WO2022155783A1 - 一种抗菌肽液体组合物及其制剂 - Google Patents

一种抗菌肽液体组合物及其制剂 Download PDF

Info

Publication number
WO2022155783A1
WO2022155783A1 PCT/CN2021/072665 CN2021072665W WO2022155783A1 WO 2022155783 A1 WO2022155783 A1 WO 2022155783A1 CN 2021072665 W CN2021072665 W CN 2021072665W WO 2022155783 A1 WO2022155783 A1 WO 2022155783A1
Authority
WO
WIPO (PCT)
Prior art keywords
infection
composition
buffer system
antimicrobial peptide
composition according
Prior art date
Application number
PCT/CN2021/072665
Other languages
English (en)
French (fr)
Inventor
陈育新
陈明侠
陈文学
黄晓雪
Original Assignee
江苏普莱医药生物技术有限公司
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 江苏普莱医药生物技术有限公司 filed Critical 江苏普莱医药生物技术有限公司
Priority to JP2023543109A priority Critical patent/JP2024503875A/ja
Priority to US18/272,989 priority patent/US20240075096A1/en
Priority to EP21920188.6A priority patent/EP4282400A1/en
Priority to PCT/CN2021/072665 priority patent/WO2022155783A1/zh
Publication of WO2022155783A1 publication Critical patent/WO2022155783A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/02—Local antiseptics

