WO2015161820A1 - 两亲性合成抗菌肽、其药物组合物及其用途 - Google Patents

两亲性合成抗菌肽、其药物组合物及其用途 Download PDF

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WO2015161820A1
WO2015161820A1 PCT/CN2015/077337 CN2015077337W WO2015161820A1 WO 2015161820 A1 WO2015161820 A1 WO 2015161820A1 CN 2015077337 W CN2015077337 W CN 2015077337W WO 2015161820 A1 WO2015161820 A1 WO 2015161820A1
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derivative
pharmaceutically acceptable
acceptable salt
peptide
antimicrobial peptide
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English (en)
French (fr)
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刘克良
邹存彬
孟庆斌
刘兴东
王晨宏
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中国人民解放军军事医学科学院毒物药物研究所
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Priority to CN201580020639.9A priority Critical patent/CN106232616B/zh
Publication of WO2015161820A1 publication Critical patent/WO2015161820A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of biomedicine and relates to a kind of amphiphilic synthetic antimicrobial peptide having a free thiol group or a dimer thereof, a derivative thereof, a pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof, and a preparation thereof for use in prevention, control or The use of a medicament for treating infectious diseases caused by Gram-positive bacteria, Gram-negative bacteria, and even resistant bacteria, fungi, and viruses.
  • Antimicrobial Peptides are broadly defined in the biological defense system and are widely found in microorganisms, animals and plants, including bacteria, fungi, insects, tunicates, amphibians, crustaceans, birds, In fish, mammals (including humans), plants and other organisms, there is a kind of positively charged amphiphilic small molecule antimicrobial peptide that resists external microbial attack and removes mutant cells in vivo. Its molecular mass is about 4KD, which is a creature. An important component of innate immunity.
  • CATH-2 Chicken cathelicidin-2
  • CMAP27 MICROstatic peptide 27
  • Yanjing Xiao et al. identified the tertiary structure of CATH-2 by nuclear magnetic resonance spectroscopy, which consists of two ⁇ -helices linked by a long hinge region with proline. Further studies have shown that the N-terminal helix of CATH-2 is more important than the C-helix in antibacterial activity, probably due to the presence of a net cationic charge at the N-terminus, the N-terminal helix is an amphiphilic structure, the C-terminal helix It is a highly hydrophobic structure. Many antibacterial peptides exert antibacterial activity by blocking the integrity of the cell membrane.
  • the amphiphilicity of the peptide is particularly important in the antibacterial activity.
  • the pivotal region of CATH-2 has been shown to be essential for the biological activity of peptides, and the proline, the LPS-neutralizing activity, and the immunostimulatory activity of lysine in place of the leucine are severely reduced.
  • the first 15 amino acid sequences of the N-terminus, C1-15 are important fragments of their antibacterial activity (Xiao Y., Herrera AI, Bommineni YR, et al.
  • the Central kink region 0f fowlicidin-2, an ⁇ -helical host defense peptide is critically involved in bacterial killing and endotoxin neutralization. J. Innate Immun., 2009, 1(3): 268-280).
  • EMMolhoek et al. further modified the C1-15 to replace the phenylalanine in C1-15 with a more hydrophobic tryptophan, not only against Gram-positive or Gram-negative bacteria, but also It is also active against bacteria that may be used in bioterrorism attacks (such as Bacillus anthracis, Vibrio cholera, Yersinia pestis, etc.). Due to the addition of tryptophan, the toxicity of the polypeptide to mammalian peripheral blood mononuclear cells (PBMC) is increased.
  • PBMC peripheral blood mononuclear cells
  • antibacterial peptides like other peptide drugs, have short half-lives and are easily Defects such as degradation, so it is of great significance to obtain antimicrobial peptides with good activity and high stability.
  • the problem to be solved by the present invention is to obtain an antimicrobial peptide having high antibacterial activity, high stability in plasma, and low hemolytic side effects.
  • the present inventors have found that, in the case where the number of fixed positive charges and the kind of amino acids are constant, changing the order of amino acids while maintaining the uniform dispersion of charged amino acids in the sequence has no significant effect on the antibacterial activity, and based on this, a series of new ones are synthesized.
  • Cationic antimicrobial peptides The introduction of cysteine in the sequence enables a significant increase in the plasma stability of the peptide but is related to the introduction position and is more stable in the middle of the sequence than at both ends.
  • the present inventors have screened out a series of antimicrobial peptides having high activity, good stability, and low hemolytic property, thereby completing the present invention.
  • the present invention provides an antimicrobial peptide having high antibacterial activity and stability by introducing or forming a dimer of a free sulfhydryl group, and a pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof, and They are useful for the prevention, treatment or adjunctive treatment of infectious diseases caused by Gram-positive bacteria, Gram-negative bacteria or even resistant bacteria, fungi or viruses.
  • a first aspect of the invention relates to an amphiphilic synthetic antimicrobial peptide, a derivative thereof or a pharmaceutically acceptable salt thereof, which has a structure represented by the formula (0):
  • the fifteen peptide consists of 8 basic amino acids and 7 hydrophobic amino acids, wherein the basic amino acid is uniformly dispersed in the sequence of the fifteen peptide, and the uniform dispersion means one, two or three
  • the hydrophobic amino acid is distributed with one or two basic amino acids (ie, there are no consecutive four hydrophobic amino acids or three consecutive basic amino acids);
  • C represents the Cys inserted into the pentapeptide
  • n represents the number of Cys inserted
  • n 0, 1 or 2
  • r represents the position of the inserted Cys in the peptide chain represented by the formula (0)
  • Cys can be located in the formula (0)
  • the sequence of the pentapeptide is: KRIGW RWRRW PRLRK (SEQ ID NO: 10). In another embodiment of the invention, the sequence of the pentapeptide is: PKRWG RWLRK IRRWR (SEQ ID NO: 11).
  • amphiphilic synthetic antimicrobial peptide, a derivative thereof, or a pharmaceutically acceptable salt thereof has the amino acid sequence represented by the general formula (1):
  • X 1 -X 8 are each independently selected from basic amino acids, such as Lys (K), Arg (R) or His (H);
  • Y 1 -Y 7 are each independently selected from hydrophobic amino acids, such as Leu (L), Ile (I), Trp (W), Pro (P), Gly (G), Val (V), Ala (A) or Met(M),;
  • r is selected from 1, 9, or 11.
  • amphiphilic synthetic antimicrobial peptide, a derivative thereof or a pharmaceutically acceptable salt thereof is selected from the group consisting of:
  • KRIGW RWRCR WPRLRK-NH 2 (SEQ ID NO: 2);
  • amphiphilic synthetic antimicrobial peptide, a derivative thereof or a pharmaceutically acceptable salt thereof has the amino acid sequence of the formula (2):
  • X 1 -X 8 are each independently selected from basic amino acids, such as Lys (K), Arg (R) or His (H);
  • Y 1 -Y 7 are each independently selected from hydrophobic amino acids, such as Leu (L), Ile (I), Trp (W), Pro (P), Gly (G), Val (V), Ala (A) or Met(M);
  • r is selected from 1, 9, or 16.
  • the antimicrobial peptide, a derivative thereof or a pharmaceutically acceptable salt thereof is selected from the group consisting of
  • a second aspect of the invention relates to an antimicrobial peptide, a derivative thereof or a pharmaceutically acceptable salt thereof, which comprises the amphiphilic synthetic antimicrobial peptide of any one of the first aspects of the invention, a derivative thereof or a pharmaceutically acceptable salt thereof, preferably,
  • There are one to several C-terminals of the amphiphilic synthetic antimicrobial peptide Such as 10-18, such as 12-16, such as 12-14) amino acids, more preferably, the one to several (eg 10-18, eg 12-16, eg 12-14) amino acids
  • a hydrophobic helix can be formed.
  • the antibacterial peptide of the first aspect of the present invention is mainly obtained by modifying the N-terminal sequence of the natural antibacterial peptide, and those skilled in the art can firstly according to the characteristics of the C-terminal sequence of the natural antibacterial peptide (for example, CATH-2).
  • the C-terminus of the antimicrobial peptide is linked to one or more amino acid or polypeptide sequences having the C-terminal sequence of the natural antimicrobial peptide to obtain an antimicrobial peptide having the same or similar function as the antimicrobial peptide of the first aspect of the invention;
  • the C-terminus is linked to one to several (eg, 10-18, eg, 12-16, eg, 12-14) amino acids (eg, hydrophobic amino acids), preferably, the one to several (eg, 10-18, For example, 12-16, such as 12-14) amino acids can form a hydrophobic helix.
  • a third aspect of the invention relates to a dimeric antibacterial peptide formed by a disulfide bond of two antimicrobial peptides, a derivative thereof or a pharmaceutically acceptable salt thereof, a derivative thereof or a pharmaceutically acceptable salt thereof, each of said two antimicrobial peptides
  • An antimicrobial peptide independently selected from any one of the first aspects of the invention, wherein n is 1 or 2.
  • the antimicrobial peptide as defined in any one of the first aspects of the invention refers to an antimicrobial peptide as defined by formula (0), formula (1) or formula (2).
