Polypeptides and their combination with cefotaxime and sulbactam

Abstract

This invention provides a polypeptide comprising the sequence shown in any one of SEQ ID NO:1-8. It also provides a composition comprising the polypeptide with the sequence shown in SEQ ID NO:7, cefotaxime, and sulbactam. The polypeptide with the sequence SEQ ID NO:7 exhibits strong inhibitory activity against *Escherichia coli* or *Listeria monocytogenes*. When used in combination with cefotaxime and sulbactam, this polypeptide exhibits strong inhibitory activity against β-lactamase-producing *Escherichia coli*. Therefore, the polypeptide and composition have significant 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 present invention, the polypeptide comprises the sequence shown 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 present invention, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:1-8.

[0021] In some embodiments of the present invention, the amino acid sequence of the polypeptide is shown in SEQ ID NO:7.

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

[0023] In a third aspect of the invention, a composition is provided, characterized in that the composition comprises a polypeptide as described in any one of the inventions.

[0024] In some embodiments of the present invention, the composition comprises a polypeptide, the amino acid sequence of which is shown in any one of SEQ ID NO:1-8.

[0025] 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.

[0026] In some embodiments of the present invention, the composition comprises a β-lactam antibiotic or a β-lactamase inhibitor.

[0027] In some embodiments of the present invention, the composition comprises a β-lactam antibiotic.

[0028] In some embodiments of the present invention, 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 invention, the use of the polypeptide described in any one of the invention in the preparation of an antibacterial agent is provided.

[0040] In some embodiments of the present invention, the antimicrobial agent is used to treat bacterial infections.

[0041] In some embodiments of the present invention, the antimicrobial agent is used to treat infections caused by *Escherichia coli* or *Listeria monocytogenes*.

[0042] In some embodiments of the present invention, the antimicrobial agent is used to treat infections caused by *Escherichia coli* ML-35p or *Listeria monocytogenes* EGDe.

[0043] In some embodiments of the present invention, the use of the polypeptide shown in any one of SEQ ID NO:1-8 is provided in the preparation of an antibacterial agent for treating Escherichia coli or Listeria monocytogenes infection.

[0044] In some embodiments of the present invention, the use of the polypeptide shown in SEQ ID NO:7 is provided in the preparation of an antibacterial agent for treating Escherichia coli or Listeria monocytogenes infections.

[0045] In some embodiments of the invention, the antimicrobial agent comprises pharmaceutically acceptable excipients.

[0046] In a fifth aspect of the invention, the use of the composition according to any one of the invention in the preparation of an antibacterial agent is provided.

[0047] In some embodiments of the present invention, the antimicrobial agent is used to treat bacterial infections.

[0048] In some embodiments of the present invention, the antimicrobial agent is used to treat infections caused by *Escherichia coli* or *Listeria monocytogenes*.

[0049] In some embodiments of the present invention, the antimicrobial agent is used to treat infections caused by *Escherichia coli* ML-35p or *Listeria monocytogenes* EGDe.

[0050] In some embodiments of the present invention, the antimicrobial agent is used to treat bacterial infections that produce β-lactamases.

[0051] In some embodiments of the present invention, the antibacterial agent is used to treat β-lactamase-producing Escherichia coli infections.

[0052] In some embodiments of the present invention, the use of compositions comprising any one of the polypeptides, cephalosporins and / or β-lactamase inhibitors shown in SEQ ID NO:1-8 in the preparation of antimicrobial agents for treating Escherichia coli or Listeria monocytogenes infections is provided.

[0053] In some embodiments of the present invention, the use of a composition comprising any one of the polypeptides, cephalosporins, and β-lactamase inhibitors shown in SEQ ID NO:1-8 in the preparation of an antimicrobial agent for treating Escherichia coli or Listeria monocytogenes infections is provided.

[0054] In some embodiments of the present invention, the use of a composition comprising the polypeptide shown in SEQ ID NO:7, a cephalosporin, and a β-lactamase inhibitor is provided in the preparation of an antimicrobial agent for treating Escherichia coli or Listeria monocytogenes infections is provided.

[0055] In some embodiments of the present invention, the use of a composition of the polypeptide shown in SEQ ID NO:7, cefotaxime, and sulbactam in the preparation of an antibacterial agent for treating Escherichia coli infection is provided.

[0056] In some embodiments of the present invention, the use of a composition of the polypeptide shown in SEQ ID NO:7, cefotaxime, and sulbactam in the preparation of an antibacterial agent for treating β-lactamase-producing Escherichia coli infections is provided.

[0057] In some embodiments of the present invention, the use of a composition of the polypeptide shown in SEQ ID NO:7, cefotaxime sodium, and sulbactam sodium in the preparation of an antibacterial agent for treating β-lactamase-producing Escherichia coli infections is provided.

