WO2020175936A1 - 항균활성을 갖는 폴리펩타이드, 이를 포함하는 패혈증 예방 또는 치료용 조성물, 및 항균용 조성물 - Google Patents

항균활성을 갖는 폴리펩타이드, 이를 포함하는 패혈증 예방 또는 치료용 조성물, 및 항균용 조성물 Download PDF

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Publication number
WO2020175936A1
WO2020175936A1 PCT/KR2020/002826 KR2020002826W WO2020175936A1 WO 2020175936 A1 WO2020175936 A1 WO 2020175936A1 KR 2020002826 W KR2020002826 W KR 2020002826W WO 2020175936 A1 WO2020175936 A1 WO 2020175936A1
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Prior art keywords
polypeptide
arginine
acid
lysine
general formula
Prior art date
Application number
PCT/KR2020/002826
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English (en)
French (fr)
Inventor
박영민
김양미
정인덕
이승현
Original Assignee
단디바이오사이언스 주식회사
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020200023629A external-priority patent/KR102368983B1/ko
Application filed by 단디바이오사이언스 주식회사 filed Critical 단디바이오사이언스 주식회사
Priority to AU2020227591A priority Critical patent/AU2020227591B2/en
Priority to CA3168643A priority patent/CA3168643A1/en
Priority to CA3139464A priority patent/CA3139464A1/en
Priority to US17/434,538 priority patent/US20220144892A1/en
Priority to EP20763798.4A priority patent/EP3932936A4/en
Priority to CN202080017455.8A priority patent/CN113544139A/zh
Priority to JP2021549548A priority patent/JP7219512B2/ja
Priority to BR112021016996A priority patent/BR112021016996A8/pt
Publication of WO2020175936A1 publication Critical patent/WO2020175936A1/ko

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Polypeptide having antibacterial activity Polypeptide having antibacterial activity, composition for preventing or treating sepsis, and composition for antibacterial
  • the present invention relates to a polypeptide having antibacterial activity, a composition for preventing or treating sepsis, and a composition for antibacterial including the same.
  • Sepsis ( 8 ⁇ ) is when pathogenic Gram-negative bacteria are infected in a living body, the cell wall component lipolysaccharide (1 0) 301) 3 ⁇ 4& ⁇ 11&acid (no,) 3 ⁇ 4) acts as a toxin and the body's immunity As an inflammatory reaction caused by excessive activation of the system, it may infect the whole body or accompany shock if the symptoms are severe.
  • sepsis is malignant tumor, leukemia, malignant lymphoma, acquired immune insufficiency syndrome (normal), collagen disease, renal failure.
  • Patients with basic diseases such as liver disease, cerebrovascular disorders, diabetes, etc., patients with humoral immunity or cellular immunity such as premature infants, weak-resistive host with adrenal steroids or anti-tumor drugs, radiation such as cobalt irradiation Treatment or induction Catheter, hemodialysis, organ transplantation, heart surgery, etc. It usually occurs in the case of treatment or surgery. Sepsis is a major cause of death for patients admitted to the hospital intensive care ward, and the mortality rate is usually 30 It is a very serious disease with more than %%. Despite advances in medical technology, surgery is still worldwide.
  • Sepsis often occurs due to infection due to sequelae, and sepsis often develops when people with weak immunity, such as newborns and elderly people, are infected. Representatively, in the case of neonatal sepsis, about 3 out of 1,000 full-term infants occur. Is known to occur, and premature infants are known to increase the incidence rate by 3 to 4 times.
  • antibacterial agent-resistant bacteria show resistance to specific antibacterial agents, so that the drug does not work.
  • penicillin-resistant Staphylococcus aureus which does not work with penicillin drugs at all. In addition to this, it was the first time in the academic world in 1961.
  • the present inventors have repeatedly studied to meet the above requirements, and as a result, a peptide having a specific amino acid sequence not only inhibits the growth of bacteria, but also has an excellent effect of removing endotoxins isolated from dead bacteria.
  • the present invention was completed by confirming that it is effective in treating sepsis and shows excellent antibacterial activity selectively against Gram-positive bacteria.
  • the task to be solved by the present invention is to provide a peptide having antibacterial activity, a composition for preventing or treating sepsis, and a composition for antibacterial including the same.
  • n 0 or 1
  • V is valine
  • XI is lysine (K) or arginine (R);
  • X2 is glycine (G) or arginine (R);
  • X3 is glutamic acid (E) or lysine (K);
  • X4 is alanine (A) or leucine (L);
  • X5 is lysine (K) or arginine (R);
  • X6 is tyrosine (Y), alanine (A), tryptophan (W), lysine (K) or aspartic acid (D); and
  • X7 is aspartic acid (D) or arginine (R),
  • any one of the nine polypeptides consisting of the following 1) to 9) is provided:
  • n 0;
  • X2 is glycine (G);
  • X3 is glutamic acid (E);
  • X4 is an alanine (A);
  • X5 is lysine (K);
  • X6 is tyrosine (Y)
  • X7 is arginine (R)
  • n 1;
  • XI is arginine (R);
  • X2 is glycine (G);
  • X3 is glutamic acid (E);
  • X4 is alanine (A);
  • X5 is lysine (K);
  • X6 is tyrosine (Y)
  • X7 is arginine (R),
  • n 1;
  • XI is arginine (R); [57] X2 is glycine (G);
  • X3 is glutamic acid (E);
  • X4 is leucine (L);
  • X5 is lysine (K);
  • X6 is tyrosine (Y)
  • n 0;
  • X2 is glycine (G);
  • X3 is glutamic acid (E);
  • P is X4 is alanine (A);
  • X5 is leucine (L);
  • X6 is tyrosine (Y)
  • X7 is aspartic acid (D), 4] a polypeptide
  • n 0;
  • X2 is arginine (R);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • X6 is tyrosine (Y)
  • n 0;
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • X6 is tyrosine (Y), and 0 2020/175936 1»(:1 ⁇ 1 ⁇ 2020/002826
  • n 0;
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • X6 is alanine (A)
  • n 0;
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • n 0;
  • XI is lysine (K);
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • X6 is lysine (K)
  • the polypeptide is L-type, D-type, and
  • a peptide mimetic or a non-natural amino acid containing a peptoid Provides a polypeptide.
  • polypeptide obtained by alkylating, PEGylating, or amidating the end of the polypeptide.
  • polypeptide is one or more of the following characteristics.
  • polypeptide which is characterized by:
  • the present invention comprises a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula as an active ingredient, preventing, improving or treating sepsis Provides a dragon composition.
  • the present invention comprises a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula as an active ingredient, preventing, improving or treating sepsis for
  • the present invention is a polypeptide represented by the above sequence general formula or the above sequence
  • It provides a method for preventing or treating sepsis, comprising administering an acetate salt substitution of trifluacetic acid of a polypeptide represented by the general formula to a subject in need thereof.
  • the present invention is a polypeptide represented by the above sequence general formula or
  • Acetate salt substitutes are used to prevent, improve or treat sepsis.
  • the present invention provides a use of a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of a polypeptide represented by the above sequence general formula for the manufacture of a preparation for preventing, improving or treating sepsis. to provide .
  • the present invention is a polypeptide represented by the above sequence general formula or the above sequence It provides an antibacterial composition comprising an acetate salt substitution of trifluacetic acid of a polypeptide represented by the general formula as an active ingredient.
  • the present invention comprises as an active ingredient the acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula or the polypeptide represented by the above sequence general formula,
  • the present invention is a polypeptide represented by the above sequence general formula or the above sequence
  • It provides an antibacterial method comprising the step of administering an acetate salt substitution of trifluacetic acid of a polypeptide represented by the general formula to a subject in need thereof.
  • the present invention provides an antibacterial use of the polypeptide represented by the above sequence general formula or the acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula.
  • the present invention is represented by the above sequence general formula for the manufacture of an antibacterial agent.
  • the antibacterial target bacteria of the antibacterial composition are Escherichia coli DH5a, Escherichia coli Kl, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Klebsiella pneumoniae,
  • the antibacterial target bacteria of the antibacterial composition [155] in one embodiment of the present invention, the antibacterial target bacteria of the antibacterial composition
  • an antibacterial composition which is one or more antibiotic-resistant bacteria selected from the group consisting of beta-lactam antibiotic-resistance (ESBL)-resistant bacteria, carbafemem-resistant bacteria, and colistin-resistant bacteria.
  • ESBL beta-lactam antibiotic-resistance
  • the antibiotic-resistant bacteria are ESBL (E. coli), Carbapenem resistant (CR)- Acinetobactor baumannii, CR- Klebsiella pneumoniae, CR- Pseudomonas aeruginosa, 3 ⁇ 4 Colistin resistant Acinetobactor baumannii £.
  • ESBL E. coli
  • Carbapenem resistant (CR)- Acinetobactor baumannii CR- Klebsiella pneumoniae
  • CR- Pseudomonas aeruginosa CR- Pseudomonas aeruginosa
  • 3 ⁇ 4 Colistin resistant Acinetobactor baumannii £ Provides an antibacterial composition, which is at least one selected from the following groups.
  • the present invention comprises a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula as an active ingredient, preventing, improving or It provides a composition for treatment.
  • the present invention comprises a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula as an active ingredient, preventing, improving or Provides therapeutic pharmaceutical/food/health food/cosmetics/quasi-drugs/feed compositions.
  • the present invention is a polypeptide represented by the above sequence general formula or the above sequence
  • It provides a method for preventing or treating an infectious disease comprising administering an acetate salt substitution of trifluacetic acid of a polypeptide represented by the general formula to a subject in need thereof.
  • the present invention relates to a polypeptide represented by the above sequence formula or trifluacetic acid of a polypeptide represented by the above sequence formula.
  • the present invention is a polypeptide represented by the above sequence general formula for the manufacture of a preparation for preventing, improving or treating infectious diseases, or an acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula. Provides a use.
  • the infectious disease is, pneumonia, peritonitis, meningitis, wound infection, osteoarthritis, cholecystitis, urinary tract infection, meningitis, endocarditis, myocarditis, epicarditis, arthritis, population inflammation, gonorrhea, bacterial dysentery , Enteritis, conjunctivitis, gastritis, otitis media, cystitis, and provides a composition, which is an infectious disease caused by Gram-negative bacteria selected from the group consisting of lymphangitis.
  • the infectious disease is, sore throat, impetigo, rheumatic fever, glomerulonephritis, neonatal sepsis, meningitis, pharyngitis, pneumonia, endocarditis, scarlet fever,
  • composition an infectious disease caused by Gram-positive bacteria selected from the group consisting of skin soft tissue infection, deep soft tissue infection, empyema and vaginitis.
  • the present invention encodes a polypeptide represented by the above sequence general formula
  • the present invention provides a recombinant vector comprising the polynucleotide.
  • the present invention provides a method for producing a polypeptide represented by the above sequence general formula, comprising the step of culturing host cells transformed with the recombinant vector.
  • the peptide according to the present invention not only suppresses the proliferation of standard bacteria and antibiotic-resistant bacteria, but also has an excellent effect of removing bacterial-derived endotoxins, so it has excellent sepsis treatment effect. When used in combination with antibiotics, the As side effects can be minimized, it can be usefully used in the prevention or treatment of sepsis.
  • the peptide according to the present invention has excellent antibacterial activity selectively against Gram-positive bacteria, so it is useful for the prevention or treatment of various infectious diseases caused by Gram-positive bacteria or Gram-positive bacteria. Can be used.
  • the peptide according to the present invention is not only safe for human body, but also protein
  • Example 1 is a graph showing the results of measuring hemolytic activity in Example 4 of the present invention.
  • Example 2 is a graph showing the results of measuring the secondary structure of the peptide in the circularly polarized dichroic component light in Example 5 of the present invention.
  • FIG. 3 is a diagram showing the results of measuring the destructive ability of a peptide against liposomes that mimic the bacterial cell membrane (top) and the red blood cell membrane (bottom) of Example 6 of the present invention.
  • Figure 5a is a comparison of the antibiotic effect against Escherichia coli (ESBL), AllD FP13-NH 2
  • Fig. 5c is a comparison of anti-inflammatory cytokine (IL-6) inhibitory effects, AllD FP13-NH 2 (TFA) (top) and AllD FP13-NH 2 (AcOH) (bottom).
  • FIG. 6 is a diagram showing the results of measuring the destruction ability of a peptide for a liposome that mimics the Gram-positive bacterial cell membrane of Example 11 of the present invention.
  • the present invention provides a polypeptide represented by the following sequence general formula:
  • n is 0 or 1;
  • V is valine
  • R is arginine
  • XI is lysine (K) or arginine (arginine: R);
  • X2 is glycine (G) or arginine (R);
  • X3 is glutamic acid (E) or lysine (K);
  • X4 is alanine (A) or leucine (L);
  • X5 is lysine (K) or arginine (arginine: R);
  • X7 is aspartic acid (D) or arginine (arginine: R),
  • any one or more of the nine polypeptides consisting of the following 1) to 9) is provided:
  • n 0;
  • X2 is glycine (G);
  • X3 is glutamic acid (E);
  • X4 is alanine (A);
  • X5 is lysine (K);
  • X6 is tyrosine (Y)
  • n 1;
  • XI is arginine (R);
  • X2 is glycine (G);
  • X3 is glutamic acid (E);
  • X4 is alanine (A);
  • X5 is lysine (K);
  • n 1;
  • XI is arginine (R);
  • X2 is glycine (G);
  • X3 is glutamic acid (E);
  • X4 is leucine (L);
  • X5 is lysine (K);
  • X6 is tyrosine (Y)
  • X7 is arginine (R)
  • [24 is a polypeptide having an amino acid sequence of LRLGVELKRYLR (SEQ ID NO: 4);
  • n 0;
  • XI is lysine (K);
  • X2 is glycine (G);
  • X3 is glutamic acid (E);
  • X4 is an alanine (A);
  • X5 is leucine (L);
  • X6 is tyrosine (Y)
  • n 0;
  • XI is arginine (R);
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • X6 is tyrosine (Y)
  • polypeptide [264] That is, a polypeptide having an amino acid sequence of RLRVKLRRYLR (SEQ ID NO: 15);
  • n 0;
  • XI is lysine (K);
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • X7 is arginine (R),
  • n 0;
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • X6 is alanine (A)
  • X7 is arginine (R),
  • n 0;
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • n 0;
  • X2 is arginine (R);
  • X3 is lysine (K);
  • X4 is leucine (L);
  • X5 is arginine (R);
  • X6 is lysine (K)
  • polypeptide refers to a linear molecule formed by bonding of amino acid residues to each other by peptide bonds.
  • the polypeptide of the present invention is used in the art together with molecular biology methods. It can be prepared according to known chemical synthesis methods (e.g., solid-phase synthesis techniques) (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963); Stewart, et al. al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111 (1984)).
  • the scope of the polypeptide of the present invention may include a biological functional equivalent having a mutation in the amino acid sequence that exhibits biological activity equivalent to that of the polypeptide of the present invention.
  • a mutation in the amino acid sequence is an amino acid side chain substituent. It can be made on the basis of approximate similarity, such as hydrophobicity, hydrophilicity, charge and size.
  • arginine, lysine and histidine are all positively charged residues; alanine, glycine Guarine has similar sizes; it can be seen that phenylalanine, trimtophan and tyrosine have a similar shape; therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, trimtophan and tyrosine; Guatyrosine is a biologically functional equivalent.
  • hydrophobic index of amino acids can be considered.
  • Each amino acid is given a hydrophobicity index according to its hydrophobicity and charge, and it is also known that substitution between amino acids with similar hydrophilicity values leads to peptides with equal biological activity.
  • the present invention The polypeptide is interpreted to include a sequence that shows a substantial identity with the sequence listed in the sequence list.
  • the substantial identity of the above is to align the sequence of the present invention with any other sequence so as to correspond as much as possible, and In the case of analyzing the aligned sequence using an algorithm commonly used in, it can mean a sequence showing at least 80% homology, 90% homology, or 95% homology.
  • Known in the industry Huang et al., Comp. Appl. BioSci. 8:155-65 (1992); Pearson et al., Meth. Mol. Biol. 24:307-31 (1994)).
  • the polypeptide may be a peptide mimic or non-natural amino acid including L-type, D-type and peptoids.
  • the D-type polypeptide has the same amino acid sequence as the L-type polypeptide.
  • [322] provides in specific embodiments of the invention, for instance, FP12-NH 2 (SEQ ID NO: 6) and amino acid sequence allD FP12-NH 2 (SEQ ID NO: 8) as the polypeptide of the same type allomeric.
  • peptide mimetics or non-natural amino acid polypeptides including L-type, D-type and peptoids may be prepared by various methods known in the art according to the defined amino acid sequence.
  • the end of the polypeptide may be alkylated, PEGylated, or amidated.
  • the technician can adjust the conditions for alkylation or amidation, and the method of alkylation or amidation can be according to various methods known in the art.
  • 2020/175936 1 (:1 ⁇ 1 ⁇ 2020/002826
  • polypeptide according to the present invention in the polypeptide according to the present invention is a polypeptide with an amine gear 11 2 added at the end-NH 2 (SEQ ID NO: 6) ).
  • polypeptide of the present invention may include a pharmaceutically acceptable salt thereof.
  • the term “pharmaceutically acceptable”, is a reasonable benefit/risk ratio without excessive toxicity, irritation, allergic reactions, or other problems or complications, and contact with the tissues of the subject (eg human).
  • the pharmaceutically acceptable salt is, for example, a pharmaceutically acceptable salt.
  • acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, Formic acid, benzoic acid, malonic acid, gluconic acid,
  • Acid addition salts are used in the usual way, e.g., the compound is dissolved in an excess acid aqueous solution, and the dichloride is water-miscible, such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation using an organic solvent. In addition, it can be prepared by heating an equimolar amount of a compound and an acid or alcohol in water, followed by evaporating the mixture to dry it, or by suction filtration of the precipitated salt.
  • Salts derived from suitable bases may include, but are not limited to, alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.
  • Alkali metal or alkaline earth metal salts can be obtained, for example, by dissolving the compound in an excess amount of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-dissolving compound salt, and evaporating and drying the filtrate. It is pharmaceutically suitable to prepare a potassium or calcium salt, and the corresponding silver salt can be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).
  • a suitable silver salt eg, silver nitrate
  • the present invention provides an acetate salt of the above-described polypeptide.
  • the present invention is a polypeptide represented by the above-described sequence general formula
  • L-form, D-form, and peptide mimetics including peptoids or non-natural amino acids, polypeptides; Alkylation,
  • a PEGylated or amidated polypeptide; or an acetate salt of a polypeptide having an amine group (NH 2 ) added at the C-terminus is provided.
  • Polypeptides according to the present invention and salt substituents thereof have one or more of the following characteristics:
  • [35 is the cell membrane disruption ability of Gram-positive bacteria.
  • polypeptide and its salt-substituted product according to the present invention have the ability to destroy bacterial cell membranes (Examples 6 and 11).
  • red blood cell membranes In particular, red blood cell membranes. The safety was proven because it did not show any destructive power (Example 6).
  • the salt substitution product thereof with the polypeptide according to the present invention has excellent resistance to proteolytic enzymes (Example 7). Treatment with proteolytic enzymes was performed. Also, it was confirmed that stability was secured against proteolytic enzymes, which showed excellent antibacterial activity.
  • the polypeptide and the salt substitution product of the present invention are LTA (lipotechoic acid), which is a cell wall component of Gram-positive bacteria, and
  • the present invention comprises a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula as an active ingredient, preventing, improving or treating sepsis Provides a dragon composition.
  • the present invention comprises the polypeptide represented by the above sequence general formula or the acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula as an active ingredient, preventing, improving or treating sepsis for
  • the present invention is a polypeptide represented by the above sequence general formula or the above sequence
  • It provides a method for preventing or treating sepsis, comprising administering an acetate salt substitution of trifluacetic acid of a polypeptide represented by the general formula to a subject in need thereof.
  • the present invention is a polypeptide represented by the above sequence general formula or of trifluacetic acid of the polypeptide represented by the above sequence
  • Acetate salt substitutes are used to prevent, improve or treat sepsis.
  • the present invention provides a use of a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of a polypeptide represented by the above sequence general formula for the manufacture of a preparation for preventing, improving or treating sepsis. to provide.
  • spontaneous inflammatory reaction refers to a condition in which a serious inflammatory reaction occurs throughout the body due to infection with microorganisms. Fever symptoms in which the body temperature rises above 38 O C or hypothermia, which decreases below 36 O C, respiratory tract More than 24 times per minute
  • SIRS systemic inflammatory response syndrome
  • the causative bacteria include Staphylococcus, Streptococcus, Escherichia coli, Pseudomonas aeruginosa, Mycobacterium tuberculosis, Bacillus pneumonia, fungi, and anaerobic bacteria, but are not limited thereto.
  • the present invention provides a composition for antibacterial, comprising as an active ingredient the acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula or the polypeptide represented by the above sequence general formula.
  • the present invention is an acetate salt of trifluacetic acid of a polypeptide represented by the above sequence general formula or a polypeptide represented by the above sequence general formula For antibacterial use, containing a substitute as an active ingredient
  • the present invention is a polypeptide represented by the above sequence general formula or the above sequence
  • It provides an antibacterial method comprising the step of administering an acetate salt substitution of trifluacetic acid of a polypeptide represented by the general formula to a subject in need thereof.
  • the present invention relates to a polypeptide represented by the above sequence formula or trifluacetic acid of a polypeptide represented by the above sequence formula
  • the present invention is represented by the above sequence general formula for the manufacture of an antibacterial agent.
  • antibacterial or "antibacterial activity,” mean a property that resists microorganisms such as bacteria or gomworms, and more specifically, antibiotics, etc., prevent the growth or proliferation of bacteria. It means a suppressive property.
  • antibacterial composition used in this specification is
  • composition having an activity that inhibits the growth of microorganisms may include all forms used in various fields requiring antibacterial effect, for example, pharmaceuticals, cosmetics, quasi-drugs, food additives or feed additives.
  • the antibacterial targets of the antibacterial composition according to the present invention include gram-negative bacteria, gram-positive bacteria, antibiotic-resistant gram-negative bacteria, and antibiotic-resistant gram-positive bacteria.
  • the bacteria that are dyed red when dyed with the Gram staining method and generally have strong pigment resistance and strong surfactant resistance.
  • the gram-negative bacteria of the present invention include all types of gram-negative bacteria containing endotoxins, and , For example, Escherichia, Pseudomonas,
  • Acinetobacter genus Salmonella genus, Klebsiella genus, Neisseria genus, Enterobacter genus, Shigella genus, Moraxella genus, Helicobacter genus,
  • Stenotrophomonas Stenotrophomonas
  • Bdello vibrio Bdello vibrio
  • Legionella Legionella
  • Gram-negative bacteria include Escherichia coli, Pseudomonas
  • Pseudomonas aeruginosa Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas putida
  • Haemolyticus (Acinetobacter haemolyticus), Salmonella enterica (Salmonella bongori), Salmonella
  • Salmonella enteritidis Salmonella enteritidis
  • Salmonella typhimurium Salmonella typhimurium
  • Salmonella g allinarum Salmonella typhimurium
  • Salmonella g allinarum Salmonella g allinarum
  • Salmonella g allinarum Salmonella g allinarum
  • Salmonella pullomm Salmonella mbandaka, Salmonella choleraesuls, Salmonella thompson, Salmonella infamtis, Salmonella infamtis, Salmonella derby, Salmonella derby, Salmonella derby Pneumonia (Klebsiella pneumonia), Klebsiella
  • Granulomatis (Klebsiella granulomatis), Hebsiella oxytoca (Hebsiella oxytoca), Klebsiella terrigena (Klebsiella terrigena), Neisseria
  • Neisseria gonorrhoeae Neisseria meningitidis, Enterobacter aerogenes, Enterobacter cloacae, Shigellaboydii, Shigella
  • Discenteriae (Shigelladysenteriae), Shigellaflexneri, Shigellasonnei, Moraxella catarrhalis, Moraxella lacunata, Moraxella lacunata, Moraxella bovis, Moraxella pylori Helicobacter pylori), Helicobacter heilmannii, Helicobacter felis, Helicobacter
  • Helicobacter mustelae, Helicobacter fenelliae, Helicobacterrappini, Helicobacter
  • Pneumophila (Legionellapneumophila), Legionallaanisa, Legionallabirminghamensis, Legionella
  • Wardsworth (Legional Lawadsworthii), etc., including, but not limited to.
  • n gram positive bacteria means gram
  • Bacteria stained with navy blue or purple in staining include, but are not limited to, Staphylococcus genus, Enterococcus genus, Streptococcus genus, Clostridium genus strains, etc.
  • Gram-positive bacteria include Staphylococcus
  • Epidermidis Staphylococcus epidermidis
  • Enterococcus faecium Enterococcus faecium
  • Streptococcus pneumoniae S ⁇ eptococcus pneumoniae
  • Bacillus anthracis Bacillus anthracis
  • antibiotic-resistant gram-positive bacteria' include, for example,
  • Methicillin-resistant Gram-positive bacteria Carbapenem-resistant Gram-positive bacteria, Vancomycin-resistant Gram-positive bacteria, Macrolide, and multidrug-resistant Gram-positive bacteria are included, but are not limited thereto.
  • Antibiotic-resistant Gram-negative bacteria for example:
  • Streptomycin-resistant Gram-negative bacteria Colistin-resistant Gram-negative bacteria, Carbapenem-resistant Gram-negative bacteria, Chloramphenicol-resistant Gram-negative bacteria, Tetracyclines-resistant Gram-negative bacteria, Cefotaxime-resistant Gram-negative bacteria, Enem-resistant Gram-negative bacteria, Wide-spectrum betalactam-based antibiotic degrading enzymes (ESBL; Beta Lactamase) resistant gram-negative bacteria, Tigecycline-resistant gram-negative bacteria, and multidrug-resistant gram-negative bacteria are included, but are not limited thereto.
  • the antibacterial target bacteria of the antibacterial composition are listed er/c/
  • It may be one or more selected from the group consisting of Staphylococcus aureus and Staphylococcus epidermidis.
  • the antibacterial target bacteria of the antibacterial composition is in the group consisting of a broad spectrum beta-lactam-based antibiotic degrading enzyme (ESBL; Extended Spectrum Beta Lactamase)-resistant bacteria, carbafemem-resistant bacteria, and colistin-resistant bacteria. It may be one or more antibiotic-resistant bacteria selected.
  • the antibiotic-resistant bacteria are ESBL (E. coli), Carbapenem resistant (CR)-Acinetobactor baumannii, CR- Klebsiella pneumoniae, CR- Pseudomonas aeruginosa, 3 ⁇ 4 Colistin resistant Acinetobactor baumannii 3 ⁇ 4 "r 1 but not limited to this.
  • the present invention comprises a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula as an active ingredient, preventing, improving or It provides a composition for treatment.
  • the present invention comprises a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula as an active ingredient, preventing, improving or Provides therapeutic pharmaceutical/food/health food/cosmetics/quasi-drugs/feed compositions.
  • the present invention is a polypeptide represented by the above sequence general formula or the above sequence
  • It provides a method for preventing or treating an infectious disease, comprising administering an acetate salt substitution of trifluacetic acid of a polypeptide represented by the general formula to a subject in need thereof.
  • the present invention provides the prevention, improvement or treatment of infectious diseases of the polypeptide represented by the above sequence general formula or the acetate salt substitution of trifluacetic acid of the polypeptide represented by the above sequence general formula. .
  • the present invention is the use of a polypeptide represented by the above sequence general formula or an acetate salt substitution of trifluacetic acid of a polypeptide represented by the above sequence general formula for the manufacture of a preparation for preventing, improving or treating infectious diseases Provides.
  • ⁇ infectious disease refers to a disease caused by the transmission and invasion of pathogens that cause diseases such as viruses, bacteria, gomworms, and parasites to animals or humans. It means any infectious disease caused by Sanggram-negative bacteria or Gram-positive bacteria.
  • infectious diseases include gram-positive bacteria such as sore throat, impetigo, rheumatic fever, glomerulonephritis, neonatal sepsis, meningitis, pharyngitis, pneumonia, endocarditis, scarlet fever, skin soft tissue infection, deep soft tissue infection, empyema and vaginitis.
  • Infectious diseases caused by, but are not limited to.
  • the pharmaceutical composition of the present invention may further contain suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • Pharmaceutical according to the present invention The composition can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to each conventional method.
  • Lactose Lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, Methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.
  • the pharmaceutical composition of the present invention is prepared using commonly used diluents or excipients such as heavy weight agents, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Solid preparations for oral administration include tablets, extenders, binders, and diluents or excipients. Pills, powders, granules, capsules, etc. are included, and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. to the active ingredient.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., which are commonly used simple diluents, liquid paraffin, and various excipients, e.g. For example, it may contain wetting agents, sweeteners, fragrances, preservatives, etc.
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspensions include propylene. Vegetable oils such as glycol (propylene glycol), polyethylene glycol, olive oil, injectable esters such as ethyl oleate, etc. can be used.
  • As the base of suppositories weepsol, macrogol, tween ) 61, cacao butter, laurin paper, glycerogelatin, etc. can be used.
  • prevention means any action that blocks, inhibits or delays symptoms caused by disease by administration of a composition according to the present invention.
  • treatment means any action in which symptoms are improved or beneficially altered by the administration of a composition according to the present invention.
  • the term "subject” refers to a subject in need of prevention or treatment of a disease.
  • the subject is a human or non-human primate, mouse, or dog. May be mammals, including cats, horses, sheep and cattle.
  • the pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount.
  • the "pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment,
  • the effective dose level is well suited for the type of disease, severity, drug activity, drug sensitivity, administration time, route and rate of administration, duration of treatment, factors including concurrent drugs and other medical fields. It can be determined based on known factors.
  • the preferred dosage can be selected according to the condition and weight of the individual, the severity of the disease, the type of drug, the route and duration of the administration.
  • the pharmaceutical composition is 0.001 to 1000 mg/kg, An amount of 0.01 to 100 mg/kg, 0.01 to 10 mg/kg, 0.1 to 10 mg/kg, or 0.1 to 1 mg/kg can be administered once or several times a day. All of the above factors are considered and there is no side effect. It is important to administer the minimum amount to achieve the maximum effect, and this can be determined by a person skilled in the art.
  • the effective amount of the pharmaceutical composition according to the present invention is the age, sex, condition, weight, and body weight of the patient. The degree of absorption of the active ingredient, the rate of inactivation and excretion rate, the type of disease, and the drug used in combination may vary.
  • composition of the present invention can be administered to an individual by various routes. All modes of administration can be expected, for example, oral, rectal or intravenous, muscle, subcutaneous, intrauterine dura mater or cerebrovascular injection. Can be administered by any route.
  • the pharmaceutical composition according to the present invention is septic or septic in addition to the above active ingredients.
  • It may further contain one or more known substances that have the effect of improving, preventing or treating shock; antibacterial; infectious diseases.
  • Antihistamines or anti-inflammatory analgesics may additionally be included.
  • bronchodilator three agonists, anticholinergics, methylxanthine, etc.
  • antihistamines acrivastine, cetirizine, desloratadine ( desloratadine), fexofenadine,
  • Levocetirizine, loratadine, mizolastine, ailmemazine, chlocertirizine, clemastine, cypropheptadine, hydride Hydroxyzine, ketotifen, promenthazine, etc. can be used.
  • Rofecoxib, etc. can be used.
  • the present invention provides a polynucleotide encoding the polypeptide.
  • polynucleotide exists in the form of single-stranded or double-stranded oxyribonucleotides or
  • RNA genome sequence DNA (gDNA and cDNA), and the RNA sequence transcribed therefrom, and includes analogs of natural polynucleotides unless specifically stated otherwise.
  • the polynucleotide is not only the nucleotide sequence, but also the
  • complementary sequences are also included.
  • the complementary sequence includes not only a perfectly complementary sequence, but also a substantially complementary sequence. This is, under stringent conditions known in the art, with the nucleotide sequence. It refers to a sequence that can be hybridized.
  • polynucleotide may be modified.
  • the modification includes addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.
  • the polynucleotide encoding the amino acid sequence is substantially identical to the nucleotide sequence. It is interpreted to include a nucleotide sequence representing The actual identity is at least 80% homology, at least 90% when the nucleotide sequence and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. It may be a sequence showing% homology or at least 95% homology.
  • the present invention provides a recombinant vector comprising the polynucleotide.
  • vector used in this specification refers to the gene of interest in the host cell.
  • Means a means for expression including viral vectors such as plasmid vectors, cozmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated virus vectors. Can be used as the recombinant vector.
  • Available vectors include plasmids commonly used in the art (e.g., pSClOl, pGV1106, pACYC177, ColEl, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX series, pET series And pUC19), phage (eg, UXkA and M13, etc.) or viruses (eg, CMV, SV40, etc.).
  • plasmids commonly used in the art e.g., pSClOl, pGV1106, pACYC177, ColEl, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX series, pET series And pUC19
  • phage
  • the polynucleotide encoding the peptide is
  • “Operatively linked” refers to a functional linkage between a nucleotide expression regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence.
  • a nucleotide expression regulatory sequence e.g., a promoter sequence
  • the regulatory sequence is thus the transcription of the other nucleotide sequence. And/or translation can be adjusted.
  • the recombination vector is typically a vector for cloning or for expression.
  • the expression vector is a plant, animal, or Conventional ones used to express foreign proteins in microorganisms can be used.
  • the recombination vector can be constructed through a variety of methods known in the art.
  • the present invention provides a host cell transformed with the recombinant vector.
  • any host cell known in the art can be used.
  • Cells include, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, and Bacillus.
  • Bacillus strains such as thuringiensis, and intestinal bacteria and strains such as Salmonella typhimurium, Serratia marcessons, and various Pseudomonas spp.
  • yeast Sacharomyce cerevisiae
  • Kunjung cells plant cells and animal cells, for example, SP2/0, CHO (Chinese hamster ovary) Kl, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines and the like
  • SP2/0 yeast
  • CHO Choinese hamster ovary
  • the present invention provides a method for producing a polypeptide represented by a sequence general formula according to the present invention, comprising the step of culturing the host cell.
  • Insertion of the polynucleotide or a recombinant vector containing the same into a host cell can be performed using an insertion method widely known in the art. For example, when the host cell is a prokaryotic cell, CaCl 2 Method or electroporation method, etc., if the host cell is a eukaryotic cell, microinjection method, calcium
  • Phosphate precipitation method electroporation method, liposome-mediated transfection method, and gene balm badment may be used, but are not limited thereto.
  • the method for selecting the transformed host cells is expressed by a selection label.
  • a phenotype it can be easily carried out according to methods widely known in the art. For example, if the above-mentioned selection marker is a specific antibiotic resistance gene, transformation by culturing the transformant in a medium containing the antibiotic Sieve can be easily selected.