  • the dimeric antimicrobial peptide, a derivative thereof, or a pharmaceutically acceptable salt thereof is selected from the group consisting of
  • An antibacterial peptide obtained by the two SEQ ID NO: 2 sequences forming an interchain disulfide bond by a cysteine at the 9th position;
  • An antibacterial peptide obtained by the two sequences of SEQ ID NO: 3 forming an interchain disulfide bond by a cysteine at position 11;
  • An antibacterial peptide obtained by the two SEQ ID NO: 4 sequences forming an interchain disulfide bond by the cysteine at the first position.
  • a fourth aspect of the invention relates to the antimicrobial peptide of any one of the first to third aspects of the invention, or a derivative thereof, or a pharmaceutically acceptable salt thereof, wherein some or all of the L-amino acid is replaced with a corresponding one D-amino acid.
  • the fifth aspect of the invention relates to the antimicrobial peptide of any one of the first to third aspects of the invention, or a derivative thereof, or a pharmaceutically acceptable salt thereof, which is a cyclized antimicrobial peptide, a derivative thereof or a pharmaceutically acceptable salt thereof, for example It is a loop between the N-terminus and the C-terminus of the antimicrobial peptide.
  • the invention relates to a kind of amphiphilic synthetic antimicrobial peptide which is designed based on the analysis of the sequence structure of the natural antimicrobial peptide.
  • the amino acid sequence of the antimicrobial peptide is shown in SEQ ID NO: 1 to SEQ ID NO: 12, and the structure is shown in Table 1.
  • the antibacterial peptide of the present invention is composed of a positively charged amino acid (ie, a basic amino acid) such as Arg, Lys, His, and a hydrophobic amino acid such as Trp, Leu, Ile, Pro, Gly, Val, Ala or Met.
  • a positively charged amino acid ie, a basic amino acid
  • Arg, Lys, His a positively charged amino acid
  • a hydrophobic amino acid such as Trp, Leu, Ile, Pro, Gly, Val, Ala or Met.
  • the amphiphilic peptide chain, and the positively charged amino acid is uniformly dispersed throughout the peptide chain.
  • the introduction of a cysteine with a free sulfhydryl group at various positions of the antimicrobial peptide can significantly improve plasma stability and is a simple and effective method for increasing plasma stability.
  • the cysteine-bearing antimicrobial peptide can form a dimer through a disulfide bond, which is superior to the antimicrobial peptide with a free sulfhydryl group.
  • Another aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the antimicrobial peptide according to any one of the first to fifth aspects of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof;
  • the composition also contains a pharmaceutically acceptable carrier or adjuvant.
  • the pharmaceutical composition of the present invention usually contains 0.1 to 90% by weight of the antimicrobial peptide of any one of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof.
  • Pharmaceutical compositions can be prepared according to methods known in the art. When used for this purpose, the antimicrobial peptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, may be combined with one or more solid or liquid pharmaceutical excipients and/or adjuvants, if desired. A suitable form of administration or dosage form for human use.
  • the antimicrobial peptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present invention may be administered in a unit dosage form, which may be enterally or parenterally, such as orally, muscle, subcutaneous, Nasal cavity, oral mucosa, skin, peritoneum or rectum.
  • Formulations such as tablets, capsules, pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, lyophilized powders Wait. It may be a general preparation, a sustained release preparation, a controlled release preparation, and various microparticle delivery systems.
  • carriers In order to form a unit dosage form into tablets, various carriers well known in the art can be widely used.
  • carriers are, for example, diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, silicic acid.
  • wetting agent and binder such as water, glycerin, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, gum arabic, gelatin paste, sodium carboxymethyl cellulose , shellac, methyl cellulose, potassium phosphate, polyvinyl pyrrolidone, etc.
  • disintegrating agents such as dried starch, alginates, agar powder, brown algae starch, sodium bicarbonate and tannic acid, calcium carbonate, polyoxyethylene, Sorbitol fatty acid ester, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.
  • disintegration inhibitors such as sucrose, glyceryl tristearate, cocoa butter, hydrogenated oil, etc.
  • absorption promotion Agents such as quaternary ammonium salts, sodium lauryl sulfate, and the like
  • lubricants such as talc, silica,
  • Tablets may also be further formed into coated tablets, such as sugar coated tablets, film coated tablets, enteric coated tablets, or bilayer tablets and multilayer tablets.
  • various carriers known in the art can be widely used.
  • the carrier are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, talc, etc.; binders such as acacia, tragacanth, gelatin , ethanol, honey, liquid sugar, rice paste or batter; etc.; disintegrating agents, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, and the like.
  • the drug delivery unit as a suppository, various carriers well known in the art can be widely used.
  • the carrier are, for example, polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like.
  • the active ingredient of the polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof is mixed with the above various carriers, and the mixture thus obtained is placed in a hard gelatin capsule or soft capsule. in.
  • the polypeptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof may be formulated into a microcapsule, suspended in an aqueous medium to form a suspension, or may be enclosed in a hard capsule or used as an injection.
  • an injection preparation such as a solution, an emulsion, a lyophilized powder injection and a suspension
  • all diluents conventionally used in the art for example, water, ethanol, polyethylene glycol, 1, 3 may be used.
  • - propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, and the like in order to prepare an isotonic injection, an appropriate amount of sodium chloride, glucose or glycerin may be added to the preparation for injection, and a conventional solubilizer, a buffer, a pH adjuster or the like may be added.
  • coloring agents may also be added to the pharmaceutical preparation.
  • coloring agents may also be added to the pharmaceutical preparation.
  • flavoring agents may also be added to the pharmaceutical preparation.
  • sweeteners may also be added to the pharmaceutical preparation.
  • the dose of the antimicrobial peptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention depends on many factors such as the nature and severity of the disease to be prevented or treated, the sex of the patient or animal, Age, body weight and individual response, the specific active ingredients used, the route of administration and the number of doses administered.
  • the above dosages may be administered in a single dosage form or divided into several, for example two, three or four dosage forms.
  • composition as used herein is intended to include a product comprising specified amounts of each of the specified ingredients, as well as any product that results, directly or indirectly, from the specified combination of the specified ingredients.
  • each active ingredient in the pharmaceutical compositions of the present invention can be varied so that the amount of active ingredient obtained is effective to the particular patient, and the compositions and modes of administration provide the desired therapeutic response.
  • the dosage level will be selected based on the activity of the particular active ingredient, the route of administration, the severity of the condition being treated, and the condition and past medical history of the patient to be treated. However, it is a practice in the art to dose the active ingredient from a level below that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved.
  • a further aspect of the invention relates to the antibacterial peptide according to any one of the first to fifth aspects of the invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, for the preparation of a therapeutic and/or prophylactic and/or adjuvant treatment of a bacterium (for example, leather Use in drugs for diseases caused by infection by Gram-positive or Gram-negative bacteria, fungi or viruses.
  • a bacterium for example, leather Use in drugs for diseases caused by infection by Gram-positive or Gram-negative bacteria, fungi or viruses.
  • a further aspect of the invention relates to a method of treating and/or preventing and/or adjunctively treating a disease caused by a bacterium, such as a Gram-positive or Gram-negative bacterium, a fungus, a viral infection, the method comprising Step of administering to a subject in need thereof a therapeutically and/or prophylactically and/or adjunctive therapeutically effective amount of the antimicrobial peptide, derivative, or pharmaceutically acceptable salt thereof according to any one of the first to fifth aspects of the present invention .
  • a bacterium such as a Gram-positive or Gram-negative bacterium, a fungus, a viral infection
  • the therapeutically and/or prophylactically effective amount of the antimicrobial peptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, when used in the above therapeutic and/or prophylactic or adjunctive treatment, may be applied in pure form or as a pharmaceutically acceptable ester. Or a prodrug form (in the presence of these forms).
  • the pharmaceutical composition containing the antimicrobial peptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients may be administered. It will be appreciated, however, that the total daily usage of the antimicrobial peptides, derivatives thereof, or pharmaceutically acceptable salts thereof, or pharmaceutical compositions of the present invention, will be determined by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend on a number of factors, including the disorder being treated and the severity of the disorder; the activity of the particular active ingredient employed; the particular composition employed. The age, weight, general health, sex and diet of the patient; the time of administration, the route of administration and the rate of excretion of the particular active ingredient employed; the duration of treatment; in combination with or in combination with the particular active ingredient employed Drugs; and similar factors well known in the medical field. For example, it is the practice in the art that the dosage of the active ingredient be started from a level lower than that required to achieve the desired therapeutic effect, gradually increasing the dosage until the desired effect is achieved.
  • the antimicrobial peptides, derivatives thereof, or pharmaceutically acceptable salts thereof of the present invention may be used in mammals, particularly humans, at a dose of from 0.001 to 1000 mg/kg body weight per day, for example from 0.01 to 100 mg/kg body weight. /day, for example between 0.01-10 mg/kg body weight/day.
  • the antimicrobial peptide of the present invention, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of the present invention can effectively prevent and/or treat and/or adjuvant the treatment of various diseases or conditions described herein.
