Polypeptides and their combination with cefotaxime and sulbactam

Abstract

The present application provides a kind of polypeptide, the polypeptide includes the sequence shown in any one of SEQ ID NO:1-8.The present application also provides a kind of composition, which includes the polypeptide of the sequence shown in SEQ ID NO:7, cefotaxime and sulbactam.The polypeptide of sequence SEQ ID NO:7 is strong in activity for inhibiting escherichia coli or listeria monocytogenes.The polypeptide is combined with cefotaxime sulbactam, and is strong in activity for inhibiting escherichia coli producing beta-lactamase.Therefore the polypeptide and composition have important clinical value.

Description

[0001] This application is a divisional application of the patent application with the application number 202410253895.3, the application date of 2024.03.06, and the invention title of "Polypeptide and its combination with cefotaxime sulbactam". TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to a polypeptide and its combination with an antibiotic. BACKGROUND

[0003] Antibacterial peptides are a series of polypeptides with antibacterial activity. Such polypeptides play an important role in the innate immunity of mammals against invasive bacterial infection. The length of antibacterial peptides ranges widely, generally from 12 to 80 amino acid residues. Their structures are also diverse.

[0004] Although antibacterial peptides have antibacterial activity, they usually have strong cytotoxicity, such as causing red blood cell lysis, which limits their practical application to some extent.

[0005] Cefotaxime is a third-generation cephalosporin antibacterial drug. Many drugs are made with cefotaxime as the main ingredient. Cefotaxime is suitable for respiratory tract infections, urinary tract infections, gastrointestinal infections, meningitis, septicemia, soft tissue infections, ear-nose-throat infections, genital tract infections, orthopedic infections, etc. caused by sensitive bacteria. Cefotaxime sodium can also be used as the first choice for meningitis, especially infantile meningitis.

[0006] The incidence of side effects of cefotaxime is 3-5%, skin rash and drug fever accounts for about 2-5%, phlebitis, diarrhea, nausea, vomiting, loss of appetite and other digestive tract reactions account for about 1%, about 3% of patients have mild increases in alkaline phosphatase or serum transaminase, and 0.7% and 0.3% of patients have temporary increases in blood urea nitrogen and creatinine, respectively. Leukopenia, acidophilic granulocytosis or thrombocytopenia can be seen.

[0007] In addition, patients with severe renal function decline should appropriately reduce the dosage of cefotaxime. When the serum creatinine value exceeds 424 μmol / L (4.8 mg) or the creatinine clearance rate is less than 20 ml / min, the maintenance dose of cefotaxime should be halved; when the serum creatinine exceeds 751 μmol / L (8.5 mg), the maintenance dose is 1 / 4 of the normal dose.

[0008] Sulbactam sodium is a semisynthetic β-lactamase inhibitor. The incidence of injection site pain is about 3.6%, and reactions such as phlebitis, diarrhea, and nausea occasionally occur, with a skin rash incidence of 1% to 6%. In very rare cases, exfoliative dermatitis and anaphylactic shock occur. Patients with reduced renal function need to extend the dosing interval and reduce the dosing frequency. SUMMARY

[0009] There is an urgent need for antibacterial drugs with strong antibacterial activity and high safety in the clinic, and there is also an urgent need to reduce the amount of antibiotics used and solve the problem of antibiotic resistance.

[0010] The inventors have designed and identified antibacterial peptides with high activity and good safety through a large number of experimental explorations, and thus completed the present application.

[0011] In a first aspect of the present application, a polypeptide is provided, characterized in that the polypeptide comprises a sequence as shown in any one of SEQ ID NO: 1-8, and the length of the amino acid sequence of the polypeptide is 18-25 amino acids.

[0012] In some embodiments of the present application, the polypeptide comprises a sequence as shown in SEQ ID NO: 1, and the length of the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids.

[0013] In some embodiments of the present application, the polypeptide comprises a sequence as shown in SEQ ID NO: 2, and the length of the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids.