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

[0059] In some embodiments of the invention, the antimicrobial agent comprises pharmaceutically acceptable excipients.

[0060] The technical solution of the present invention has at least one of the following technical effects:

[0061] (1) The polypeptides of the present invention have improved antibacterial activity;

[0062] (2) The polypeptides of the present invention are non-toxic or have very low toxicity to mammalian cells;

[0063] (3) The polypeptides of the present invention have strong antibacterial activity and are non-toxic or have very low toxicity to mammalian cells.

[0064] (4) The preparation process of the polypeptide molecules of the present invention is simple and low in cost;

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

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

[0067] (7) The polypeptides of the present invention have strong inhibitory activity against Escherichia coli or Listeria monocytogenes, producing unexpected technical effects.

[0068] (8) The composition of the present invention has a strong inhibitory effect on the activity of Escherichia coli that produces β-lactamase, resulting in unexpected technical effects. Detailed Implementation

[0069] As used herein, the term “β-lactam antibiotics” includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salt of any chemical purity, polymorph, solvate, and hydrate.

[0070] 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 salts, potassium salts), polymorphs, solvates, and hydrates.

[0071] 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 or potassium salts), polymorphs, solvates, and hydrates.

[0072] As used herein, the term "cefotaxime" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salts (e.g., sodium and potassium salts) of any chemical purity, polymorphs, solvates, hydrates, active metabolites, and prodrugs. In some specific embodiments, cefotaxime in the embodiments of the present invention may be cefotaxime sodium ((6R,7R)-3-[(acetoxy)methyl]-7-[2-(2-aminothiazolyl-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 "sulbactam" includes not only the compound molecule itself, but also its free acid, pharmaceutically acceptable salts of any chemical purity (e.g., sodium salts, potassium salts), polymorphs, solvates, and hydrates. In some specific embodiments, sulbactam in the embodiments of the present invention may be sulbactamic acid ((2S,5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid-4,4-dioxide), sulbactam sodium ((2S,5R)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid sodium-4,4-dioxide), etc.

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

[0075] Example

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

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

[0078] Table 1. Designed peptides

[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 Shuyu. Application and mechanism study of bioactive peptide FK18 in neurological injury diseases [D]. Shanghai Jiaotong University, 2017.), the reference peptide and crude peptides 1-8 were obtained by Fmoc solid-phase synthesis, using the C-terminal amino acids of the peptide protected by Fmoc as starting materials.

[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 per well in 96-well plates and incubated at 37°C and 5% CO2 for 12 h. Then, cells were incubated at 37°C for 24 h in medium supplemented with different concentrations of peptides (the control group was replaced with an equal volume of medium without peptides), with five replicates for each concentration. After incubation, cells were treated with 20 μL LMTS-PMS reagent for 2 h, and then the absorbance at 490 nm was measured. Relative cell viability (%) was calculated, and results are expressed as mean ± SD. See Table 3.

[0090] The results showed that peptide 8 was cytotoxic at high concentrations, with its toxicity at 100 μg / mL being significantly greater than that of the control group (P<0.01). Peptide 7, on the other hand, was non-toxic and exhibited good biocompatibility.

[0091] Table 3. Results of peptide cytotoxicity tests

[0092]

[0093]

[0094] Example 4. Combination of peptides and antibiotics

[0095] Antimicrobial susceptibility testing was performed on 24 clinically isolated β-lactamase-producing *Escherichia coli* strains using the CLSI microbroth dilution method, and the MIC value of each peptide was determined. The drug dilution concentration ranged from 0.0039 μg / mL to 512 μg / mL, and the MIC was defined as the interval [a]-[b], where [a] is the highest concentration at which visible bacterial growth was observed after 24 hours of incubation at 37°C, and [b] is the lowest concentration at which no visible bacterial growth was observed. Cefotaxime and sulbactam were sodium salts purchased from Sigma.

[0096] The results are shown in Table 4.

[0097] Table 4. Test results of the combination of peptide and cefotaxime / sulbactam

[0098]

[0099] Although the invention has been disclosed with reference to specific embodiments, other embodiments and variations of the invention can be devised by those skilled in the art without departing from the true spirit and scope of the invention, 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 in SEQ ID NO:

6.

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

3. The antibacterial composition according to claim 2, characterized in that, The antibacterial composition contains β-lactam antibiotics or β-lactamase inhibitors.

4. The antibacterial composition according to claim 3, characterized in that, The antibacterial composition contains cefotaxime and sulbactam.

5. The use of the polypeptide as described in claim 1 in the preparation of antibacterial drugs; The antibiotic is used to treat infections caused by Escherichia coli ML-35p or Listeria monocytogenes EGDe.

Images