  • a further aspect of the invention relates to a method of inhibiting a bacterial infection in vivo or in vitro, the method comprising using an effective amount of the antibacterial agent, a derivative thereof, or the like thereof according to any one of the first to fifth aspects of the invention
  • the step of medicinal salts is not limited to any one of the first to fifth aspects of the invention.
  • the invention also relates to a recombinant vector comprising the nucleic acid molecule of any of the invention.
  • the vector is, for example, a prokaryotic expression vector or a eukaryotic expression vector.
  • the invention also relates to a recombinant cell comprising the recombinant cell of any of the invention.
  • the cells are, for example, prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., yeast cells, insect cells, mammalian cells).
  • prokaryotic cells e.g., E. coli
  • eukaryotic cells e.g., yeast cells, insect cells, mammalian cells.
  • antibacterial peptide means a polypeptide having antibacterial, antifungal and/or antiviral activity.
  • polypeptide has the general meaning well-known to those skilled in the art, for example, it is usually 10-100 amino acids in length, and also includes derivatives, modifications and the like of the polypeptide.
  • the term "effective amount” includes a dose that can achieve treatment, prevention, alleviation and/or alleviation of the disease or condition of the present invention in a subject.
  • the term "subject” may refer to a patient or other pharmaceutical composition according to any one of the present invention, which comprises the antimicrobial peptide, a derivative thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of the present invention.
  • An animal particularly a mammal, such as a human, a dog, a monkey, a cow, a horse, or the like, for treating, preventing, ameliorating, and/or ameliorating the disease or condition of the present invention.
  • disease and/or condition refers to a physical state of the subject that is associated with the disease and/or condition of the present invention.
  • the basic amino acid is selected from the group consisting of Arg, Lys, His, preferably Arg, Lys;
  • the hydrophobic amino acid is selected from the group consisting of Trp, Leu, Ile, Pro, Gly, Val, Ala or Met, preferably Ile, Gly , Trp, Pro, Leu.
  • C 1-20 alkylamido means C 1-20 alkyl-CO-NH
  • the C 1-20 alkyl group means a straight chain having 1 to 20 carbon atoms or A branched monovalent saturated hydrocarbon group, for example, a C 1-18 alkyl group, a C 1-16 alkyl group, a C 1-14 alkyl group, a C 1-12 alkyl group, a C 1-6 alkyl group, for example, a methyl group, a Base, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, pentyl, hexyl, dodecyl, tetradecyl, hexadecyl, octadecyl and the like.
  • X 1 -X8 includes X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 .
  • Y 1 -Y7 includes Y 1 , Y 2 , Y 3 , Y 4 , Y 5 , Y 6 , Y 7 .
  • an amino acid means an L-form amino acid.
  • the antimicrobial peptide of any one of the first aspects does not contain a cysteine, it is a fifteen peptide, and when it contains a cysteine, it is a sixteen or seventeen peptide.
  • each formula is removed Cysteine is not included in the polypeptide sequence, and when the antibacterial peptide sequence contains a cysteine, an appropriate amount of cysteine can be inserted in the polypeptide sequence and corresponding position according to the values of n and r.
  • the preparation method of the antimicrobial peptide provided by the present invention is a known solid phase synthesis method.
  • the prepared antimicrobial peptide was confirmed by mass spectrometry.
  • antibacterial activity of antimicrobial peptides was determined by 96-well plate method (Park IY, Park CB, Kim MS, et al. Parasin I, an antimicrobial pores derived from histone H2A in the catfish, Parasilurus asotus. FEBS Letters, 1998, 437(3) :258-262.)
  • the natural antimicrobial peptide F 2,5,12 W was used as a control.
  • the synthetic antimicrobial peptide of the present invention can maintain the high antibacterial activity of the natural antimicrobial peptide.
  • Plasma stability is an important indicator for evaluating antimicrobial agents.
  • the stability of the antimicrobial peptide in human plasma was examined, and the results showed that the synthetic antibacterial peptide having a free thiol group in the present invention has significantly higher antibacterial stability than the natural antimicrobial peptide.
  • antimicrobial peptides Since the main mode of action of the antimicrobial peptide is to dissolve the cell membrane, extravasation of its cellular contents exerts antibacterial activity. Antimicrobial peptides may also act on higher organisms, including human cells, while sterilizing, so whether antibacterial peptides can cause leakage of red blood cells is a criterion for their toxicity.
  • the synthetic antimicrobial peptides of the present invention are less toxic to human red blood cells.
  • Figures 1 (A) - (L) are mass spectra of antimicrobial peptides. among them:
  • Figures 1(A)-(L) are mass spectra of antimicrobial peptides 1-12, respectively.
  • Figures 2(A)-(C) are plasma stability profiles, where:
  • Figure 2 (A) is an antibacterial stability map of antimicrobial peptide 1-4
  • Figure 2 (B) is an antibacterial stability map of antimicrobial peptide 5-9
  • Figure 2 (C) is an antibacterial stability map of antimicrobial peptide 10-12
  • Figure 3 (A) - (B) is a spectrum of hemolytic activity of antimicrobial peptides, wherein:
  • Figure 3 (A) is a map of hemolytic activity of antimicrobial peptide 1-3
  • Figure 3 (B) is a map of hemolytic activity of antimicrobial peptides 5 and 9.
  • the solid phase synthesis carrier Rink amide resin used in the examples is Tianjin Nankai Synthetic Co., Ltd.; the natural amino acids protected by HBTU, HOBt, DIEA and Fmoc and the unnatural amino acids of type D are products of Shanghai Jill Biochemical Co., Ltd. and Chengdu Nuoxin Technology Co., Ltd. .
  • TFA is a product of Beijing Bomai Technology Co., Ltd.; DMF and DCM are products of Bomaijie; and chromatographic pure acetonitrile is a product of Fisher Company.
  • Other reagents are domestically produced pure products if they are not described.
  • the peptide synthesis steps were as follows: 1 1.14 g of Rink-Amide resin (load: 0.44 mmol/g) was weighed into a silanized polypeptide reactor, and swelled with DCM for 30 min while stirring to disperse uniformly. Wash with DMF, DCM, MeOH, DCM (2 ⁇ 2 min) and drain. Fmoc-protection (5 min, 25 min) was removed by addition of 20% (v/v) piperidine/DMF, the amino group was freed, the resin was washed and dried.
  • the prepared lysate was added to the peptide resin under ice bath conditions, electromagnetically stirred, the resin turned orange-red, and reacted under ice bath for 30 min, then the ice bath was removed, and the reaction was further continued at room temperature for 90 min to complete the reaction.
  • 200 ml of cold diethyl ether was added to the reactor under vigorous stirring, and a white precipitate was precipitated, and stirring was continued for 30 min; the precipitate was filtered off with a G4 sand core filter funnel, washed repeatedly with cold diethyl ether for 3 times, and dried.
  • 50 ml of double distilled water was added to fully dissolve the solid, suction filtration, and the filtrate was lyophilized to obtain 1.13 g of a crude peptide.
  • the crude peptide obtained was purified by medium pressure or high pressure chromatography.
  • the column was a C8 column and the eluent was acetonitrile, water and a small amount of acetic acid.
  • Specific procedure 1 g of crude peptide was weighed, dissolved in 20 ml of water, centrifuged at 3000 rpm for 10 min, and the supernatant was applied for loading.
  • the column was pre-equilibrated with 200 ml of 15% acetonitrile/water/0.1% glacial acetic acid solution. After loading, the mixture was further equilibrated with 200 ml of the same eluent, and the eluent component was detected by HPLC.
  • the antimicrobial peptide 2-9 can be prepared by a method similar to the antimicrobial peptide 1.
  • the sequences of the antimicrobial peptides 1-9 correspond to SEQ ID NOS: 1 to 9, respectively.
  • the preparation of dimer 10-12 was obtained by oxidation with 20% DMSO/H 2 O.
  • the antibacterial peptides 10-12 are the antibacterial peptides obtained by the two SEQ ID NO: 2 sequences by the cysteine at the 9th position to form an interchain disulfide bond, and the two sequences shown by SEQ ID NO: 3 are passed.
  • the antibacterial peptide obtained by the cysteine at the 11th position forms an interchain disulfide bond
  • the antibacterial peptide obtained by the two SEQ ID NO: 4 sequences form an interchain disulfide bond by the cysteine at the 1st position.
  • the specific method is as follows:
  • the pure peptide was determined by MALDI-TOF-MS for its molecular weight (see Table 2).
  • strains used in the following examples were purchased from the China National Institute for the Control of Pharmaceutical and Biological Products.
  • the antibacterial activity of the synthetic antimicrobial peptide was evaluated by a 96-well plate method.
  • the antibacterial activity evaluation steps are as follows: bacterial resuscitation, take 10mL sterile centrifuge tubes, add 5ml nutrient broth, add 2 ⁇ l Escherichia coli (E.coli) glycerol solution, Bacillus subtilis (B) .subtilis) glycerol solution, Staphylococcus aureus (S. aureus) glycerol solution. Incubate at 37 ° C in a constant temperature shaker for 18-24 h at 180 rpm. Nutritional broth was added to a 96-well plate at 100 ⁇ l/well.
