Use of antibacterial peptide BMAP-28 in combination with antibiotics for preparing antibacterial drugs

Abstract

The application discloses application of antibacterial peptide BMAP-28 and antibiotics in combined preparation of antibacterial drugs and belongs to the technical field of biological medicine. The antibiotics are selected from one or more of the following: ciprofloxacin, gentamicin and cefixime. The application combines the antibacterial peptide BMAP-28 with antibiotics such as ciprofloxacin and gentamicin, finds that the combination has a significant synergistic antibacterial effect on Enterobacter sakazii, can greatly save the dosage of the drug, reduce the toxic side effect and block the evolution process of drug resistance. Through the destruction of the bacterial cell membrane and the assistance of the antibiotic effect, the combination can effectively overcome the multiple drug resistance, widen the antibacterial spectrum and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the antimicrobial peptide BMAP-28 in the combined preparation of antimicrobial drugs with antibiotics. Background Technology

[0002] Since the discovery of penicillin in 1928, antibiotics, as the cornerstone of modern medicine, have made indelible contributions to treating bacterial infections, protecting human health, and promoting livestock development. However, in the past century, the overuse and abuse of antibiotics has triggered a global public health crisis—antibiotic resistance. The emergence and spread of multidrug-resistant bacteria and even "superbugs" have rendered many once-effective antibiotics ineffective, leading to prolonged treatment times, soaring medical costs, and increased risk of death for patients. The World Health Organization has listed antibiotic resistance as one of the most serious threats to human health in the 21st century.

[0003] The emergence of drug-resistant bacteria is the result of the combined effects of natural selection and antibiotic selection pressure. Bacteria acquire resistance genes through gene mutation or horizontal gene transfer and develop various complex resistance mechanisms, mainly including: ① producing antibiotic-inactivating or modifying enzymes, such as β-lactamases that hydrolyze penicillin and cephalosporin antibiotics; ② altering the target site of antibiotics, making them unable to recognize and bind to them, such as the resistance of methicillin-resistant Staphylococcus aureus to β-lactam antibiotics; ③ reducing cell membrane permeability to antibiotics or actively effluxing antibiotics, reducing drug accumulation within the bacteria; ④ forming biofilms. Biofilms are three-dimensional microbial communities encapsulated by extracellular polymers (such as polysaccharides, proteins, and nucleic acids) secreted by bacteria. Their physical barrier function and the presence of slow-metabolizing "persistent bacteria" can increase their resistance to antibiotics by 10-1000 times.

[0004] Faced with the increasingly severe situation of drug resistance, developing new antibacterial drugs is the fundamental solution. However, the development cycle of new antibiotics is long, the investment is large, and the success rate is low, far behind the rate at which drug-resistant bacteria emerge. Therefore, finding new anti-infection strategies is urgent. Among them, combination therapy, which utilizes the synergistic effect of two or more drugs with different mechanisms of action, is considered one of the effective means to combat drug resistance. The advantages of combination therapy are: ① By attacking multiple targets, it is difficult to induce bacteria to develop multiple drug resistance mechanisms simultaneously; ② It can reduce the dosage of single drugs, thereby reducing their toxic side effects and selective pressure; ③ It may restore the sensitivity of drug-resistant bacteria to the original antibiotics.

[0005] Antimicrobial peptides (AMPs), as an important component of the innate immune system, are considered one of the most promising antibiotic alternatives in the post-antibiotic era. Antimicrobial peptides typically consist of 12-50 amino acid residues, and their mechanism of action differs significantly from traditional antibiotics. Most antimicrobial peptides electrostatically adsorb onto the negatively charged bacterial cell membrane, disrupting membrane integrity through methods such as the "bucket-plate model," "carpet model," or "ring-pore model," leading to leakage of cell contents and bacterial death. This physical membrane-disrupting mechanism makes it difficult for bacteria to develop resistance. Furthermore, some antimicrobial peptides can penetrate the cell membrane and act on intracellular targets, such as inhibiting nucleic acid and protein synthesis or interfering with enzyme activity.

