CN118666968A - Polypeptides and combinations thereof with cefotaxime sulbactam - Google Patents

Abstract

The present invention provides a polypeptide, the polypeptide comprising a sequence shown in any one of SEQ ID NO:1-8. A composition is also provided, which comprises a polypeptide having a sequence shown in SEQ ID NO:7, cefotaxime and sulbactam. The polypeptide having a sequence of SEQ ID NO:7 has a strong activity in inhibiting Escherichia coli or Listeria monocytogenes. After the polypeptide is used in combination with cefotaxime and sulbactam, it has a strong activity in inhibiting β-lactamase-producing Escherichia coli. Therefore, the polypeptide and the composition have important clinical value.

Description

Peptide and combination thereof with ceftriaxone and sulbactam

This application is a divisional application of a patent application with an application date of March 6, 2024, an application number of 202410253895.3, and an invention name of “Polypeptide and its combination with cefotaxime sulbactam”.

Technical Field

The present invention relates to the field of biomedicine, and in particular to a polypeptide and a combination thereof with an antibiotic.

Background Art

Antimicrobial peptides are a series of polypeptides with antimicrobial activity. Such polypeptides play an important role in the innate immunity of mammals against invasive bacterial infections. Antimicrobial peptides have a wide range of lengths, ranging from 12 to 80 amino acid residues. Their structures are also varied.

Although antimicrobial peptides have antibacterial activity, they usually also have strong cytotoxicity, for example, they can cause red blood cell lysis, which leads to certain limitations in their practical applications.

Cefotaxime is a third-generation cephalosporin antibiotic, and many medicines are made with it as the main ingredient. Cefotaxime is suitable for respiratory tract infections, urinary tract infections, gastrointestinal infections, meningitis, sepsis, soft tissue infections, ENT infections, reproductive tract infections, orthopedic infections, etc. caused by sensitive bacteria. Cefotaxime sodium can also be used as the first choice drug for meningitis, especially meningitis in infants and young children.

The incidence of side effects of cefotaxime is as high as 3-5%, rash and drug fever account for about 2-5%, gastrointestinal reactions such as phlebitis, diarrhea, nausea, vomiting, loss of appetite, etc. account for about 1%, mild elevation of alkaline phosphatase or serum transaminase accounts for about 3%, temporary increase of blood urea nitrogen and creatinine accounts for 0.7% and 0.3% respectively, and leukopenia, acidosis or thrombocytopenia can be seen.

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

Sulbactam sodium is a semi-synthetic β-lactamase inhibitor. The incidence of injection site pain is about 3.6%, phlebitis, diarrhea, nausea and other reactions occasionally occur, and the incidence of rash is 1% to 6%. Exfoliative dermatitis and anaphylactic shock occur in very rare cases. Patients with impaired renal function need to extend the dosing interval and reduce the frequency of dosing.

Summary of the invention

Currently, there is an urgent need in clinical practice for antimicrobial drugs with strong antimicrobial activity and high safety. There is also an urgent need to reduce the dosage of antibiotics and solve the problem of antibiotic resistance.

The inventors designed and identified antimicrobial peptides with high activity and good safety through a large number of experimental explorations, thus completing the present invention.

In a first aspect of the present invention, a polypeptide is provided, characterized in that the polypeptide comprises a sequence shown in any one of SEQ ID NOs: 1-8, and the length of the amino acid sequence of the polypeptide is 18-25 amino acids.

In some embodiments of the present invention, the polypeptide comprises the sequence 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.

In some embodiments of the present invention, the polypeptide comprises the sequence 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.

In some embodiments of the present invention, the polypeptide comprises the sequence 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.

In some embodiments of the present invention, the polypeptide comprises the sequence 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.

In some embodiments of the present invention, the polypeptide comprises the sequence 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.

In some embodiments of the present invention, the polypeptide comprises the sequence 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.

In some embodiments of the present invention, the polypeptide comprises the sequence 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.

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.

In some embodiments of the present invention, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 1-8.

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

In a second aspect of the present invention, a polypeptide is provided, wherein the amino acid sequence of the polypeptide is as shown in SEQ ID NO:7.