  • Simulated vision example 1 Table. LPS and functional measurement of ADK-derived pentide postbodum against coli K1
  • FP1 (ADK 44-54) is indicated as WT (wild type), and in various parts of WT
  • Peptides with point mutations were prepared (FP3, FP5, FP6, and FP9). Peptides were designed based on the FP3 and LPS binding model (FP12-NH 2 and FP13-NH 2 ), and non-natural amino acids and amino acid isomers (allomeric D-type amino acids) were added to enhance the interaction with LPS in the residue sequence of FP3. Introduced to design a peptide (allD FP12-NH 2 , allD FP13-NH 2 , allD FP-13-9a, allD FP-13-9w, allD FP-13-9k, and allD FP13-NH 2 (AcOH)) Also, the N-terminal
  • a pegylation peptide (PEG-alld FP13-NH 2 (AcOH)) was additionally designed and provided after synthesis from Anygen.
  • LPS Lipopolysaccharide
  • ITC Isothermal Titration Calorimeter
  • the sample volume and shape of the cell were 300 id, coin-shaped, fixed-in-place; the syringe rotation rate was 1200 RPM; and the temperature was 30 O C, 35 O C, and 25 O C.
  • LPS and peptide were diluted with PBS to produce 2mM LPS and 0.2mM peptide. After washing, 300uL of peptide was put in the cell of the ITC device, and 40uL of LPS was put in the syringe. After setting the measurement conditions of the ITC (temperature, number of injections), the syringe was inserted into the cell and the ITC measurement started. When the measurement was completed, the Kd values of LPS and peptide were calculated through analysis.
  • PEG polyethylene glycol
  • Mw molecular weight
  • the concentration of antibiotics and peptides having an OD __ value of 50% of the growth inhibition rate was checked and calculated as MIC 5 °, and the growth inhibition rate of 80% was calculated as the concentration (ug/ml) corresponding to MIC.
  • the 96-well micro titration plate was centrifuged for 5 minutes. 100 supernatant was taken and transferred to another 96-well micro titration plate, and the absorbance at 405 11111 was measured. At this time, when treated with 0.1% Triton -100 The value was calculated as 100% hemolysis, and the hemolytic activity of the peptide was calculated by Equation 1 below as% 1 1110 ⁇ 8.
  • melittin an antibacterial peptide that has strong antibacterial activity and at the same time, exhibits strong hemolytic activity, was used as a control peptide.
  • the circular dichroic component light method shows a characteristic absorption pattern according to the secondary structure of the main skeleton of the polypeptide.
  • FP12-NH 2 and FP13-NH 2 peptides do not have secondary structures in aqueous solutions, but In 111 ⁇ 6116 (3 ⁇ 4-1161), there were two minimum points at wavelengths near 208 and 22211111, which are characteristic absorption patterns of the urine structure (Fig. 2).
  • Liposome composed of EYPC/EYPG (7:3, w/w) and mimicking human red blood cells
  • EYPC/CH (1:10, w/w) was prepared and the following fluorescent dye release test (dye leakage assay) was conducted.
  • EYPG is an abbreviation for egg yolk L-a-phosphatidyl-DL-glycerol
  • EYPC is an abbreviation for egg yolk l,2-diacyl-sn-glycero-3-phosphocholine
  • CH is an abbreviation for cholesterol.
  • the wavelength was set to 520 nm, and the peptide was administered to the liposome, and the 100% dye leakage was expressed as the fluorescence intensity when 0.01% Triton-X 1QQ was added to measure the relative dye emission.
  • the percentage dye leakage for EYPC/EYPG (7:3, w/w) liposomes that mimics bacterial cells, which captures the fluorescent dye calcein, is expressed according to the concentration of the peptide.
  • the antibacterial peptide melittin was used as a control group (peptide showing the mechanism of action of cell membrane targets).
  • the lower drawing of FIG. 3 is a neutral mimic human red blood cell trapped by a fluorescent dye calcein by the peptide.
  • the percent dye release (dye leakage) for the lipid EYPC/CH (10:1, w/w) liposome was expressed according to the peptide concentration, and the antibacterial peptide melittin was used as a control group (peptide indicating the mechanism of action of the cell membrane target).
  • FP12-NH 2 , FP13-NH 2 , allD FP12-NH 2 and allD FP13-NH 2 peptides are large dyes for PC/PG (7:3, w/w) liposomes that mimic bacterial cell membranes. It was found that the ability to destroy the bacterial cell membrane was high by causing the release.
  • the FP12-NH 2 , FP13-NH 2 , allD FP12-NH 2 , allD FP13-NH 2 peptides selectively mimic the red blood cell membrane of mammals, which is almost a dye for PC/CH (1: 1, w/w) liposomes. The results were consistent with low hemolytic activity due to no release. Therefore, it was found that the peptides according to the present invention have a mechanism of action of selectively destroying the bacterial cell membrane.
  • the vaccination was divided into 6 doses of 3 hours, 4 hours and 5 hours, and the survival rate of 3 days was observed at 12 hour intervals.
  • Cardiac vision example 9 Acetate salt hydrate of pentide
  • test method is as follows:
  • a 6-week-old female ICR mouse (SPF mouse, Sam Taco) with an average weight of 25 g was used as an experimental animal.
  • the experimental animals were provided with sterilized feed and water, and were bred as less than W animals per cage, and day and night cycles. Was set to 12 hours.
  • test strain After incubation for 2 days in a 37 O C incubator, prepare the test strain according to the inoculation strain (1x10 8 ) in [BAP (blood agar plate)] MHB (Mueller Hinton Broth) liquid medium.
  • AllD FP13-NH 2 (TFA) and allD FP13-NH 2 (AcOH) mixed with sol (vehicle) were inoculated in 6 doses of 0 hour, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours into the mouse Then, the survival rate was observed every 12 hours.
  • the vaccination was divided into 6 doses of 5 hours and the survival rate was observed at 12 hour intervals and 72 hours.
  • BMDC bone marrow-derived dendritic cells
  • Cardiac Visual Example 11 Measurement of the ability to destroy pentide against liposomes that mimic Gram-positive bacterial cell membranes
  • EYPG/EYPC (6:4, w/w), which mimics the cell membrane of Gram-positive bacteria trapped with a fluorescent dye, calcein, in order to conduct an experiment to confirm whether it exhibits a mechanism of action that kills bacteria. ), and a fluorescence dyeing test was performed (dye leakage as say).
  • FP12-NH 2 , FP13-NH 2 , allD FP12-NH 2 , and allD FP13-NH 2 peptides are a lot of EYPG/EYPC (6:4, w/w) liposomes that mimic bacterial cell membranes. It was found that the ability to destroy the bacterial cell membrane was excellent by causing the dye to be released. Accordingly, FP12-NH 2 , FP13-NH 2 , allD FP12-NH 2 , allD FP13-NH 2 according to the present invention
  • LTA lipotechoic acid
  • LPP lipoprotein
  • LTA contains LPP and does not bind to LPP containing mutant LTA, and LPP is expected to play a more important role in the binding of FP13-NH 2 and LTA.
  • LPP lipoprotein binding
  • the peptide according to the present invention not only inhibits the growth of standard bacteria and antibiotic-resistant bacteria, but also has an excellent effect of removing bacterial-derived endotoxins, so it has excellent sepsis treatment effect. When used in combination with antibiotics, Since side effects can be minimized, it can be usefully used in the prevention or treatment of sepsis.
  • the peptide according to the present invention has excellent antibacterial activity selectively against Gram-positive bacteria, and is an antibacterial composition or gram amount against Gram-positive bacteria. It can be usefully used for the prevention or treatment of various infectious diseases caused by negative bacteria.
  • the peptide according to the present invention is not only safe for the human body, but also shows stability against proteolytic enzymes, so it can be used for various purposes. .

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Abstract

본 발명은 항균활성을 갖는 펩타이드, 이를 포함하는 패혈증 예방 또는 치료용 조성물, 및 항균용 조성물 등을 제공한다. 보다 구체적으로, 본 발명에 따른 펩타이드는 박테리아의 증식 억제뿐 아니라 박테리아 유래의 내독소를 제거하는 우수한 효과를 가지고 있어 뛰어난 패혈증 치료 효과를 발휘하는바 패혈증의 예방 또는 치료에 유용하게 이용할 수 있다. 또한, 본 발명에 따른 펩타이드는 그람 양·음성균에 대해 선택적으로 우수한 항균활성을 가지고 있어, 그람 양·음성균에 대한 항균용 조성물 또는 그람 양·음성균에 의해 유발되는 다양한 감염성 질환들의 예방 또는 치료에 유용하게 이용될 수 있다. 또한, 본 발명에 따른 펩타이드는 단백질 분해효소에 대해서 우수한 저항성을 나타내므로 높은 안정성을 가져 약학적 조성물, 화장학적 조성물, 식품 조성물, 건강식품 조성물, 사료 조성물 및 의약외품 조성물 등 다양한 형태로 활용가능할 것으로 기대된다.

Description

2020/175936 1»（：1/10公020/002826 명세서
발명의 명칭 :항균활성을갖는폴리펩타이드，이를포함하는 패혈증예방또는치료용조성물，및항균용조성물 기술분야
[1] 본발명은항균활성을갖는폴리펩타이드,이를포함하는패혈증예방또는 치료용조성물,및항균용조성물등에관한것이다.
[2] 본출원은 2019년 2월 28일에출원된한국특허출원제 10-2019-0023534호및 2020년 2월 26일에출원된한국특허출원제 10-2020-0023629호에기초한 우선권을주장하며,해당출원의명세서및도면에개시된모든내용은본 출원에원용된다.
[3]
배경기술
[4] 패혈증（8明企）은병원성그람음성세균이생체에감염된경우세포벽성분인 리포를리사카라이드（1 0]301）¾&¥11&산（노, ] ¾）가독소로작용하여생체의 면역체계가과도하게활성화됨으로써유발되는염증반응으로서,전신에 감염증을일으키거나증상이심할경우쇼크를동반하기도한다.구체적으로 패혈증은악성종양,백혈병,악성림프종,후천성면역부전증후군 正 ），교원병, 신부전,간질환,뇌혈관장해,당뇨병등의기초질환을가진환자나고령자, 미숙아같은체액성면역부전또는세포성면역부전을가진저항력이약한 숙주가부신스테로이드나항종양제의화학요법,코발트조사등의방사선치료 또는유치카테테르,혈액투석 ,장기이식 ,심장수술등의처치 ,수술을받은 경우에주로발병한다.패혈증은병원중환자병동에입원한환자가사망하는 주요한원인이되며,이에의한치사율이보통 30%이상인매우심각한 질병이다.의학기술의발전에도불구하고아직도전세계적으로수술
후유증으로감염이되어패혈증이발생하는경우가많으며,신생아나노인들과 같이체내의면역력이약한사람이감염되었을경우패혈증으로진행되는 경우도많다.대표적으로,신생아패혈증의경우만삭아 1,000명중 3명정도가 발생하는것으로알려져 있고,미숙아는발병률이 3내지 4배증가하는것으로 알려져 있다.
[5] 패혈증에걸렸을경우대개항생제치료를받게되지만,적절한처치가늦어져 균들이많이증식했을경우나항생제에내성이강한균주에의해감염이되었을 경우에는항생제만으로는효과적인치료를할수없으며 ,다양한항생제에대한 내성을갖는병원균이점차증가하고있어새로운패혈증치료제의개발이 시급한실정이다.
[6]
[7] 한편,항균제내성세균이란특정항균제에내성을보여약효가듣지않는 세균을말한다.예를들어,페니실린의약효가전혀듣지않는페니실린내성 황색포도상구균이이에해당된다.이외에도, 1961년최초로학계에
보고되었으며,그후로전세계적으로주요병원성감염균이되고있는메티실린 내성황색포도상구균 (Me比 licillin-Resistant Staphylococcus aureus, MRS A)이 보고된바있고, 1988년반코마이신에내성을보이는장구균 (vancomycin resistant enierococc , VRE)이유럽에서처음으로발견되었고,또한,인체감염의가장 흔한원인균인황색포도상구균의마지막치료제로알려진반코마이신에고도의 내성을보이는포도상구균 (vancomycin-resistant staphylococcus aureus, VRSA)이 2002년미국질병통제국 (Centers for Disease Control)에서세계최초로
보고됨으로써소위슈퍼박테리아의확산가능성이매우높아지고있다.
[8]
발명의상세한설명
기술적과제
[9] 이에,본발명자들은상기와같은요구를충족시키기위하여연구를거듭한 결과,특정아미노산서열을가지는펩타이드가박테리아의증식억제뿐아니라, 죽은박테리아로부터분리된내독소를제거하는우수한효과를가지고있어 패혈증치료에효과적이라는점과,그람양음성균에대해선택적으로우수한 항균활성을보임을확인함으로써,본발명을완성하였다.
[1이 따라서 ,본발명이해결하고자하는과제는항균활성을갖는펩타이드,이를 포함하는패혈증예방또는치료용조성물,항균용조성물등을제공하는 것이다.
[11]
[12] 그러나본발명이이루고자하는기술적과제는이상에서언급한과제에
제한되지않으며,언급되지않은또다른과제들은아래의기재로부터본발명이 속하는기술분야의통상의지식을가진자에게명확하게이해될수있을 것이다.
[13]
과제해결수단
[14] 본발명의목적을달성하기위하여 ,본발명은하기의서열일반식으로
표시되는폴리펩타이드를제공한다:
[15] [일반식]
[ 16] Ln-X 1 -L-X2-V-X3-X4-X5-R-X6-L-X7
[17] 상기일반식에서,
[18] n은 0또는 1;
[19] L은류신 (leucine);
[2이 V는발린 (valine);
[21] 요은아르기닌 (arginine); 2020/175936 1»（：1/10公020/002826
3
XI은라이신 (lysine: K)또는아르기닌 (arginine: R);
X2는글리신 (glycine: G)또는아르기닌 (arginine: R);
X3은글루탐산 (glutamic acid: E)또는라이신 (lysine: K);
X4는알라닌 (alanine: A)또는류신 (leucine: L);
X5는라이신 (lysine: K)또는아르기닌 (arginine: R);
X6은티로신 (tyrosine: Y),알라닌 (alanine: A),트립토판 (tryptophan: W), 라이신 (lysine: K)또는아스파르트산 (aspartic acid: D);및
[28] X7은아스파르트산 (aspartic acid: D)또는아르기닌 (arginine: R),
[29] 단,상기일반식에서 K-L-G-V-E-A-K-R-Y-L-D의서열로표시되는
폴리펩타이드는제외함.
[30]
[31] 본발명의일구현예에 있어서,하기 1)내지 9)로이루어진 9종의폴리펩타이드 중어느하나의폴리펩타이드를제공한다 :
[32] 1)상기일반식에서 ,
[33] n은 0;
[34] XI은라이신 (lysine: K);
[35] X2는글리신 (glycine: G);
[36] X3은글루탐산 (glutamic acid: E);
[37] X4는알라닌 (alanine: A);
[38] X5는라이신 (lysine: K);
[39] X6은티로신 (tyrosine: Y),및
[40] X7은아르기닌 (arginine: R)인,
[41] 폴리펩타이드;
[42]
247356
[ 22222243] 2)상기일반식에서 ,
[44] n은 1 ;
[45] XI은아르기닌 (arginine: R);
[46] X2는글리신 (glycine: G);
[47] X3은글루탐산 (glutamic acid: E);
[48] X4는알라닌 (alanine: A);
[49] X5는라이신 (lysine: K);
[50] X6은티로신 (tyrosine: Y),및
[51] X7은아르기닌 (arginine: R)인,
[52] 폴리펩타이드;
[53]
[54] 3)상기일반식에서 ,
[55] n은 1 ;
[56] XI은아르기닌 (arginine: R); [57] X2는글리신 (glycine: G) ;
[58] X3은글루탐산 (glutamic acid: E);
[59] X4는류신 (leucine: L);
[60] X5는라이신 (lysine: K);
[61] X6은티로신 (tyrosine: Y),및
[62] X7은아르기닌 (arginine: R)인,
[63] 폴리펩타이드;
[64]
[65] 4)상기일반식에서 ,
[66] n은 0;
[67] XI은라이신 (lysine: K);
[68] X2는글리신 (glycine: G);
[69] X3은글루탐산 (glutamic acid: E);
P이 X4는알라닌 (alanine: A);
pi] X5는류신 (leucine: L);
[72] X6은티로신 (tyrosine: Y),및
[73] X7은아스파르트산 (aspartic acid: D)인, 4] 폴리펩타이드;
P5]
[76] 5)상기일반식에서 ,
7] n은 0;
[78] XI은아르기닌 (arginine: R);
R9] X2는아르기닌 (arginine: R);
[8이 X3은라이신 (lysine: K);
[81] X4는류신 (leucine: L);
[82] X5는아르기닌 (arginine: R);
[83] X6은티로신 (tyrosine: Y),및
[84] X7은아르기닌 (arginine: R)인,
[85] 폴리펩타이드;
[86]
[87] 6)상기일반식에서 ,
[88] n은 0;
[89] XI은라이신 (lysine: K);
[90] X2는아르기닌 (arginine: R);
[91] X3은라이신 (lysine: K);
[92] X4는류신 (leucine: L);
[93] X5는아르기닌 (arginine: R);
[94] X6은티로신 (tyrosine: Y),및 0 2020/175936 1»(：1^1{2020/002826
5
[95] X7은아르기닌 (arginine: R)인,
[96] 폴리펩타이드;
[97]
[98] 7)상기일반식에서 ,
[99] n은 0;
[100] XI은라이신 (lysine: K);
[101] X2는아르기닌 (arginine: R);
[102] X3은라이신 (lysine: K);
[103] X4는류신 (leucine: L);
[104] X5는아르기닌 (arginine: R);
[105] X6은알라닌 (alanine: A),및
[106] X7은아르기닌 (arginine: R)인,
[107] 폴리펩타이드;
[108]
[109] 8)상기일반식에서 ,
[110] n은 0;
[111] XI은라이신 (lysine: K);
[112] X2는아르기닌 (arginine: R);
[113] X3은라이신 (lysine: K);
[114] X4는류신 (leucine: L);
[115] X5는아르기닌 (arginine: R);
[116] X6은트립토판 (tryptophan: W) ,및
[117] X7은아르기닌 (arginine: R)인,
[118] 폴리펩타이드;및
[119]
[12이 9)상기일반식에서 ,
[121] n은 0;
[122] XI은라이신 (lysine: K);
[123] X2는아르기닌 (arginine: R);
[124] X3은라이신 (lysine: K);
[125] X4는류신 (leucine: L);
[126] X5는아르기닌 (arginine: R);
[127] X6은라이신 (lysine: K),및
[128] X7은아르기닌 (arginine: R)인,
[129] 폴리펩타이드.
