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CN118702776A - Polypeptides and combinations thereof with cefotaxime sulbactam - Google Patents

Polypeptides and combinations thereof with cefotaxime sulbactam Download PDF

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Publication number
CN118702776A
CN118702776A CN202410960351.0A CN202410960351A CN118702776A CN 118702776 A CN118702776 A CN 118702776A CN 202410960351 A CN202410960351 A CN 202410960351A CN 118702776 A CN118702776 A CN 118702776A
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polypeptide
cefotaxime
seq
sulbactam
polypeptides
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Inventor
王勇
易祥
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Guangdong Medical Insurance Pharmaceutical Co ltd
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Guangdong Medical Insurance Pharmaceutical Co ltd
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/429—Thiazoles condensed with heterocyclic ring systems
    • A61K31/43—Compounds containing 4-thia-1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula, e.g. penicillins, penems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/542—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/545—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine
    • A61K31/546—Compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins, cefaclor, or cephalexine containing further heterocyclic rings, e.g. cephalothin
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
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  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Communicable Diseases (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention provides a polypeptide comprising a sequence as set forth in any one of SEQ ID NOs 1 to 8. Also provided is a composition comprising a polypeptide having the sequence shown in SEQ ID No. 7, cefotaxime and sulbactam. The polypeptide with the sequence of SEQ ID NO. 7 has strong activity for inhibiting escherichia coli or listeria monocytogenes. After the polypeptide is combined with cefotaxime sulbactam, the activity of the polypeptide for inhibiting the escherichia coli producing beta-lactamase is strong. The polypeptides and compositions are therefore of great clinical value.

Description

Polypeptides and combinations thereof with cefotaxime sulbactam
The application is a divisional application of patent application with the application date of 2024, 03 and 06, the application number of 202410253895.3 and the application and creation name of 'polypeptide and combination of cefotaxime and sulbactam'.
Technical Field
The invention relates to the field of biological medicine, in particular to a polypeptide and a combination of the polypeptide and antibiotics.
Background
Antimicrobial peptides are a series of polypeptides having antimicrobial activity. Such polypeptides play an important role in the innate immunity of mammals to invasive bacterial infections. Antimicrobial peptides are of a wide range of lengths, typically ranging from 12 amino acid residues to 80 amino acid residues. Their structures are also becoming ever-changing.
Antibacterial peptides, although having antibacterial activity, generally have strong cytotoxicity, for example, can cause erythrocyte lysis, which results in certain limitation in practical application.
Cefotaxime (cefotaxin) is a third generation cephalosporin antibacterial drug, and many drugs are prepared by taking Cefotaxime (cefotaxin) as a main component. Cefotaxime is suitable for respiratory tract infection, urinary tract infection, gastrointestinal tract infection, meningitis, septicemia, soft tissue infection, otorhinolaryngological infection, genital tract infection, orthopedics infection, etc. caused by sensitive bacteria. The cefotaxime sodium can also be used as a first-choice medicament for meningitis, especially for infant meningitis.
The incidence of side effects of cefotaxime is 3-5%, rash and drug fever are about 2-5%, digestive tract reactions such as phlebitis, diarrhea, nausea, vomiting, inappetence are about 1%, mild elevation of alkaline phosphatase or serum transaminase is about 3%, temporary blood urea nitrogen and creatinine elevation are 0.7% and 0.3%, respectively, and leukopenia, acid granulocyte or thrombocytopenia are visible.
In addition, the patient with severe renal dysfunction should be suitably reduced in the administration of cefotaxime. Serum creatinine values above 424. Mu. Mol/L (4.8 mg) or creatinine clearance below 20 ml/min, the maintenance amount of cefotaxime should be halved; when serum creatinine exceeds 751. Mu. Mol/L (8.5 mg), the maintenance amount is 1/4 of the normal amount.
Sulbactam sodium is a semisynthetic beta-lactamase inhibitor. Pain incidence rate at the injection site is about 3.6%, reactions such as phlebitis, diarrhea, nausea and the like occur occasionally, and rash incidence rate is 1% -6%. Very few cases develop exfoliative dermatitis and anaphylactic shock. For patients with renal hypofunction, the administration interval should be prolonged and the administration frequency should be reduced.