In the third aspect of the present invention, a composition is provided, characterized in that the composition comprises the polypeptide described in any one of the present invention.

In some embodiments of the present invention, the composition comprises a polypeptide, and the amino acid sequence of the polypeptide is shown in any one of SEQ ID NOs: 1-8.

In some embodiments of the present invention, the composition comprises a polypeptide, and the amino acid sequence of the polypeptide is 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 a cephalosporin.

In some embodiments of the present invention, the cephalosporin is selected from cefuroxime, cefoperazone, cefuroxime, cefuroxime, cefuroxime, cefotaxime, cefotaxime, cefazolin, cefuroxime, cefuroxime, cefuroxime, cefotax ... Spodopime, Cefditoren, Ceftibuten, Ceftiofur, Ceftiolin, Cefazolin, Cefazolidin, Ceftriaxone, Ceftazidime, Cefpiramide, Cefsulodin, Latamoxef, Cefclidine, Cefepime, Cefuroxime, Cefazolin, Ceftriaxone, Cefpirome, Cefquinome, Fluoxetine, Cefclozine, Cefdrol, Cefoperazone, Cefoperazone, Cefoperando, Cefaclan, Cefdrolol, Cefoperazone, Ceftriaxone, Ceftriaxone, Cefmatilen, Cefmepidium, Cefovecin, Cefoxazole, Cefuroxime, Cefuroxime, Cefsumaquine, Ceftioxime, Ceftobiprole, Ceftobiprole, Cefuroxime.

In some embodiments of the invention, the composition comprises a β-lactamase inhibitor.

In some embodiments of the present invention, the β-lactamase inhibitor is 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 present invention, the composition comprises cefotaxime and sulbactam, and the mass ratio of the polypeptide, cefotaxime and sulbactam is 0.2:2:1.

In some embodiments of the present invention, the composition comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 7;

The composition comprises ceftriaxone sodium and sulbactam sodium;

The mass ratio of the polypeptide, ceftriaxone sodium and sulbactam sodium is 0.2:2:1.

In some embodiments of the present invention, the composition comprises a pharmaceutically acceptable excipient.

In a fourth aspect of the present invention, there is provided use of any one of the polypeptides of the present invention in the preparation of antibacterial drugs.

In some embodiments of the invention, the antibacterial drug is used to treat bacterial infection.

In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Escherichia coli or Listeria monocytogenes. In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Escherichia coli. In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Listeria monocytogenes.

In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Escherichia coli ML-35p or Listeria monocytogenes EGDe. In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Escherichia coli ML-35p. In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Listeria monocytogenes EGDe.

In some embodiments of the present invention, there is provided a use of a polypeptide shown in any one of SEQ ID NOs: 1-8 in the preparation of an antibacterial drug for treating Escherichia coli or Listeria monocytogenes infection.

In some embodiments of the present invention, there is provided use of the polypeptide shown in SEQ ID NO: 7 in the preparation of an antibacterial drug for treating Escherichia coli or Listeria monocytogenes infection.

In some embodiments of the present invention, the antibacterial drug comprises a pharmaceutically acceptable excipient.

In the fifth aspect of the present invention, there is provided use of the composition described in any one of the present invention in the preparation of an antibacterial drug.

In some embodiments of the invention, the antibacterial drug is used to treat bacterial infection.

In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Escherichia coli or Listeria monocytogenes. In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Escherichia coli. In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Listeria monocytogenes.

In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Escherichia coli ML-35p or Listeria monocytogenes EGDe. In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Escherichia coli ML-35p. In some embodiments of the invention, the antibacterial agent is used to treat infections caused by Listeria monocytogenes EGDe.

In some embodiments of the invention, the antibacterial drug is used to treat β-lactamase-producing bacterial infections.

In some embodiments of the present invention, the antibacterial drug is used to treat β-lactamase-producing Escherichia coli infection.

In some embodiments of the present invention, there is provided a use of a composition comprising a polypeptide as shown in any one of SEQ ID NOs: 1-8, a cephalosporin and/or a β-lactamase inhibitor in the preparation of an antibacterial drug for treating Escherichia coli or Listeria monocytogenes infection.