[13이
[131] 본발명의일구현예에서,상기폴리펩타이드는 L형, D형및
펩토이드 (peptoid)를포함하는펩타이드모방체또는비천연아미노산인, 폴리펩타이드를제공한다.
[132] 본발명의일구현예에서 ,상기폴리펩타이드의말단을알킬레이션 (Alkylation), 페길레이션 (PEGylation),또는아미데이션 (Amidation)한것인,폴리펩타이드를 제공한다.
[133] 본발명의일구현예에서,상기폴리펩타이드의 C말단에아민기 (NH 2)가
추가된것인,폴리펩타이드를제공한다.
[134] 본발명의일구현예에서 ,상기폴리펩타이드는하기특징중하나이상의
특징을가지는것인,폴리펩타이드를제공한다 :
[135] 그람음성균의리포폴리사카라이드 (LPS)에의결합능;
[136] 박테리아세포막의선택적파괴능;
[137] 단백질분해효소에대한저항성 ;
[138] 그람양성균의세포막파괴능;및
[139] 그람양성균유래 LTA(lipotechoic acid)또는 LPP(lipoprotein)에의결합능.
[14이
[141] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드의
트리늘루아세트산 (Trifluoroacetic acid: TFA)의아세테이트염치환물을
제공한다.
[142]
[143] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,패혈증의 예방,개선또는치료용조성물을 제공한다.
[144] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,패혈증의 예방,개선또는치료용
약학적/식품/건강식품/화장품/의약외품/사료조성물을제공한다.
[145] 본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열
일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을이를필요로하는개체에투여하는단계를포함하는패혈증의 예방 또는치료방법을제공한다.
[146] 뿐만아니라,본발명은상기서열일반식으로표시되는폴리펩타이드또는
상기서열일반식으로표시되는폴리펩타이드의트리플루아세트산의
아세테이트염치환물의패혈증에대한예방,개선또는치료용도를제공한다.
[147] 나아가,본발명은패혈증의예방,개선또는치료용제제의제조를위한상기 서열일반식으로표시되는폴리펩타이드또는상기서열일반식으로표시되는 폴리펩타이드의트리플루아세트산의아세테이트염치환물의용도를제공한다 .
[148]
[149] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,항균용조성물을제공한다.
[15이 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,항균용
약학적/식품/건강식품/화장품/의약외품/사료조성물을제공한다.
[151] 본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열
일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을이를필요로하는개체에투여하는단계를포함하는항균방법을 제공한다.
[152] 뿐만아니라,본발명은상기서열일반식으로표시되는폴리펩타이드또는 상기서열일반식으로표시되는폴리펩타이드의트리플루아세트산의 아세테이트염치환물의항균용도를제공한다.
[153] 나아가,본발명은항균제의제조를위한상기서열일반식으로표시되는
폴리펩타이드또는상기서열일반식으로표시되는폴리펩타이드의
트리플루아세트산의아세테이트염치환물의용도를제공한다.
[154] 본발명의일구현예에서,상기항균용조성물의항균대상균은 Escherichia coli DH5a, Escherichia coli Kl, Acinetobacter baumannii, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Klebsiella pneumoniae,
Staphylococcus aureus및 Staphylococcus epidermidis £-이루어진군에서선택된 1이상인,항균용조성물을제공한다.
[155] 본발명의일구현예에서 ,상기항균용조성물의항균대상균은광범위
베타락탐계항생제분해효소 (ESBL;Extended Spectrum Beta Lactamase)내성균, 카바페멤내성균및콜리스틴내성균으로이루어진군에서선택된 1이상의 항생제내성균인,항균용조성물을제공한다.
[156] 본발명의일구현예에서 ,상기항생제내성균은 ESBL (E. coli), Carbapenem resistant (CR)- Acinetobactor baumannii, CR- Klebsiella pneumoniae, CR- Pseudomonas aeruginosa, ¾ Colistin resistant Acinetobactor baumannii £.이早어진 군에서선택된 1이상인,항균용조성물을제공한다.
[157]
[158] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,감염성질환의예방,개선또는치료용 조성물을제공한다.
[159] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,감염성질환의예방,개선또는치료용 약학적/식품/건강식품/화장품/의약외품/사료조성물을제공한다. 2020/175936 1»（：1^1{2020/002826
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[16이 본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열
일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을이를필요로하는개체에투여하는단계를포함하는감염성 질환의 예방또는치료방법을제공한다.
[161] 뿐만아니라,본발명은상기서열일반식으로표시되는폴리펩타이드또는 상기서열일반식으로표시되는폴리펩타이드의트리플루아세트산의
아세테이트염치환물의감염성질환에 대한예방,개선또는치료용도를 제공한다.
[162] 나아가,본발명은감염성질환의 예방,개선또는치료용제제의 제조를위한 상기서열일반식으로표시되는폴리펩타이드또는상기서열일반식으로 표시되는폴리펩타이드의트리플루아세트산의 아세테이트염치환물의용도를 제공한다.
[163] 본발명의 일구현예에서 ,상기감염성질환은,폐렴 ,복막염 ,뇌막염 ,창상감염 , 골관절염，담낭염,요로감염증,뇌수막염,심내막염,심근염,심외막염,관절염, 인구염,임질,세균성 이질,장염 ,결막염，위염,중이염,방광염 ,및림프관염으로 이루어지는군으로부터선택되는그람음성균에 의해유발되는감염성질환인, 조성물을제공한다.
[164] 본발명의 일구현예에서,상기감염성질환은,인후염,농가진,류마티스열, 사구체신염,신생아패혈증,수막염，인두염,폐렴,심내막염,성홍열,
피부연조직감염,심부연조직감염,농흉및질염으로이루어지는군으로부터 선택되는그람양성균에 의해유발되는감염성질환인,조성물을제공한다.
[165]
[166] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드를코딩하는
폴리뉴클레오티드를제공한다.
[167]
[168] 또한,본발명은상기폴리뉴클레오티드를포함하는재조합벡터를제공한다.
[169]
[170] 또한,본발명은상기 재조합벡터로형질전환된숙주세포를배양하는단계를 포함하는,상기서열일반식으로표시되는폴리펩타이드의 제조방법을 제공한다.
[171]
발명의효과
[172] 본발명에 따른펩타이드는,표준박테리아및항생제내성 박테리아의증식 억제뿐아니라,박테리아유래내독소를제거하는우수한효과를가지고있어 우수한패혈증치료효과를가지고있고,항생제와병용할경우항생제에 의한 부작용을최소화할수있어패혈증의 예방또는치료에유용하게 이용할수 있다. [173] 또한,본발명에따른펩타이드는그람양음성균에대해선택적으로우수한 항균활성을가지고있어,그람양음성균에대한항균용조성물또는그람 양음성균에의해유발되는다양한감염성질환들의예방또는치료에유용하게 이용될수있다.
[174] 또한,본발명에따른펩타이드는인체에안전할뿐만아니라,단백질
분해효소에대해서도안정성을나타내므로,다양한용도로활용될수있다.
[175]
도면의간단한설명
[176] 도 1은,본발명실시예 4의용혈활성측정결과를나타낸그래프이다.
[177] 상단좌측부터우측방향으로, FP12-NH 2（서열번호 6） / allD_FP12-NH 2
（서열번호 8）;및 FP13-NH 2 P1열번호 7） / allD_FP13-NH 2（서열번호 9）.
[178] 하단좌측부터우측방향으로, allD_FP13_9A（서열번호 10）/ allD_FP13_9D
（서열번호 18）;및 allD_FP13_9W（서열번호 11）/ allD_FP13_9K（서열번호 12）.
[179] 도 2는,본발명실시예 5의원편광이색성분광기에서펩타이드의 2차구조를 측정한결과를보여주는그래프이다.
[18이 도 3은,본발명실시예 6의박테리아세포막（상단）과적혈구세포막（하단）을 모방하는리포좀에대한펩타이드의파괴능측정결과를나타낸도면이다.
[181] 도 4는,본발명실시예 8의 allD FP13-NH 2펩타이드의항생효과를나타낸
도면이다.각각좌즉에서우즉방향으로,표준균주 Escherichia coli K1에대한 allD FP13-NH 2펩타이드의항생효과;임상균주 Escherichia coli（ESBL）에대한 allD FP13-NH 2펩타이드의항생효과;및임상균주 Carbapenem-resistant Aczneto公 acie厂 baummanii에대한 allD FP13-NH 2펩타이드의항생효과를나타낸것이다.
[182] 도 5a내지 5c는,본발명실시예 9의 allD FP13-NH 2（AcOH）펩타이드의
항생효과,독성효과및항염증싸이토카인억제효과확인결과를나타낸것이다.
[183] 도 5a는, Escherichia coli（ ESBL）에대한항생효과비교, AllD FP13-NH 2
（TFA）（위）및 AllD FP13-NH 2（AcOH）（아래）;
[184] 도 5b는, in vivo독성비교, AllD FP13-NH 2（TFA）（위）및 AllD FP13-NH 2
（AcOH）（아래）;및
[185] 도 5c는,항염증싸이토카인（IL-6）억제효과비교, AllD FP13-NH 2（TFA）（위）및 AllD FP13-NH 2（ AcOH）（아래）.
[186] 도 6은,본발명실시예 11의그람양성균세포막을모방하는리포좀에대한 펩타이드의파괴능측정결과를나타낸도면이다.
[187]
발명의실시를위한형태
[188] 본발명은하기의서열일반식으로표시되는폴리펩타이드를제공한다 :
[189] [일반식]
[ 19이 Ln-X 1 -L-X2-V-X3-X4-X5-R-X6-L-X7 [191] 상기일반식에서,
[192] n은 0또는 1;
[193] L은류신 (leucine);
[194] V는발린 (valine);
[195] R은아르기닌 (arginine);
[196] XI은라이신 (lysine: K)또는아르기닌 (arginine: R);
[197] X2는글리신 (glycine: G)또는아르기닌 (arginine: R);
[198] X3은글루탐산 (glutamic acid: E)또는라이신 (lysine: K);
[199] X4는알라닌 (alanine: A)또는류신 (leucine: L);
[200] X5는라이신 (lysine: K)또는아르기닌 (arginine: R);
[201] X6은티로신 (tyrosine: Y),알라닌 (alanine: A),트립토판 (tryptophan: W),
라이신 (lysine: K)또는아스파르트산 (aspartic acid: D);및
[202] X7은아스파르트산 (aspartic acid: D)또는아르기닌 (arginine: R),
[203] 단,상기일반식에서 K-L-G-V-E-A-K-R-Y-L-D의서열로표시되는
폴리펩타이드는제외함.
[204]
[205] 본발명의일구현에에 있어서,하기 1)내지 9)로이루어진 9종의폴리펩타이드 중어느하나이상의폴리펩타이드를제공한다 :
[206] 1)상기일반식에서 ,
[207] n은 0;
[208] XI은라이신 (lysine: K);
[209] X2는글리신 (glycine: G) ;
[210] X3은글루탐산 (glutamic acid: E);
[211] X4는알라닌 (alanine: A);
[212] X5는라이신 (lysine: K);
[213] X6은티로신 (tyrosine: Y),및
[214] X7은아르기닌 (arginine: R)인,
[215] 폴리펩타이드로,
[216] 즉, KLGVEAKRYLR (서열번호 2)의아미노산서열을갖는폴리펩타이드 ;
[217]
[218] 2)상기일반식에서 ,
[219] n은 1;
[220] XI은아르기닌 (arginine: R);
[221 ] X2는글리신 (glycine: G) ;
[222] X3은글루탐산 (glutamic acid: E);
[223] X4는알라닌 (alanine: A);
[224] X5는라이신 (lysine: K);
[225] X6은티로신 (tyrosine: Y),및 [226] X7은아르기닌 (arginine: R)인,
[227] 폴리펩타이드로,
[228] 즉, LRLGVEAKRYLR (서열번호 3)의아미노산서열을갖는폴리펩타이드 ;
[229]
[23이 3)상기일반식에서 ,
[231] n은 1;
[232] XI은아르기닌 (arginine: R);
[233] X2는글리신 (glycine: G) ;
[234] X3은글루탐산 (glutamic acid: E);
[235] X4는류신 (leucine: L);
[236] X5는라이신 (lysine: K);
[237] X6은티로신 (tyrosine: Y),및
[238] X7은아르기닌 (arginine: R)인,
[239] 폴리펩타이드로
[24이 즉, LRLGVELKRYLR (서열번호 4)의아미노산서열을갖는폴리펩타이드 ;
[241]
[242] 4)상기일반식에서 ,
[243] n은 0;
[244] XI은라이신 (lysine: K);
[245] X2는글리신 (glycine: G) ;
[246] X3은글루탐산 (glutamic acid: E);
[247] X4는알라닌 (alanine: A);
[248] X5는류신 (leucine: L);
[249] X6은티로신 (tyrosine: Y),및
[250] X7은아스파르트산 (aspartic acid: D)인,
[251] 폴리펩타이드로
[252] 즉, KLGVEALRYLD (서열번호 5)의아미노산서열을갖는폴리펩타이드 ;
[253]
[254] 5)상기일반식에서 ,
[255] n은 0;
[256] XI은아르기닌 (arginine: R);
[257] X2는아르기닌 (arginine: R);
[258] X3은라이신 (lysine: K);
[259] X4는류신 (leucine: L);
[260] X5는아르기닌 (arginine: R);
[261] X6은티로신 (tyrosine: Y),및
[262] X7은아르기닌 (arginine: R)인,
[263] 폴리펩타이드로 [264] 즉, RLRVKLRRYLR (서열번호 15)의아미노산서열을갖는폴리펩타이드 ;
[265]
[266] 6)상기일반식에서 ,
[267] n은 0;
[268] XI은라이신 (lysine: K);
[269] X2는아르기닌 (arginine: R);
[27이 X3은라이신 (lysine: K);
[271] X4는류신 (leucine: L);
[272] X5는아르기닌 (arginine: R);
[273] X6은티로신 (tyrosine: Y),및
[274] X7은아르기닌 (arginine: R)인,
[275] 폴리펩타이드로
[276] 즉, KLRVKLRRYLR (서열번호 16)의아미노산서열을갖는폴리펩타이드 ;
[277]
[278] 刀상기일반식에서 ,
[279] n은 0;
[28이 XI은라이신 (lysine: K);
[281] X2는아르기닌 (arginine: R);
[282] X3은라이신 (lysine: K);
[283] X4는류신 (leucine: L);
[284] X5는아르기닌 (arginine: R);
[285] X6은알라닌 (alanine: A),및
[286] X7은아르기닌 (arginine: R)인,
[287] 폴리펩타이드로
[288] 즉, KLRVKLRRALR (서열번호 10)의아미노산서열을갖는폴리펩타이드 ;
[289]
[29이 8)상기일반식에서 ,
[291] n은 0;
[292] XI은라이신 (lysine: K);
[293] X2는아르기닌 (arginine: R);
[294] X3은라이신 (lysine: K);
[295] X4는류신 (leucine: L);
[296] X5는아르기닌 (arginine: R);
[297] X6은트립토판 (tryptophan: W) ,및
[298] X7은아르기닌 (arginine: R)인,
[299] 폴리펩타이드로
[300] 즉, KLRVKLRRWLR (서열번호 11)의아미노산서열을갖는폴리펩타이드 ;및
[301] [302] 9)상기일반식에서 ,
[303] n은 0;
[304] XI은라이신 (lysine: K);
[305] X2는아르기닌 (arginine: R);
[306] X3은라이신 (lysine: K);
[307] X4는류신 (leucine: L);
[308] X5는아르기닌 (arginine: R);
[309] X6은라이신 (lysine: K),및
[31이 X7은아르기닌 (arginine: R)인,
[311] 폴리펩타이드로
[312] 즉, KLRVKLRRKLR (서열번호 12)의아미노산서열을갖는폴리펩타이드.
[313]
[314] 본명세서에서사용된용어,”폴리펩타이드”는펩타이드결합에의해아미노산 잔기들이서로결합되어형성된선형 (linear)의분자를의미한다.본발명의 폴리펩타이드는분자생물학적인방법과함께당업계에공지된화학적합성 방법 (예를들어,고상합성기술 (solid-phase synthesis techniques))에따라제조될 수있다 (Merrifield, J. Amer. Chem. Soc. 85: 2149-54(1963); Stewart, et al., Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co.: Rockford, 111(1984)).