[0014] In some embodiments of the present application, the polypeptide comprises a sequence as shown in SEQ ID NO: 3, and the length of the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids.

[0015] In some embodiments of the present application, the polypeptide comprises a sequence as shown in SEQ ID NO: 4, and the length of the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids.

[0016] In some embodiments of the present application, the polypeptide comprises a sequence as shown in SEQ ID NO: 5, and the length of the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids.

[0017] In some embodiments of the present application, the polypeptide comprises a sequence as shown in SEQ ID NO: 6, and the length of the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids.

[0018] In some embodiments of the present application, the polypeptide comprises a sequence as shown in SEQ ID NO: 7, and the length of the amino acid sequence of the polypeptide is 18-25, 18-20 or 18 amino acids.

[0019] In some embodiments of the application, the polypeptide comprises the sequence set forth in SEQ ID NO: 8, and the length of the amino acid sequence of the polypeptide is 18-25, 18-20, or 18 amino acids.

[0020] In some embodiments of the application, the amino acid sequence of the polypeptide is set forth in any one of SEQ ID NOs: 1-8.

[0021] In some embodiments of the application, the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 7.

[0022] In a second aspect of the application, there is provided a polypeptide, characterized in that the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 7.

[0023] In a third aspect of the application, there is provided a composition, characterized in that the composition comprises a polypeptide according to any one of the application.

[0024] In some embodiments of the application, the composition comprises a polypeptide, and the amino acid sequence of the polypeptide is set forth in any one of SEQ ID NOs: 1-8.

[0025] In some embodiments of the application, the composition comprises a polypeptide, and the amino acid sequence of the polypeptide is set forth in SEQ ID NO: 7.

[0026] In some embodiments of the application, the composition comprises a beta-lactam antibiotic or a beta-lactamase inhibitor.

[0027] In some embodiments of the application, the composition comprises a beta-lactam antibiotic.

[0028] In some embodiments of the application, the composition comprises a cephalosporin.

[0029] In some embodiments of the present invention, the cephalosporin is optionally selected from cefoperazone, cefotaxime, cefotaxime, cefotaxime, cefotaxime, cefospirin, cefotracin, cefotaxime, cefoxitin, cefoxitin, cefotaxime, cefoxitin, cefotaxime, cefotaxime, cefotiazole, cefotaxime, cefotaxime, cefotiam, cefoproxetine, cefotiam, cefotiam, cefotaxime ... Cefpodoxime, Cefterenol, Cefbuprofen, Ceftiofur, Cefthiarin, Cefazolin, Ceftriaxone, Ceftazidime, Cefpirome, Cefsulfuron-methyl, Latamoxef, Cefclidine, Cefepime, Cefrenaline, Ceftiofur, Cefazolin, Cefpirome, Cefquinoxime, Cefoxycephalosporin, Ceflorazine, Cefloren, Cefparo, Cefcanena, Cefdrolone, Cefpyridamole, Ceftriazole, Cefvetriazole, Cefmatilen, Cefmepidium, Cefovecin, Cefoxazole, Cefrotitol, Cefsuccinylcholine, Ceftobiprole, Ceftobiprole, Ceffuran.

[0030] In some embodiments of the present invention, the composition comprises a β-lactamase inhibitor.

[0031] In some embodiments of the present invention, the β-lactamase inhibitor is optionally selected from clavulanic acid, sulbactam, and tazobactam.

[0032] In some embodiments of the present invention, the composition comprises a β-lactam antibiotic and a β-lactamase inhibitor.

[0033] In some embodiments of the present invention, the composition comprises cefotaxime and sulbactam.

[0034] In some embodiments of the present invention, the composition comprises cefotaxime and sulbactam, wherein the mass ratio of the polypeptide, cefotaxime, and sulbactam is 0.2:2:1.

[0035] In some embodiments of the present invention, the composition comprises a polypeptide, the amino acid sequence of which is shown in SEQ ID NO:7;

[0036] The composition comprises cefotaxime sodium and sulbactam sodium;

[0037] The mass ratio of the polypeptide, cefotaxime sodium, and sulbactam sodium is 0.2:2:1.