[0006] Bovine bone marrow antimicrobial peptide BMAP-28 is a member of the cathelicidin family, composed of 28 amino acid residues with a net positive charge of +7. Its secondary structure features an amphiphilic α-helix at the N-terminus (residues 1-17), a β-turn formed by proline-glycine (PG) in the middle (residues 18-19), and a hydrophobic sequence at the C-terminus (residues 20-28). BMAP-28 exhibits potent cytotoxic activity against Gram-positive bacteria, Gram-negative bacteria, fungi, and even tumor cells. Studies have shown that it can effectively treat Pseudomonas aeruginosa infection in mice at doses of 0.2-0.8 mg / kg, significantly improving survival rates.

[0007] However, the further application of BMAP-28 is limited by its low cell selectivity, high production cost, and stability in complex physiological environments. More importantly, despite the promising prospects of antimicrobial peptide monotherapy, its efficacy in vivo, particularly against deep infections and biofilm-associated infections, still faces challenges. Summary of the Invention

[0008] The purpose of this invention is to provide the application of the antimicrobial peptide BMAP-28 in the combined preparation of antimicrobial drugs with antibiotics, thereby addressing the problems existing in the prior art. This invention combines the antimicrobial peptide BMAP-28 with antibiotics such as ciprofloxacin and gentamicin, and found that it has a significant synergistic antimicrobial effect against *Cronobacter sakazakii*, which can significantly reduce drug dosage, decrease toxic side effects, and inhibit the evolution of drug resistance. By disrupting the bacterial cell membrane and enhancing the action of antibiotics, it can effectively overcome multidrug resistance, broaden the antimicrobial spectrum, and has broad prospects for clinical application.

[0009] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of the antimicrobial peptide BMAP-28 in the combined preparation of antimicrobial drugs with antibiotics, wherein the antibiotics are selected from one or more of ciprofloxacin, gentamicin, and cefixime.

[0010] Furthermore, the amino acid sequence of the antimicrobial peptide BMAP-28 is shown in SEQ ID NO.1.

[0011] Furthermore, the antimicrobial drug is used to treat infections caused by Gram-negative bacteria and / or Gram-positive bacteria.

[0012] Furthermore, the Gram-negative bacterium is Cronobacter sakazakii.

[0013] The present invention also provides an antibacterial drug, wherein the drug comprises an antimicrobial peptide BMAP-28 and an antibiotic as the active ingredient.

[0014] Furthermore, the amino acid sequence of the antimicrobial peptide BMAP-28 is shown in SEQ ID NO.1.

[0015] Furthermore, the antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

[0016] Furthermore, the drug also contains a pharmaceutically acceptable carrier or excipient.

[0017] The present invention discloses the following technical effects: This invention has revealed that the antimicrobial peptide BMAP-28 exhibits a synergistic antibacterial effect when used in combination with antibiotics such as ciprofloxacin, gentamicin, and cefixime. In particular, when BMAP-28 is used in combination with ciprofloxacin or gentamicin, the partial inhibitory concentration index (FIC index) against *Cronobacter sakazakii* ATCC 12868 is 0.5, clearly indicating a significant synergistic antibacterial effect. This suggests that while achieving the same or better therapeutic effect, the dosage of antibiotics and antimicrobial peptides can be significantly reduced. This not only lowers treatment costs but also significantly reduces the potential toxic side effects and nephrotoxicity associated with high-dose antibiotics, and alleviates the evolutionary pressure of bacterial resistance.