[315] 또한,본발명의폴리펩타이드의범위에는본발명의폴리펩타이드와균등한 생물학적활성을발휘하는아미노산서열의변이를갖는생물학적기능 균등물이포함될수있다.이러한아미노산서열의변이는아미노산곁사슬 치환체의상대적유사성,예컨대,소수성,친수성,전하및크기등에기초하여 이루어질수있다.아미노산곁사슬치환체의크기 ,모양및종류에대한분석에 의하여 ,아르기닌,라이신과히스티딘은모두양전하를띤잔기이고;알라닌, 글라이신과세린은유사한크기를가지며 ;페닐알라닌,트림토판과타이로신은 유사한모양을갖는다는것을알수있다.따라서,이러한고려사항에기초하여, 아르기닌,라이신과히스티딘;알라닌,글라이신과세린;그리고페닐알라닌, 트림토판과타이로신은생물학적으로기능균등물이라할수있다.
[316] 변이를도입하는데있어서,아미노산의소수성인덱스가고려될수있다. 각각의아미노산은소수성과전하에따라소수성인덱스가부여되어있다.또한, 유사한친수성값 (hydrophilicity value)을가지는아미노산사이의치환이균등한 생물학적활성을갖는펩타이드를초래한다는것도알려져 있다.
[317] 분자의활성을전체적으로변경시키지않는펩타이드에서의아미노산교환은 당해분야에공지되어 있다 (H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979).가장통상적으로일어나는교환은아미노산잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/V al, Ala/Glu, Asp/Gly간의교환이다.
[318] 상술한생물학적균등활성을갖는변이를고려한다면,본발명의 폴리펩타이드는서열목록에기재된서열과실질적인동일성 (substantial identity)을나타내는서열도포함하는것으로해석된다.상기의실질적인 동일성은,본발명의서열과임의의다른서열을최대한대응되도록얼라인하고, 당업계에서통상적으로이용되는알고리즘을이용하여얼라인된서열을분석한 경우에,최소 80%의상동성, 90%의상동성또는 95%의상동성을나타내는 서열을의미할수있다.서열비교를위한얼라인먼트방법은당업계에공지되어 있다 (Huang et al., Comp. Appl. BioSci. 8:155-65(1992); Pearson et al., Meth. Mol. Biol. 24:307-31(1994)).
[319]
[32이 또한,본발명의일구현예에서 ,본발명에따른서열일반식으로표시되는
폴리펩타이드는 L형, D형및펩토이드 (peptoid)를포함하는펩타이드모방체 또는비천연아미노산일수있다.
[321] 상기 D형폴리펩타이드는 L형폴리펩타이드와아미노산서열이동일한
거울상이성질체로서 allomeric type의폴리펩타이드를의미한다.
[322] 본발명의구체적실시예에서,예를들어, FP12-NH 2(서열번호 6)와아미노산 서열이동일한 allomeric type의폴리펩타이드로서 allD FP12-NH 2(서열번호 8)을 제공한다.
[323] 본명세서에서폴리펩타이드를명명하며사용된용어,’’allD”는 D형
폴리펩타이드를지칭한다.
[324] 상기 L형 , D형및펩토이드 (peptoid)를포함하는펩타이드모방체또는비천연 아미노산의폴리펩타이드는정해진아미노산서열에따라당업계공지된 다양한방법으로제조될수있다.
[325]
[326] 본발명의일구현예에서 ,본발명에따른서열일반식으로표시되는
폴리펩타이드의말단을알킬레이션 (Alkylation),페길레이션 (PEGylation)또는 아미데이션 (Amidation)한것일수있다.
[327] 본발명의구체적실시예에서 ,본발명에따른폴리펩타이드중 allD FP13-NH 2 (AcOH) (서열번호 13)의 N말단을페길레이션하여 , PEG-allD FP13-NH 2 (AcOH) (서열번호 14)를제공한다.이에제한되지않지만,상기페길레이션을위하여본 발명에따른폴리펩타이드와 Fmoc-NH-PEG2-CH 2COOH을반응시킬수있으며， 이때 polyethylene glycol의 Mw (분자량)은 385.4Da이다.
[328] 선택되는폴리펩타이드에따라통상의기술자가페길레이션을위한조건을 조절할수있으며 ,페길레이션하는방법은당업계공지된다양한방법에따를수 있다.
[329] 알킬레이션또는아미데이션,역시,선택되는폴리펩타이드에따라통상의
기술자가알킬레이션또는아미데이션을위한조건을조절할수있으며 , 알킬레이션또는아미데이션하는방법은당업계공지된다양한방법에따를수 있다. 2020/175936 1»（：1^1{2020/002826
15
[33이
[331] 본발명의일구현예에서,본발명에따른서열일반식으로표시되는
폴리펩타이드의 0말단에아민기어112）가추가된것일수있다.
[332] 본발명의구체적실시예에서,본발명에따른폴리펩타이드중므 2（서열번호 15）의（:말단에아민기어11 2）가추가된폴리펩타이드로서므 요 - NH 2（서열번호 6）을제공한다.
[333] 본명세서에서폴리펩타이드를명명하며사용된용어,”- NH 2”는
폴리펩타이드의（:말단에아민기어112）가추가된것을지칭한다.
[334] 폴리펩타이드의 0말단에아민기（ 1 2）를추가하는것은당업계공지된
다양한방법에따를수있다.
[335]
[336] 또한,본발명의폴리펩타이드의범위에는이의약학적으로허용가능한염도 포함될수있다.
[337] 본명세서에서사용된용어,”약학적으로허용가능한”이라는용어는과도한 독성,자극,알러지반응또는기타문제점이나합병증없이이득/위험비가 합리적이어서대상체（예:인간）의조직과접촉하여사용하기에적합하며,건전한 의학적판단의범주이내인펩타이드를의미한다.상기약학적으로허용가능한 염은,예를들어약학적으로허용가능한
Figure imgf000017_0001
형성된산 부가염및약학적으로허용가능한금속염을포함한다.
[338] 적합한산의 예로는염산,브롬산,황산,질산,과염소산,푸마르산,말레산, 인산,글리콜산,락트산,살리실산,숙신산,톨루엔- 설폰산,타르타르산, 아세트산,시트르산,메탄설폰산,포름산,벤조산,말론산,글루콘산,
나프탈렌 -2 -설폰산,벤젠설폰산등을들수있다.산부가염은통상의방법,예를 들면화합물을과량의산수용액에용해시키고,이염을메탄올,에탄올,아세톤 또는아세토니트릴과같은수혼화성유기용매를사용하여침전시켜서제조할 수있다.또한,동몰량의화합물및물중의산또는알코올을가열하고이어서 상기혼합물을증발시켜서건조시키거나,또는석출된염을흡인여과시켜 제조할수있다.
[339] 적합한염기로부터유도된염은나트륨,칼륨등의알칼리금속,마그네슘등의 알칼리토금속,및암모늄등을포함할수있으나,이에제한되는것은아니다. 알칼리금속또는알칼리토금속염은,예를들면화합물을과량의알칼리금속 수산화물또는알칼리토금속수산화물용액중에용해하고,비용해화합물염을 여과한후여액을증발,건조시켜얻을수있다.이때,금속염으로서는특히 나트륨,칼륨또는칼슘염을제조하는것이제약상적합하며，또한이에 대응하는은염은알칼리금속또는알칼리토금속염을적당한은염（예, 질산은）과반응시켜얻을수있다.
[34이
[341] 상술한폴리펩타이드의약학적으로허용가능한염의일환으로,본발명은 상술한폴리펩타이드의아세테이트염을제공한다.
[342] 보다구체적으로,본발명에따른폴리펩타이드의
트리늘루아세트산 (Trifluoroacetic acid: TFA)의아세테이트염치환물을 제공한다.
[343] 보다구체적으로,본발명은상술한서열일반식으로표시되는폴리펩타이드;
L형, D형및펩토이드 (peptoid)를포함하는펩타이드모방체또는비천연 아미노산인폴리펩타이드;말단이알킬레이션 (Alkylation),
페길레이션 (PEGylation)또는아미데이션 (Amidation)된폴리펩타이드;또는 C 말단에아민기 (NH 2)가추가된폴리펩타이드의아세테이트염을제공한다.
[344] 본명세서에서폴리펩타이드를명명하며사용된용어 ,” (AcOH)”는
폴리펩타이드의아세테이트염을의미한다.
[345]
[346] 본발명에따른폴리펩타이드와이의염치환물은다음과같은특징중하나 이상의특징을가진다:
[347] 그람음성균의리포폴리사카라이드 (LPS)에의결합능;
[348] 박테리아세포막의선택적파괴능;
[349] 단백질분해효소에대한저항성 ;
[35이 그람양성균의세포막파괴능;및
[351] 그람양성균유래 LTA(lipotechoic acid)또는 LPP(lipoprotein)에의결합능.
[352] 본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의염
치환물의후술하는바와같은특징을확인하였으나,이들특징에제한되는것은 아니다.
[353] 본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의염
치환물이리포폴리사카라이드 (LPS)에강한결합력을가짐을확인하였다 (실시예 1).
[354] 또한,본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의염 치환물이박테리아세포막에대한파괴능을가짐을확인하였다 (실시예 6및 실시예 11).특히,적혈구세포막에대해서는파괴능을나타내지않는바, 안전성이입증되었다 (실시예 6).
[355] 또한,본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의염 치환물이단백질분해효소에대한우수한저항성을가짐을확인하였다 (실시예 7).단백질분해효소와함께처리하여도,우수한항균활성을나타내는바,단백질 분해효소에대해안정성을확보하고있음을확인하였다.
[356] 또한,본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의염 치환물이그람양성균의세포벽성분인 LTA(lipotechoic acid)와
LPP(lipoprotein)에우수한결합력을가짐을확인하였다 (실시예 12).
[357] 또한,본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의염 치환물이미미한용혈활성을나타내므로매우독성이낮음을확인하였다 （실시예 4）.
[358]
[359] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,패혈증의 예방,개선또는치료용조성물을 제공한다.
[36이 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,패혈증의 예방,개선또는치료용
약학적/식품/건강식품/화장품/의약외품/사료조성물을제공한다.
[361] 본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열
일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을이를필요로하는개체에투여하는단계를포함하는패혈증의 예방 또는치료방법을제공한다.
[362] 뿐만아니라,본발명은상기서열일반식으로표시되는폴리펩타이드또는 상기서열일반식으로표시되는폴리펩타이드의트리플루아세트산의
아세테이트염치환물의 패혈증에 대한예방,개선또는치료용도를제공한다.
[363] 나아가,본발명은패혈증의 예방,개선또는치료용제제의제조를위한상기 서열일반식으로표시되는폴리펩타이드또는상기서열일반식으로표시되는 폴리펩타이드의트리플루아세트산의아세테이트염치환물의용도를제공한다.
[364]
[365] 본명세서에서사용된용어,”패혈증”이란미생물에감염되어 전신에심각한 염증반응이나타나는상태를말한다.체온이 38OC이상으로올라가는발열증상 혹은 36OC이하로내려가는저체온증,호흡수가분당 24회 이상으로
증가（빈호흡）,분당 90회 이상의심박수（빈맥）,혈액검사상백혈구수의증가 혹은현저한감소중두가지 이상의증상을보이는경우,이를전신성 염증반응 증후군（systemic inflammatory response syndrome; SIRS）이라고부른다.이러한 전신성 염증반응증후군이미생물의감염에의한것일때패혈증이라고한다. 신체의감염병소에서 병원균이지속적또는단속적으로혈류에들어와여러 장기조직에 정착하여 병소를만들고심한전신증상을보이는것이다.
원인균으로는포도상구균,연쇄상구균,대장균,녹농균,결핵균,폐렴간균,진균, 혐기성균등이 있으며,이에 제한되지 않는다.
[366]
[367] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,항균용조성물을제공한다.
[368] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,항균용
약학적/식품/건강식품/화장품/의약외품/사료조성물을제공한다.
[369] 본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열
일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을이를필요로하는개체에투여하는단계를포함하는항균방법을 제공한다.
[37이 뿐만아니라,본발명은상기서열일반식으로표시되는폴리펩타이드또는 상기서열일반식으로표시되는폴리펩타이드의트리플루아세트산의
아세테이트염치환물의항균용도를제공한다.
[371] 나아가,본발명은항균제의제조를위한상기서열일반식으로표시되는
폴리펩타이드또는상기서열일반식으로표시되는폴리펩타이드의
트리플루아세트산의아세테이트염치환물의용도를제공한다.
[372] 본명세서에서사용된용어, "항균”또는 "항균활성”이란,세균이나곰광이와 같은미생물에대해저항하는성질을의미하며 ,보다상세하게는항생물질등이 세균의성장또는증식을억제하는특성을의미한다.
[373] 본명세서에서사용된용어, "항균용조성물”은세균이나곰광이와같은
미생물의생육을저해하는활성을가진조성물로서 ,항균효과가요구되는 다양한분야에사용되는모든형태가포함될수있으며,예를들어,의약품, 화장품,의약외품,식품첨가제또는사료첨가제등의형태일수있다.
구체적으로,의약에 있어서는항생제나오염방지제와같은목적으로,식품에 있어서는방부나항균목적으로,농업에 있어서는항균,살균,소독의목적으로, 화장품이나생활용품에있어서는비듬억제용,무좀방지용,겨드랑이
채취억제용,항여드름용등미생물과직접연관된제품에사용되거나청소용 세정제나식기세척용세정제등에방부나항균또는살균목적으로사용되어질 수있으며,이러한목적으로만한정되는것은아니다.
[374] 본발명의일구현예에서,본발명에따른항균용조성물의항균대상은그람 음성균,그람양성균,항생제내성그람음성균및항생제내성그람양성균등이 포함된다.
[375] 본명세서에서사용된용어,’’그람음성균 (gram negative bacteria)’’은
그람염색법으로염색했을때붉은색으로염색되는세균을의미하는것으로, 일반적으로색소저항력이강하고,계면활성제내성이강하다.본발명의그람 음성균에는엔도톡신 (endotoxin)을보유한모든종류의그람음성균이포함되며, 예를들어,에스케리아 (Escherichia)속,슈도모나스 (Pseudomonas)속,
아시네토박터 (Acinetobacter)속,살모넬라 (Salmonella)속,크렙시엘라 (Klebsiella) 속,나이세리아 (Neisseria)속,엔테로박터 (Enterobacter)속,시겔라 (Shigella)속, 모락셀라 (Moraxella)속,헬리코박터 (Helicobacter)속,
스테노트로포모나스 (Stenotrophomonas)속,브델로비브리오 (Bdello vibrio)속, 레지오넬라 (Legionella)속균주등이포함되며 ,이에제한되지않는다. 2020/175936 1»（：1/10公020/002826
19 구체적으로,그람음성균에는대장균 (Escherichia coli),슈도모나스
애루지노사 (Pseudomonas aeruginosa),슈도모나스물루오레슨스 (Pseudomonas fluorescens),슈도모나스푸티다 (Pseudomonas putida),슈도모나스
클로로라피스 (Pseudomonas chlororaphis),슈도모나스퍼투시노제나 (Pseudomanas pertucinogena),슈도모나스스투트제리 (Pseudomanas stutzeri),슈도모나스 시린개 (Pseudomanas syringae),아시네토박터바우마니 (Acinetobacter baumannii), 아시네토박터류오피이 (Acinetobacter lwoffii),아시네토박터
칼코아세티쿠스 (Acinetobacter calcoaceticus),아시네토박터
헤모리티쿠스 (Acinetobacter haemolyticus),살모넬라엔테리카 (Salmonella enterica),살모넬라봉고리 (Salmonella bongori),살모넬라
엔테리티디스 (Salmonella enteritidis),살모넬라티피뮤리움 (Salmonella
typhimurium),살모넬라갈리나룸 (Salmonella g allinarum),살모넬라
풀로룸 (Salmonella pullomm),살모넬라엠반다카 (Salmonella mbandaka),살모넬라 콜레라에슐스 (Salmonella choleraesuls),살모넬라톰슨 (Salmonella thompson), 살모넬라인팜티스 (Salmonella infamtis),살모넬라더비 (Salmonella derby), 크렙시엘라뉴모이아 (Klebsiella pneumonia),크렙시엘라
그라눌로마티스 (Klebsiella granulomatis),크렙시엘라옥시토카 (Hebsiella oxytoca),크렙시엘라테리게나 (Klebsiella terrigena),나이세리아
고노레아 (Neisseria gonorrhoeae),나이세리아메닌자이티디스 (Neisseria meningitidis),엔테로박터에어로게네스 (Enterobacter aerogenes),엔테로박터 클로아케 (Enterobacter cloacae),시겔라보이디 (Shigellaboydii),시겔라
디센테리아에 (Shigelladysenteriae),시겔라물렉스네리 (Shigellaflexneri),시겔라 손네이 (Shigellasonnei),모락셀라카타할리스 (Moraxellacatarrhalis),모락셀라 라쿠나타 (Moraxellalacunata),모락셀라보비스 (Moraxellabovis),헬리코박터 파일로리 (Helicobacterpylori),헬리코박터헤일마니 (Helicobacterheilmannii), 헬리코박터펠리스 (Helicobacterfelis),헬리코박터
무스틸레 (Helicobactermustelae),헬리코박터페넬리에 (Helicobacterfenelliae), 헬리코박터라피니 (Helicobacterrappini),헬리코박터
헤파티쿠스 (Helicobacterhepaticus),헬리코박터빌리스 (Helicobacterbilis), 헬리코박터풀로룸 (Helicobacterpullorum),스테노트로포모나스
말토필리아 (Stenotrophomonasmaltophilia),스테노트로포모나스
나이트리트리더센스 (Stenotrophomonasnitritireducens),브델로비브리오
박테리오보러스 (Bdellovibriobacteriovoms),레지오넬라
뉴모필라 (Legionellapneumophila),레지오넬라아니사 (Legionallaanisa), 레지오넬라비르밍하멘시스 (Legionallabirminghamensis),레지오넬라
보제마니 (Legionallabozemanii),레지오넬라
신신나티엔시스 (Legionallacincinnatiensis),레지오넬라
두모피 (Legionalladumoffii) ,레지오넬라필레이 (Legionallafeeleii),레지오넬라 2020/175936 1»（：1/10公020/002826
20 고르마니 (Legionallagormanii),레지오넬라하케리애 (Legionallahackeliae) , 레지오넬라이스라엘렌시스 (Legionallaisraelensis),레지오넬라
조르다니스 (Legionallajordanis),레지오넬라란신젠시스 (Legionallalansingensis), 레지오넬라롱베아채 (Legionallalongbeachae),레지오넬라
마세아체르니 (Legionallamaceachernii),레지오넬라믹다데이 (Legionallamicdadei), 레지오넬라오크리드젠시스 (Legionallaoakridgensis),레지오넬라
사인텔렌시 (Legionallasainthelensi),레지오넬라
툭소넨시스 (Legionallatucsonensis),레지오넬라
와드스워티 (Legionallawadsworthii)등이포함되며 ,이에제한되지않는다.