Disclosure of Invention
At present, there is urgent need for antibacterial drugs with strong antibacterial activity and high safety in clinic, and there is urgent need for reducing the dosage of antibiotics and solving the problem of antibiotic drug resistance.
The inventor designs and identifies the antibacterial peptide with high activity and good safety through a large amount of experimental exploration, thereby completing the invention.
In a first aspect of the invention there is provided a polypeptide comprising a sequence as set out in any one of SEQ ID NOS.1 to 8 and the amino acid sequence of the polypeptide being 18 to 25 amino acids in length.
In some embodiments of the invention, the polypeptide comprises the sequence set forth in SEQ ID NO. 1 and the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids in length.
In some embodiments of the invention, the polypeptide comprises the sequence set forth in SEQ ID NO. 2 and the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids in length.
In some embodiments of the invention, the polypeptide comprises the sequence set forth in SEQ ID NO. 3 and the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids in length.
In some embodiments of the invention, the polypeptide comprises the sequence set forth in SEQ ID NO. 4 and the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids in length.
In some embodiments of the invention, the polypeptide comprises the sequence set forth in SEQ ID NO. 5 and the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids in length.
In some embodiments of the invention, the polypeptide comprises the sequence set forth in SEQ ID NO. 6 and the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids in length.
In some embodiments of the invention, the polypeptide comprises the sequence set forth in SEQ ID NO. 7 and the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids in length.
In some embodiments of the invention, the polypeptide comprises the sequence set forth in SEQ ID NO. 8 and the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids in length.
In some embodiments of the invention, the amino acid sequence of the polypeptide is as set forth in any one of SEQ ID NOs 1-8.
In some embodiments of the invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO. 7.
In a second aspect of the invention, there is provided a polypeptide, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO. 7.
In a third aspect of the invention there is provided a composition characterised in that the composition comprises a polypeptide according to any one of the invention.
In some embodiments of the invention, the composition comprises a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs 1 to 8.
In some embodiments of the invention, the composition comprises a polypeptide having the amino acid sequence shown in SEQ ID NO. 7.
In some embodiments of the invention, the composition comprises a β -lactam antibiotic or a β -lactamase inhibitor.
In some embodiments of the invention, the composition comprises a β -lactam antibiotic.
In some embodiments of the invention, the composition comprises cephalosporin.
In some embodiments of the present invention, the cephalosporin is selected from the group consisting of cephalexin, cefadroxil, cephalexin, cefalexin, cefalotin, ceftiodine, cefalotin, cefpiralin, ceftriaxone, cefazedone, cefazolin, cefradine, ceftezole, cefaclor, cefnixie, cefradine, and a mixture of cefradine and cefradine Cefprozil, cefuroxime, cefazolin, cefamandole, cefradit, cefotiam, chlorocarbon, cefbuperazone, cefmetazole, cefminox, cefotetan, cefoxitin, cefcapene, cefdaxime, cefdinir, ceftolam, cefetamet, cefixime, ceftizoxime, pharmaceutical composition, and pharmaceutical composition Cefprozil, cefuroxime, cefazolin, cefamandole, cefradite, cefotiam, chlorocarbon, cefbuperazone, cefmetazole Cefminox, cefotetan, cefoxitin, cefcapene, cefdaxime, cefdinir, ceftolam, cefetamet, cefixime.
In some embodiments of the invention, the composition comprises a beta-lactamase inhibitor.
In some embodiments of the invention, the β -lactamase inhibitor is optionally selected from clavulanic acid, sulbactam, and tazobactam.
In some embodiments of the invention, the composition comprises a β -lactam antibiotic and a β -lactamase inhibitor.
In some embodiments of the invention, the composition comprises cefotaxime and sulbactam.
In some embodiments of the invention, the composition comprises cefotaxime and sulbactam, the mass ratio of the polypeptide, cefotaxime, sulbactam being 0.2:2:1.