Definitions

  • the present invention relates to the field of biotechnology, in particular, to an antimicrobial peptide liquid composition and a preparation thereof.
  • Antibacterial drugs mainly include antibiotics and synthetic antibacterial drugs. Since the 1920s and 1930s, the discovery and application of antibiotics has saved countless lives. Antibiotics are substances produced by bacteria or other microorganisms in the process of life, which can inhibit or kill bacteria, spirochetes, mycoplasma, chlamydia and other pathogenic microorganisms. Listed antibiotics mainly include ⁇ -lactams, macrolides, polysaccharides (vancomycin, teicoplanin), aminoglycosides, tetracyclines, chloramphenicol, peptide lactones (up to tropomycin), etc.
  • antibiotic substitutes have become a hot topic in the field of biomedicine.
  • antimicrobial peptides have become the "stars of hope” for antibiotic alternatives because of their broad-spectrum and efficient bactericidal activity against bacteria, and they are not easy to develop drug resistance.
  • the cell membrane is the main target of antimicrobial peptides, and the aggregation of antimicrobial peptide molecules on the cell membrane can lead to an increase in the permeability of the cell membrane and cause the cell membrane to lose its barrier function.
  • the development of microbial resistance to antimicrobial peptides requires substantial changes in the lipid composition of microbial cell membranes.
  • Antibacterial peptides are small molecular polypeptides with biological activity that are induced in vivo, with a molecular weight of about 2000 to 7000 and composed of 20 to 60 amino acid residues. According to the source, antimicrobial peptides can be divided into: plant antimicrobial peptides, animal antimicrobial peptides, bacterial antimicrobial peptides (also known as bacteriocins, including cationic peptides and neutral peptides, secreted by both Gram-positive and Gram-negative bacteria) and Human-derived antimicrobial peptides, etc.
  • antimicrobial peptides have broad-spectrum antibacterial effects, which can regulate wound inflammation and promote angiogenesis and epithelial tissue regeneration at wound sites.
  • antimicrobial peptides are one of the key signaling molecules of the endogenous immune system. They interact with various cells and various growth factors to achieve a balanced state and achieve wound repair.
  • CN101111256A discloses the amino acid sequences Ac-Lys-Trp-Lys-Ser-Phe-Leu-Lys-Thr-Phe-Lys-Ser-Ala-Ala-Lys-Thr-Val-Leu of antimicrobial peptides NA L and D-NA L -His-Thr-Ala-Leu-Lys-Ala-Ile-Ser-Ser- NH2 .
  • the antimicrobial peptides have antimicrobial activity, desired levels of hemolytic activity, and broad-spectrum therapeutic coefficients against Gram-positive and Gram-negative bacteria and other microorganisms with cellular or structural components of lipid bilayer membranes.
  • the main problem of protein-based drug formulations is poor drug stability.
  • excipients such as polyols (such as sorbitol, mannitol, glycerol and propylene glycol, etc.), sugars (such as lactose, gum, dextrin, glucose and trehalose, etc.), amino acids (such as glycine, serine, glutamic acid and lysine, etc.), salts (such as citrate, acetate and phosphate, etc.), surfactants, etc., thereby increasing its stability.
  • the freeze-dried preparation has the defects of complicated prescription and complex process, so it will be of great significance if a stable protein liquid preparation can be provided.
  • the present invention aims to provide an antibacterial peptide liquid composition, in order to solve the increasingly serious drug resistance problem of bacteria and the pain caused by stubborn infection to the majority of patients.
  • the antimicrobial peptide liquid composition provided by the present invention includes an antimicrobial peptide, at least one stabilizer and a buffer system; the mass concentration of the antimicrobial peptide in the composition is 0.1 ⁇ to 10 ⁇ ; the mass concentration of the stabilizer is 0.5% to 5%, and the buffer system is a phosphate buffer system or an acetate buffer system.
  • the antimicrobial peptide is a polypeptide of the following general formula:
  • I is selected from any of the following amino acid residues: L-leucine, D-leucine, L-valine, D-valine, L-alanine, D-alanine, glycine, L- Serine, D-serine, L-lysine and D-lysine (L, A, S, V and K in L- and D-optical isomeric forms, and G);
  • amino acid residue II is selected from any of the following amino acid residues: selected from any of the following amino acid residues: L-leucine, D-leucine, L-valine, D-valine, L-alanine, D- - Alanine, Glycine, L-Serine, D-Serine, L-Lysine and D-Lysine (L, A, S, V and K in L- and D-optical isomeric forms, and G) ;
  • Section A is shown in SEQ ID No: 1, and its sequence is: KWKSFLKTFK;
  • Section B is shown in SEQ ID No: 2, and its sequence is: KTVLHTALKAISS;
  • A represents an alanine residue
  • the direction is from N-terminal to C-terminal.
  • the antimicrobial peptides are NAL and D-NA L .
  • the NA L has the sequence of SEQ ID No: 3, and its amino acid sequence is: KWKSFLKTFKSAAKTVLHTALKAISS;
  • the D-NA L has the sequence of SEQ ID No: 4, and its amino acid sequence is: KWKSFLKTFKSAAKTVLHTALKAISS (all amino acids are D-configuration except A at position 13 is L configuration).
  • the capital letter D- indicates that the antimicrobial peptides are all composed of D-amino acids except for the specified site (for example: D-NA L means that the antimicrobial peptides contain non-polar amino acids except for the specified site). All but the L-alanine substitution in the center of the surface consists of D-amino acids, and the non-polar surface is represented by a capital letter N).
  • the concentration of the antimicrobial peptide is 0.5 ⁇ 6 ⁇ , more preferably 1 ⁇ 4 ⁇ , further preferably 1 ⁇ 2 ⁇ , specifically 0.2 ⁇ , 0.5 ⁇ , 1 ⁇ , 2 ⁇ .
  • the buffer system is a buffer system of disodium hydrogen phosphate-citric acid, wherein the ion concentration of the buffer system in the composition is 0.01M-0.1M, preferably the ion concentration is 0.01M-0.02M, more The preferred ion concentration is 0.015M.
  • the pH value of the composition is 3.5-5.5, specifically 3.5, 4.5 or 5.5.
  • the stabilizer in the present invention can be selected from at least one of the following: polyols (such as sorbitol, mannitol, glycerol, and propylene glycol, etc.), sugars (such as lactose, gum, dextrin, glucose, trehalose, etc.) , amino acids (such as glycine, serine, glutamic acid and lysine, etc.), salts (such as citrate, acetate and phosphate, etc.), surfactants, etc., not only stabilize antimicrobial peptides, but also maintain Antibacterial ability of antimicrobial peptides.
  • polyols such as sorbitol, mannitol, glycerol, and propylene glycol, etc.
  • sugars such as lactose, gum, dextrin, glucose, trehalose, etc.
  • amino acids such as glycine, serine, glutamic acid and lysine, etc.
  • salts such as
  • the stabilizer is mannitol, wherein the concentration of mannitol in the liquid antimicrobial peptide composition is 0.5% to 5%, preferably the concentration of mannitol is 1% to 2%, more preferably the concentration of mannitol is 1%, specifically 0.5%, 1%, 1.25%, 1.5% or 5%.
  • the stability test shows that the antimicrobial peptide composition provided by the present invention is stable for at least 24 months (4°C condition);
  • the antimicrobial peptide composition provided by the present invention is mainly suitable for treating skin infectious diseases. Compared with gels and creams, sprays can effectively avoid direct contact with skin wounds, and act directly on the lesions, reducing systemic toxic reactions, and have good stability and easy absorption, which improves the safety and efficacy of clinical medication. Compliance, more suitable for clinical needs.
  • composition preparation form provided by the present invention is a liquid preparation, and its dosage form can be a spray, a solution, a gel, an emulsion, a sol, a drop, a syrup, a suspension, an oral liquid, a lotion or a wipe agent, etc., preferably a spray.
  • the present invention also provides a preparation method of the above-mentioned antimicrobial peptide liquid composition.
  • the preparation method of the external composition of the present invention is simple, and the raw and auxiliary materials required for each composition can be selected and prepared according to the conventional preparation method of each dosage form disclosed in the prior art.
  • the preparation method of the antimicrobial peptide liquid composition provided by the present invention comprises the following steps:
  • step 2) Weigh the antibacterial peptide and add it to the solution of step 1), stir to dissolve, and make up to the full amount with water for injection.
  • the method further includes the step of filtering the above-mentioned antimicrobial peptide liquid composition with a 0.22 micron microporous membrane.
  • a specific preparation method of a spray formulation is disclosed. After the antimicrobial peptide liquid composition is prepared by the above method, 5ml/bottle is filled into a spray bottle, and the spray pump is tightened to obtain the antimicrobial peptide. spray.
  • the antibacterial peptide used in the present invention is a brand-new broad-spectrum anti-infective drug that destroys the bacterial cell membrane to achieve the effect of killing bacteria. It is a very effective way and method to solve the problem of bacterial resistance.
  • the main problem of protein-based drug formulations is poor drug stability.
  • excipients such as polyols (such as sorbitol, mannitol, glycerol and propylene glycol, etc.), sugars (such as lactose, gum, dextrin, glucose and trehalose, etc.), amino acids (such as glycine, serine, glutamic acid and lysine, etc.), salts (such as citrate, acetate and phosphate, etc.), surfactants, etc., thereby increasing its stability.
  • the stability of the protein-based liquid composition is more difficult to guarantee than the stability of the lyophilized powder.
  • the present invention focuses on the stability of the antimicrobial peptide liquid composition.
  • the research team of the present invention found that compared with other stabilizers (such as glycerol, lactose, glycine), mannitol can significantly increase the stability of antimicrobial peptides.
  • stabilizers such as glycerol, lactose, glycine