[376] 본명세서에서사용된용어, n그람양성균 (gram positive bacteria)’’은그람
염색에서감청색또는보라색으로염색이되는세균으로, Staphylococcus속, Enterococcus속, Streptococcus속, Clostridium속균주등이포함되며 ,이에 제한되지않는다.구체적으로,그람양성균에는스타필로코커스
아우레우스 (Staphylococcus aureus),스타필로코커스
에피데르미디스 (Staphylococcus epidermidis)엔테로코커스패시움 (Enterococcus faecium),스트렙토코커스뉴모니아 (S仕 eptococcus pneumoniae),바실러스 안트라시스 (Bacillus anthracis)등이포함되며 ,이에제한되지않는다.
[377] 본명세서에서사용된용어,’，항생제내성그람양성균’，에는예를들어,
Methicillin내성그람양성균, Carbapenem내성그람양성균, Vancomycin내성 그람양성균, Macrolide및다제내성그람양성균등이포함되며,이에제한되지 않는다.
[378] 본명세서에서사용된용어,”항생제내성그람음성균”에는예를들어,
Streptomycin내성그람음성균, Colistin내성그람음성균, Carbapenem내성그람 음성균, Chloramphenicol내성그람음성균, Tetracyclines내성그람음성균, Cefotaxime내성그람음성균,ᄂ enem내성그람음성균,광범위베타락탐계 항생제분해효소 (ESBL;Extended Spec仕 um Beta Lactamase)내성그람음성균, Tigecycline내성그람음성균및다제내성그람음성균등이포함되며,이에 제한되지않는다.
[379] 본발명의일구현예에서,상기항균용조성물의항균대상균은표 er/c/
coli DH5a, Escherichia coli Kl, Acinetobacter baumannii Pseudomonas aeruginosa， Salmonella enteritidis， Salmonella typhimurium， Klebsiella pneumoniae，
Staphylococcus aureus및 Staphylococcus epidermidis로이루어진군에서선택된 1이상일수있다.
[38이 또한,본발명의일구현예에서 ,상기항균용조성물의항균대상균은광범위 베타락탐계항생제분해효소 (ESBL;Extended Spectrum Beta Lactamase)내성균, 카바페멤내성균및콜리스틴내성균으로이루어진군에서선택된 1이상의 항생제내성균일수있다.예를들어,상기항생제내성균은 ESBL (E. coli), Carbapenem resistant (CR)- Acinetobactor baumannii， CR- Klebsiella pneumoniae, CR- Pseudomonas aeruginosa, ¾ Colistin resistant Acinetobactor baumannii ¾ "r1 있으나,이에제한되지않는다.
[381]
[382] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,감염성질환의예방,개선또는치료용 조성물을제공한다.
[383] 또한,본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열 일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을유효성분으로포함하는,감염성질환의예방,개선또는치료용 약학적/식품/건강식품/화장품/의약외품/사료조성물을제공한다.
[384] 본발명은상기서열일반식으로표시되는폴리펩타이드또는상기서열
일반식으로표시되는폴리펩타이드의트리플루아세트산의아세테이트염 치환물을이를필요로하는개체에투여하는단계를포함하는감염성질환의 예방또는치료방법을제공한다.
[385] 뿐만아니라,본발명은상기서열일반식으로표시되는폴리펩타이드또는 상기서열일반식으로표시되는폴리펩타이드의트리플루아세트산의 아세테이트염치환물의감염성질환에대한예방,개선또는치료용도를 제공한다.
[386] 나아가,본발명은감염성질환의 예방,개선또는치료용제제의제조를위한 상기서열일반식으로표시되는폴리펩타이드또는상기서열일반식으로 표시되는폴리펩타이드의트리플루아세트산의아세테이트염치환물의용도를 제공한다.
[387] 본명세서에서사용된용어,’’감염성질환’’은바이러스,세균,곰광이,기생충과 같이질병을일으키는병원체가동물이나인간에게전파,침입하여일으킨 질환을의미하며，본발명의목적상그람음성균또는그람양성균에의해 유발되는모든감염성질환을의미한다.
[388] 예를들어,상기감염성질환에는,폐렴,복막염 ,뇌막염 ,창상감염 ,골관절염 , 담낭염 ,요로감염증,뇌수막염，심내막염,심근염,심외막염，관절염,인구염, 임질,세균성이질,장염,결막염,위염,중이염,방광염,림프관염등의그람 음성균에의해유발되는감염성질환이포함되나,이에제한되지않는다.
[389] 예를들어,상기감염성질환에는,인후염,농가진,류마티스열,사구체신염, 신생아패혈증,수막염，인두염,폐렴,심내막염,성홍열,피부연조직감염 , 심부연조직감염,농흉및질염등의그람양성균에의해유발되는감염성질환이 포함되나,이에제한되지않는다.
[39이
[391] 본발명의약학적조성물은약학적조성물의제조에통상적으로사용하는 적절한담체,부형제및희석제를더포함할수있다.본발명에따른약학적 조성물은각각통상의방법에따라산제,과립제,정제,캡슐제,현탁액,에멀젼, 시럽,에어로졸등의경구형제형,외용제,좌제및멸균주사용액의형태로 제형화하여사용될수있다.
[392] 본발명의약학적조성물에포함될수있는담체，부형제및희석제로는
락토오스,덱스트로오스,수크로오스,솔비톨,만니톨,자일리톨,에리스리톨, 말티톨,전분,아카시아고무,알지네이트,젤라틴,칼슘포스페이트,칼슘 실리케이트,셀룰로오스,메틸셀룰로오스,미정질셀룰로오스,폴리비닐 피롤리돈,물,메틸히드록시벤조에이트,프로필히드록시벤조에이트,탈크, 마그네슘스테아레이트및광물유를들수있다.
[393] 본발명의약학적조성물을제제화할경우에는보통사용하는중진제 ,증량제 , 결합제,습윤제,붕해제,계면활성제등의희석제또는부형제를사용하여 조제된다.경구투여를위한고형제제에는정제,환제,산제,과립제,캡슐제등이 포함되며 ,이러한고형제제는상기유효성분에적어도하나이상의부형제예를 들면,전분,칼슘카보네이트,수크로오스,락토오스,젤라틴등을섞어조제된다. 또한단순한부형제이외에마그네슘스테아레이트,탈크같은윤활제들도 사용된다.경구투여를위한액상제제로는현탁제,내용액제,유제,시럽제등이 해당되는데흔히사용되는단순희석제인물,리퀴드파라핀이외에여러가지 부형제,예를들면습윤제,감미제,방향제,보존제등이포함될수있다.비경구 투여를위한제제에는멸균된수용액,비수성용제,현탁제,유제,동결건조제제, 좌제가포함된다.비수성용제 ,현탁제로는프로필렌글라이콜 (propylene glycol), 폴리에틸렌글라이콜,올리브오일과같은식물성기름,에틸올레이트와같은 주사가능한에스테르등이사용될수있다.좌제의기제로는위텝솔 (witepsol), 마크로골,트윈 (tween) 61,카카오지,라우린지,글리세로제라틴등이사용될수 있다.
[394] 본명세서에서사용된용어,”예방”이란본발명에따른조성물의투여에의해 질환에의한증상을차단하거나,억제또는지연시키는모든행위를의미한다.
[395] 본명세서에서사용된용어,”치료”또는”개선”이란본발명에따른조성물의 투여에의해증세가호전되거나이롭게변경되는모든행위를의미한다.
[396] 본명세서에서사용되는용어, "투여 "는임의의적절한방법으로개체에게
소정의본발명의조성물을제공하는것을의미한다.
[397] 본명세서에서사용된용어, "개체”란질병의예방또는치료를필요로하는 대상을의미한다.예를들어,상기개체는인간,또는비-인간인영장류, 생쥐 (mouse),개,고양이,말,양및소를포함하는포유류일수있다.
[398] 본발명에따른약학적조성물은약학적으로유효한양으로투여된다.상기 "약학적으로유효한양”은의학적치료에적용가능한합리적인수혜/위험 비율로질환을치료하기에충분한양을의미하며,유효용량수준은환자의 질환의종류,중증도,약물의활성,약물에대한민감도,투여시간,투여경로및 배출비율,치료기간,동시사용되는약물을포함한요소및기타의학분야에잘 알려진요소에따라결정될수있다.바람직한투여량은개체의상태및체중, 질병의정도,약물형태,투여경로및기간에따라선택될수있다.구체적인 예로,상기약학적조성물은 0.001내지 1000 mg/kg, 0.01내지 100 mg/kg, 0.01 내지 10 mg/kg, 0.1내지 10 mg/kg또는 0.1내지 1 mg/kg의양을 1일 1회내지 수회로나누어투여할수있다.상기한요소들을모두고려하여부작용없이 최소한의양으로최대효과를얻을수있는양을투여하는것이중요하며,이는 당업자에의해결정될수있다.구체적으로,본발명에따른약학적조성물의 유효량은환자의연령,성별,상태,체중,체내에서활성성분의흡수도,불활성율 및배설속도,질병종류,병용되는약물에따라달라질수있다.
[399] 본발명의약학적조성물은개체에게다양한경로로투여될수있다.투여의 모든방식은예상될수있는데,예를들면,경구,직장또는정맥,근육,피하,자궁 내경막또는뇌혈관내주사에의해투여될수있다.
[400] 본발명에따른약학적조성물은상기유효성분외에패혈증또는패혈증성
쇼크;항균;감염성질환의개선,예방또는치료효과를갖는공지된물질을하나 이상추가로포함할수있다.
[401] 본발명에따른약학적조성물은상기유효성분외에기관지확장제 ,
항히스타민제또는소염진통제를추가로포함할수있다.
[402] 예를들어 ,상기기관지확장제로는 (3효능제 ,항콜린제 ,메틸잔틴등을사용할 수있고,상기항히스타민제로는아크리바스틴 (acrivastine),세티리진 (cetirizine), 데슬로라타딘 (desloratadine) ,펙소페나딘 (fexofenadine) ,
레보세티리진 (levocertirizine),로라타딘 (loratadine),미졸라스틴 (mizolastine), 아일메마진 (ailmemazine),클로세티리진 (chlocertirizine),클레마스틴 (clemastine), 시프로펩타딘 (cypropheptadine),하이드록시진 (hydroxyzine),케토티펜 (ketotifen), 프로멘타진 (promenthazine)등을사용할수있고,상기소염진통제로는
아스피린 (aspirin),디클로페낙 (diclofenac),페노프로펜 (fenoprofen),
늘루비프로펜 (flurbiprofen),이부프로펜 (ibuprofen),인도메타신 (indomethacin), 케토프로펜 (ketoprofen),나프록센 (naproxen),피록시캄 (piroxicam),
술린닥 (sulindac),셀레콕시브 (celecoxib),발데콕시브 (valdecoxib),
로페콕시브 (rofecoxib)등을사용할수있다.
[403]
[404] 본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의염의
LPS와의결합능을측정하여,본발명에따른폴리펩타이드및이의염이
LPS와의결합력이높음을확인하였다 (실시예 1).
[405] 또한,본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의 염이우수한항균활성을나타냄을확인하였다 (실시예 2,실시예 3및실시예 10).
[406] 또한,본발명의구체적실시예에서는,본발명에따른폴리펩타이드와이의 염이강한항생효과를나타냄을확인하였다 (실시예 8및실시예 9).
[407] [408] 또한,본발명은상기폴리펩타이드를코딩하는폴리뉴클레오티드를제공한다.
[409] 본명세서에서사용된용어, "폴리뉴클레오티드 (polynucleotide)”는단일가닥 또는이중가닥형태로존재하는데옥시리보뉴클레오티드또는
리보뉴클레오티드의중합체이다. RNA게놈서열, DNA(gDNA및 cDNA)및 이로부터전사되는 RNA서열을포괄하며,특별하게다른언급이없는한천연의 폴리뉴클레오티드의유사체를포함한다.
[410] 상기폴리뉴클레오티드는상기뉴클레오티드서열뿐만아니라,그서열에
상보적인 (complementary)서열도포함한다.상기상보적인서열은완벽하게 상보적인서열뿐만아니라,실질적으로상보적인서열도포함한다.이는 당업계에공지된가혹조건 (stringent conditions)하에서,상기뉴클레오티드 서열과혼성화될수있는서열을의미한다.
[411] 또한,상기폴리뉴클레오티드는변형될수있다.상기변형은뉴클레오티드의 추가,결실또는비보존적치환또는보존적치환을포함한다.상기아미노산 서열을코딩하는폴리뉴클레오티드는상기뉴클레오티드서열에대하여 실질적인동일성을나타내는뉴클레오티드서열도포함하는것으로해석된다. 상기의실질적인동일성은,상기뉴클레오티드서열과임의의다른서열을 최대한대응되도록얼라인하고,당업계에서통상적으로이용되는알고리즘을 이용하여얼라인된서열을분석한경우에,최소 80%의상동성,최소 90%의 상동성또는최소 95%의상동성을나타내는서열일수있다.
[412]
[413] 또한,본발명은상기폴리뉴클레오티드를포함하는재조합벡터를제공한다.
[414] 본명세서에서사용된용어, "벡터 (vector)”는숙주세포에서목적유전자를
발현시키기위한수단을의미한다.예를들어,플라스미드벡터,코즈미드벡터 및박테리오파아지벡터,아데노바이러스벡터,레트로바이러스벡터및 아데노-연관바이러스벡터와같은바이러스벡터를포함한다.상기재조합 벡터로사용될수있는벡터는당업계에서종종사용되는플라스미드 (예를 들면, pSClOl, pGV1106, pACYC177, ColEl, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFRl, pHV14, pGEX시리즈, pET시리즈및 pUC19등),파지 (예를들면, UXkA및 M13등)또는바이러스 (예를들면, CMV, SV40등)를조작하여제작될수있다.
[415] 상기재조합벡터에서상기펩타이드를코딩하는폴리뉴클레오티드는
프로모터에작동적으로연결될수있다.본명세서에서사용된용어,
"작동적으로연결된 (operatively linked)”은뉴클레오티드발현조절서열 (예를 들면,프로모터서열)과다른뉴클레오티드서열사이의기능적인결합을 의미한다.따라서，이에의해상기조절서열은상기다른뉴클레오티드서열의 전사및/또는해독을조절할수있다.
[416] 상기재조합벡터는,전형적으로클로닝을위한벡터또는발현을위한
벡터로서구축될수있다.상기발현용벡터는당업계에서식물,동물또는 미생물에서외래의단백질을발현하는데사용되는통상의것을사용할수있다. 상기재조합벡터는당업계에공지된다양한방법을통해구축될수있다.
[417]
[418] 또한,본발명은상기재조합벡터로형질전환된숙주세포를제공한다.
[419] 숙주세포는당업계에공지된어떠한숙주세포도이용할수있으며,원핵
세포로는,예를들어 , E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110,바실러스서브틸리스,바실러스츄린겐시스와 같은바실러스속균주,그리고살모넬라티피무리움,세라티아마르세슨스및 다양한슈도모나스종과같은장내균과균주등이 있으며,진핵세포에형질 전환시키는경우에는숙주세포로서 ,효모 (Saccharomyce cerevisiae),곤중세포, 식물세포및동물세포,예를들어 , SP2/0, CHO(Chinese hamster ovary) Kl, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN및 MDCK 세포주등이이용될수있다.
[42이 본발명은상기숙주세포를배양하는단계를포함하는,본발명에따른서열 일반식으로표시되는폴리펩타이드의제조방법을제공한다.
[421] 상기폴리뉴클레오티드또는이를포함하는재조합벡터의숙주세포내로의 삽입은,당업계에널리알려진삽입방법을사용할수있다.상기운반방법은 예를들어,숙주세포가원핵세포인경우, CaCl 2방법또는전기천공방법등을 사용할수있고,숙주세포가진핵세포인경우에는,미세주입법,칼슘
포스페이트침전법,전기천공법,리포좀-매개형질감염법및유전자밤바드먼트 등을사용할수있으나,이에한정하지는않는다.