[0038] In some embodiments of the invention, the composition comprises pharmaceutically acceptable excipients.

[0039] In a fourth aspect of the application, there is provided use of a polypeptide according to any one of the preceding items in the manufacture of an antibacterial agent.

[0040] In some embodiments of the application, the antibacterial agent is for use in the treatment of a bacterial infection.

[0041] In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by E. coli or L. monocytogenes. In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by E. coli. In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by L. monocytogenes.

[0042] In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by E. coli ML-35p or L. monocytogenes EGDe. In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by E. coli ML-35p. In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by L. monocytogenes EGDe.

[0043] In some embodiments of the application, there is provided use of a polypeptide according to any one of SEQ ID NOs: 1-8 in the manufacture of an antibacterial agent for the treatment of an infection caused by E. coli or L. monocytogenes.

[0044] In some embodiments of the application, there is provided use of a polypeptide according to SEQ ID NO: 7 in the manufacture of an antibacterial agent for the treatment of an infection caused by E. coli or L. monocytogenes.

[0045] In some embodiments of the application, the antibacterial agent comprises a pharmaceutically acceptable excipient.

[0046] In a fifth aspect of the application, there is provided use of a composition according to any one of the preceding items in the manufacture of an antibacterial agent.

[0047] In some embodiments of the application, the antibacterial agent is for use in the treatment of a bacterial infection.

[0048] In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by E. coli or L. monocytogenes. In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by E. coli. In some embodiments of the application, the antibacterial agent is for use in the treatment of an infection caused by L. monocytogenes.

[0049] In some embodiments of the present application, the antibacterial agent is used for treating infection caused by E. coli ML-35p or L. monocytogenes EGDe. In some embodiments of the present application, the antibacterial agent is used for treating infection caused by E. coli ML-35p. In some embodiments of the present application, the antibacterial agent is used for treating infection caused by L. monocytogenes EGDe.

[0050] In some embodiments of the present application, the antibacterial agent is used for treating infection caused by β-lactamase producing bacteria.

[0051] In some embodiments of the present application, the antibacterial agent is used for treating infection caused by β-lactamase producing bacteria.

[0052] In some embodiments of the present application, there is provided use of a composition comprising a polypeptide as set forth in any one of SEQ ID NOs: 1-8, a cephalosporin and / or a β-lactamase inhibitor in the preparation of an antibacterial agent for treating infection caused by E. coli or L. monocytogenes.

[0053] In some embodiments of the present application, there is provided use of a composition comprising a polypeptide as set forth in any one of SEQ ID NOs: 1-8, a cephalosporin and a β-lactamase inhibitor in the preparation of an antibacterial agent for treating infection caused by E. coli or L. monocytogenes.

[0054] In some embodiments of the present application, there is provided use of a composition comprising a polypeptide as set forth in SEQ ID NO: 7, a cephalosporin and a β-lactamase inhibitor in the preparation of an antibacterial agent for treating infection caused by E. coli or L. monocytogenes.

[0055] In some embodiments of the present application, there is provided use of a composition comprising a polypeptide as set forth in SEQ ID NO: 7, cefotaxime and sulbactam in the preparation of an antibacterial agent for treating infection caused by E. coli.

[0056] In some embodiments of the present application, there is provided use of a composition comprising a polypeptide as set forth in SEQ ID NO: 7, cefotaxime and sulbactam in the preparation of an antibacterial agent for treating infection caused by β-lactamase producing E. coli.

[0057] In some embodiments of the present application, there is provided use of a composition comprising a polypeptide as set forth in SEQ ID NO: 7, cefotaxime sodium and sulbactam sodium in the preparation of an antibacterial agent for treating infection caused by β-lactamase producing E. coli.

[0058] The mass ratio of the polypeptide, cefotaxime sodium, sulbactam sodium is 0.2:2:1.