  • the acidic pH buffer system can make the antimicrobial peptide more stable, and the buffer system can consider a phosphate buffer system (such as disodium hydrogen phosphate + citric acid, or disodium hydrogen phosphate + sodium dihydrogen phosphate, etc.) , acetate buffer system (such as sodium acetate + phosphoric acid, or sodium acetate + acetic acid, etc.), citrate buffer system (such as sodium citrate + acetic acid, or sodium citrate + citric acid, etc.), combined with Stability and bacteriostatic test of antimicrobial peptides, preferably phosphate buffer system and acetate buffer system (including disodium hydrogen phosphate, citric acid, sodium acetate, glacial acetic acid), more preferably disodium hydrogen phosphate and citric acid composition buffer system.
  • a phosphate buffer system such as disodium hydrogen phosphate + citric acid, or disodium hydrogen phosphate + sodium dihydrogen phosphate, etc.
  • acetate buffer system such
  • the ion concentration of the buffer system also has a certain influence on the stability of the antimicrobial peptide.
  • the inventors have found that the ion concentration of the buffer system is 0.01M-0.1M, preferably 0.01M-0.02M, More preferably, the ion concentration is 0.015M.
  • the antibacterial peptide spray of the present invention exhibits good bioavailability and safety in use; can reduce irritation to the skin and/or wound surface; has high stability, and can improve the microenvironment inhibition of the wound surface in the long-term coverage of the wound surface.
  • the antimicrobial peptide composition of the present invention can be used to prepare a broad-spectrum anti-infective pharmaceutical preparation for topical use; it can also be used to treat local infection, or to treat diseases caused by local infection.
  • Suitable for pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), erythromycin-resistant and susceptible strains of Streptococcus pyogenes, Pseudomonas aeruginosa IPM- R and IPM-S strains, especially various primary skin infections caused by drug-resistant bacteria, as well as secondary skin infections such as eczema co-infection, ulcer co-infection, including diabetic foot, burn wound infection, bedsore infection and other stubborn infections Sexual diseases, has a wide range of application prospects.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MSSA methicillin-sensitive Staphylococcus aureus
  • the present invention also protects a broad-spectrum anti-infective product for topical external use, the active ingredient of which includes the antimicrobial peptide composition provided by the present invention.
  • the product may be a drug or a pharmaceutical formulation.
  • the present invention also provides a method for local anti-infection, comprising the steps of: administering the antimicrobial peptide composition of the present invention to a recipient animal to fight local infection.
  • the present invention also provides a method for treating diseases caused by local infection, comprising the steps of: administering the antimicrobial peptide composition of the present invention to a recipient animal to treat diseases caused by local infection.
  • the animals can be mammals, such as humans; the animals can also be other animals other than mammals, such as mice, which are infected with the infection.
  • the above-mentioned infections are caused by at least one of the following pathogenic bacteria: Methicillin-resistant Staphylococcus aureus, Methicillin-sensitive Staphylococcus aureus, Streptococcus pyogenes erythromycin-resistant strains, Streptococcus pyogenes erythromycin Susceptible strains, Pseudomonas aeruginosa IPM-R and IPM-S strains; the above-mentioned infections include at least one of the following: various primary skin infections caused by drug-resistant bacteria, as well as eczema co-infection, ulcer co-infection and other secondary skin infections, including diabetic foot, burn wound infection, bedsore infection and other stubborn infectious diseases.
  • the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.
  • the methods are conventional methods unless otherwise specified.
  • the raw materials can be obtained from open commercial sources unless otherwise specified.
  • sequences of the antimicrobial peptides used in the following examples (having the sequence of SEQ ID No: 3) can be synthesized artificially.
  • Mannitol, glycerol, lactose and glycine were selected as stabilizers for antimicrobial peptides, and aqueous solutions of antimicrobial peptide compositions were prepared respectively (the concentration of antimicrobial peptides was 2 ⁇ ), and the stability of antimicrobial peptide compositions was investigated.
  • the detection indicators included properties, related substances ( Maximum single impurity and total impurity), content, etc.
  • antimicrobial peptide (2 ⁇ ) + mannitol (1%) composition
  • Antimicrobial peptide (2 ⁇ )+glycine (1%) composition
  • the concentration of antimicrobial peptides is 2 ⁇ and the concentration of mannitol in the composition system is 0.5%, the content of antimicrobial peptides in the composition solution of antimicrobial peptides at 6 months (25°C condition) is 97.55%;
  • the concentration of mannitol in the system reaches more than 1%, the content of the antimicrobial peptide composition solution can be maintained at more than 98% in 6 months (25°C condition). Therefore, the mannitol concentration is preferably 1%.
  • Embodiment 2 Antibacterial peptide composition buffer system and molar concentration investigation of buffer system
  • the antibacterial peptide composition buffer system was investigated according to the combination in the following table, and the mass concentration of the antibacterial peptide in the antibacterial peptide composition buffer system in the following table was all 4 ⁇ .
  • disodium hydrogen phosphate and citric acid buffer system can ensure that the antimicrobial peptide composition solution can still have more than 98% antimicrobial peptide content in 3 months (25°C condition). Therefore, disodium hydrogen phosphate and citric acid buffer system is the preferred buffer system.
  • the ion concentration is about 0.015M, which can ensure that the antibacterial peptide composition solution can be in 3 months (25 °C conditions)
  • the content of antibacterial peptides Still has 98%.
  • Embodiment 3 Investigation of pH value of antimicrobial peptide composition
  • Mannitol (1%) was selected as stability, disodium hydrogen phosphate and citric acid as buffer system (0.015M) to prepare antimicrobial peptide (2 ⁇ ) composition solutions with different pH values (3.0, 3.5, 4.5, 5.5, 6.5).
  • pH values 3.0, 3.5, 4.5, 5.5, 6.5.
  • hydrochloric acid was selected as the pH adjuster, the pH value of the antimicrobial peptide aqueous solution was adjusted to 4.5, the stability of the antimicrobial peptide was investigated, and compared with the antimicrobial peptide composition solution with disodium hydrogen phosphate and citric acid as the buffer system, the results are shown in the table. 8.
  • the antimicrobial peptide composition can ensure that the antimicrobial peptide composition solution can still have more than 98% antimicrobial peptide content in 3 months (25°C condition) under the condition of pH 3.5-5.5.
  • the pH value of the antimicrobial peptide aqueous solution was adjusted to 4.5 with hydrochloric acid, the antimicrobial peptide content in the antimicrobial peptide aqueous solution decreased to below 95% within 3 months (25° C.).
  • 5ml/bottle is filled in a spray bottle, and the spray pump is tightened to obtain an antimicrobial peptide spray.
  • Relevant substances According to the test of high performance liquid chromatography (Appendix V D of Chinese Pharmacopoeia 2015 edition), use octadecylsilane-bonded silica gel as a filler, inject 20 ⁇ L of the solution into a liquid chromatograph, and record the chromatogram. Relevant substances and limits: known impurities A, B, C, D and unknown single impurities, each impurity shall not exceed 1.0%; total impurities include the sum of impurities A, B, C, D and other unknown single impurities, total impurities Must not exceed 3.0%.
  • the antimicrobial peptide compositions were tested against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant coagulase-negative staphylococci (MRSCNS) and In vitro bacteriostatic and bactericidal efficacy of methicillin-sensitive coagulase-negative staphylococcus (MSSCNS) clinical isolates.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MSSA methicillin-sensitive Staphylococcus aureus
  • MRSCNS methicillin-resistant coagulase-negative staphylococci
  • MSSCNS methicillin-resistant coagulase-negative staphylococcus
  • the control drug was vancomycin. The results are shown in Table 10 and Table 11.
  • MRSA, MSSA, MRSCNS, and MSSCNS strains are all from the Clinical Laboratory Center of Jiangsu Provincial People's Hospital and preserved by Nanjing Dermatology Research Institute.
  • Staphylococcus aureus and Streptococcus pyogenes which are the most common in clinical local skin bacterial infections
  • Pseudomonas aeruginosa which are common in clinical refractory infections
  • Establishments include MRSA (methicillin-resistant Staphylococcus aureus), MSSA (methicillin-sensitive Staphylococcus aureus), erythromycin-resistant and susceptible strains of Streptococcus pyogenes, Pseudomonas aeruginosa IPM-R and IPM- Local skin infection model of S-strain clinical strains [1-3] , and 0.5% ofloxacin gel was used as the control drug, and the vehicle group (1% mannitol, 0.015M disodium hydrogen phosphate and citric acid) Buffer system, pH 4.5) was used as a negative control group to quantitatively evaluate the efficacy of antimicrobial peptide sprays on local skin infections of the above strains. The results are shown in Tables 12-14.
  • test strains were all from the Clinical Laboratory Center of Jiangsu Provincial People's Hospital and preserved by Nanjing Skin Research Institute, and the strain codes were compiled by Nanjing Skin Research Institute.
  • the statistical summary of the bacteriostatic rate after treatment shows that the dosage of antimicrobial peptides is far lower than ofloxacin when the same therapeutic effect is achieved.
  • the antibacterial peptide composition of the invention can be used to prepare a broad-spectrum anti-infective drug preparation for topical use, and is suitable for pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), pyogenic bacteria Streptococcus erythromycin-resistant and sensitive strains, Pseudomonas aeruginosa IPM-R and IPM-S strains, especially various primary skin infections caused by drug-resistant bacteria, as well as eczema co-infection, ulcer co-infection, etc. Secondary skin infections, including diabetic foot, burn wound infection, bedsore infection and other intractable infectious diseases, have broad application prospects.
  • MRSA methicillin-resistant Staphylococcus aureus
  • MSSA methicillin-sensitive Staphylococcus aureus
  • pyogenic bacteria Streptococcus erythromycin-resistant and sensitive strains Pseudomon