  • the antibacterial peptide sample solution was added to the first row of the 96-well plate, 100 ⁇ l/well, and each sample was repeated three wells, with no sample solution as the positive control well. Dilution method is used to dilute one by one to prepare different concentration of sample solution. A 96-fold diluted bacterial suspension was added to a 96-well plate at 10 ⁇ l/well. Incubate at 37 ° C in a constant temperature shaker for 16-18 h at 180 rpm. The clarification was observed and its OD600 value was determined.
  • the result is judged by visual inspection.
  • the turbidity of the solution in the pore indicates that the growth rate of the bacteria is above 50%.
  • the clear and transparent solution in the pore means that the growth rate of the bacteria is less than 50%, and the minimum concentration corresponding to the transparent pore is the MIC of the sample.
  • One of the three wells in each sample was considered to be turbid and the bacterial survival rate was considered to be greater than 50% at this sample concentration.
  • the results of the antibacterial activity are shown in Table 3.
  • the synthesized antimicrobial peptide has high antibacterial activity.
  • Control peptide F 2 5, 12 W (sequence: RWGRW LRKIR RWRPK).
  • the plasma stability evaluation method is as follows:
  • the antibacterial peptide solution was incubated with an equal volume of human plasma for 1 h, 3 h, 6 h, and 18 h, and the antibacterial activity was further evaluated according to the antibacterial activity evaluation method (for example, Bacillus subtilis), and the specific method was evaluated with reference to the antibacterial activity.
  • the results are shown in Table 4 and Figures 2(A)-(C).
  • the stability of the dimer depends on the monomer, that is, the stability of the monomer is good, the stability of the dimer is also good, the stability of the monomer is not good, and the stability of the dimer is not good.
  • the free sulfhydryl group is more active, considering its stability, it is of certain significance to oxidize it to form a dimer.
  • the lytic activity of the synthetic antimicrobial peptide with good antibacterial peptide activity and plasma stability was determined, and the natural antimicrobial peptide F 2, 5, 12 W was used as a control.
  • the blood sample used was taken from normal human blood.
  • the hemolytic activity assay procedure was as follows: human blood (containing anticoagulant heparin) was washed with PBS (NaCl 8 g, KCl 0.2 g, Na 2 HPO 4 1.44 g, KH 2 PO 4 0.24 g, pH 7.4), centrifuged at 1000 rpm for 10 min, discarded. The supernatant is repeated three times. Human red blood cells were diluted to a 10% (v/v) solution by dilution with PBS buffer. Different concentrations of the antimicrobial peptide sample solution were dispensed into a centrifuge tube, 200 ⁇ L/tube, and 50 ⁇ L of the diluted hRBCs were added to the centrifuge tube, three times each.
  • the mixture was shaken at 37 rpm in a 37 ° C incubator and shaken for 1 h. After taking out, it was placed in a low-temperature high-speed centrifuge, centrifuged at 3500 rpm for 5 min at 4 ° C, and the supernatant was aspirated into a 96-well plate, and the OD value at a wavelength of 414 nm was measured using a microplate reader.
  • the red blood cells were suspended in PBS buffer as a blank, and the red blood cells were suspended in 0.1% Triton X-100 for 100% hemolysis. The percentage of hemolysis is calculated by:

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Abstract

本发明属于生物医药领域，提供了一类具有自由巯基或其二聚体的抗菌肽、其药物组合物及用途。

Description

两亲性合成抗菌肽、其药物组合物及其用途 技术领域
本发明属于生物医药领域，涉及一类具有自由巯基或其二聚体的两亲性合成抗菌肽及其衍生物、可药用盐、其药物组合物，及其在制备用于预防、控制或治疗由革兰氏阳性菌、革兰氏阴性菌、甚至耐药菌、真菌、病毒引起的感染性疾病的药物中的用途。
背景技术
对抗生素的耐药性是一种普遍的自然现象，细菌的抗生素耐药率及耐药谱正在以惊人的速度增长和扩展，给感染性疾病的控制带来了潜在的危机。寻找和开发新型的细菌不易对其产生耐药性的抗感染药物成为了全世界医学和药学研究领域的热点课题。
抗菌肽(Antimicrobial Peptides，AMPs)广义上是指生物防御***中产生的、广泛存在于从微生物到动植物，包括细菌、真菌、昆虫、被囊动物、两栖类动物、甲壳类动物、鸟类、鱼类、哺乳动物(包括人类)、植物等所有生物体内，具有抵御外界微生物侵害，清除体内突变细胞的一类带正电荷的两亲性小分子抗菌肽，其分子质量在4KD左右，是生物先天免疫的重要组成成分。
近年来的研究表明，抗菌肽主要通过破坏细胞膜的完整性，使细胞内容物外渗，从而杀死细菌(Zasloff M.Antimicrobial peptides of multicenular organisms.Nature，2002，415(6870)：389-395.)。这使得细菌难以对其产生耐药性。
由于天然生物抗菌肽具有高效广谱的抗菌活性，特别是对多重耐药菌有杀伤作用，而且细菌难以对其产生耐药性，天然生物抗菌肽的开发利用有望成为人类摆脱耐药菌危机的新途径，因此抗菌肽的潜在应用价值受到了国内外学者的广泛关注，是目前学术研究的活跃领域之一。
