER BV, NL, vol. 12, no. 7, 14 March 2018 (2018-03-14), NL , pages 860 - 869, XP009516289, ISSN: 1873-9946, DOI: 10.1093/ecco-jcc/jjy034 *
KIM YE NA, JUNG YEONSOON: "Renal and Urinary Manifestations of Inflammatory Bowel Disease", KOREAN JOURNAL OF GASTROENTEROLOGY, KOREAN SOCIETY OF GASTROENTEROLOGY, KR, vol. 73, no. 5, 1 January 2019 (2019-01-01), KR , pages 260, XP093310059, ISSN: 1598-9992, DOI: 10.4166/kjg.2019.73.5.260 *
LEE SANG-OK, TAE-HO LEE: " Study on the Intracellular Superoxide Dismutase Produced by Bacillus circulans", KOR. J. APPL. MICROBIOL. BIOENG., vol. 15, no. 6, 1 January 1987 (1987-01-01), pages 381 - 387, XP093310063 *
YOUNG EUN CHOI, JIHYUN YANG, SE WON OH, MYUNG-GYU KIM, TAI YEON KOO, SUKMIN CHUNG, SUNGYEON KIM, YOOKYUNG JANG, SANG-KYUNG JO: "A-1362 Dietary Supplementation with Bacillus Superoxide Dismutase Protects Against Contrast-Induced Acute Kidney Injury In Mice", APCN KSN 2024 COEX SEOUL KOREA, THE 22ND ASIA PACIFIC CONGRESS OF NEPHROLOGY AND 44TH ANNUAL MEETING OF THE KOREA SOCIETY OF NEPHROLOGY. 13-16 JUNE 2024, KOREA SOCIETY OF NEPHROLOGY, KOREA, 13 June 2024 (2024-06-13) - 16 June 2024 (2024-06-16), Korea, pages 1, XP009563283 *

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