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dispersion Chemistry (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Dermatology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

提供了一种抗菌肽组合物，包括抗菌肽、至少一种稳定剂以及缓冲体系；所述组合物中所述抗菌肽的质量浓度为0.1‰～10‰；所述缓冲体系为磷酸盐缓冲体系或醋酸盐缓冲体系；所述抗菌肽的氨基酸序列为：KWKSFLKTFaAbKTVLHTALKAISS。抗菌肽组合物为局部外用广谱抗感染药物，适用于致病菌尤其是耐药菌引起的各种原发性皮肤感染，以及湿疹合并感染、溃疡合并感染等继发性皮肤感染，包括糖尿病足、烧伤创面感染、褥疮感染等顽固感染性疾病，具有广泛的应用前景。

Description

一种抗菌肽液体组合物及其制剂 技术领域
本发明涉及生物技术领域，具体而言，涉及一种抗菌肽液体组合物及其制剂。
背景技术
抗菌药物主要包括抗生素类以及合成抗细菌药物。上世纪二三十年代以来，抗生素的发现和应用拯救了无数人的生命。抗生素是由细菌或其他微生物在生活过程中所产生的物质，具有抑制或杀灭细菌、螺旋体、支原体、衣原体等致病微生物的作用。已上市的抗生素主要包括β-内酰胺类，大环内酯类，多糖类(万古霉素，替考拉宁)，氨基糖苷类，四环素类，氯霉素类，肽内酯类(达托霉素)等。
如果早前人类因其受益，那么，如今全世界则不得不致力解决抗生素滥用所导致的后果——耐药细菌的出现及传播。超级细菌产生的对抗生素不敏感现象，“超级细菌”之外还有“超超级细菌”，随着一个个“超级细菌”被发现，耐药细菌的阵容愈发强大。
抗生素替代品的研发已成为生物医药领域的热点话题。其中，抗菌肽因其对细菌具有广谱高效杀菌活性，且又不易产生耐药性而成为抗生素替代品的“希望之星”。已报道细胞膜是抗菌肽的主要靶点，抗菌肽分子在细胞膜上的聚集会导致细胞膜的通透性增加，使细胞膜丧失其屏障功能。微生物产生对抗菌肽的抗药性需要对微生物细胞膜脂成分产生实质性改变。
抗菌肽是生物体内经诱导产生的一种具有生物活性的小分子多肽，分子量在2000～7000左右，由20～60个氨基酸残基组成。按照来源，抗菌肽可分为：植物抗菌肽，动物抗菌肽，细菌抗菌肽(又称细菌素，包括阳离子肽和中性肽，革兰氏阳性菌和革兰氏阴性菌均可分泌)以及人源性抗菌肽等。
2013年11月，中科院昆明动物研究所研究员张云课题组发现，天然抗菌肽具有选择性免疫激活和调节功能，对败血症有良好的预防和治疗作用。
另有文献报道，人源性抗菌肽具有广谱抗菌作用，可调节创伤炎症，促进创伤部位血管生成及其上皮组织再生。在人体创伤愈合过程中，抗菌肽是内生型免疫系统的关键信号分子之一，与各类细胞及各种生长因子相互作用，协调达到平衡状态，实现创伤修复。
CN101111256A公开了抗菌肽NA L以及D-NA L的氨基酸序列Ac-Lys-Trp- Lys-Ser-Phe-Leu-Lys-Thr-Phe-Lys-Ser-Ala-Ala-Lys-Thr-Val-Leu-His-Thr-Ala-Leu-Lys-Ala-Ile-Ser-Ser-NH 2。该抗菌肽具有抗微生物活性、期望的溶血活性水平及针对革兰氏阳性细菌和革兰氏阴性细菌以及其它带有脂双层膜的细胞成分或结构组分的微生物的广谱治疗系数。
蛋白质类药物制剂的主要问题是药物稳定性差。改善蛋白类药物的稳定性，很容易可以想到通过将蛋白类药物制备成无菌冻干粉末，从而增强蛋白类药物的稳定性。针对液体组合物，可通过加入辅料(稳定剂)如多元醇(如山梨醇、甘露醇、甘油以及丙二醇等)、糖类(如乳糖、胶质、糊精、葡萄糖和海藻糖等)、氨基酸(如甘氨酸、丝氨酸、谷氨酸和赖氨酸等)、盐类(如枸橼酸盐，醋酸盐和磷酸盐等)、表面活性剂等，从而增加其稳定性。但是冻干制剂存在处方、工艺复杂的缺陷，因此如果能提供稳定的蛋白类液体制剂将具有重要的意义。
发明公开
本发明旨在提供一种抗菌肽液体组合物，以期望解决细菌等日益严重的抗药性问题及顽固感染对广大患者造成的痛苦。
本发明所提供的抗菌肽液体组合物，包括抗菌肽、至少一种稳定剂以及缓冲体系；所述组合物中所述抗菌肽的质量浓度为0.1‰～10‰；所述稳定剂的质量浓度为0.5％～5％，所述缓冲体系为磷酸盐缓冲体系或醋酸盐缓冲体系。
所述抗菌肽为如下通式的多肽：
区段甲-Ⅰ-A-Ⅱ-区段乙；
Ⅰ选自下述任意氨基酸残基：L-亮氨酸、D-亮氨酸、L-缬氨酸、D-缬氨酸、L-丙氨酸、D-丙氨酸、甘氨酸、L-丝氨酸、D-丝氨酸、L-赖氨酸及D-赖氨酸(L-和D-光学异构形式的L、A、S、V和K，以及G)；
Ⅱ选自下述任意氨基酸残基：选自下述任意氨基酸残基：L-亮氨酸、D-亮氨酸、L-缬氨酸、D-缬氨酸、L-丙氨酸、D-丙氨酸、甘氨酸、L-丝氨酸、D-丝氨酸、L-赖氨酸及D-赖氨酸(L-和D-光学异构形式的L、A、S、V和K，以及G)；
区段甲如SEQ ID No：1所示，其序列为：KWKSFLKTFK；
区段乙如SEQ ID No：2所示，其序列为：KTVLHTALKAISS；
A代表丙氨酸残基；
所述通式中，方向为自N端至C端。
优选的，所述抗菌肽为NA L以及D-NA L。
所述NA L具有SEQ ID No：3的序列，其氨基酸序列为：KWKSFLKTFKSAAKTVLHTALKAISS；
所述D-NA L具有SEQ ID No：4的序列，其氨基酸序列为：KWKSFLKTFKSAAKTVLHTALKAISS(除了第13位的A为L构型外，所有氨基酸都是D-构型)。
在抗菌肽名称中，大写母D-(非下标)表示除特指位点外，该抗菌肽全部由D-型氨基酸所组成(例如:D-NA L代表该抗菌肽除了含有非极性表面中心的L-丙氨酸取代之外全部由D-型氨酸所组成，非极性表面用大写字母N代表)。
优选的，抗菌肽的浓度为0.5‰～6‰，更优选1‰～4‰，进一步优选为1‰～2‰，具体可为0.2‰、0.5‰、1‰、2‰。
优选的，所述缓冲体系为磷酸氢二钠-枸橼酸的缓冲体系，其中，所述组合物中缓冲体系的离子浓度为0.01M～0.1M，优选离子浓度为0.01M～0.02M，更优选离子浓度为0.015M。
优选的，所述组合物的pH值为3.5～5.5，具体如3.5、4.5或5.5。
本发明中所述稳定剂可选自下述至少一种：多元醇(如山梨醇、甘露醇、甘油以及丙二醇等)、糖类(如乳糖、胶质、糊精、葡萄糖和海藻糖等)、氨基酸(如甘氨酸、丝氨酸、谷氨酸和赖氨酸等)、盐类(如枸橼酸盐，醋酸盐和磷酸盐等)、表面活性剂等，不仅使抗菌肽稳定，且能够维持抗菌肽的抑菌能力。
优选的，所述稳定剂为甘露醇，其中，所述抗菌肽液体组合物中甘露醇的浓度为0.5％～5％，优选甘露醇的浓度为1％～2％，更优选甘露醇的浓度为1％，具体如0.5％、1％、1.25％、1.5％或5％。
稳定性试验表明，本发明提供的抗菌肽组合物至少在24个月内稳定(4℃条件)；
本发明提供的抗菌肽组合物主要适用于治疗皮肤感染性疾病。喷雾剂用药相比凝胶剂和乳膏剂等可有效避免与皮肤创面直接接触，直接作用于病变部位，减少了全身的毒性反应，且稳定性好，易吸收，提高了临床用药的安全性和顺应性，更适于临床需要。
因此，本发明提供的组合物制剂形式为液体制剂，其剂型可为喷雾剂、溶液剂、凝胶剂、乳剂、溶胶剂、滴剂、糖浆剂、混悬剂、口服液、洗剂或擦剂等，优选为喷雾剂。
本发明还提供了上述抗菌肽液体组合物的制备方法。
进一步的，本发明外用组合物制备方法简单，选择各组合物所需的原辅料，按照现有技术中公开的各剂型的常规制备方法制备即可。