2005年A.van Dijk等报道，在鸡骨髓细胞里发现了一种新型的抗菌肽Chicken cathelicidin-2(CATH-2)，又名Chicken myeloid  antimicrobial peptide 27(CMAP27)。CATH-2对多种细菌呈现出抗菌活性，同时还具有中和脂多糖(lipopolysaccharide)活性(van Dijk A.，Veldhuizen E.J.，van Asten A.J.，et al.CMAP27，a novel chicken cathelicidin-like antimicrobial protein.Vet Immunol Immunopath0l.2005，106(3)：321-327.)。
Yanjing Xiao等经核磁共振光谱确定了CATH-2的三级结构，其由一个带有脯氨酸的长枢纽区域所连结的两个α-螺旋组成。进一步研究显示，CATH-2的N-端螺旋在抗菌活性中比C-螺旋更重要，这可能是由于其N端高的净阳离子电荷的存在，N端螺旋是两亲性结构，C端螺旋是高度疏水性结构。许多抗菌肽发挥抗菌活性是通过阻断细胞膜的完整性，为了能够接近并进入细胞膜，肽的两亲性在抗菌活性中就显得尤为重要。此外，CATH-2的枢纽区域经证实是肽生物活性所必需的，用亮氨酸替代枢纽区的脯氨酸，其抗菌活性、LPS-中和活性、免疫刺激活性都严重降低。综上所述，N端的前15个氨基酸序列C1-15(即N-端α-螺旋和枢纽区域)是其发挥抗菌活性的重要片段(Xiao Y.，Herrera A.I.，Bommineni Y.R.，et al.The central kink region 0f fowlicidin-2，an α-helical host defense peptide，is critically involved in bacterial killing and endotoxin neutralization.J.Innate Immun.，2009，1(3)：268-280)。
E.M.Molhoek等对C1-15做了进一步结构改造，将C1-15中的苯丙氨酸用疏水性更大的色氨酸替换，不仅能够抗革兰氏阳性菌、革兰氏阴性菌，而且对那些可能被用于生物恐怖袭击的细菌(如炭疽芽孢杆菌Bacillus anthracis、霍乱弧菌Vibrio cholera、耶尔森氏鼠疫杆菌Yersinia pestis等)也有活性。由于色氨酸的加入，多肽对哺乳动物细胞外周血单核细胞(PBMC)的毒性增加。但是，发挥广谱抗菌作用以及LPS-中和作用的有效浓度要比对PBMC产生毒性的浓度要低的多(Molhoek E.M.，van Dij k A.，Veldhuizen E.J.A.，et al.A cathelicidin-2-derived peptide effectively impairs Staphylococcus epidermidis biofilms.Int.J.Antimicrob.Agents，2011，37(5)：476-479.)。
但是抗菌肽与其它肽类药物一样，具有诸如半衰期短、在体内易被 降解等缺点，因此获得活性好、稳定性高的抗菌肽具有十分重要的意义。
发明内容
本发明需要解决的问题是获得抗菌活性高，在血浆中具有高稳定性以及低溶血性副作用的抗菌肽。本发明人发现，在固定正电荷数量和氨基酸种类不变的情况下，改变氨基酸的顺序同时保持带电荷氨基酸均匀分散在序列中，对抗菌活性无明显影响，并基于此合成了一系列新的阳离子抗菌肽。序列中引入半胱氨酸能够使肽的血浆稳定性显著提高但与引入位置有关，在序列中间比两端的稳定性高。本发明人筛选得到了活性高，稳定性好且溶血性低的一系列抗菌肽，由此完成了本发明。
具体地，为了解决天然抗菌肽稳定性差的问题，本发明通过自由巯基的引入或形成二聚体，提供了抗菌活性高、稳定性好的抗菌肽，及其可药用盐，药物组合物以及它们用于预防、治疗或辅助治疗由革兰氏阳性菌、革兰氏阴性菌甚至耐药菌、真菌或病毒引起的感染性疾病中的用途。
本发明第一方面涉及两亲性合成抗菌肽、其衍生物或其可药用盐，其具有如通式(0)所示的结构：
Figure PCTCN2015077337-appb-000001
其中，十五肽由8个碱性氨基酸和7个疏水性氨基酸组成，其中碱性氨基酸均匀地分散在十五肽的序列中，所述均匀地分散是指隔1个、2个或3个疏水性氨基酸即分布有1个或2个碱性氨基酸(即不存在连续4个疏水性氨基酸或连续3个碱性氨基酸)；
C代表***十五肽的Cys，n代表***的Cys数量，n＝0，1或2，r代表***的Cys在式(0)所示的肽链中的位置，Cys可以位于式(0)所示的肽链的N末端(r＝1)、C末端(当n＝1时r＝16，当n＝2时，r＝17)或式(0)所示的肽链中的任意位置(r＝2-15或2-16中的任意数值)；
Z代表N末端基团，例如Z＝NH2或C1-20烷基酰胺基(例如AcNH)；
B代表C末端基团，例如B＝COOH或羧基衍生物，例如B＝CONH2。
在本发明的一个实施方案中，所述十五肽的序列为：KRIGW RWRRW PRLRK(SEQ ID NO：10)。在本发明的另一个实施方案中，所述十五肽的序列为：PKRWG RWLRK IRRWR(SEQ ID NO：11)。
在本发明的一个实施方案中，所述两亲性合成抗菌肽、其衍生物或其可药用盐具有通式(1)所示的氨基酸序列：
Figure PCTCN2015077337-appb-000002
其中，
X1-X8各自独立地选自碱性氨基酸，例如Lys(K)，Arg(R)或His(H)；
Y1-Y7各自独立地选自疏水性氨基酸，例如Leu(L)，Ile(I)，Trp(W)，Pro(P)，Gly(G)，Val(V)，Ala(A)或Met(M)，；
C代表***十五肽的Cys，n代表***的Cys数量，n＝0，1或2；
r代表***的Cys在式(1)所示的肽链中的位置，Cys可以位于式(1)所示的肽链的N末端(r＝1)、C末端(当n＝1时r＝16，当n＝2时，r＝17)或式(1)所示的肽链中的任意位置(r＝2-15或2-16中的任意数值)；
Z代表N末端基团，例如Z＝NH2或C1-20烷基酰胺基(例如AcNH)；
B代表C末端基团，例如B＝COOH或羧基衍生物，例如B＝CONH2。
在本发明的具体实施方案中，r选自1，9或11。
在本发明的具体实施方案中，所述两亲性合成抗菌肽、其衍生物或其可药用盐选自：
KRIGW RWRRW PRLRK-NH2(SEQ ID NO：1)；
KRIGW RWRCR WPRLRK-NH2(SEQ ID NO：2)；
KRIGW RWRRW CPRLRK-NH2(SEQ ID NO：3)；
CKRIGW RWRRW PRLRK-NH2(SEQ ID NO：4)。
在本发明的另一个实施方案中，所述两亲性合成抗菌肽、其衍生物或其可药用盐具有通式(2)所示的氨基酸序列：
Figure PCTCN2015077337-appb-000003
其中，X1-X8各自独立地选自碱性氨基酸，例如Lys(K)，Arg(R)或His(H)；
Y1-Y7各自独立地选自疏水性氨基酸，例如Leu(L)，Ile(I)，Trp(W)，Pro(P)，Gly(G)，Val(V)，Ala(A)或Met(M)；
C代表***十五肽的Cys，n代表***的Cys数量，n＝0，1或2；
r代表***的Cys在式(2)所示的肽链中的位置，Cys可以位于式(2)所示的肽链的N末端(r＝1)、C末端(当n＝1时r＝16，当n＝2时，r＝17)或式(2)所示的肽链中的任意位置(r＝2-15或2-16中的任意数值)；
Z代表N末端基团，例如Z＝NH2或C1-20烷基酰胺基(例如AcNH)；
B代表C末端基团，例如B＝COOH或羧基衍生物，例如B＝CONH2。
在本发明的具体实施方案中，r选自1，9或16。
在本发明的具体实施方案中，所述抗菌肽、其衍生物或其可药用盐选自：
PKRWG RWLRK IRRWR-NH2(SEQ ID NO：5)；
CPKRWG RWLRK IRRWR-NH2(SEQ ID NO：6)；
PKRWG RWLRK IRRWRC-NH2(SEQ ID NO：7)；
CPKRWG RWLRK IRRWRC-NH2(SEQ ID NO：8)；
PKRWG RWLCRK IRRWR-NH2(SEQ ID NO：9)。
本发明第二方面涉及抗菌肽、其衍生物或其可药用盐，其含有本发明第一方面任一项的两亲性合成抗菌肽、其衍生物或其可药用盐，优选地，在所述两亲性合成抗菌肽的C端还连接有一个至数个(例 如10-18个，例如12-16个，例如12-14个)氨基酸，更优选地，所述一个至数个(例如10-18个，例如12-16个，例如12-14个)氨基酸可以形成疏水螺旋。
本发明第一方面的抗菌肽主要是在天然抗菌肽的N端序列基础上改造获得的，本领域技术人员可以根据天然抗菌肽(例如CATH-2)C端序列的特点，在本发明第一方面的抗菌肽的C端连接上具有天然抗菌肽C端序列特点的一个或多个氨基酸或多肽序列，以获得与本发明第一方面的抗菌肽具有相同或相近功能的抗菌肽；例如可以在C端连接上一个至数个(例如10-18个，例如12-16个，例如12-14个)氨基酸(例如疏水氨基酸)，优选地，所述一个至数个(例如10-18个，例如12-16个，例如12-14个)氨基酸可以形成疏水螺旋。
本发明第三方面涉及由两条抗菌肽、其衍生物或其可药用盐通过二硫键形成的二聚体抗菌肽、其衍生物或其可药用盐，所述两条抗菌肽各自独立地选自本发明第一方面任一项所定义的抗菌肽，其中n为1或2。
在本发明的实施方案中，所述本发明第一方面任一项所定义的抗菌肽是指式(0)、式(1)或式(2)所定义的抗菌肽。
在本发明的实施方案中，所述二聚体抗菌肽、其衍生物或其可药用盐，其选自：
两条SEQ ID NO：2所示的序列通过第9位的半胱氨酸形成链间二硫键得到的抗菌肽；
两条SEQ ID NO：3所示的序列通过第11位的半胱氨酸形成链间二硫键得到的抗菌肽；
两条SEQ ID NO：4所示的序列通过第1位的半胱氨酸形成链间二硫键得到的抗菌肽。
本发明第四方面涉及本发明第一至第三方面任一项的抗菌肽、其衍生物或其可药用盐，其中的部分或全部L-氨基酸被替换为相应的 D-氨基酸。
本发明第五方面涉及本发明第一至第三方面任一项的抗菌肽、其衍生物或其可药用盐，其为环合的抗菌肽、其衍生物或其可药用盐，例如为抗菌肽的N末端和C末端之间环合。
本发明涉及一类两亲性合成抗菌肽，其是在对天然抗菌肽的序列结构分析总结基础上设计而成的。在本发明的实施方案中，所述抗菌肽的氨基酸序列如SEQ ID NO：1至SEQ ID NO：12所示，结构见表1.
