WO2015161820A1 - Peptide antimicrobien synthétique amphiphile, composition pharmaceutique et leur utilisation - Google Patents

Peptide antimicrobien synthétique amphiphile, composition pharmaceutique et leur utilisation Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
derivative
pharmaceutically acceptable
acceptable salt
peptide
antimicrobial peptide
Prior art date
Application number
PCT/CN2015/077337
Other languages
English (en)
Chinese (zh)
Inventor
刘克良
邹存彬
孟庆斌
刘兴东
王晨宏
Original Assignee
中国人民解放军军事医学科学院毒物药物研究所
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
Application filed by 中国人民解放军军事医学科学院毒物药物研究所 filed Critical 中国人民解放军军事医学科学院毒物药物研究所
Priority to CN201580020639.9A priority Critical patent/CN106232616B/zh
Publication of WO2015161820A1 publication Critical patent/WO2015161820A1/fr

Links

Images

Classifications

    • 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:

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Physics & Mathematics (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention appartient au domaine de la biomédecine et concerne une classe de peptides antimicrobiens possédant des triols libres ou leurs dimères, des compositions pharmaceutiques et leur utilisation.
PCT/CN2015/077337 2014-04-24 2015-04-24 Peptide antimicrobien synthétique amphiphile, composition pharmaceutique et leur utilisation WO2015161820A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201580020639.9A CN106232616B (zh) 2014-04-24 2015-04-24 两亲性合成抗菌肽、其药物组合物及其用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410166654 2014-04-24
CN201410166654.1 2014-04-24

Publications (1)

Publication Number Publication Date
WO2015161820A1 true WO2015161820A1 (fr) 2015-10-29

Family

ID=54331770

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/077337 WO2015161820A1 (fr) 2014-04-24 2015-04-24 Peptide antimicrobien synthétique amphiphile, composition pharmaceutique et leur utilisation

Country Status (2)

Country Link
CN (1) CN106232616B (fr)
WO (1) WO2015161820A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018197445A1 (fr) * 2017-04-25 2018-11-01 Universiteit Utrecht Holding B.V. Peptides antimicrobiens et leur utilisation
CN110128544A (zh) * 2019-04-03 2019-08-16 中国农业大学 一种具有免疫调节和抗炎功能的杂合肽及其制备方法与应用
WO2021003413A3 (fr) * 2019-07-02 2021-03-04 Fred Hutchinson Cancer Research Center Protéines de répétition en tandem circulaires à auto-assemblage présentant une stabilité accrue
US11274126B2 (en) 2016-03-21 2022-03-15 University Of Rhode Island Board Of Trustees pH-sensitive cyclic peptides
CN114989254A (zh) * 2022-06-17 2022-09-02 中山大学 一种多肽及其设计方法和在制备抑制具核梭杆菌产品或预防结直肠癌药物中的应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113121649B (zh) * 2019-12-26 2022-10-04 李瑛� 一种新型两亲性蛋白、其制备方法及用途
CN114805821B (zh) * 2022-04-28 2023-02-03 上海摩漾生物科技有限公司 一种基于纤维素的可降解抗菌肽及其制备方法
CN116332777A (zh) * 2023-02-20 2023-06-27 华中科技大学 二芳基苯甲胺类化合物、制备及作为载体合成多肽的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528248A (zh) * 2006-07-10 2009-09-09 奥地利科学院 抗微生物肽
CN101801995A (zh) * 2007-07-23 2010-08-11 Amp医疗有限两合公司 抗菌肽

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101040848B (zh) * 2007-04-19 2010-05-19 上海交通大学 两亲性聚合物载药纳米粒子的制备方法
CN101570569B (zh) * 2008-04-29 2012-01-11 昆山博青生物科技有限公司 合成抗菌肽、其制备方法及应用
US8258250B2 (en) * 2009-10-07 2012-09-04 Johnson & Johnson Consumer Companies, Inc. Compositions comprising superhydrophilic amphiphilic copolymers and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101528248A (zh) * 2006-07-10 2009-09-09 奥地利科学院 抗微生物肽
CN101801995A (zh) * 2007-07-23 2010-08-11 Amp医疗有限两合公司 抗菌肽