[0018] The combined drug strategy of this invention can greatly increase the difficulty for bacteria to develop drug resistance mechanisms through a multi-target attack strategy, providing an effective "drug repurposing" strategy to address the increasingly serious problem of bacterial drug resistance. Through ingenious combination, it reactivates the activity of existing antibiotics, offering advantages such as short development cycles, high success rates, and relatively low costs. It provides a new strategy for treating various bacterial infections and has a very broad clinical application prospect. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] The sequence of the antimicrobial peptide BMAP-28 in the following embodiments of the present invention is: GGLRSLGRKILRAWKKYGPIIVPIIRIG (SEQ ID NO.1). This peptide can be synthesized by Fmoc-solid phase synthesis and purified by high performance liquid chromatography (HPLC) and verified by mass spectrometry (MS) to obtain a product with a purity of over 95%.

[0025] The test strain in the following examples of the present invention is Cronobacter sakazakii ( Cronobacter sakazakii ATCC12868).

[0026] Example 1: Determination of the antimicrobial activity of antimicrobial peptides / antibiotics 1. Experimental Methods The minimal inhibitory concentration (MIC) method recommended by the Clinical and Laboratory Standards Institute (CLSI) was adopted, and the specific steps are as follows: (1) Cell culture: The -20 ℃ frozen bacterial solution was inoculated into MHB medium at 2% (bacterial solution volume / medium medium volume) and cultured overnight in a shaker at 168 rpm and 37 °C to allow it to grow to the logarithmic growth phase. Then, the second generation strain was inoculated and cultured again, and the second generation strain was adjusted to OD using MHB. 600nm =0.4, diluted 100 times as the bacterial solution to be tested.

[0027] (2) Peptide dilution: Add 95 μL and 50 μL of BSA solution to row A and other rows respectively. Then, add 5 μL of antimicrobial peptide solution to row A. Gradually dilute 50 μL from top to bottom. After gradient dilution in row G, discard 50 μL of the mixture.

[0028] (3) Inoculation of bacterial culture: 50 μL of diluted second-generation bacterial culture was inoculated into the AG row and the first 6 wells of the H row, and 50 μL of sterile MHB medium was added to the last 6 wells of the H row. The first 6 wells and the last 6 wells of the H row were used as positive and negative control groups, respectively. After 24 h of incubation, the negative control wells served as the contamination group of the 96-well plate. If no turbidity was observed, the absorbance was measured at 600 nm using a microplate reader. The concentration of antimicrobial peptide at which no obvious turbidity was observed in the solution in the wells was taken as the minimum inhibitory concentration. This experiment was independently repeated three times.

[0029] 2. Experimental Results The MIC values ​​of the selected test antibiotics against Cronobacter sakazakii ATCC 12868 were determined using the microdilution method, and the results are shown in Table 1. Ciprofloxacin, gentamicin, and cefixime showed good antibacterial effects, with MIC values ​​all lower than BMAP-28.

[0030] Table 1. MIC values ​​of antimicrobial peptides or antibiotics against Cronobacter sakazakii ATCC 12868 Example 2: Antimicrobial peptide combined with antibiotic antimicrobial assay 1. Experimental Methods Based on a modification of the Pankey method, a standard chessboard method is used for identification. The specific process is as follows: (1) The bacterial culture and preparation of the bacterial inoculum are the same as in Example 1; (2) Drug preparation: In MHB medium, the antibiotic was diluted to twice the maximum detectable concentration, and the antimicrobial peptide to be tested was diluted to four times the maximum detectable concentration. The maximum detectable concentration for both the antibiotic and the antimicrobial peptide was 2 × MIC of the corresponding test bacteria. (3) Drug dilution: Antibiotic dilution: Add 50 μL of MHB medium to each well of the 96-well plate. Add 50 μL of the antibiotic solution prepared in (2) to wells A12-G12 in columns 11 and 12. Add an additional 50 μL of antibiotic solution to well H11. Mix the solution in column 11 thoroughly and aspirate 50 μL to column 10 for a 2-fold serial dilution. Continue this process until column 2 is reached. Aspirate 50 μL and discard the solution. At this point, the concentration of the antibiotic decreases by a factor of 2 along the X-axis from column 12 to column 2. Dilution of antimicrobial peptides: Add 50 μL of the antimicrobial peptide solution prepared in (2) to wells H1-H11 in row H, mix well, and add 50 μL to row G. Repeat this process to make a 2-fold serial dilution to row B. Discard 50 μL. At this time, the concentration of antimicrobial peptides decreases by 2-fold along the Y-axis from row H to row B. The concentration of antibiotics in each column of the diluted 96-well plate is the same, and the concentration of antimicrobial peptides in each row is the same. (4) Bacterial inoculation: Add 50 μL of the bacterial inoculation solution prepared in (1) (except for H12) to the 96-well plate. Add 100 μL of culture medium to the H12 well as a negative control. At this time, the A1 well of the 96-well plate does not contain antibiotics and antimicrobial peptides and serves as a positive control. After incubating the 96-well plate at 37℃ for 18-20 h, the negative control wells serve as the contamination group of the 96-well plate. If no turbidity is observed, the absorbance is measured at 600 nm using an ELISA reader. The concentration at which the solution in the wells does not show obvious turbidity is taken as the minimum inhibitory concentration when used in combination. This experiment was independently repeated three times.