本发明所提供的抗菌肽液体组合物的制备方法包括下述步骤：
1)量取70～90％用量的注射用水，分别加入形成所述缓冲体系的物质，搅拌至溶解；再将稳定剂加入上述溶液中，搅拌至全部溶解；
2)称取抗菌肽加入步骤1)的溶液中，搅拌溶解，注射用水补至全量，即得。
所述方法还包括用0.22微米的微孔滤膜过滤上述抗菌肽液体组合物的步骤。
根据本发明的一个实施例，公开了一种喷雾制剂的具体制备方式，采用上述方法制备得到抗菌肽液体组合物后，以5ml/瓶灌装于喷雾瓶中，旋紧喷雾泵，得到抗菌肽喷雾剂。
本发明中采用的抗菌肽是以破坏细菌细胞膜，从而达到杀灭细菌效用的全新广谱抗感染药物，具有独特的药理作用和强效的杀菌效果，使其不同于传统的抗生素，不易产生耐药性，同时和传统的抗生素基本没有交叉耐药现象，是解决病菌耐药性问题非常有效的途径和方法。
蛋白质类药物制剂的主要问题是药物稳定性差。改善蛋白类药物的稳定性，很容易可以想到通过将蛋白类药物制备成无菌冻干粉末，从而增强蛋白类药物的稳定性。针对液体组合物，可通过加入辅料(稳定剂)如多元醇(如山梨醇、甘露醇、甘油以及丙二醇等)、糖类(如乳糖、胶质、糊精、葡萄糖和海藻糖等)、氨基酸(如甘氨酸、丝氨酸、谷氨酸和赖氨酸等)、盐类(如枸橼酸盐，醋酸盐和磷酸盐等)、表面活性剂等，从而增加其稳定性。无疑蛋白类液体组合物的稳定性相比冻干粉末的稳定性更难以保证。本发明着重解决抗菌肽液体组合物的稳定性。
本发明的研究团队发现，与其它稳定剂(如甘油、乳糖、甘氨酸)相比，甘露醇能显著增加抗菌肽的稳定性。
本发明的研究团队发现，酸性pH缓冲体系能使抗菌肽更加稳定，缓冲体系可考虑磷酸盐缓冲体系(如磷酸氢二钠+枸橼酸，或者磷酸氢二钠+磷酸二氢钠，等)，醋酸盐缓冲体系(如醋酸钠+磷酸，或者醋酸钠+醋酸，等)，枸橼酸缓冲 体系(如枸橼酸钠+醋酸，或者枸橼酸钠+枸橼酸，等)，结合抗菌肽的稳定性及抑菌试验，优选磷酸盐缓冲体系和醋酸盐缓冲体系(包含磷酸氢二钠、枸橼酸、醋酸钠、冰醋酸)，更优选磷酸氢二钠与枸橼酸组成的缓冲体系。
本发明的组合物中，缓冲体系的离子浓度对抗菌肽的稳定性也存在一定的影响，发明人研究发现缓冲体系的离子浓度为0.01M～0.1M，优选离子浓度为0.01M～0.02M，更优选离子浓度为0.015M。
本发明涉及的抗菌肽喷雾剂，表现出良好的生物利用度及使用安全性；能够减弱对皮肤和/或创面的刺激性；具有高稳定性，在长期覆盖创面中可改善创面的微环境抑制细菌的生长；使创伤快速修复；能促进愈合，缩短伤口愈合时间，在临床创伤护理应用领域前景广泛。
本发明的抗菌肽组合物可用于制备局部外用广谱抗感染药物制剂；还可用于治疗局部感染，或用于在治疗由局部感染引起的疾病。适用于致病菌包括耐甲氧西林金黄色葡萄球菌(MRSA)、甲氧西林敏感金黄色葡萄球菌(MSSA)、化脓性链球菌红霉素耐药和敏感株、铜绿假单胞菌IPM-R和IPM-S株，尤其是耐药菌引起的各种原发性皮肤感染，以及湿疹合并感染、溃疡合并感染等继发性皮肤感染，包括糖尿病足、烧伤创面感染、褥疮感染等顽固感染性疾病，具有广泛的应用前景。
本发明还保护一种局部外用广谱抗感染产品，其活性成分包括本发明所提供的抗菌肽组合物。
示例性的，所述产品可为药物或药物制剂。
本发明还提供了局部抗感染的方法，包括如下步骤：给受体动物施用本发明所述的抗菌肽组合物以抗局部感染。
本发明还提供了治疗由局部感染引起的疾病的方法，包括如下步骤：给受体动物施用本发明所述的抗菌肽组合物以治疗由局部感染引起的疾病。
本发明中，所述动物可为哺乳动物，如人；所述动物还可为除哺乳动物以外的所述感染的其他动物，如鼠。
以上所述感染由下述至少一种致病菌引起：耐甲氧西林金黄色葡萄球菌、甲氧西林敏感金黄色葡萄球菌、化脓性链球菌红霉素耐药株、化脓性链球菌红霉素敏感株、铜绿假单胞菌IPM-R和IPM-S株；以上所述感染包括下述至少一种：耐药菌引起的各种原发性皮肤感染，以及湿疹合并感染、溃疡合并感染等继发性 皮肤感染，包括糖尿病足、烧伤创面感染、褥疮感染等顽固感染性疾病。
具体实施方式
下面结合具体实施例对本发明作进一步阐述，但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
下述实施例中使用的抗菌肽的序列(具有SEQ ID No：3的序列)能够通过人工合成。
实施例1、抗菌肽组合物稳定剂筛选
选择甘露醇，甘油，乳糖和甘氨酸等作为抗菌肽的稳定剂，分别制备抗菌肽组合物水溶液(抗菌肽浓度为2‰)，考察抗菌肽组合物的稳定性，检测指标包括性状、有关物质(最大单杂及总杂)、含量等。
有关物质检测方法：
照高效液相色谱法(中国药典2015年版二部附录V D)试验，用十八烷基硅烷键合硅胶为填充剂，取溶液20μL注入液相色谱仪，记录色谱图。有关物质及限度：已知杂质A、B、C、D及未知单杂，每个杂质都不得超过1.0％，最大单杂以所有单个杂质的最大值进行汇总统计；总杂包括杂质A，B，C和D以及其他未知单杂的总和，总杂质不得超过3.0％。
下表中：抗菌肽(2‰)+甘露醇(1％)组合物；
抗菌肽(2‰)+甘油(1％)组合物；
抗菌肽(2‰)+乳糖(1％)组合物；
抗菌肽(2‰)+甘氨酸(1％)组合物。
表1抗菌肽组合物(25℃条件下)考察结果
Figure PCTCN2021072665-appb-000001
Figure PCTCN2021072665-appb-000002
表2抗菌肽组合物(4℃条件下)试验考察结果
Figure PCTCN2021072665-appb-000003
Figure PCTCN2021072665-appb-000004
由表1和表2抗菌肽组合物稳定性考察结果可知，甘露醇(1％)作为抗菌肽的稳定剂，可以保证抗菌肽组合物溶液能够在36个月(4℃条件)抗菌肽的含量仍具有97％以上。
考虑到不同抗菌肽浓度在不同甘露醇体系的稳定性，进一步考察了不同抗菌肽浓度(2‰和0.2‰)在不同甘露醇(0.5％～5％)体系的稳定性，结果见表3。
表3不同甘露醇体系抗菌肽组合物的稳定性考察结果
Figure PCTCN2021072665-appb-000005
由表3可知，当抗菌肽浓度为2‰时，组合物体系中甘露醇浓度为0.5％时，抗菌肽组合物溶液在6个月(25℃条件)抗菌肽的含量为97.55％；当组合物体系中甘露醇浓度达到1％以上时，抗菌肽组合物溶液在6个月(25℃条件)抗菌 肽的含量可保持在98％以上。故而优选甘露醇浓度为1％。
实施例2抗菌肽组合物缓冲体系及缓冲体系的摩尔浓度考察
抗菌肽组合物缓冲体系按照下表组合考察，下表各抗菌肽组合物缓冲体系中抗菌肽的质量浓度均为4‰。
表4抗菌肽组合物缓冲体系
Figure PCTCN2021072665-appb-000006
表5抗菌肽组合物缓冲体系试验考察结果
Figure PCTCN2021072665-appb-000007
由表5可知，选择磷酸氢二钠与枸橼酸缓冲体系，可以保证抗菌肽组合物溶液能够在3个月(25℃条件)抗菌肽的含量仍具有98％以上。因此，磷酸氢二钠与枸橼酸缓冲体系为优选的缓冲体系。
针对优选的磷酸氢二钠与枸橼酸缓冲体系，考察缓冲体系离子浓度(0.01M～0.1M)对抗菌肽稳定性的影响，考察结果见表6。
表6缓冲体系离子浓度对抗菌肽稳定性的影响
Figure PCTCN2021072665-appb-000008
由表6可知，缓冲体系离子浓度对抗菌肽组合物溶液稳定性的考察结果可见，离子浓度约为0.015M，可以保证抗菌肽组合物溶液能够在3个月(25℃条件)抗菌肽的含量仍具有98％。
实施例3、抗菌肽组合物pH值考察