[0059] In some embodiments of the present application, the antibacterial agent comprises a pharmaceutically acceptable excipient.

[0060] The technical solutions of the present application have at least one of the following technical effects:

[0061] (1) The polypeptide of the present application has improved antibacterial activity;

[0062] (2) The polypeptide of the present application has no or very low toxicity to mammalian cells;

[0063] (3) The polypeptide of the present application has strong antibacterial activity and no or very low toxicity to mammalian cells;

[0064] (4) The polypeptide molecule of the present application is simple to prepare and has low cost;

[0065] (5) The composition of the polypeptide + cephalosporin + β-lactamase of the present application has strong antibacterial activity;

[0066] (6) The composition of the polypeptide + cephalosporin + β-lactamase of the present application has strong antibacterial activity against enzyme-producing drug-resistant bacteria;

[0067] (7) The polypeptide of the present application has strong activity in inhibiting E. coli or Listeria monocytogenes, and unexpected technical effects are produced;

[0068] (8) The composition of the present application has strong activity in inhibiting β-lactamase-producing E. coli, and unexpected technical effects are produced. DETAILED DESCRIPTION

[0069] As used herein, the term "β-lactam antibiotic" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salt of any chemical purity (such as sodium salt, potassium salt), polymorph, solvate, hydrate.

[0070] As used herein, the term "cephalosporin" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salt of any chemical purity (such as sodium salt, potassium salt), polymorph, solvate, hydrate.

[0071] As used herein, the term "β-lactamase inhibitor" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salt of any chemical purity (such as sodium salt, potassium salt), polymorph, solvate, hydrate.

[0072] As used herein, the term "ceftazidime" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salts of any chemical purity (such as sodium salt, potassium salt), polymorphs, solvates, hydrates, active metabolites, prodrugs. In some specific embodiments, the ceftazidime in embodiments of the present application can be ceftazidime sodium ((6R,7R)-3-[(acetyloxy)methyl]-7-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate sodium salt).

[0073] As used herein, the term "ceftazidime" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salts of any chemical purity (such as sodium salt, potassium salt), polymorphs, solvates, hydrates, active metabolites, prodrugs. In some specific embodiments, the ceftazidime in embodiments of the present application can be ceftazidime sodium ((6R,7R)-3-[(acetyloxy)methyl]-7-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate sodium salt).

[0074] Some schemes of the present application will be described below in exemplary embodiments using specific language. However, it should be understood that these embodiments are not intended to limit the scope of the present application. The experimental methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

[0075] Examples

[0076] Example 1. Sequence design and preparation of polypeptides

[0077] To obtain highly active antibacterial peptides, the inventors designed a series of new polypeptides with reference to the polypeptide with the sequence of SEQ ID NO: 9, as shown in Table 1.

[0078] Table 1. Designed polypeptides

[0079] Peptide 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

[0080] Using the literature method (Xiong S. Application and mechanism of biological peptide FK18 in nerve injury diseases [D]. Shanghai Jiaotong University, 2017.), the crude reference polypeptide and polypeptides 1-8 were obtained by Fmoc solid-phase synthesis method using the Fmoc-protected C-terminal amino acid of the polypeptide as the starting material.

[0081] Using XBridge BEH C18 OBD Prep Column ( Purification was performed using a 5 μm (30 mm x 50 mm) HPLC column. The mobile phase consisted of phosphate aqueous solution and acetonitrile, with a flow rate of 25 mL / min and a column temperature of 30 °C. The injection volume was 0.5 mL, and gradient elution was used, with acetonitrile levels ranging from 5% to 90%. The eluent was collected, lyophilized, and the resulting peptides were purified.

[0082] HPLC analysis (Xbridge C18 column, mobile phase: phosphate aqueous solution and acetonitrile) determined the purity of the reference peptide and peptides 1-8 to be 98.1%-99.6%. ESI-MS results showed that the peptides were the target peptides and conformed to the theoretical molecular weight.