[0031] (5) Results Analysis: The interaction between the tested drugs was evaluated by calculating the partial inhibitory concentration index (FIC index). The formula for calculating the FIC index is as follows: .

[0032] Evaluation criteria: FIC index ≤0.5, 0.5-4 and >4 represent synergistic effect, no interaction and antagonistic effect, respectively.

[0033] 2. Experimental Results The checkerboard microdilution method was used to combine different antimicrobial peptides with different antibiotics in pairs to test the effect of combined antimicrobial peptides and antibiotics on the MIC value of *Cronobacter sakazakii* ATCC 12868, in order to further explore the synergistic effect of antimicrobial peptides and antibiotics. The experimental results are expressed using the Partially Known Concentration Index (FIC index), where FIC index ≤0.5, 0.5-4, and >4 represent synergistic effect, no interaction, and antagonistic effect, respectively. The results are shown in Table 2. Table 2 shows that BMAP-28 only exhibits synergistic effects with ciprofloxacin and gentamicin. Although BMAP-28 showed no significant synergistic effect with cefixime, the combined use of both antibiotics and peptides decreased the MIC concentration. Notably, BMAP-28 showed an antagonistic effect when combined with polymyxin B sulfate. This may be because both are membrane-targeting bactericidal substances, and there may be competition for bacterial binding sites, leading to antagonistic effects. This finding can provide a supplementary explanation for the reduced bactericidal effect of BMAP-28 when used in combination with other membrane-targeted bactericides.

[0034] Table 2. MIC values ​​of BMAP-28 in combination with antibiotics against Cronobacter sakazakii ATCC 12868 The above experimental results show that BMAP-28, when used in combination with ciprofloxacin and gentamicin, exhibits a clear synergistic antibacterial effect against Cronobacter sakazakii ATCC12868 (FIC index = 0.5), which provides a solid basis for the development of novel antibacterial drugs based on antimicrobial peptide-antibiotic combinations.

[0035] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an antimicrobial peptide BMAP-28 in the combined preparation of antimicrobial drugs with antibiotics, characterized in that, The antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

2. The application according to claim 1, characterized in that, The amino acid sequence of the antimicrobial peptide BMAP-28 is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The antimicrobial drug is used to treat infections caused by Gram-negative and / or Gram-positive bacteria.

4. The application according to claim 3, characterized in that, The Gram-negative bacterium mentioned is Cronobacter sakazakii.

5. An antibacterial drug, characterized in that, The drug uses a combination of the antimicrobial peptide BMAP-28 and an antibiotic as its active ingredient.

6. The antibacterial drug according to claim 5, characterized in that, The amino acid sequence of the antimicrobial peptide BMAP-28 is shown in SEQ ID NO.

1.

7. The antibacterial drug according to claim 5, characterized in that, The antibiotic is selected from one or more of ciprofloxacin, gentamicin, and cefixime.

8. The antibacterial drug according to claim 5, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

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