选择甘露醇(1％)作为稳定性，磷酸氢二钠与枸橼酸作为缓冲体系(0.015M)配制不同pH值(3.0，3.5，4.5，5.5，6.5)抗菌肽(2‰)组合物溶液，考察抗菌肽组合物在不同pH值条件下的稳定性，结果见表7。
同时选择盐酸作为pH调节剂，调节抗菌肽水溶液的pH值至4.5，考察抗菌肽的稳定性，并与磷酸氢二钠与枸橼酸作为缓冲体系的抗菌肽组合物溶液进行对比，结果见表8。
表7组合物pH值对抗菌肽稳定性的影响
Figure PCTCN2021072665-appb-000009
Figure PCTCN2021072665-appb-000010
表8不同pH调节剂对抗菌肽稳定性的影响(pH4.5)
Figure PCTCN2021072665-appb-000011
由表7和表8可知，抗菌肽组合物在pH值3.5～5.5条件下，可以保证抗菌肽组合物溶液能够在3个月(25℃条件)抗菌肽的含量仍具有98％以上。当以盐酸调节抗菌肽水溶液pH值为4.5时，在3个月(25℃条件)抗菌肽水溶液中抗菌肽的含量已下降至95％以下。
实施例4抗菌肽喷雾组合物
(1)处方
处方1、处方组成
Figure PCTCN2021072665-appb-000012
处方2、处方组成
Figure PCTCN2021072665-appb-000013
处方3、处方组成
Figure PCTCN2021072665-appb-000014
处方4、处方组成
Figure PCTCN2021072665-appb-000015
处方5、处方组成
Figure PCTCN2021072665-appb-000016
处方6、处方组成
Figure PCTCN2021072665-appb-000017
(2)制备工艺
①量取70～90％处方量的注射用水，分别加入处方量磷酸氢二钠和枸橼酸，搅拌15分钟，搅拌至溶解；再将处方量甘露醇加入上述溶液中，搅拌10分钟，至全部溶解。
②称取处方量的抗菌肽加入上述溶液中，搅拌溶解，注射用水补至全量。
③用0.22微米的微孔滤膜过滤，取样测定中间体性状、含量、pH值等。
以5ml/瓶灌装于喷雾瓶中，旋紧喷雾泵，得到抗菌肽喷雾剂。
灌装后将抽取适量的成品按照质量标准项下各检测方法进行检验。
(3)质量要求
1)有关物质：照高效液相色谱法(中国药典2015年版二部附录V D)试验，用十八烷基硅烷键合硅胶为填充剂，取溶液20μL注入液相色谱仪，记录色谱图。有关物质及限度：已知杂质A、B、C、D及未知单杂，每个杂质都不得超过1.0％；总杂包括杂质A，B，C、D以及其他未知单杂的总和，总杂质不得超过3.0％。
2)pH：应符合规定pH 3.5～5.5。
3)含量：照高效液相色谱法(中国药典2015年版四部通则0512)测定，精密量取本品适量，加水溶解并定量稀释制成每1ml中含0.25mg的溶液，作为供试品溶液，精密量取10μl注入液相色谱仪，记录色谱图；另取抗菌肽对照品适量，同法测定。
以处方1及处方6进行了稳定性考察，结果见表9。
表9抗菌肽组合物(25℃条件下)考察结果
Figure PCTCN2021072665-appb-000018
由表9可见，处方1和处方6在25℃条件下，在6个月内抗菌肽的含量均能够维持在98％以上。
(4)体外抑菌和杀菌试验
测试抗菌肽组合物(处方2与处方4)对耐甲氧西林金黄色葡萄球菌(MRSA)、甲氧西林敏感金黄色葡萄球菌(MSSA)、耐甲氧西林凝固酶阴性葡萄球菌(MRSCNS)和甲氧西林敏感凝固酶阴性葡萄球菌(MSSCNS)临床分离菌株的体外抑菌作用和杀菌效力。对照药为万古霉素。结果见表10和表11。
试验菌株：
标准株：金黄色葡萄球菌ATCC29213(来源于江苏省人民医院临床检验中心，南京皮肤研究所保存)；
临床株：MRSA、MSSA、MRSCNS、以及MSSCNS株均来源于江苏省人民医院临床检验中心，南京皮肤研究所保存。
表10抗菌肽和万古霉素对金黄色葡萄球菌、凝固酶阴性葡萄球菌的体外抑菌作用
Figure PCTCN2021072665-appb-000019
表11抗菌肽和万古霉素对金黄色葡萄球菌、凝固酶阴性葡萄球菌的体外杀菌作用
Figure PCTCN2021072665-appb-000020
(5)体内药效试验
选择临床上局部皮肤细菌感染中最常见的金黄色葡萄球菌和化脓性链球菌以及临床顽固性感染中常见的铜绿假单胞菌作为感染菌，分别通过小鼠皮肤局部感染或轻度破损感染，建立包括MRSA(耐甲氧西林金黄色葡萄球菌)、MSSA(甲氧西林敏感金黄色葡萄球菌)、化脓性链球菌红霉素耐药和敏感株、铜绿假单胞菌IPM-R和IPM-S株临床株的局部皮肤感染模型 [1～3]，并以0.5％氧氟沙星 凝胶作对照药，溶媒组(1％的甘露醇，0.015M的磷酸氢二钠与枸橼酸的缓冲体系，pH4.5)作为阴性对照组，定量评价抗菌肽喷雾剂对上述菌株局部皮肤感染的药效。结果见表12～14。
试验菌株均来源于江苏省人民医院临床检验中心，南京皮肤研究所保存，菌株代码由南京皮研所编制。
1‰(处方1)，0.5‰(处方4)和0.2‰抗菌肽喷雾剂(处方6)对金黄色葡萄球菌(MRSA和MSSA)、化脓性链球菌(红霉素敏感和耐药株)引起的小鼠局部皮肤感染有明显的抑制作用。1‰抗菌肽喷雾剂对铜绿假单胞菌(IPM-R和IPM-S)引起的小鼠局部皮肤感染亦有抑制作用(抗菌肽组抑制率的计算＝[1-(给药组匀浆中的菌液浓度/溶媒组匀浆中的菌液浓度)]×100％；氧氟沙星凝胶组抑制率的计算＝[1-(给药组匀浆中的菌液浓度/模型组匀浆中的菌液浓度)]×100％)。通过对治疗后的抑菌率统计汇总，表明达到相同的治疗效应情况下，抗菌肽的给药量远远低于氧氟沙星。
表12-1抗菌肽喷雾剂治疗小鼠浅表皮肤局部感染MRSA后皮损匀浆培养计算抑制率结果
Figure PCTCN2021072665-appb-000021
Figure PCTCN2021072665-appb-000022
表12-2抗菌肽喷雾剂治疗小鼠浅表皮肤局部感染MSSA后皮损匀浆培养计算抑制率结果
Figure PCTCN2021072665-appb-000023
Figure PCTCN2021072665-appb-000024
表13抗菌肽喷雾剂治疗小鼠局部皮肤感染化脓性链球菌后皮损匀浆培养计算抑制率结果
Figure PCTCN2021072665-appb-000025
Figure PCTCN2021072665-appb-000026
表14-1抗菌肽喷雾剂治疗小鼠局部皮肤感染IPM-R后皮损匀浆培养计算抑制率结果
Figure PCTCN2021072665-appb-000027
Figure PCTCN2021072665-appb-000028
表14-2抗菌肽喷雾剂治疗小鼠局部皮肤感染IPM-S后皮损匀浆培养计算抑制率结果
Figure PCTCN2021072665-appb-000029
Figure PCTCN2021072665-appb-000030
参考文献：
1、单纯性和复杂性皮肤感染抗菌药物临床研究指导原则，1998年7月美国FDA发布，2009年药审中心组织翻译
2、徐叔云等。药理实验方法学。人民卫生出版社，2006，11，第三版3、新药(西药)临床前研究指导原则汇编，中华人民共和国卫生部药政局，1993.7
工业应用
本发明的抗菌肽组合物可用于制备局部外用广谱抗感染药物制剂，适用于致病菌包括耐甲氧西林金黄色葡萄球菌(MRSA)、甲氧西林敏感金黄色葡萄球菌(MSSA)、化脓性链球菌红霉素耐药和敏感株、铜绿假单胞菌IPM-R和IPM-S株，尤其是耐药菌引起的各种原发性皮肤感染，以及湿疹合并感染、溃疡合并感染等继发性皮肤感染，包括糖尿病足、烧伤创面感染、褥疮感染等顽固感染性疾病，具有广泛的应用前景。