In some embodiments of the present invention, there is provided a use of a composition comprising a polypeptide as shown in any one of SEQ ID NOs: 1-8, a cephalosporin and a β-lactamase inhibitor in the preparation of an antibacterial drug for treating Escherichia coli or Listeria monocytogenes infection.

In some embodiments of the present invention, there is provided use of a composition comprising a polypeptide as shown in SEQ ID NO: 7, a cephalosporin and a β-lactamase inhibitor in the preparation of an antibacterial drug for treating Escherichia coli or Listeria monocytogenes infection.

In some embodiments of the present invention, there is provided use of a combination of a polypeptide as shown in SEQ ID NO: 7, cefotaxime and sulbactam in the preparation of an antibacterial drug for treating Escherichia coli infection.

In some embodiments of the present invention, there is provided use of a combination of a polypeptide as shown in SEQ ID NO: 7, cefotaxime and sulbactam in the preparation of an antibacterial drug for treating β-lactamase-producing Escherichia coli infection.

In some embodiments of the present invention, there is provided a use of a combination of a polypeptide as shown in SEQ ID NO: 7, ceftriaxone sodium and sulbactam sodium in the preparation of an antibacterial drug for treating β-lactamase-producing Escherichia coli infection;

The mass ratio of the polypeptide, ceftriaxone sodium and sulbactam sodium is 0.2:2:1.

In some embodiments of the present invention, the antibacterial drug comprises a pharmaceutically acceptable excipient.

The technical solution of the present invention has at least one technical effect selected from the following:

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

(2) The polypeptides of the present invention have no toxicity or very low toxicity to mammalian cells;

(3) The polypeptide of the present invention has strong antibacterial activity and is non-toxic or has very low toxicity to mammalian cells;

(4) The preparation process of the polypeptide molecule of the present invention is simple and low-cost;

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

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

(7) The polypeptide of the present invention has strong activity in inhibiting Escherichia coli or Listeria monocytogenes, producing unexpected technical effects;

(8) The composition of the present invention has strong activity in inhibiting β-lactamase-producing Escherichia coli, producing unexpected technical effects.

DETAILED DESCRIPTION

As used herein, the term "β-lactam antibiotics" 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 (eg, sodium salt, potassium salt) of any chemical purity, 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 (eg, sodium salt, potassium salt), polymorphs, solvates, and 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 salt, potassium salt), polymorphs, solvates, hydrates, active metabolites, and prodrugs. In some specific embodiments, cefotaxime in the embodiments of the present invention can be cefotaxime sodium ((6R, 7R)-3-[(acetyloxy)methyl]-7-[2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetylamino]-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 its free acid, pharmaceutically acceptable salts of any chemical purity (e.g., sodium salt, potassium salt), polymorphs, solvates, and hydrates. In some specific embodiments, sulbactam in the embodiments of the present invention may be sulbactam 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), and the like.

Below, some schemes of the present invention will be described in this article using specific language in exemplary embodiments. However, it should be understood that these embodiments are not intended to limit the scope of the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used, unless otherwise specified, can all be reagents and materials obtained from commercial sources.

Example

Example 1. Sequence design and preparation of polypeptide

In order to obtain highly active antimicrobial peptides, the inventors designed a series of new peptides based on the polypeptide with the sequence of SEQ ID NO: 9 as a reference, as shown in Table 1.

Table 1. Designed peptides

Peptides Amino acid sequence SEQ ID NO Reference peptide RKRIHIGPGRAFYTT 9 Peptide 1 RKRIHIGPGFAFYTT 1 Peptide 2 KARKRIHIGPGRAFYTTN 2 Peptide 3 KARKRIHIGPGFAFYTTN 3 Peptide 4 RRKRIHIGPGRAFYTTT 4 Peptide 5 RRKRIHIGPGFAFYTTT 5 Peptide 6 RARKRIHIGPGRAFYTTQ 6 Peptide 7 RARKRIHIGPGFAFYTTQ 7 Peptide 8 RARKRIHIGPGFAFYTTRR 8

Using the literature method (Xiong Shuyu. Application and mechanism research of biological peptide FK18 in neurological damage diseases [D]. Shanghai Jiaotong University, 2017.), the Fmoc-protected C-terminal amino acid of the peptide was used as the starting material, and the crude products of the reference peptide and peptides 1-8 were obtained by Fmoc solid phase synthesis.