In some embodiments of the invention, the composition comprises a polypeptide having an amino acid sequence as set forth in SEQ ID NO. 7;
The composition comprises cefotaxime sodium and sulbactam sodium;
The mass ratio of the polypeptide to the cefotaxime sodium to the sulbactam sodium is 0.2:2:1.
In some embodiments of the invention, the composition comprises a pharmaceutically acceptable adjuvant.
In a fourth aspect of the invention there is provided the use of a polypeptide according to any one of the invention in the manufacture of an antibacterial agent.
In some embodiments of the invention, the antibacterial agent is used to treat a bacterial infection.
In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by E.coli or Listeria monocytogenes. In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by E.coli. In some embodiments of the invention, the antibacterial agent is used to treat infection caused by listeria monocytogenes.
In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by E.coli ML-35p or Listeria monocytogenes EGDe. In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by escherichia coli ML-35 p. In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by listeria monocytogenes EGDe.
In some embodiments of the invention, there is provided the use of a polypeptide as set forth in any one of SEQ ID NOs 1-8 for the manufacture of an antibacterial agent for the treatment of an E.coli or Listeria monocytogenes infection.
In some embodiments of the invention, there is provided the use of a polypeptide as set forth in SEQ ID NO. 7 for the manufacture of an antibacterial agent for the treatment of an E.coli or Listeria monocytogenes infection.
In some embodiments of the invention, the antimicrobial agent comprises a pharmaceutically acceptable adjuvant.
In a fifth aspect of the invention there is provided the use of a composition according to any of the invention in the manufacture of an antibacterial agent.
In some embodiments of the invention, the antibacterial agent is used to treat a bacterial infection.
In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by E.coli or Listeria monocytogenes. In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by E.coli. In some embodiments of the invention, the antibacterial agent is used to treat infection caused by listeria monocytogenes.
In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by E.coli ML-35p or Listeria monocytogenes EGDe. In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by escherichia coli ML-35 p. In some embodiments of the invention, the antibacterial agent is used to treat an infection caused by listeria monocytogenes EGDe.
In some embodiments of the invention, the antibacterial agent is used to treat a bacterial infection that produces beta-lactamase.
In some embodiments of the invention, the antibacterial agent is used to treat a beta-lactamase-producing E.coli infection.
In some embodiments of the invention there is provided the use of a composition comprising a polypeptide as set forth in any one of SEQ ID NOs 1 to 8, a cephalosporin and/or a beta-lactamase inhibitor for the manufacture of an antibacterial agent for the treatment of an E.coli or Listeria monocytogenes infection.
In some embodiments of the invention there is provided the use of a composition comprising a polypeptide as set forth in any one of SEQ ID NOs 1 to 8, a cephalosporin and a beta-lactamase inhibitor in the manufacture of an antibacterial agent for the treatment of an E.coli or Listeria monocytogenes infection.
In some embodiments of the invention, there is provided the use of a composition comprising a polypeptide as set forth in SEQ ID NO. 7, a cephalosporin and a beta-lactamase inhibitor in the manufacture of an antibacterial agent for the treatment of an E.coli or Listeria monocytogenes infection.
In some embodiments of the present invention, there is provided the use of a composition of a polypeptide as set forth in SEQ ID NO. 7, cefotaxime and sulbactam for the manufacture of an antibacterial agent for the treatment of E.coli infections.
In some embodiments of the present invention, there is provided the use of a composition of a polypeptide as set forth in SEQ ID NO. 7, cefotaxime and sulbactam for the manufacture of an antibacterial agent for the treatment of E.coli infection producing beta-lactamase.
In some embodiments of the present invention, there is provided the use of a composition of a polypeptide as set forth in SEQ ID NO. 7, cefotaxime sodium and sulbactam sodium for the manufacture of an antibacterial agent for the treatment of E.coli infection producing beta-lactamase;
The mass ratio of the polypeptide to the cefotaxime sodium to the sulbactam sodium is 0.2:2:1.
In some embodiments of the invention, the antimicrobial agent comprises a pharmaceutically acceptable adjuvant.