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MOLHOEK EM ET AL.: "Chicken cathelicidin-2-derived peptides with enhanced immunomodulatory and antibacterial activities against biological warfare agents", INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS, vol. 36, no. 3, 30 September 2010 (2010-09-30), XP027147675 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11274126B2 (en) 2016-03-21 2022-03-15 University Of Rhode Island Board Of Trustees pH-sensitive cyclic peptides
WO2018197445A1 (fr) * 2017-04-25 2018-11-01 Universiteit Utrecht Holding B.V. Peptides antimicrobiens et leur utilisation
US11130781B2 (en) 2017-04-25 2021-09-28 Universiteit Utrecht Holding B.V. Antimicrobial peptides and their use
CN110128544A (zh) * 2019-04-03 2019-08-16 中国农业大学 一种具有免疫调节和抗炎功能的杂合肽及其制备方法与应用
CN110128544B (zh) * 2019-04-03 2020-12-01 中国农业大学 一种具有免疫调节和抗炎功能的杂合肽及其制备方法与应用
WO2021003413A3 (fr) * 2019-07-02 2021-03-04 Fred Hutchinson Cancer Research Center Protéines de répétition en tandem circulaires à auto-assemblage présentant une stabilité accrue
CN114989254A (zh) * 2022-06-17 2022-09-02 中山大学 一种多肽及其设计方法和在制备抑制具核梭杆菌产品或预防结直肠癌药物中的应用
CN114989254B (zh) * 2022-06-17 2023-11-03 中山大学 一种多肽及其设计方法和在制备抑制具核梭杆菌产品或预防结直肠癌药物中的应用

Also Published As

Publication number Publication date
CN106232616A (zh) 2016-12-14
CN106232616B (zh) 2019-10-25

Similar Documents

Publication Publication Date Title
WO2015161820A1 (fr) Peptide antimicrobien synthétique amphiphile, composition pharmaceutique et leur utilisation
ES2532392T3 (es) Péptidos antimicrobianos
RU2472805C2 (ru) Антибиотические пептиды
US11186619B2 (en) Antimicrobial peptides based on CMAP27
US7745390B2 (en) Antimicrobial peptides
KR102555177B1 (ko) 항균 펩타이드 및 이의 사용 방법
KR101341210B1 (ko) 신규한 항균 펩타이드 및 이의 용도
CN117586352B (zh) 一种基于宽体金线蛭唾液腺的抗菌多肽aph220及其应用
US7262163B2 (en) Short amphipathic peptides with activity against bacteria and intracellular pathogens
US8076284B2 (en) Analogues of antimicrobial and anticancer peptide synthesized and produced from Gaegurin 5
CN102939301A (zh) 作为抗菌剂使用的肽化合物
CN106986927A (zh) 修饰肽及其用于治疗自体免疫疾病的用途
US10905735B2 (en) Chemosynthetic cyclo-hepta modified peptide capable of inhibiting toxin of Staphylococcus aureus and use thereof
WO2019085926A1 (fr) Analogue de polymyxine et son procédé de préparation
KR20150038612A (ko) 백본-고리형 펩타이드와의 조합물
TWI577697B (zh) 耐高鹽及抗蛋白之抗菌胜肽及其製造方法
JP7046990B2 (ja) 医薬的特性が改善されたプロドラッグペプチド
CN107216369A (zh) 一种鱼精蛋白类似活性肽及其制备方法与应用
JP2001526537A (ja) 敗血症の治療法
CN114989246B (zh) Fk3多肽类似物及其应用
CN113698460B (zh) 一种大肠杆菌lipid A结合基元PCK及其制备方法与应用
JP2001510164A (ja) 動物に対する毒性を減じた生物活性ペプチドおよび同ペプチドを調製する方法。
EP4396196A1 (fr) Peptides ayant des activités antimicrobiennes
AU2022340625A1 (en) Peptides with antimicrobial activities
CN118005740A (zh) 一种高稳定高活性的抗菌多肽aph318及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15783594

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15783594

Country of ref document: EP

Kind code of ref document: A1