[422] 상기형질전환된숙주세포를선별하는방법은선택표지에의해발현되는
표현형을이용하여,당업계에널리알려진방법에따라용이하게실시할수 있다.예를들어,상기선택표지가특정항생제내성유전자인경우에는,상기 항생제가함유된배지에서형질전환체를배양함으로써형질전환체를용이하게 선별할수있다.
[423]
[424]
[425] 이하,본발명의이해를돕기위하여바람직한실시예를제시한다.그러나
하기의실시예는본발명을보다쉽게이해하기위하여제공되는것일뿐,하기 실시예에의해본발명의내용이한정되는것은아니다.
[426]
[427] 심시예 1 :표. coli K1에 대한 ADK유래펜타이드후보듬의 LPS겸함능측정
[428] FP1 (ADK 44-54)을 WT(wild type)으로표기하며， WT에서다양한부분들에
대하여점돌연변이 (point mutation)된펩타이드를제작하였다 (FP3, FP5, FP6,및 FP9). FP3의잔기서열에서 LPS와의상호작용을높이기위해 FP3와 LPS 결합모델을기반하여펩타이드를설계하였고 (FP12-NH 2및 FP13-NH 2), 비자연계아미노산과아미노산이성질체 (allomeric D형아미노산)을추가 도입하여펩타이드를설계하였다 (allD FP12-NH 2, allD FP13-NH 2, allD FP-13-9a, allD FP-13-9w, allD FP-13-9k,및 allD FP13-NH 2 (AcOH)).또한, N-말단
페길화 (pegylation)한펩타이드 (PEG-alld FP13-NH 2(AcOH))를추가설계하여 (주)애니젠에서합성후제공받았다.제공받은각각의펩타이드들과
리포를리사카라이드 (lipopolysaccharide, LPS),두물질간의 Binding affinity (Kd)를즉정하는 Isothermal Titration Calorimeter (ITC)를이용하여 LPS와의 binding affinity를확인하였다.
[429] 구체적으로,펩타이드와 LPS간결합력 (Binding affinity (Kd))분석방법은
다음과같다.
[430] Malvern microcal peaq-itc cell장비를사용하여즉정하였고, LPS와의
interaction을확인하기위해하기와같은전처리를하였다. Cell의샘플양및 모양은 300 id, coin- shaped, fixed-in-place;실린지회전률은 1200 RPM;그리고 온도는 30OC, 35OC, 25OC로사용하였다.
[431] 시험전에미리 PBS로 LPS와 peptide를희석시켜 LPS 2mM과 peptide 0.2mM을 만들었다. Wash가끝난 ITC기기의 cell안에 peptide 300uL를넣고 syringe에는 LPS 40uL를넣어두었다. ITC의측정조건 (온도, injection횟수)를설정한후 Syringe를 cell안에꽂고 ITC측정을시작하였다.측정이완료되면분석을통해 LPS와 peptide의 Kd값을산출하였다.
[432] 측정결과, FP3, FP5, FP6, FP9, FP12-NH 2, FP13-NH 2, allD FP12-NH 2, allD
FP13-NH 2, allD FP-13-9a, allD FP-13-9w,및 allD FP-13-9k,및 allD FP13-NH 2 (AcOH)의모든종류의펩타이드에서 LPS binding affinity가있음이
확인되었으며 , FPl(wild type, WT)와동등하거나그이상의강한 LPS binding affinity를보임을알수있었다.
[433]
[표 1]
Figure imgf000029_0001
[434] [표 2]
Figure imgf000029_0002
[435] (상기표 1및표 2에서 , allD는 D형아미노산을의미함.)
[436] (상기표 1및표 2에서, (AcOH)는펩타이드의 N말단으로부터 9번째 아미노산의말단의 Trifluoroacetic acid(TFA)을 acetate염으로치환한염 치환물을의미함.)
[437] (상기표 2에서 , PEG는페길화 (pegylation)를의미함.펩타이드 N-말단 pegylation의정보: PEG(polyethylene glycol), Mw (분자량) =385.4Da, Fmoc-NH-PEG2-CH 2COOH) [438]
[439] 심시예 2 : allD FP13-NH 2과시판중이 항생제 4종의그람양음성표주규주에 대한항규활성비교평가
[44이 실시예 1에서설계한펩타이드중 LPS에강한 affinity를확인한펩타이드 1종
(allD FP13-NH 2)과시판중인항생제 4종의그람양음성표준균주에대한 항균활성을비교확인하였다.
[441] 종 (Ampicillin, Gentamicin, Levofloxacin, Imipenem)과 allD FP13-NH 2 타이드의항균활성을비교하기위하여,그람음성및그람양성 Escherichia coli DH5a, Escherichia coli Kl, Acinetobacterbaumannii,
Figure imgf000030_0001
as aeruginosa, Salmonella enteritidis, Salmonella typhimurium,및 Klebsiella pneumoniae)에대한최소저해농도 (Minimum Inhibitory Concentration, MIC 5。과 MIC 8Q)측정을통한항균활성실험법으로평가하였다.
[442] 보다구체적으로, 96-well plate의각 well에배지 lOOuL와첫번째열에는배지 200uL와항생제및펩타이드를분주하였다.첫번째열부터열한번째열까지 ½씩 serial dilution하고미리배양한그람음성및그람양성박테리아를희석하여
(희석시 OD 6M_의값은약 0.0025)각 well에분주하였다. Plate를
37°Cincubator에서 4시간동안배양한후 OD 6Mnm에서측정하였다. blank (배지와 균만들어간 well)의 OD __값으로균생장율을 100%로산정하고,
생장억제율이 50%의 OD __값을가지는항생제및펩타이드의농도를 확인하여 MIC 5。로산출하고생장억제율 80%는 MIC 에해당하는농도 (ug/ml) 로산출하였다.
[443] 그결과, allD FP13-NH 2 (AcOH)펩타이드는시판중인항생제 4종 (Ampicillin, Gentamicin, Levofloxacin, Imipenem)과비교해 ,동등하거나더우수한정도로 그람양음성균주 7종전체에대해서항균활성을나타냄을확인할수있었다.
[444]
[ ,11.3]
Figure imgf000031_0001
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©eis½(sicsri: 8.1¾~12S <S.5 <18 i.svo6o>;：;.：n 8725-128 <5.5
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Afafecife 0.125-128 < 10 <2.0 Gents idrs 0 2S 128 3 §. 7» bsisflisiaife 0125 ·· 128 1：8 ίίί·ΐ8¾¾ίTί： S:125-12S <4:8 <S：8 aiiiipPbTiSS: Q128 - 128 <¾,8 *48 kssietls pmomniaeAiec 13883]
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Figure imgf000032_0002
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3§gX| ¾I91 ) ISA/KR 2020/175936 1»（：1^1{2020/002826
31 로희석한 8.0%적혈구용액을 96 -웰마이크로적정플레이트에 100 씩 로딩한후,펩타이드용액 100 씩섞어준후, 37ᄋ（：에서 1시간동안배양한후,
96 -웰마이크로적정플레이트를 5분간원심분리하였다.상층액을 100 씩 취하여다른 96 -웰마이크로적정플레이트에옮긴후, 405 11111에서의흡광도를 측정하였다.이때, 0.1%트리톤 -100로처리하였을경우의 값을 100%용혈로 계산하고,펩타이드의용혈활성을 % 1 1110切 8로서하기수학식 1에의하여 산출하였다.
[456] 여기서,쇼는펩타이드용액에서의 405 11111의흡광도이고,：8는 0.1%트리톤
X- 100에서의 405 ·의흡광도이며 ,（：는
Figure imgf000033_0001
용액에서의 405■의흡광도를 나타낸다.
[457] 이 때 대조군의 펩타이드로서 강한항균활성을가짐과동시에 강한용혈활성을 나타내는항균펩타이드인멜리틴을사용하였다.
[458]
[459] [수학식 1]
[46이
« 31 11111
Figure imgf000033_0002
[461]
[462] 그결과,도 1에나타낸바와같이본발명에 따른펩타이드들은 100uM
고농도에서도거의용혈활성을나타내지 않아독성이낮은것을알수있었다. 반면에,대조군인멜리틴은낮은농도에서도 100%용혈활성을보임으로서 독성이 매우높음을알수있었다.
[463]
[464] 심시예 5 :워편광이색성분광범을이용한펜타이드 2차구조측정
[465] 므 요 !! 2와므 3- 1 2펩타이드의 2차구조를측정하기위하여다음을
실시하였다.
[466] 생체막유사환경에서의 펩타이드의 이차구조를연구하기 위해수용액,
100mM쬬 ， 50mM이 에서 펩타이드시료당 1001^가되도록용매 0.31111에 녹였다. 1111111 path !^!!의。 11을이용하여 0 1-720원편광이색성분광기에서 의주사속도로 0.111111마다흡수값이 얻어졌으며 6번의주사가평균이 되어측정값을얻었다.
[467] 원편광이색성분광법은폴리펩타이드의주골격의 2차구조에 따라특징적인 흡수형태를보인다. FP12-NH 2와 FP13-NH 2펩타이드는수용액에서는 이차구조를가지지 않으나생체막유사환경인
Figure imgf000033_0003
111止6116에서는（¾-1161뇨구조의 특징적인흡수형태인 208, 22211111근처의파장에서두개의 최소점을가졌다（도 2）.
[468] 상기결과로부터 이들펩타이드는박테리아의 생체막에서 01-11£1뇨구조를 형성하면서항균활성을나타낼것임을예측할수있다.반면에 D-amino acid 치환을한 all_D(FP12-NH 2), all_D(FP13-NH 2)펩타이드들은정확히모체 펩타이드의거울상을가짐을알수있었다.모체펩타이드의 CD spectrum이 right-handed a-helix (우선성알파힐릭스)구조를가지는것과는반대로 negative intensity를가짐으로서 left-handed a-helix구조를가짐을확인하였다 (도 2).
[469]
[47이 심시예 6 :그람음성 박테리아세포막과적혐구세포막을모방하는리포좀에 대한펜타이드의파괴능측정
[471] FP12-NH 2, FP13-NH 2, allD FP12-NH 2, allD FP13-NH 2펩타이드를대상으로 작용기전을연구하기위해박테리아세포막과적혈구세포막을모방하는 리포좀에대한펩타이드의파괴능을측정하였다.
[472] 펩타이드의항균작용이박테리아의세포막을표적으로하여세균을죽이는 작용기전을나타내는것인지를확인하기위하여 ,형광염료 (fluorescent dye)인 칼세인 (calcein)을포획시킨그람음성박테리아의세포막을모방하는
EYPC/EYPG (7:3, w/w)로구성된리포좀과인간의적혈구를모방하는
EYPC/CH(1:10, w/w)을제작하여다음의형광염료방출실험 (dye leakage assay)을 행하였다.
[473] EYPG는 egg yolk L-a-phosphatidyl-DL-glycerol의약어이며 , EYPC는 egg yolk l,2-diacyl-sn-glycero-3-phosphocholine의약어이다. CH는콜레스테롤의약자이다. 각구성성분의함량대로구성된인지질을클로로포름에녹인다음, rotary evaporator로클로로포름을제거하고,하룻밤동안동결건조시켰다.이후, 건조된지질필름을트리스-완충용액에칼세인을녹여서 액체질소로
얼림-녹임을반복한후 extruder장치를이용하여칼세인을포획한리포좀을 제작하였다·그리고나서 Spectrofluorimeter (Shimadzu RF 5301 PC
spectrofluorimeter, Japan)의 excitation waveleng仕 l을 490nm에 emission
wavelength를 520nm에맞추고,리포좀에펩타이드를투여하여 100 % dye leakage는 0.01 % Triton-X 1QQ을넣었을때의형광세기로표시하여상대적인 염료방출을측정하였다.
[474] 그결과를도 3에도시하였다.도 3의상단도면은펩타이드에의한
형광염료 (fluorescent dye)칼세인 (calcein)을포획시킨,박테리아세포를모방하는 EYPC/EYPG (7:3, w/w)리포좀에대한 %염료방출 (dye leakage)을펩타이드 농도에따라나타낸것이고,여기서는대조군 (세포막표적의작용기전을 나타내는펩타이드)으로항균펩타이드 melittin을사용하였다.도 3의하단 도면은펩타이드에의한형광염료 (fluorescent dye)칼세인 (calcein)을포획시킨 인간의적혈구를모방하는중성지질인 EYPC/CH (10:1, w/w)리포좀에대한 % 염료방출 (dye leakage)을펩타이드농도에따라나타낸것이고,여기서는대조군 (세포막표적의작용기작을나타내는펩타이드)으로항균펩타이드 melittin을 사용하였다. [475] 도 3에보이듯이대조펩타이드인 melittin은낮은농도에서도박테리아의 세포막을모방하는 PC/PG (7:3, w/w)로구성된리포좀에서칼세인을방출시켰다. 인간의적혈구를모방하는 PC/CH (10: 1, w/w)리포좀에서 Melittin은
Figure imgf000035_0001
서
W0%에가까운칼세인방출을일으켜서전혀박테리아세포에대한선택성을 보이지않고독성이매우높을것임을예측할수있다.
[476] 반면에 FP12-NH 2, FP13-NH 2, allD FP12-NH 2, allD FP13-NH 2펩타이드들은 박테리아세포막을모방하는 PC/PG (7:3, w/w)리포좀에대해큰염료방출을 일으켜서박테리아세포막파괴능이높음을알수있었다. FP12-NH 2, FP13-NH 2, allD FP12-NH 2, allD FP13-NH 2펩타이드들은선택적으로포유동물의적혈구 세포막을모방하는 PC/CH (1: 1, w/w)리포좀에대해서는거의염료방출을 보이지않아낮은용혈활성과일치하는결과를보였다.따라서본발명에따른 펩타이드들은박테리아세포막을선택적으로파괴하는작용기전을가짐을알 수있었다.
[477]
[478] 심시예 7 :펜타이드의다백짐분해효소저항성측정을통하아정성평가
[479] 설계된 allD FP12-NH 2, allD FP13-NH 2펩타이드의단백질분해효소저항성을 측정을통하여혈액내안정성을확인하였다.
[480] 보다구체적으로,세가지단백질분해효소인 trypsin, protease K, chymotrypsin에 대한저항성을확인하기위해 ,단백질분해효소를처리한후 coli，A. baumannii 및 S. aureus에대한펩타이드의항균활성을즉정하였다.
[481] 그결과,비천연아미노산인 D-amino acid치환을거쳐설계된 allD FP12-NH 2, allD FP13-NH 2펩타이드들은이들단백질분해효소에대한저항성이매우 증진되었음을확인할수있었다.
[482] 표 5에서의及. coli’A. baumannii에대한실험에서 , allD FP12-NH 2, allD FP13-NH 2펩타이드들은단백질분해효소를처리하지않은경우와비교하여비슷하거나 2-4배정도감소되는것에불과한항균활성을나타냄으로서안정성이매우높은 고기능성펩타이드임을확인하였다.
[483] 또한,표 6에서의 S. aureus에대한실험에서도, allD FP12-NH 2, allD FP13-NH 2 펩타이드들은안정성이매우높은고기능성펩타이드임을확인하였다.
[484] [IB]
Figure imgf000036_0002
4B5j |ϊ£6]
Figure imgf000036_0003
4 6]
Figure imgf000036_0001
¾
## «^UMedler Hinton Bmft) 10 ml# *#：## #¾¾# ¾#«H 3TC
3§gX| ¾I91 ) ISA/KR 인큐베이터에서 12시간동안배양하였다.배양액은 1 ml씩멸균된 cryo tube에 담아 - 80OC에서보관하였다.
[49이 시험균주는 37°C인큐베이터에서 2일간배양후 [BAP (blood agar plate)] MHB
(Mueller Hinton Broth)액체배지에접종균주양 (1x10 8또는 8x10 7)에맞추어 준비하고 6주령마우스의복강내로 (1 mL)주입하여감염을유도하였다.실험 별그룹으로생쥐 (그룹당 n=5또는 6)를구분하고 HARTMANN'S DEX solution (vehicle)와혼합한 allD FP13-NH 2를마우스복강내로 0시간, 1시간, 2시간,
3시간, 4시간, 5시간으로 6회로나눠접종하고 12시간간격으로 3일생존율 (survival rate)을관찰하였다.
[491] 그 ¾ ^1-, Escherichia coli K1 Escherichia coli (ESBL), Carbapenem-resistant Acinetobacter baumannii을각각감염시킨패혈증동물모델에 kg당종 3mg이상 12mg의농도범위에서생존율이증가 (항생효과)하는것을확인하였다 (도 4). 실시예 1에서설계한펩타이드가표준균주및임상균주감염동물모델에서도 m vivo상항생및항패혈증효과를나타냄을검증한것이다.
[492]
[493] 심시예 9 :펜타이드의 acetate염치화물
[494] allD FP13-NH 2의 acetate염치환물의치료효과및약물학적효과를확인하기 위하여 , Escherichia coli (표준균주및 ESBL)균주를각각복강투여하여유도한 패혈증동물모델혹은정상마우스에펩타이드 allD FP13-NH 2 (AcOH)을 처리하여 ,염치환물의항생효과와독성 ,항염증싸이토카인 IL-6의억제활성을 Trifluoroacetic acid(TFA)말단의동일펩타이드와비교하였다.
[495] 실험방법은다음과같다:
[496] 6주령의암컷평균체중 25g의 ICR마우스 (SPF mouse,샘타코)를실험동물로 사용하였다.실험동물은멸균사료와물을제공하고케이지별 W마리미만의 동물로사육하였고,밤낮주기는 12시간으로하였다.