表1合成抗菌肽序列
Figure PCTCN2015077337-appb-000004
Figure PCTCN2015077337-appb-000005
本发明的抗菌肽是由带有正电荷氨基酸(即碱性氨基酸)，如Arg、Lys、His，及疏水性氨基酸，如Trp、Leu、Ile、Pro、Gly、Val、Ala或Met等，组成的两亲性肽链，且带有正电荷的氨基酸均匀分散在整条肽链中。
在本发明的一些实施方案中，在抗菌肽的不同位置引入带有自由巯基的半胱氨酸可显著提高血浆稳定性，是一种简单有效的提高血浆稳定性的方法。
在本发明的一些实施方案中，带有半胱氨酸的抗菌肽可通过二硫键形成二聚体，其稳定性优于带有自由巯基的抗菌肽。
本发明的另一方面涉及一种药物组合物，其包含本发明第一至第五方面任一项所述的抗菌肽、其衍生物、或其可药用盐；可选地，所述药物组合物还包含药学上可接受的载体或辅料。
通常本发明药物组合物含有0.1-90重量％的本发明任一项所述的抗菌肽、其衍生物、或其可药用盐。药物组合物可根据本领域已知的方法制备。用于此目的时，如果需要，可将本发明的抗菌肽、其衍生物、或其可药用盐与一种或多种固体或液体药物赋形剂和/或辅剂结合，制成可作为人用的适当的施用形式或剂量形式。
本发明的抗菌肽、其衍生物、或其可药用盐或者本发明的药物组合物可以以单位剂量形式给药，给药途径可为肠道或非肠道，如口服、肌肉、皮下、鼻腔、口腔粘膜、皮肤、腹膜或直肠等。给药剂型例如片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、***片、栓剂、冻干粉针剂等。可以是普通制剂、缓释制剂、控释制剂及各种微粒给药***。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸 铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、***胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。为了将给药单元制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、Gelucire、高岭土、滑石粉等；粘合剂如***胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。为了将给药单元制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。为了将给药单元制成胶囊，将有效成分本发明的多肽、其衍生物、或其可药用盐与上述的各种载体混合，并将由此得到的混合物置于硬的明明胶囊或软胶囊中。也可将有效成分本发明的多肽、其衍生物、或其可药用盐制成微囊剂，混悬于水性介质中形成混悬剂，亦可装入硬胶囊中或制成注射剂应用。为了将给药单元制成注射用制剂，如溶液剂、乳剂、冻干粉针剂和混悬剂，可以使用本领域常用的所有稀释剂，例如，水、乙醇、聚乙二醇、1，3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、 矫味剂、甜味剂或其它材料。
本发明的抗菌肽、其衍生物、或其可药用盐或者本发明的药物组合物的给药剂量取决于许多因素，例如所要预防或治疗疾病的性质和严重程度，患者或动物的性别、年龄、体重及个体反应，所用的具体活性成分，给药途径及给药次数等。上述剂量可以单一剂量形式或分成几个，例如二、三或四个剂量形式给药。
本文所用的术语“组合物”意指包括包含指定量的各指定成分的产品，以及直接或间接从指定量的各指定成分的组合产生的任何产品。
可改变本发明药物组合物中各活性成分的实际剂量水平，以便所得的活性成分的量能有效针对具体患者，并且组合物和给药方式得到所需的治疗反应。剂量水平须根据具体活性成分的活性、给药途径、所治疗病况的严重程度以及待治疗患者的病况和既往病史来选定。但是，本领域的做法是，活性成分的剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。
本发明的再一方面涉及本发明第一至第五方面任一项所述的抗菌肽、其衍生物、或其可药用盐在制备治疗和/或预防和/或辅助治疗细菌(例如革兰氏阳性菌或革兰氏阴性菌)、真菌或病毒感染所致的疾病的药物中的用途。
本发明的再一方面涉及一种治疗和/或预防和/或辅助治疗细菌(例如革兰氏阳性菌或革兰氏阴性菌)、真菌、病毒感染所引起的疾病的方法，所述方法包括给与有需要的受试者治疗和/或预防和/或辅助治疗有效量的本发明第一至第五方面任一项所述的抗菌肽、其衍生物、或其可药用盐的步骤。
当用于上述治疗和/或预防或辅助治疗时，治疗和/或预防有效量的本发明的抗菌肽、其衍生物、或其可药用盐可以以纯形式应用，或者以药学可接受的酯或前药形式(在存在这些形式的情况下)应用。或 者，可以以含有本发明的抗菌肽、其衍生物、或其可药用盐与一种或多种药物可接受赋形剂的药物组合物给药。但应认识到，本发明的抗菌肽、其衍生物、或其可药用盐或者本发明的药物组合物的总日用量须由主诊医师在可靠的医学判断范围内作出决定。对于任何具体的患者，具体的治疗有效剂量水平须根据多种因素而定，所述因素包括所治疗的障碍和该障碍的严重程度；所采用的具体活性成分的活性；所采用的具体组合物；患者的年龄、体重、一般健康状况、性别和饮食；所采用的具体活性成分的给药时间、给药途径和***率；治疗持续时间；与所采用的具体活性成分组合使用或同时使用的药物；及医疗领域公知的类似因素。例如，本领域的做法是，活性成分的剂量从低于为得到所需治疗效果而要求的水平开始，逐渐增加剂量，直到得到所需的效果。一般说来，本发明的抗菌肽、其衍生物、或其可药用盐用于哺乳动物特别是人的剂量可以介于0.001-1000mg/kg体重/天，例如介于0.01-100mg/kg体重/天，例如介于0.01-10mg/kg体重/天。
本发明的抗菌肽、其衍生物、或其可药用盐或者本发明的药物组合物可以有效地预防和/或治疗和/或辅助治疗本发明所述的各种疾病或病症。
本发明的再一方面涉及一种在体内或体外抑制细菌感染的方法，所述方法包括使用有效量的本发明第一至第五方面任一项所述的抗菌、其衍生物、或其可药用盐的步骤。
本发明还涉及核酸分子，其编码本发明第一方面任一项的两亲性合成抗菌肽、其衍生物或其可药用盐中所述的多肽(十五肽(n＝0)、十六肽(n＝1)或十七肽(n＝2))。
本发明还涉及重组载体，其含有本发明任一项所述的核酸分子。
在本发明中，所述载体例如为原核表达载体或真核表达载体。
本发明还涉及重组细胞，其含有本发明任一项所述的重组细胞。
在本发明中，所述细胞例如为原核细胞(例如大肠杆菌)或真核细胞(例如酵母细胞、昆虫细胞、哺乳动物细胞)。
在本发明中，术语“抗菌肽”是指具有抗细菌、抗真菌和/或抗病毒活性的多肽。
在本发明中，术语“多肽”具有本领域人员公知的一般含义，例如其长度通常为10-100个氨基酸，并且还包括多肽的衍生物、修饰物等等。
在本发明中，术语“有效量”包括可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。
在本发明中，术语“受试者”可以指患者或者其它接受本发明任一项所述的抗菌肽、其衍生物、或其可药用盐或者本发明任一项所述的药物组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。
在本发明中，术语“疾病和/或病症”是指所述受试者的一种身体状态，该身体状态与本发明所述疾病和/或病症有关。
在本发明中，所述碱性氨基酸选自Arg、Lys、His，优选Arg、Lys；所述疏水性氨基酸选自Trp、Leu、Ile、Pro、Gly、Val、Ala或Met，优选Ile、Gly、Trp、Pro、Leu。
在本发明中，术语“C1-20烷基酰胺基”是指C1-20烷基-CO-NH，所述C1-20烷基是指含有1-20个碳原子的直链或支链一价饱和烃基，例如为C1-18烷基、C1-16烷基、C1-14烷基、C1-12烷基、C1-6烷基，例如为甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、戊基、己基、十二烷基、十四烷基、十六烷基、十八烷基等。
在本发明的实施方案中，当所述抗菌肽具体序列的N端没有特别注明时，N端即为NH2，也就是Z＝NH2；在本发明的实施方案中，当所述抗菌肽具体序列的C端没有特别注明时，C端即为COOH，也就是B＝COOH，当C端序列显示-NH2时，C端即为CONH2，也就是B＝CONH2。
在本发明中，X1-X8包括X1、X2、X3、X4、X5、X6、X7、X8。
在本发明中，Y1-Y7包括Y1、Y2、Y3、Y4、Y5、Y6、Y7。
在本发明中，除特别注明外，氨基酸是指L型氨基酸。
在本发明中，当第一方面任一项的抗菌肽中不含有半胱氨酸时，其为十五肽，当含有半胱氨酸时，其为十六或十七肽。
在本发明中，各通式中除去
Figure PCTCN2015077337-appb-000006
的多肽序列中不包括半胱氨酸，当抗菌肽序列中含有半胱氨酸时，可以根据n和r的数值在多肽序列和相应位置处***适当数量的半胱氨酸。
在本发明中，n＝0时，r不存在；n＝1时，r选自1-16中的任意一个数值；n＝2时，r选自1-17中的任意两个数值。
在本发明中，r＝2-15是指r为选自2、3、4、5、6、7、8、9、10、11、12、13、14或15的任意数值，例如为其中一个或两个数值；r＝2-16是指r为选自2、3、4、5、6、7、8、9、10、11、12、13、14、15或16的任意数值，例如为其中一个或两个数值。
本发明提供的抗菌肽的制备方法是已知的固相合成法。制备的抗菌肽经质谱确认结构。
利用96孔板法检测抗菌肽的抗菌活性(Park I Y，Park C B，Kim M S，et al.Parasin I，an antimicrobial peptide derived from histone H2A in the catfish，Parasilurus asotus.FEBS Letters，1998，437(3)：258-262.)以天然抗菌肽F2，5，12W为对照。结果发现本发明合成抗菌肽能够保持天然抗菌肽的高抗菌活性。