The technical scheme of the invention has at least one technical effect selected from the following:
(1) The polypeptides of the invention have improved antibacterial activity;
(2) The polypeptide of the invention has no toxicity or low toxicity to mammalian cells;
(3) The polypeptide of the invention has strong antibacterial activity and has no toxicity or low toxicity to mammalian cells;
(4) The polypeptide molecule of the invention has simple preparation process and low cost;
(5) The composition of the polypeptide, the cephalosporin and the beta-lactamase has strong antibacterial activity;
(6) The composition of the polypeptide, the cephalosporin and the beta-lactamase has strong antibacterial activity on enzyme-producing drug-resistant bacteria;
(7) The polypeptide of the invention has strong activity for inhibiting escherichia coli or listeria monocytogenes, and unexpected technical effects are generated;
(8) The composition of the invention has strong activity for inhibiting the escherichia coli producing beta-lactamase, and has unexpected technical effects.
Detailed Description
As used herein, the term "β -lactam antibiotic" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salts of any chemical purity, polymorphs, solvates, hydrates.
As used herein, the term "cephalosporin" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salts of any chemical purity (e.g. sodium, potassium), polymorphs, solvates, hydrates.
As used herein, the term "β -lactamase inhibitor" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salts of any chemical purity (e.g., sodium, potassium), polymorphs, solvates, hydrates.
As used herein, the term "cefotaxime" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salts of any chemical purity (e.g., sodium, potassium salts), polymorphs, solvates, hydrates, active metabolites, prodrugs. In some embodiments, the cefotaxime in embodiments of the present invention may be cefotaxime sodium ((6R, 7R) -3- [ (acetoxy) methyl ] -7- [2- (2-aminothiazol-4-yl) -2- (methoxyimino) acetamido ] -8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid sodium salt).
As used herein, the term "sulbactam" includes not only the compound molecule itself, but also the free acid, pharmaceutically acceptable salts of any chemical purity (e.g., sodium, potassium), polymorphs, solvates, hydrates thereof. In some embodiments, sulbactam may be sulbactam acid ((2 s,5 r) -3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid-4, 4-dioxide), sulbactam sodium ((2 s,5 r) -3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid sodium-4, 4-dioxide), and the like.
In the following, some embodiments of the invention will be described in an exemplary embodiment, using specific language herein. However, it should be understood that these embodiments are not intended to limit the scope of the invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Examples
Example 1 sequence design and preparation of Polypeptides
In order to obtain high-activity antibacterial peptide, the inventor designs a series of novel polypeptides by taking the polypeptide with the sequence of SEQ ID NO. 9 as a reference, and the novel polypeptides are shown in a table 1.
TABLE 1 designed polypeptides
Peptides Amino acid sequence SEQ ID NO
Reference polypeptide RKRIHIGPGRAFYTT 9
Polypeptide 1 RKRIHIGPGFAFYTT 1
Polypeptide 2 KARKRIHIGPGRAFYTTN 2
Polypeptide 3 KARKRIHIGPGFAFYTTN 3
Polypeptide 4 RRKRIHIGPGRAFYTTT 4
Polypeptide 5 RRKRIHIGPGFAFYTTT 5
Polypeptide 6 RARKRIHIGPGRAFYTTQ 6
Polypeptide 7 RARKRIHIGPGFAFYTTQ 7
Polypeptide 8 RARKRIHIGPGFAFYTTRR 8
Reference polypeptides and crude products of polypeptides 1-8 were obtained by Fmoc solid phase synthesis using the Fmoc protected polypeptide C-terminal amino acid as starting material using literature methods (Xiong Shu. Application of biopeptides FK18 in nerve injury diseases and mechanism study [ D ]. Shanghai university of transportation, 2017.).
XBIridge BEH C18 OBD Prep Column using XBIridge5 Μm,30mmX50 mm) HPLC chromatography column, the mobile phase was phosphate aqueous solution and acetonitrile, the flow rate was 25mL/min, the column temperature was 30 ℃. Sample injection amount is 0.5mL, gradient elution is carried out, and acetonitrile is from 5% to 90%. Collecting the eluent, and freeze-drying to obtain the pure product of each polypeptide.
HPLC analysis determines (Xbridge C18 column, mobile phase is phosphate aqueous solution and acetonitrile) that the purity of reference polypeptide and polypeptide 1-8 is 98.1% -99.6%. ESI-MS results showed that the target polypeptide was of theoretical molecular weight.