Claims (14)

  1. 一种抗菌肽液体组合物，包括抗菌肽、至少一种稳定剂以及缓冲体系；所述组合物中所述抗菌肽的质量浓度为0.1‰～10‰；所述缓冲体系为磷酸盐缓冲体系或醋酸盐缓冲体系；
    所述抗菌肽为如下通式的多肽：
    区段甲-Ⅰ-A-Ⅱ-区段乙；
    Ⅰ选自下述任意氨基酸残基：L-亮氨酸、D-亮氨酸、L-缬氨酸、D-缬氨酸、L-丙氨酸、D-丙氨酸、甘氨酸、L-丝氨酸、D-丝氨酸、L-赖氨酸及D-赖氨酸；
    Ⅱ选自下述任意氨基酸残基：选自下述任意氨基酸残基：L-亮氨酸、D-亮氨酸、L-缬氨酸、D-缬氨酸、L-丙氨酸、D-丙氨酸、甘氨酸、L-丝氨酸、D-丝氨酸、L-赖氨酸及D-赖氨酸；
    区段甲如SEQ ID No：1所示，其序列为：KWKSFLKTFK；
    区段乙如SEQ ID No：2所示，其序列为：KTVLHTALKAISS；
    A代表丙氨酸残基；
    所述通式中，方向为自N端至C端。
  2. 根据权利要求1所述的组合物，其特征在于：所述抗菌肽的质量浓度为0.5‰～6‰，优选1‰～4‰，更优选为1‰～2‰。
  3. 根据权利要求1或2所述的组合物，其特征在于：所述缓冲体系为磷酸氢二钠-枸橼酸的缓冲体系，其中，所述组合物中缓冲体系的离子浓度为0.01M～0.1M，优选离子浓度为0.01M～0.02M，更优选离子浓度为0.015M。
  4. 根据权利要求1-3中任一项所述的组合物，其特征在于：所述抗菌肽为NA L或D-NA L；
    所述NA L具有SEQ ID No:3的序列，其氨基酸序列为：KWKSFLKTFKSAAKTVLHTALKAISS；
    所述D-NA L具有SEQ ID No：4的序列，其氨基酸序列为：KWKSFLKTFKSAAKTVLHTALKAISS；且除了第13位的A外，所有氨基酸都是D-构型。
  5. 根据权利要求1-4中任一项所述的组合物，其特征在于：所述稳定剂选自下述至少一种：多元醇、氨基酸、盐类、表面活性剂；
    优选的，所述稳定剂为甘露醇，其中，所述组合物中甘露醇的质量浓度为 0.5％～5％，优选甘露醇的质量浓度为1％～2％，更优选甘露醇的质量浓度为1％。
  6. 根据权利要求1-5中任一项所述的组合物，其特征在于：所述组合物的pH值为3.5～5.5。
  7. 根据权利要求1-6中任一项所述的组合物，其特征在于：所述组合物制剂形式为液体制剂，其剂型为喷雾剂、溶液剂、凝胶剂或乳剂，溶胶剂，滴剂，糖浆剂，混悬剂，口服液，洗剂，擦剂，优选为喷雾剂。
  8. 权利要求1-7中任一项所述的组合物在制备局部外用广谱抗感染产品中的应用。
  9. 权利要求1-7中任一项所述的组合物在治疗局部感染和/或在治疗由局部感染引起的疾病中的应用。
  10. 根据权利要求8或9所述的应用，其特征在于：所述产品为药品；
    所述感染由下述至少一种致病菌引起：耐甲氧西林金黄色葡萄球菌、甲氧西林敏感金黄色葡萄球菌、化脓性链球菌红霉素耐药株、化脓性链球菌红霉素敏感株、铜绿假单胞菌IPM-R和IPM-S株；
    所述感染包括下述至少一种：耐药菌引起的各种原发性皮肤感染，以及湿疹合并感染、溃疡合并感染等继发性皮肤感染，包括糖尿病足、烧伤创面感染、褥疮感染等顽固感染性疾病。
  11. 一种局部外用广谱抗感染产品，其活性成分包括权利要求1-7中任一项所述的组合物。
  12. 根据权利要求11所述的产品，其特征在于：所述产品为药品；
    所述感染由下述至少一种致病菌引起：耐甲氧西林金黄色葡萄球菌、甲氧西林敏感金黄色葡萄球菌、化脓性链球菌红霉素耐药株、化脓性链球菌红霉素敏感株、铜绿假单胞菌IPM-R和IPM-S株；
    所述感染包括下述至少一种：耐药菌引起的各种原发性皮肤感染，以及湿疹合并感染、溃疡合并感染等继发性皮肤感染，包括糖尿病足、烧伤创面感染、褥疮感染等顽固感染性疾病。
  13. 一种局部抗感染的方法，包括如下步骤：给受体动物施用权利要求1-7中任一项所述的组合物或权利要求11或12所述的产品以抗局部感染。
  14. 一种治疗由局部感染引起的疾病的方法，包括如下步骤：给受体动物施用权利要求1-7中任一项所述的组合物或权利要求11或12所述的产品以治疗由局部感染引起的疾病。
PCT/CN2021/072665 2021-01-19 2021-01-19 一种抗菌肽液体组合物及其制剂 WO2022155783A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2023543109A JP2024503875A (ja) 2021-01-19 2021-01-19 抗菌ペプチド液体組成物およびその製剤
US18/272,989 US20240075096A1 (en) 2021-01-19 2021-01-19 Antimicrobial peptide liquid composition and formulation thereof
EP21920188.6A EP4282400A1 (en) 2021-01-19 2021-01-19 Antimicrobial peptide liquid composition and formulation thereof
PCT/CN2021/072665 WO2022155783A1 (zh) 2021-01-19 2021-01-19 一种抗菌肽液体组合物及其制剂