Using XBridge BEH C18 OBD Prep Column( 5μm, 30mmX50 mm) HPLC column was used for purification, the mobile phase was phosphate aqueous solution and acetonitrile, the flow rate was 25mL/min, the column temperature was 30℃. The injection volume was 0.5mL, and the gradient elution was from 5% to 90% acetonitrile. The eluate was collected and freeze-dried to obtain the pure products of each polypeptide.

HPLC analysis (Xbridge C18 column, mobile phase phosphate aqueous solution and acetonitrile) showed that the purity of the reference peptide and peptides 1-8 was 98.1%-99.6%. ESI-MS results showed that they were the target peptides, which were consistent with the theoretical molecular weight.

Example 2. Antibacterial activity test of polypeptide

Prepare the peptide solution to be tested diluted with MH broth (2-fold gradient dilution, concentration range of 0.0039μg/mL-256μg/mL), take 100μL each and add it to a 96-well plate. The negative control group uses an equal amount of MH broth. Collect bacteria in the logarithmic growth phase, dilute in 10mM NaPB (pH 7.2–7.4) to obtain a bacterial suspension of about 1×10 ⁷ CFU/mL, and then take 1μL and add it to the peptide solution. Incubate at 37°C for 24 hours under light-proof conditions. Five tests were performed in a 96-well plate, with three replicates in each group in each test. In this example, the measured minimum inhibitory concentration MIC is expressed as an interval [a]-[b], where [a] is the highest test concentration at which bacterial growth is visible to the naked eye, and [b] is the lowest concentration at which no bacterial growth is visible to the naked eye. The results are shown in Table 2.

From the data in Table 2, it can be seen that the antibacterial activity of polypeptides 1 to 8 is improved compared with the reference polypeptides. Among them, the antibacterial activity of polypeptide 7 is increased by more than 30 times, and it has strong antibacterial activity against the ampicillin-resistant strain ML-35p of Escherichia coli.

Table 2. Antibacterial activity test results of peptides

Example 3. Cytotoxicity test of polypeptide

Cytotoxicity was measured on human hepatocytes (L-O2 cells). The AQueous single solution cell proliferation detection kit (Promega) was operated according to the instructions for evaluating cell viability. 100 μL of L-O2 cells were seeded in a 96-well plate at 10 ⁴ cells/well and incubated at 37°C, 5% CO ₂ for 12 h. Then, the cells were incubated at 37°C for 24 h in a medium supplemented with different concentrations of polypeptides (the control group was replaced with an equal amount of medium without polypeptides), and 5 replicate wells were set for each concentration. After incubation, the cells were treated with 20uLMTS-PMS reagent for another 2 h, and then the absorbance value at 490 nm was measured. The relative cell viability (%) was calculated, and the results are expressed as mean ± SD. As shown in Table 3.

The results showed that peptide 8 was cytotoxic at high concentrations, and the toxicity of peptide 8 was much greater than that of the control group at 100 μg/mL (P<0.01). Peptide 7 was non-toxic and had good biocompatibility.

Table 3. Cytotoxicity test results of peptides

Example 4. Combination of polypeptides and antibiotics

The CLSI broth microdilution method was used to test the drug susceptibility of 24 clinically isolated β-lactamase-producing Escherichia coli, 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 in the interval [a]-[b], where [a] was the highest test concentration at which bacterial growth was visible to the naked eye after 24 hours of incubation at 37°C, and [b] was the lowest concentration at which no bacterial growth was visible to the naked eye. Both cefotaxime and sulbactam were sodium salts purchased from Sigma.

The results are shown in Table 4.

Table 4. Test results of peptides combined with cefotaxime and sulbactam

Although the invention has been disclosed with reference to specific embodiments, other embodiments and variations of the invention may be designed by others skilled in the art without departing from the true spirit and scope of the invention, and the appended claims are intended to be interpreted as including all such embodiments and equivalent variations.

Claims (5)

1. A polypeptide is characterized in that the amino acid sequence of the polypeptide is shown as SEQ ID NO. 4.

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.