EXAMPLE 2 antimicrobial Activity test of Polypeptides
A MH broth diluted test polypeptide solution (2-fold gradient dilution, concentration range of 0.0039. Mu.g/mL-256. Mu.g/mL) was prepared, and 100. Mu.L of each was added to a 96-well plate. The negative control group used an equal amount of MH broth. Bacteria in the logarithmic growth phase were collected, diluted in 10mM NaPB (pH 7.2-7.4) to give a bacterial suspension of about 1X 10 7 CFU/mL, and 1. Mu.L was added to the polypeptide solution. The cells were incubated at 37℃for 24 hours in the absence of light. 5 tests were performed in 96-well plates, with 3 replicates for each group in each test. In this example, the measured minimum inhibitory concentration MIC is represented by intervals [ a ] - [ b ], where [ a ] is the highest test concentration for macroscopic bacterial growth and [ b ] is the lowest concentration for no macroscopic bacterial growth. The results are shown in Table 2.
From the data in Table 2, it is clear that the antimicrobial activity of each of the polypeptides 1 to 8 was improved as compared with the reference polypeptide. Wherein the antibacterial activity of the polypeptide 7 is improved by more than 30 times, and the polypeptide has strong antibacterial activity on escherichia coli ampicillin resistant strain ML-35 p.
TABLE 2 antibacterial Activity test results of polypeptides
EXAMPLE 3 cytotoxicity test of Polypeptides
Cytotoxicity was measured on human hepatocytes (L-O2 cells). Reference CellTiterInstructions for the AQueous single solution cell proliferation assay kit (Promega) were run to assess cell viability. 100. Mu.L of L-O2 cells were seeded at 10 4 cells/well in 96-well plates and incubated at 37℃for 12h at 5% CO 2. Cells were then incubated at 37℃for 24h in medium supplemented with different concentrations of polypeptide (the control group was replaced with an equal amount of medium without added polypeptide), with 5 duplicate wells per concentration. After incubation, the cells were treated with 20uLMTS-PMS reagent for an additional 2h, and then absorbance at 490nm was measured. Relative cell viability (%) was calculated and the results were expressed as mean ± SD. As shown in table 3.
The results show that the polypeptide 8 has cytotoxicity at high concentration, and the toxicity of the polypeptide 8 is far greater than that of the control group (P < 0.01) at 100 mug/mL. While polypeptide 7 has no toxicity and good biocompatibility.
TABLE 3 cytotoxicity test results of polypeptides
Example 4 combination of Polypeptides with antibiotics
The MIC value of each polypeptide was determined by performing a drug sensitive assay on 24 clinically isolated beta-lactamase-producing E.coli cells according to the CLSI micro broth dilution method. The drug dilution concentration ranges from 0.0039 μg/mL to 512 μg/mL, and MIC is in the interval [ a ] to [ b ], wherein [ a ] is the highest test concentration at which bacterial growth is visible to the naked eye after 24 hours of incubation at 37 ℃, and [ b ] is the lowest concentration at which bacterial growth is not visible to the naked eye. Both cefotaxime and sulbactam are sodium salts, purchased from Sigma.
The results are shown in Table 4.
TABLE 4 test results of the combination of polypeptide and cefotaxime sulbactam
Although the invention has been disclosed with reference to specific embodiments, other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention, and the appended claims are to be construed as including all such embodiments and equivalents.

Claims (5)

1. A polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 8.
2. A composition comprising, as a main ingredient, the composition comprises the polypeptide of claim 1.
3. The composition of claim 2, wherein the composition comprises a β -lactam antibiotic or a β -lactamase inhibitor.
4. A composition according to claim 3, wherein the composition comprises cefotaxime and sulbactam.
5. Use of the polypeptide of claim 1 for the preparation of an antibacterial agent;
The antibacterial agent is used for treating infection caused by escherichia coli or listeria monocytogenes.
CN202410960351.0A 2024-03-06 2024-03-06 Polypeptides and combinations thereof with cefotaxime sulbactam Pending CN118702776A (en)

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CN118126132B (en) 2024-08-16
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