[497] 시험균주는 37OC인큐베이터에서 2일간배양후 [BAP (blood agar plate)] MHB (Mueller Hinton Broth)액체배지에접종균주양 (1x10 8)에맞추어준비하고
6주령생쥐의복강내로 (Ixl0 8/200 M)주입하여감염을유도하였다.실험별 그룹으로생쥐 (그룹당 n=5또는 6)를구분하고 HARTMANN'S DEX
sol(vehicle)와혼합한 allD FP13-NH 2 (TFA)와 allD FP13-NH 2 (AcOH)를마우스 복강내로 0시간, 1시간, 2시간, 3시간, 4시간, 5시간으로 6회로나눠접종하고 12시간간격으로생존율 (survival rate)을관찰하였다.
[498] in vivo독성평가를위해 ,실험별그룹으로생쥐 (그룹당 n=5또는 6)를
구분하고 HARTMANN'S DEX sol( vehicle)와혼합한 allD FP13-NH2 (TFA), allD FP13-NH2 (AcOH)를마우스복강내로 0시간, 1시간, 2시간, 3시간, 4시간,
5시간으로 6회로나눠접종하고 12시간간격 , 72시간생존율 (survival rate)을 관찰하였다.
[499] 또한, BMDC (골수유래수지상세포)에대한 IL-6분비시험은 allD FP13-NH 2
Figure imgf000038_0001
3§gXI ¾I91^) ISA/KR [508]
[509] 심시예 11 :그람양성박테리아세포막을모방하는리포좀에대한펜타이드의 파괴능측정
[510] FP12-NH 2, FP13-NH 2, allD FP12-NH 2, allD FP13-NH 2펩타이드를대상으로 작용기전을연구하기위해박테리아세포막을모방하는리포좀에대한 펩타이드의파괴능을측정하였다.
[511] 펩타이드의항균작용이그람양성박테리아의세포막을표적으로하여
박테리아를죽이는작용기전을나타내는것인지를확인하는실험을진행하기 위해,형광염료 (fluorescent dye)인칼세인 (calcein)을포획시킨그람양성 박테리아의세포막을모방하는 EYPG/EYPC (6:4, w/w)로구성된리포좀을 제작하여형광염료방줄실험 (dye leakage as say)을행하였다.
[512] 그결과, FP12-NH 2, FP13-NH 2, allD FP12-NH 2, allD FP13-NH 2펩타이드들은 박테리아세포막을모방하는 EYPG/EYPC (6:4, w/w)리포좀에대해많은 염료방출을일으켜서박테리아세포막파괴능이우수함을알수있었다.이로써, 본발명에따른 FP12-NH 2, FP13-NH 2, allD FP12-NH 2, allD FP13-NH 2
펩타이드들그람양성박테리아세포막을파괴하는작용기전도가짐을알수 있었다 (도 6).
[513]
[514] 심시예 12 : allD FP13-NH 의그람양성규유래 T.TA및 LPP경함뉴측정
[515] 펩타이드의그람양성균에대한항균활성을확인하여작용기전을확인하기 위해다음실험을진행하였다.
[516] allD FP13-NH 2를이용하여 ,그람양성균 ( 5. aureus)의세포벽성분인
LTA(lipotechoic acid)와 LPP(lipoprotein)에대한결합력을 ITC분석을통해 확인하였다.
[517] ITC분석법을이용한결합능을확인하는바,실험방법은실시예 1의방법을
준용하였다.
[518] 그결과, allD FP13-NH 2가그람양성균의세포벽성분인 LTA와 LPP에
결합하는것을확인하였다. LTA에는 LPP가포함되어 있으며,돌연변이된 LTA를포함하는 LPP에는결합하지않는것을확인하였으며, FP13-NH 2와 LTA의결합에는 LPP가더중요한역할을할것으로예상되었다.따라서
FP13-NH 2의그람양성균에대한항균활성기전은그람양성균세포벽성분인 LPP(lipoprotein)결합이중요할것으로확인하였다.
[519]
[52이 전술한본발명의설명은예시를위한것이며,본발명이속하는기술분야의 통상의지식을가진자는본발명의기술적사상이나필수적인특징을변경하지 않고서다른구체적인형태로쉽게변형이가능하다는것을이해할수있을 것이다.그러므로이상에서기술한실시예들은모든면에서예시적인것이며 한정적이아닌것으로이해해야한다. 2020/175936 1»（：1^1{2020/002826
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[521]
산업상이용가능성
[522] 본발명에 따른펩타이드는,표준박테리아및항생제내성 박테리아의증식 억제뿐아니라,박테리아유래내독소를제거하는우수한효과를가지고있어 우수한패혈증치료효과를가지고있고,항생제와병용할경우항생제에 의한 부작용을최소화할수있어패혈증의 예방또는치료에유용하게 이용할수 있다.또한,본발명에따른펩타이드는그람양음성균에 대해선택적으로 우수한항균활성을가지고있어,그람양음성균에 대한항균용조성물또는그람 양음성균에 의해유발되는다양한감염성 질환들의 예방또는치료에유용하게 이용될수있다.또한,본발명에따른펩타이드는인체에 안전할뿐만아니라, 단백질분해효소에 대해서도안정성을나타내므로,다양한용도로활용될수 있다.
[523]
Figure imgf000040_0001
[547] Lys Leu Gly Val Glu Ala Leu Arg Tyr Leu Asp
[548]
[549] 6
[550] FP12-NH2
[551] Arg Leu Arg Val Lys Leu Arg Arg Tyr Leu Arg
[552]
[553] 7
[554] FP13-NH2
[555] Lys Leu Arg Val Lys Leu Arg Arg Tyr Leu Arg
[556]
[557] 8
[558] allD FP12-NH2
[559] Arg Leu Arg Val Lys Leu Arg Arg Tyr Leu Arg
[560]
[561] 9
[562] allD FP13-NH2
[563] Lys Leu Arg Val Lys Leu Arg Arg Tyr Leu Arg
[564] 1 5 10
[565]
[566] 10
[567] allD FP-13-9a-NH2
[568] Lys Leu Arg Val Lys Leu Arg Arg Ala Leu Arg
[569]
[570] 11
[571] allD FP-13-9w-NH2
[572] Lys Leu Arg Val Lys Leu Arg Arg Trp Leu Arg
[573]
[574] 12
[575] allD FP-13-9k-NH2
[576] Lys Leu Arg Val Lys Leu Arg Arg Lys Leu Arg
[577]
[578] 13
[579] allD FP13-NH2 (AcOH)
[580] Lys Leu Arg Val Lys Leu Arg Arg Tyr Leu Arg
[581]
[582] 14
[583] PEG- allD FP13-NH2 (AcOH)
[584] Lys Leu Arg Val Lys Leu Arg Arg Tyr Leu Arg [585]
[586] 15
[587] FP12
[588] Arg Leu Arg Val Lys Leu Arg Arg Tyr Leu Arg
[589]
[590] 16
[591] FP13
[592] Lys Leu Arg Val Lys Leu Arg Arg Tyr Leu Arg
[593]
[594] 17
[595] allD FP21-NH2
[596] Arg Leu Arg Val Lys Leu Arg Arg Trp Leu Arg
[597]
[598] 18
[599] allD FP-13-9d-NH2
[600] Lys Leu Arg Val Lys Leu Arg Arg Asp Leu Arg
[601]

Claims

2020/175936 1»（：1/10公020/002826
41
청구범위
［청구항 1］ 하기의서열일반식으로표시되는폴리펩타이드:
［일반식］
Ln-X 1 -L-X2-V-X3-X4-X5-R-X6-L-X7
상기일반식에서,
n은 0또는 1;
L은류신 (leucine);
V는발린 (valine);
요은아르기닌 (arginine);
XI은라이신 (lysine: K)또는아르기닌 (arginine: R);
X2는글리신 (glycine: G)또는아르기닌 (arginine: R);
X3은글루탐산 (glutamic acid: E)또는라이신 (lysine: K);
X4는알라닌 (alanine: A)또는류신 (leucine: L);
X5는라이신 (lysine: K)또는아르기닌 (arginine: R);
X6은티로신 (tyrosine: Y),알라닌 (alanine: A),트립토판 (仕 yptophan: W), 라이신 (lysine: K)또는아스파르트산 (aspartic acid: D);및 X7은아스파르트산 (aspartic acid: D)또는아르기닌 (arginine: R), 단,상기일반식에서 K-L-G-V-E-A-K-R-Y-L-D의서열로표시되는 폴리펩타이드는제외함.
［청구항 2］ 제 1항에 있어서, 1)내지 9)로이루어진 9종의폴리펩타이드중어느 하나의폴리펩타이드:
1)제 1항의일반식에서,
n은 0;
XI은라이신 (lysine: K);
X2는글리신 (glycine: G);
X3은글루탐산 (glutamic acid: E);
X4는알라닌 (alanine: A);
X5는라이신 (lysine: K);
X6은티로신 (tyrosine: Y),및
X7은아르기닌 (arginine: R)인,
폴리펩타이드;
2)제 1항의일반식에서,
n은 1;
XI은아르기닌 (arginine: R);
X2는글리신 (glycine: G);
X3은글루탐산 (glutamic acid: E);
X4는알라닌 (alanine: A); X5는라이신 (lysine: K);
X6은티로신 (tyrosine: Y),및
X7은아르기닌 (arginine: R)인 , 폴리펩타이드;
3)제 1항의 일반식에서 ,
n은 1;
XI은아르기닌 (arginine: R);
X2는글리신 (glycine: G);
X3은글루탐산 (glutamic acid: E);
X4는류신 (leucine: L);
X5는라이신 (lysine: K);
X6은티로신 (tyrosine: Y),및
X7은아르기닌 (arginine: R)인, 폴리펩타이드；
4)제 1항의일반식에서 ,
n은 0;
XI은라이신 (lysine: K);
X2는글리신 (glycine: G);
X3은글루탐산 (glutamic acid: E);
X4는알라닌 (alanine: A);
X5는류신 (leucine: L);
X6은티로신 (tyrosine: Y),및
X7은아스파르트산 (aspartic acid: D)인, 폴리펩타이드;
5)제 1항의일반식에서，
n은 0;
XI은아르기닌 (arginine: R);
X2는아르기닌 (arginine: R);
X3은라이신 (lysine: K);
X4는류신 (leucine: L);
X5는아르기닌 (arginine: R);
X6은티로신 (tyrosine: Y),및
X7은아르기닌 (arginine: R)인, 폴리펩타이드;
6)제 1항의일반식에서 ,
n은 0;
XI은라이신 (lysine: K);
X2는아르기닌 (arginine: R); 2020/175936 1»（：1/10公020/002826
43
X3은라이신 (lysine: K);
X4는류신 (leucine: L);
X5는아르기닌 (arginine: R);
X6은티로신 (tyrosine: Y),및
X7은아르기닌 (arginine: R)인,
폴리펩타이드;
7)제 1항의일반식에서,
n은 0;
XI은라이신 (lysine: K);
X2는아르기닌 (arginine: R);
X3은라이신 (lysine: K);
X4는류신 (leucine: L);
X5는아르기닌 (arginine: R);
X6은알라닌 (alanine: A),및
X7은아르기닌 (arginine: R)인,
폴리펩타이드;
8)제 1항의일반식에서,
n은 0;
XI은라이신 (lysine: K);
X2는아르기닌 (arginine: R);
X3은라이신 (lysine: K);
X4는류신 (leucine: L);
X5는아르기닌 (arginine: R);
X6은트립토판 (tryptophan: W) ,및
X7은아르기닌 (arginine: R)인,
폴리펩타이드;및
9)제 1항의일반식에서,
n은 0;
XI은라이신 (lysine: K);
X2는아르기닌 (arginine: R);
X3은라이신 (lysine: K);
X4는류신 (leucine: L);
X5는아르기닌 (arginine: R);
X6은라이신 (lysine: K),및
X7은아르기닌 (arginine: R)인 ,
폴리펩타이드.
[청구항 3] 제 1항에 있어서,
상기폴리펩타이드는 L형, D형및펩토이드 (peptoid)를포함하는 펩타이드모방체또는비천연아미노산인,폴리펩타이드.
[청구항 4] 제 1항에 있어서,
상기폴리펩타이드의말단을알킬레이션 (Alkylation),
페길레이션 (PEGylation)또는아미데이션 (Amidation)한것인,
폴리펩타이드.
[청구항 5] 제 1항에 있어서,
상기폴리펩타이드의 C말단에아민기 (NH 2)가추가된것인,
폴리펩타이드.
[청구항 6] 제 1항에 있어서,
상기폴리펩타이드는하기특징중하나이상의특징을가지는것인, 폴리펩타이드:
그람음성균의리포폴리사카라이드 (LPS)에의결합능;
박테리아세포막의선택적파괴능;
단백질분해효소에대한저항성 ;
그람양성균의세포막파괴능;및
그람양성균유래 LT A(lipotechoic acid)또는 LPP(lipoprotein)에의결합능.
[청구항 7] 제 1항에따른쓸리펩타이드의트리늘루아세트산 (Trifluoroacetic acid:
TFA)의아세테이트염치환물.
[청구항 8] 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물을유효성분으로포함하는, 패혈증의 예방또는치료용조성물.
[청구항 9] 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물을유효성분으로포함하는, 항균용조성물.
[청구항 ] 제 9항에 있어서,
상기항균용조성물의항균대상균은 Escherichia coli DH5a, Escherichia coli Kl， Acinetobacterbaumannii， Pseudomonas aeruginosa， Salmonella enteritidiS Salmonella typhimurium， Klebsiella pneumoniae， Staphylococcus aureus및 Staphylococcus epidermidis로이루어진군에서선택된 1이상인, 항균용조성물.
[청구항 11] 제 9항에 있어서,
상기항균용조성물의항균대상균은광범위베타락탐계항생제 분해효소 (ESBL;Extended Spectrum Beta Lactamase)내성균,카바페뎀 내성균및콜리스틴내성균으로이루어진군에서선택된 1이상의 항생제내성균인,항균용조성물.
[청구항 12] 제 11항에 있어서,
상기항생제내성균은 ESBL (及. co/z·), Carbapenem resistant (CR)- Acinetobactor baumannii， CR- Klebsiella pneumoniae CR -Pseudomonas aeruginosa, ¾ Colistin resistant Acinetobactor baumannii £. 01 어 ¾ 군에서선택된 1이상인,항균용조성물.
［청구항 13］ 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물을유효성분으로포함하는, 감염성질환의예방또는치료용조성물.
［청구항 14］ 제 13항에 있어서,
상기감염성질환은,폐렴 ,복막염 ,뇌막염 ,창상감염 ,골관절염 ,담낭염 , 요로감염증,뇌수막염，심내막염,심근염,심외막염，관절염,인구염,임질, 세균성이질,장염,결막염,위염,중이염,방광염,및림프관염으로 이루어지는군으로부터선택되는그람음성균에의해유발되는감염성 질환인,조성물.
［청구항 15］ 제 13항에 있어서,
상기감염성질환은,인후염,농가진,류마티스열,사구체신염,
신생아패혈증,수막염，인두염,폐렴,심내막염,성홍열,피부연조직감염 , 심부연조직감염,농흉및질염으로이루어지는군으로부터선택되는 그람양성균에의해유발되는감염성질환인,조성물.
［청구항 16］ 제 1항의폴리펩타이드를코딩하는폴리뉴클레오티드.
［청구항 17］ 제 16항의폴리뉴클레오티드를포함하는재조합벡터 .
［청구항 18］ 제 17항의재조합벡터로형질전환된숙주세포를배양하는단계를
포함하는,
제 1항의폴리펩타이드의제조방법.
［청구항 19］ 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물을이를필요로하는개체에 투여하는단계를포함하는패혈증의치료방법.
［청구항 2이 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물의패혈증에대한예방,개선 또는치료용도.
［청구항 21］ 패혈증의 예방,개선또는치료용제제의제조를위한제 1항의
폴리펩타이드또는제 7항의폴리펩타이드의트리플루아세트산의 아세테이트염치환물의용도.
［청구항 22］ 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물을이를필요로하는개체에 투여하는단계를포함하는감염성질환의치료방법.
［청구항 23］ 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물의감염성질환에대한예방, 개선또는치료용도.
［청구항 24］ 감염성질환의예방,개선또는치료용제제의제조를위한제 1항의
폴리펩타이드또는제 7항의폴리펩타이드의트리플루아세트산의 2020/175936 1»（：1^1{2020/002826
46 아세테이트염치환물의용도.
[청구항 25] 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물을이를필요로하는개체에 투여하는단계를포함하는항균방법 .
[청구항 26] 제 1항의폴리펩타이드또는제 7항의폴리펩타이드의
트리플루아세트산의아세테이트염치환물의항균용도.
[청구항 27] 항균제의제조를위한제 1항의폴리펩타이드또는제 7항의
폴리펩타이드의트리플루아세트산의아세테이트염치환물의용도.
PCT/KR2020/002826 2019-02-28 2020-02-27 항균활성을 갖는 폴리펩타이드, 이를 포함하는 패혈증 예방 또는 치료용 조성물, 및 항균용 조성물 WO2020175936A1 (ko)

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CA3139464A CA3139464A1 (en) 2019-02-28 2020-02-27 Polypeptide having antibacterial activity, composition for preventing or treating sepsis comprising same, and antibacterial composition
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CN202080017455.8A CN113544139A (zh) 2019-02-28 2020-02-27 具有抗菌活性的多肽、包含该多肽的用于预防或治疗败血症的组合物和抗菌组合物
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