[0083] Example 2. Antibacterial activity test of peptides

[0084] Prepare MH broth solutions of the test peptides (serial dilutions of 2-fold, concentration range 0.0039 μg / mL–256 μg / mL), and add 100 μL to each well of a 96-well plate. Use an equal volume of MH broth for the negative control group. Collect bacteria in the logarithmic growth phase and dilute them in 10 mM NaPB (pH 7.2–7.4) to obtain approximately 1 × 10⁻⁶ ppm. 7 A bacterial suspension of CFU / mL was prepared, and 1 μL was added to the peptide solution. The mixture was incubated at 37°C for 24 hours in the dark. Five tests were performed in 96-well plates, with three replicates for each group in each experiment. In this embodiment, the measured minimum inhibitory concentration (MIC) is expressed as the interval [a]-[b], where [a] is the highest concentration at which visible bacterial growth was observed, and [b] is the lowest concentration at which no visible bacterial growth was observed. The results are shown in Table 2.

[0085] As shown in Table 2, peptides 1 through 8 exhibited improved antibacterial activity compared to the reference peptide. Peptide 7, in particular, showed more than a 30-fold increase in antibacterial activity and demonstrated strong inhibitory activity against the ampicillin-resistant strain ML-35p of *Escherichia coli*.

[0086] Table 2. Results of antibacterial activity test of peptides

[0087]

[0088] Example 3. Cytotoxicity test of peptides

[0089] Cytotoxicity was measured on human hepatocytes (L-O2 cells). Refer to CellTiter. The AQueous Single Solution Cell Proliferation Detection Kit (Promega) was used to assess cell viability. 100 μL of L-O2 cells were incubated at 10... 4Cells were seeded at 1 cell / well in 96-well plates and incubated at 37°C, 5% CO2 for 12 h. Then, cells were incubated in culture medium supplemented with different concentrations of polypeptides (control group replaced with equal amount of culture medium without polypeptides) at 37°C for 24 h, 5 replicates for each concentration. After incubation, cells were treated with 20 uL MTS-PMS reagent for 2 h, and then the absorbance value at 490 nm was measured. The relative cell viability (%) was calculated, and the results were expressed as mean ± SD. As shown in Table 3.

[0090] The results showed that polypeptide 8 had cytotoxicity at high concentrations, and the toxicity of polypeptide 8 at 100 μg / mL was much greater than that of the control group (P < 0.01). Polypeptide 7 had no toxicity and had good biocompatibility.

[0091] Table 3. Cytotoxicity test results of polypeptides

[0092]

[0093]

[0094] Example 4. Combination of polypeptides with antibiotics

[0095] The MIC values of each polypeptide were determined by CLSI microbroth dilution method for 24 clinical isolates of β-lactamase-producing Escherichia coli. The drug dilution concentration range was 0.0039 μg / mL-512 μg / mL, and the MIC was the interval [a]-[b], where [a] was the highest test concentration with visible bacterial growth after 24 hours of incubation at 37°C, and [b] was the lowest concentration without visible bacterial growth. Cefotaxime and sulbactam were both sodium salts, purchased from Sigma.

[0096] The results are shown in Table 4.

[0097] Table 4. Test results of polypeptide combination with cefotaxime and sulbactam

[0098]

[0099] Although the present application has been disclosed with reference to specific embodiments, other embodiments and variations of the present application can be devised by others skilled in the art without departing from the true spirit and scope of the present application, and the appended claims are intended to be construed as including all such embodiments and equivalent variations.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown as SEQ ID NO:

8.

2. A composition characterized in that, The composition comprises the polypeptide of claim 1.

3. The composition of claim 2, wherein The composition comprises a beta-lactam antibiotic or a beta-lactamase inhibitor.

4. The composition of claim 3, wherein The composition comprises cefotaxime and sulbactam.

5. Use of the polypeptide of claim 1 in the preparation of an antibacterial agent. The antibacterial agent is used for treating infection caused by E. coli ML-35p or L. monocytogenes EGDe.

Images