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/072665 WO2022155783A1 (zh) 2021-01-19 2021-01-19 一种抗菌肽液体组合物及其制剂

Publications (1)

Publication Number Publication Date
WO2022155783A1 true WO2022155783A1 (zh) 2022-07-28

Family

ID=82548364

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/072665 WO2022155783A1 (zh) 2021-01-19 2021-01-19 一种抗菌肽液体组合物及其制剂

Country Status (4)

Country Link
US (1) US20240075096A1 (zh)
EP (1) EP4282400A1 (zh)
JP (1) JP2024503875A (zh)
WO (1) WO2022155783A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116535474A (zh) * 2023-06-27 2023-08-04 深圳市森盈智能科技有限公司 一种抗黏附生物相容性抗菌肽及其在生物涂层中的应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101111256A (zh) 2004-12-15 2008-01-23 科罗拉多大学 抗菌肽及其使用方法
CN101496773A (zh) * 2009-02-25 2009-08-05 吉林大学 一种含林蛙抗菌肽的抗菌洗剂及其制备方法
CN102219831A (zh) * 2011-04-18 2011-10-19 江阴普莱医药生物技术有限公司 一种抗菌肽及其制备方法和应用
CN102731629A (zh) * 2012-05-21 2012-10-17 长春普莱医药生物技术有限公司 一种抗菌肽及其应用
CN102727866A (zh) * 2011-04-13 2012-10-17 瑞普（天津）生物药业有限公司 一种抗菌肽组合物及其制备方法
CN105017384A (zh) * 2015-07-13 2015-11-04 长春普莱医药生物技术有限公司 一种新型抗菌肽及其应用
WO2017173240A1 (en) * 2016-03-31 2017-10-05 Gojo Industries, Inc. Antimicrobial peptide stimulating cleansing composition
CN112755175A (zh) * 2021-01-19 2021-05-07 江苏普莱医药生物技术有限公司 一种抗菌肽液体组合物及其制剂

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101111256A (zh) 2004-12-15 2008-01-23 科罗拉多大学 抗菌肽及其使用方法
CN101496773A (zh) * 2009-02-25 2009-08-05 吉林大学 一种含林蛙抗菌肽的抗菌洗剂及其制备方法
CN102727866A (zh) * 2011-04-13 2012-10-17 瑞普（天津）生物药业有限公司 一种抗菌肽组合物及其制备方法
CN102219831A (zh) * 2011-04-18 2011-10-19 江阴普莱医药生物技术有限公司 一种抗菌肽及其制备方法和应用
CN102731629A (zh) * 2012-05-21 2012-10-17 长春普莱医药生物技术有限公司 一种抗菌肽及其应用
CN105017384A (zh) * 2015-07-13 2015-11-04 长春普莱医药生物技术有限公司 一种新型抗菌肽及其应用
WO2017173240A1 (en) * 2016-03-31 2017-10-05 Gojo Industries, Inc. Antimicrobial peptide stimulating cleansing composition
CN112755175A (zh) * 2021-01-19 2021-05-07 江苏普莱医药生物技术有限公司 一种抗菌肽液体组合物及其制剂

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"Chinese Pharmacopoeia", APPENDIX V D, vol. II, 2015
"Chinese Pharmacopoeia", GENERAL RULES, vol. IV, 2015, pages 0512
"Compilation of guiding principles for preclinical research of new drugs (Western drugs", PHARMACEUTICAL ADMINISTRATION BUREAU OF THE MINISTRY OF HEALTH OF THE PEOPLE'S REPUBLIC OF CHINA, vol. 7, 1993
CHEN ET AL.: "Rational Design of alpha-Helical Antimicrobial Peptides with Enhanced Activities and SpecificityfTherapeutic Index.", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, no. 13, 1 April 2005 (2005-04-01), American Society for Biochemistry and Molecular Biology, US, pages 12316 - 12329, XP008136869, ISSN: 0021-9258, DOI: 10.1074/jbc.M413406200 *
XU SHUYUN ET AL.: "Pharmacological Experimental Methodology", 2006, PEOPLE'S MEDICAL PUBLISHING HOUSE

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116535474A (zh) * 2023-06-27 2023-08-04 深圳市森盈智能科技有限公司 一种抗黏附生物相容性抗菌肽及其在生物涂层中的应用
CN116535474B (zh) * 2023-06-27 2023-09-19 深圳市森盈智能科技有限公司 一种抗黏附生物相容性抗菌肽及其在生物涂层中的应用

Also Published As

Publication number Publication date
JP2024503875A (ja) 2024-01-29
EP4282400A1 (en) 2023-11-29
US20240075096A1 (en) 2024-03-07

Similar Documents

Publication Publication Date Title
US9962419B2 (en) Compositions comprising cocktails of antibacterial phages and uses thereof for the treatment of bacterial infections
US8044022B2 (en) Hyaluronic acid binding peptides enhance host defense against pathogenic bacteria
US6025326A (en) Compositions and methods for the prevention and treatment of oral mucositis
JP6157514B2 (ja) 抗菌性ペプチド
US20070065908A1 (en) Human cathelicidin antimicrobial peptides
Isobe et al. Existence of functional lingual antimicrobial peptide in bovine milk
AU776044B2 (en) Antimcrobial activity of the first cationic cluster of human lactoferrin
WO2022155783A1 (zh) 一种抗菌肽液体组合物及其制剂
CN112755175B (zh) 一种抗菌肽液体组合物及其制剂
JPWO2008096814A1 (ja) 新規ポリペプチド及びそれを有効成分として含有する抗菌剤
JP6223444B2 (ja) 蛋白質‐脂質複合体を用いた抗生物質治療の増強
US20190241624A1 (en) Antimicrobial peptides and uses thereof
CN104524547B (zh) 菌丝霉素在抑制无乳链球菌中的应用
US20180289656A1 (en) Compositions and methods to treat urinary tract infections
US7060677B1 (en) Antimicrobial activity of the first cationic cluster of human lactoferrin
CN113209273B (zh) 一种抗菌肽涂剂及其制备方法
CN113081977B (zh) 一种抗感染的抗菌肽冻干制剂及其制备方法
KR20210031466A (ko) 상승 작용량의 붕산을 이용한 뎁시펩타이드 항생제의 항균 작용 증진
KR20190103808A (ko) 고장초 추출물을 유효성분으로 함유하는 항균용 조성물
AU704851B2 (en) Compositions and methods for the prevention and treatment of oral mucositis
BR102012017234A2 (pt) Composições farmacêuticas compreendendo peptídeos catiônicos incluídos e/ou associados à ciclodextrinas e usos
Hafeez et al. Microbiology of the Skin
CN114292323A (zh) 一种角质细胞生长因子活性多肽及其应用
WO2019079150A1 (en) COMPOSITIONS AND METHODS FOR TREATING ATOPIC DERMATITIS
KR20200062921A (ko) 탄저균 특이적 항균능을 갖는 약학적 조성물 및 이의 제조방법

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: 21920188

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023543109

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021920188

Country of ref document: EP

Effective date: 20230821