血浆稳定性是评价抗菌药物的重要指标。本发明中考察了抗菌肽在人血浆中稳定性，结果显示本发明中的带有自由巯基的合成抗菌肽其抗菌稳定性明显高于天然抗菌肽。
由于抗菌肽的主要作用方式是溶解细胞膜，使其细胞内容物外渗发挥抗菌活性。抗菌肽在杀菌的同时也有可能作用于高等有机体包括人体细胞，所以把抗菌肽能否使红细胞发生渗漏作为其是否有毒的一个标准。本发明中的合成抗菌肽对人血红细胞毒性较低。
附图说明
图1(A)-(L)是抗菌肽的质谱图。其中：
图1(A)-(L)分别是抗菌肽1-12的质谱图
图2(A)-(C)是血浆稳定性图谱，其中：
图2(A)是抗菌肽1-4抗菌稳定性图谱
图2(B)是抗菌肽5-9抗菌稳定性图谱
图2(C)是抗菌肽10-12抗菌稳定性图谱
图3(A)-(B)是抗菌肽溶血活性图谱，其中：
图3(A)是抗菌肽1-3溶血活性图谱
图3(B)是抗菌肽5和9溶血活性图谱
具体实施方式
在本发明中使用的缩写具有下面的含义：
Ac        乙酰基
Ala(A)    丙氨酸
AMPs      抗菌肽
Arg(R)    精氨酸
Boc       叔丁氧羰基
Cys(C)    半胱氨酸
DCC       二环己基碳二亚胺
DCM       二氯甲烷
DIEA      二异丙基乙胺
DMF       N，N-二甲基甲酰胺
EDT       乙二硫醇
TFA       三氟醋酸
Fmoc      芴甲氧羰基
Gly(G)    甘氨酸
HBTU      苯并三氮唑-N，N，N’，N’四甲基脲六氟磷酸盐
His(H)    组氨酸
HPLC      高效液相色谱
HOBt      1-羟基苯并***
Ile(I)    异亮氨酸
Leu(L)    亮氨酸
Met(M)    甲硫氨酸
Lys(K)    赖氨酸
MALDI-T   基质辅助激光解吸离子化飞行时间质谱
OD        光密度
Pro(P)    脯氨酸
RP-HPLC   反向高效液相色谱
TEA       三乙胺
Trp(W)    色氨酸
Val(V)    缬氨酸
本发明中，所有氨基酸构型除注明为D-型外，均为L-型。
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例所用固相合成载体Rink酰胺树脂为天津南开合成责任有限公司产品；HBTU、HOBt、DIEA及Fmoc保护的天然氨基酸和D型的非天然氨基酸为上海吉尔生化公司及成都诺新技术责任公司产品。TFA为北京博迈科技有限公司产品；DMF、DCM为博迈杰公司产品；色谱纯乙腈为Fisher公司产品。其它试剂如无说明均为国产分析纯产品。
实施例1：抗菌肽1的制备
采用标准的Fmoc固相多肽合成方法。所有多肽序列均按照多肽合成的常规将C-端酰胺化(本领域人员知晓，这些修饰对多肽活性没有根本性影响)。选用Rink Amide树脂，肽类由C-端向N-端延长。缩合剂为HBTU/HOBt/DIEA。脱保护剂为哌啶/DMF溶液。裂解剂为TFA，粗肽水溶解后冻干保存。用中压液相色谱法或HPLC进行 分离纯化，纯肽含量＞95％。基质辅助激光解析飞行时间质谱(MALDI-TOF-MS)确定多肽分子量。
多肽合成步骤如下：①称取1.14g Rink-Amide树脂(载量：0.44mmol/g)置于已硅烷化的多肽反应器内，加入DCM溶胀30min，同时搅拌，使之分散均匀。用DMF、DCM、MeOH、DCM洗涤(2×2min)，抽干。加入20％(v/v)哌啶/DMF脱除Fmoc-保护(5min，25min)，游离出氨基，洗涤树脂，抽干。②用DMF浸润树脂，加入3eqFmoc-AA-OH，3eq HOBt，加入与DMF等量的DCM溶解后，加入3eq DCC，室温搅拌反应4h。洗涤，抽干，茚三酮试剂检测为阴性。若为阳性则重复②过程。③以20％(v/v)哌啶/DMF脱除Fmoc-保护(5min，25min)，洗涤，茚三酮试剂检测为阳性，④用DMF浸润树脂，加入3eq Fmoc-AA-OH，3eq HOBt，加入与DMF等量的DCM溶解后，加入3eq DCC，室温搅拌反应4h。洗涤，抽干，茚三酮试剂检测为阴性。若为阳性则重复④过程，若为阴性则重复③④继续增长肽链。
肽树脂的裂解：肽链合成结束后，将上述合成好的肽树脂称量后放入250ml茄形瓶中，冰浴，电磁搅拌。按1g肽树脂加入10ml的量配置裂解液(三氟醋酸/乙二硫醇/苯甲醚/间甲酚/水(90：2.5：2.5：2.5：2.5，v/v))。TFA需预先冰浴降温30min或者预先放于冰箱中使用。将配制好的裂解液加入到冰浴条件下的肽树脂中，电磁搅拌，树脂变橙红色，冰浴条件下反应30min，然后撤冰浴，室温下再继续反应90min使反应完成。剧烈搅拌下向反应器中加入冷***200ml，析出白色沉淀，继续搅拌30min；用G4的砂芯抽滤漏斗滤出析出物，用冷***反复洗涤3遍，晾干。加入双蒸水50ml使固体充分溶解，抽滤，滤液冻干得粗肽1.13g。
粗肽的纯化：所得粗肽用中压或高压色谱进行纯化。色谱柱为C8柱，洗脱液为乙腈，水及少量乙酸。具体操作步骤：称取粗肽1g，加水20ml溶解，3000转/分钟下离心10min，取上清液上样。色谱柱预先用15％乙腈/水/0.1％冰乙酸溶液200ml平衡，上样后继续用200ml同样洗脱液平衡，高效液相检测洗脱液成分。根据检测结果逐步升高乙腈含量，直至所纯化的多肽峰被洗脱出来。合并同组分洗 脱液，旋转蒸发除去大部分溶剂，冻干得纯肽，HPLC检测含量＞95％。
抗菌肽2-9可以采用类似于抗菌肽1的方法制备。抗菌肽1-9的序列分别对应于SEQ ID NO：1～9。
实施例2：二聚体的制备
二聚体10-12的制备是采用20％DMSO/H2O氧化得到。抗菌肽10-12分别为两条SEQ ID NO：2所示的序列通过第9位的半胱氨酸形成链间二硫键得到的抗菌肽、两条SEQ ID NO：3所示的序列通过第11位的半胱氨酸形成链间二硫键得到的抗菌肽、两条SEQ ID NO：4所示的序列通过第1位的半胱氨酸形成链间二硫键得到的抗菌肽。。具体方法如下：
称取一定量原料肽于50mL茄形瓶中，用5％醋酸水溶液将其溶解，用(NH4)2CO3调节pH至中性(pH 6-7)，再加入20％(v/v)DMSO水溶液，原料肽浓度在3-4mM。HPLC监测反应进程，中压液相色谱分离纯化，浓缩后冻干即得到纯肽。抗菌肽1-12的质谱图见图1(A)-(L)。
纯肽经MALDI-TOF-MS确定其分子量(见表2)。
表2
Figure PCTCN2015077337-appb-000007
实施例3：抗菌活性评价
以下实施例中所使用的菌株购于中国药品生物制品检定所。
采用96孔板法对合成抗菌肽的抗菌活性进行评价。
抗菌活性评价步骤如下：细菌复苏，取10mL灭菌离心管三个，分别加入5ml营养肉汤，依次加入2μl大肠杆菌(Escherichia coli，E.coli)甘油菌液，枯草芽孢杆菌(Bacillus subtilis，B.subtilis)甘油菌液，金黄葡萄球菌(Staphylococcus aureus，S.aureus)甘油菌液。于37℃恒温振荡器中孵育18-24h，180转/分。96孔板中加入营养肉汤，100μl/孔。向96孔板第一排加入抗菌肽样品溶液，100μl/孔，每个样品重复三孔，以不加样品溶液为阳性对照孔。采用倍稀法，逐排稀释，配成不同浓度的样品溶液。向96孔板中加入稀释1000倍后的菌悬液，10μl/孔。于37℃恒温振荡器中孵育16-18h，180转/分。观察其澄清情况并测定其OD600值。
结果判断方法为：目测澄清情况，孔内溶液浑浊即表示细菌生长率在50％以上，孔内溶液澄清透明即表示细菌生长率小于50％，透明孔所对应的最小浓度即该样品的MIC。每个样品三个孔中有一个浑浊即认为在此样品浓度下细菌存活率大于50％。
抗菌活性结果如表3所示。所合成抗菌肽具有较高的抗菌活性。
表3抗菌肽对不同细菌的抗菌活性(MIC)
Figure PCTCN2015077337-appb-000008
Figure PCTCN2015077337-appb-000009
结果显示，所设计合成的抗菌肽具有较高的抗菌活性。对照肽F2，5，12W(序列为：RWGRW LRKIR RWRPK)。
实施例4：血浆稳定性评价
血浆稳定性评价方法如下：
将抗菌肽的溶液与等体积人血浆孵育1h，3h，6h，18h后，依照抗菌活性评价方法再评价其抗菌活性(以枯草芽孢杆菌为例)，具体方法参照抗菌活性评价。结果见表4、图2(A)-(C)。
表4
Figure PCTCN2015077337-appb-000010
结果显示，所设计合成的抗菌肽P-11、P-1血浆稳定性较差；而经过引入带有自由巯基的半胱氨酸修饰后，P-C(9)-11，P-C(11)-11以及P-C(9)-1血浆稳定性得到明显改善；但是将带有自由巯基的半胱氨酸引 入到P-11、P-1的末端，却未能对血浆稳定性产生显著影响。这就说明，带有自由巯基的半胱氨酸在提高此类抗菌肽的血浆稳定性方面有重要贡献，但是其引入位置决定了其能否发挥改善稳定性作用。由10-12血浆稳定性可以看出，二聚体的稳定性取决于单体，即单体稳定性好二聚体稳定性也好，单体稳定性不好二聚体稳定性也不好。但是鉴于自由巯基比较活泼，考虑其稳定性，将其氧化形成二聚体具有一定意义。
实施例5：溶血活性测定
本实施例测定了抗菌肽活性与血浆稳定性都较好的合成抗菌肽溶血活性，并以天然抗菌肽F2，5，12W作为对照。使用的血样取于正常人血。
溶血活性测定步骤如下：人血(含抗凝剂肝素)经PBS(NaCl 8g，KCl 0.2g，Na2HPO41.44g，KH2PO40.24g，pH 7.4)洗涤，1000rpm，离心10min，弃上清，重复操作三次。用PBS缓冲液稀释将人血红细胞稀释至10％(V/V)溶液。将不同浓度的抗菌肽样品溶液分装到离心管中，200μL/管，再向离心管中加入稀释后的hRBCs 50μL，每个重复三次。放入37℃恒温箱中振荡95转/分，振荡1h。取出后置于低温高速离心机中，在4℃下，3500rpm，离心5min，吸取上清液180μL于96孔板中，使用酶标仪检测414nm波长处的OD值。以血红细胞悬浮在PBS缓冲液中为空白，以血红细胞悬浮在0.1％TritonX-100中为100％溶血。溶血百分率由下式计算：
Figure PCTCN2015077337-appb-000011
合成抗菌肽的溶血活性结果见图3(A)和(B).
结果显示，设计合成抗菌肽P-1引入半胱氨酸后溶血活性显著增强；而P-11其溶血活性比较低，虽然引入半胱氨酸后P-C(9)-11和P-C(11)-11两条抗菌肽溶血活性增强，但是其溶血活性与天然抗菌肽相当，仍在可接受范围内。
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (19)

  1. 两亲性合成抗菌肽、其衍生物或其可药用盐，其具有如通式(0)所示的结构：
    Figure PCTCN2015077337-appb-100001
    其中，十五肽由8个碱性氨基酸和7个疏水性氨基酸组成，其中碱性氨基酸均匀地分散在十五肽的序列中，所述均匀地分散是指隔1个、2个或3个疏水性氨基酸即分布有1个或2个碱性氨基酸；
    C代表***十五肽的Cys，n代表***的Cys数量，n＝0，1或2，r代表***的Cys在式(0)所示的肽链中的位置，Cys可以位于式(0)所示的肽链的N末端(r＝1)、C末端(当n＝1时r＝16，当n＝2时，r＝17)或式(0)所示的肽链中的任意位置(r＝2-15或2-16中的任意数值)；
    Z代表N末端基团，例如Z＝NH2或C1-20烷基酰胺基(例如AcNH)；
    B代表C末端基团，例如B＝COOH或羧基衍生物，例如B＝CONH2。
  2. 权利要求1的两亲性合成抗菌肽、其衍生物或其可药用盐，其具有如通式(1)所示的氨基酸序列：
    Figure PCTCN2015077337-appb-100002
    其中X1-X8各自独立地选自碱性氨基酸，例如Lys(K)，Arg(R)或His(H)；
    Y1-Y7各自独立地选自疏水性氨基酸，例如Leu(L)，Ile(I)，Trp(W)，Pro(P)，Gly(G)，Val(V)，Ala(A)或Met(M)；
    C代表***十五肽的Cys，n代表***的Cys数量，n＝0，1或2；
    r代表***的Cys在式(1)所示的肽链中的位置，Cys可以位于式(1)所示的肽链的N末端(r＝1)、C末端(当n＝1时r＝16，当n＝2 时，r＝17)或式(1)所示的肽链中的任意位置(r＝2-15或2-16中的任意数值)；
    Z代表N末端基团，例如Z＝NH2或C1-20烷基酰胺基(例如AcNH)；
    B代表C末端基团，例如B＝COOH或羧基衍生物，例如B＝CONH2。
  3. 权利要求1或2的两亲性合成抗菌肽、其衍生物或其可药用盐，其中r选自1，9或11。
  4. 权利要求2的两亲性合成抗菌肽、其衍生物或其可药用盐，其中选自：
    KRIGW RWRRW PRLRK-NH2 (SEQ ID NO：1)；
    KRIGW RWRCR WPRLRK-NH2(SEQ ID NO：2)；
    KRIGW RWRRW CPRLRK-NH2 (SEQ ID NO：3)；
    CKRIGW RWRRW PRLRK-NH2 (SEQ ID NO：4)。
  5. 权利要求1的两亲性合成抗菌肽、其衍生物或其可药用盐，其具有如通式(2)所示的氨基酸序列：
    Figure PCTCN2015077337-appb-100003
    其中X1-X8各自独立地选自碱性氨基酸，例如Lys(K)，Arg(R)或His(H)；
    Y1-Y7各自独立地选自疏水性氨基酸，例如Leu(L)，Ile(I)，Trp(W)，Pro(P)，Gly(G)，Val(V)，Ala(A)或Met(M)；
    C代表***十五肽的Cys，n代表***的Cys数量，n＝0，1或2；
    r代表***的Cys在式(2)所示的肽链中的位置，Cys可以位于式(2)所示的肽链的N末端(r＝1)、C末端(当n＝1时r＝16，当n＝2时，r＝17)或式(2)所示的肽链中的任意位置(r＝2-15或2-16中的任意数值)；
    Z代表N末端基团，例如Z＝NH2或C1-20烷基酰胺基(例如AcNH)；
    B代表C末端基团，例如B＝COOH或羧基衍生物，例如B＝CONH2。
  6. 权利要求1或5的两亲性合成抗菌肽、其衍生物或其可药用盐，其中r选自1，9或16。
  7. 权利要求5的两亲性合成抗菌肽、其衍生物或其可药用盐，其选自：
    PKRWG RWLRK IRRWR-NH2(SEQ ID NO：5)；
    CPKRWG RWLRK IRRWR-NH2(SEQ ID NO：6)；
    PKRWG RWLRK IRRWRC-NH2(SEQ ID NO：7)；
    CPKRWG RWLRK IRRWRC-NH2(SEQ ID NO：8)；
    PKRWG RWLCRK IRRWR-NH2(SEQ ID NO：9)。
  8. 抗菌肽、其衍生物或其可药用盐，其含有权利要求1-7任一项的两亲性合成抗菌肽、其衍生物或其可药用盐，优选地，在所述两亲性合成抗菌肽的C端还连接有一个至数个(例如10-18个，例如12-16个，例如12-14个)氨基酸，更优选地，所述一个至数个(例如10-18个，例如12-16个，例如12-14个)氨基酸可以形成疏水螺旋。
  9. 由两条抗菌肽、其衍生物或其可药用盐通过二硫键形成的二聚体抗菌肽、其衍生物或其可药用盐，所述两条抗菌肽各自独立地选自权利要求1-8所定义的抗菌肽，其中n为1或2。
  10. 权利要求9的二聚体抗菌肽、其衍生物或其可药用盐，其选自：
    两条SEQ ID NO：2所示的序列通过第9位的半胱氨酸形成链间二硫键得到的抗菌肽；
    两条SEQ ID NO：3所示的序列通过第11位的半胱氨酸形成链间 二硫键得到的抗菌肽；
    两条SEQ ID NO：4所示的序列通过第1位的半胱氨酸形成链间二硫键得到的抗菌肽。
  11. 权利要求1-10任一项的抗菌肽、其衍生物或其可药用盐，其中的部分或全部L-氨基酸被替换为D-氨基酸。
  12. 权利要求1-10任一项的抗菌肽、其衍生物或其可药用盐，其为环合的抗菌肽、其衍生物或其可药用盐。
  13. 一种药物组合物，其包含权利要求1至12中任一项所述的的抗菌肽、其衍生物或其可药用盐；可选地，所述药物组合物还包含药学上可接受的载体或辅料。
  14. 权利要求1至12中任一项所述的抗菌肽、其衍生物或其可药用盐在制备治疗和/或预防和/或辅助治疗细菌(例如革兰氏阳性菌或革兰氏阴性菌)、真菌或病毒感染所引起的疾病的药物中的用途。
  15. 治疗和/或预防和/或辅助治疗细菌(例如革兰氏阳性菌或革兰氏阴性菌)、真菌或病毒感染所引起的疾病的方法，所述方法包括给予有需要的受试者治疗和/或预防和/或辅助治疗有效量的权利要求1至12中任一项的抗菌肽、其衍生物或其可药用盐的步骤。
  16. 一种在体内或体外抑制细菌感染的方法，所述方法包括使用有效量的权利要求1至12中任一项的抗菌肽、其衍生物或其可药用盐的步骤。
  17. 核酸分子，其编码权利要求1-7任一项的两亲性合成抗菌肽、 其衍生物或其可药用盐中所述的多肽(十五肽、十六肽或十七肽)。
  18. 重组载体，其含有权利要求17的核酸分子。
  19. 重组细胞，其含有权利要求18的重组载体。
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