An antibacterial peptide, composition and use thereof

Abstract

This invention discloses an antimicrobial peptide, its composition, and its applications, belonging to the field of polypeptide pharmaceuticals. This antimicrobial peptide, by introducing groups containing fluorinated phenyl, chlorophenyl, and diaminobutyric acid to form an amphiphilic molecular conformation, possesses advantages such as good biocompatibility, high stability, low synthesis cost, and low likelihood of inducing drug resistance. Experiments show that the minimum inhibitory concentration (MIC) of this antimicrobial peptide against various Gram-negative bacteria is significantly lower than that of the traditional antibiotic polymyxin B and the control peptide D65. Furthermore, in a neutropenic mouse infection model, it can significantly reduce bacterial load, demonstrating superior antimicrobial activity. Simultaneously, the antimicrobial peptide exhibits lower toxicity to human renal proximal tubular epithelial cells compared to the traditional antibiotic polymyxin B and the control peptide D65. In summary, this antimicrobial peptide holds promise as a replacement for polymyxin B in the treatment of multidrug-resistant Gram-negative bacterial infections, and is suitable for the prevention and control of various infections of the respiratory system, urinary system, and skin and soft tissues.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide medicine, and particularly relates to an antimicrobial peptide, its composition, and its application. Background Technology

[0002] In the modern medical system, antibiotics, as core drugs for treating bacterial infectious diseases, have long played a crucial role in resisting the invasion of pathogenic microorganisms. However, long-term widespread clinical use and evolutionary selection pressures have led to an exponential increase in bacterial resistance, particularly prominent in Gram-negative bacteria such as *Escherichia coli*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, and *Pseudomonas aeruginosa*. Through molecular mechanisms such as horizontal gene transfer and gene mutation, these strains have developed complex and diverse drug-resistant phenotypes, constituting a serious global public health crisis.

[0003] Polymyxins, currently the last line of defense against multidrug-resistant Gram-negative bacterial infections, exert their bactericidal effect by specifically binding to lipid A in the bacterial outer membrane lipopolysaccharide (LPS), thereby disrupting cell membrane integrity. However, this drug faces significant obstacles in clinical application: on the one hand, bacteria reduce drug affinity through LPS glycosylation and actively remove intracellular drugs using efflux pump systems, leading to rapid development of resistance; on the other hand, high concentrations of polymyxins accumulate in the kidneys, causing severe toxic reactions, with 30%-60% of patients experiencing nephrotoxicity such as renal tubular damage and decreased glomerular filtration rate, accompanied by neurotoxicity and ototoxicity. Although the global polymyxin market reached $2.5 billion in 2023, its limited efficacy and significant side effects cannot meet the urgent clinical need for safe and effective treatment options.

[0004] With the increasing prevalence of bacterial resistance and the limited use of traditional antibiotics, antimicrobial peptides (AMPs), with their unique amphiphilic structure and multi-target mechanism of action, have emerged as highly promising candidates to replace traditional antibiotics. AMPs exert their antibacterial activity by disrupting cell membrane integrity and interfering with key intracellular metabolic pathways, possessing significant advantages such as broad-spectrum antibacterial activity (covering bacteria, fungi, and viruses), excellent thermostability, and low susceptibility to inducing drug resistance. However, existing patents (such as CN106232617A) involve cyclic antimicrobial peptides, even the D65 peptide, which has shown relatively optimal antibacterial efficacy. While it has demonstrated some application potential in infection treatment, it still suffers from practical limitations such as insufficient activity against Gram-negative bacteria and high nephrotoxicity, restricting its clinical translation.

[0005] Given the challenges of polymyxin's clinical application and the serious situation of Gram-negative bacterial resistance, accelerating the development of novel antimicrobial peptide drugs is imperative. Developing antimicrobial peptides with broad-spectrum antimicrobial activity, low nephrotoxicity, and high biocompatibility is not only a key pathway to overcoming current treatment bottlenecks but also a strategic necessity to address the global bacterial resistance crisis and safeguard public health. There is an urgent need to strengthen collaborative innovation between basic research and clinical translation to promote antimicrobial peptide drugs as an effective alternative to polymyxin. Summary of the Invention

[0006] Purpose of the Invention: This invention aims to achieve two core objectives: First, to develop a series of antimicrobial peptides with novel structures and mechanisms of action to overcome the limitations of existing antimicrobial drugs in terms of drug resistance, antimicrobial spectrum, and safety; Second, to develop drug compositions adapted to these novel antimicrobial peptides and explore their applications in the treatment, prevention, and other related fields of antibacterial infections, thereby providing a safe, efficient, and low-resistance innovative solution to solve the clinical problem of drug-resistant bacterial infections and fill the gaps in existing treatment methods.

[0007] Technical solution: The antimicrobial peptide of the present invention has the following characteristics:

[0008] (1) The sequence shown is [(S)-4-amino-3-(3-(trifluoromethyl)phenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-ortholeucine-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-), and the structure is shown in formula (I):

[0009]

[0010] (2) The sequence shown is [(S)-4-amino-3-(3-(trifluoromethyl)phenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-[2-aminobutyric acid]-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-), and the structure is shown in formula (II):

[0011]

[0012] (3) The sequence shown is [(S)-4-amino-3-(3-(trifluoromethyl)phenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-valine-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-), and the structure is shown in formula (III):

[0013]

[0014] (4) The sequence shown is [(S)-4-amino-3-(4-chlorophenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-ortholeucine-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-), and the structure is shown in Formula (IV):

[0015]

[0016] (5) The sequence shown is [(S)-4-amino-3-(4-chlorophenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-leucine]-[2-aminobutyric acid]-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-), and the structure is shown in formula (V):

[0017]

[0018] The pharmaceutical composition of the present invention contains the aforementioned antimicrobial peptide or its pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug as an active ingredient.

[0019] Preferably, the pharmaceutical composition further contains pharmaceutically acceptable excipients. "Pharmaceutically acceptable" excipients are substances suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., substances with a reasonable benefit / risk ratio. They also include various excipients and diluents, and may contain liquids such as water, saline, glycerin, and ethanol, or auxiliary substances such as lubricants, glidants, wetting agents, emulsifiers, and pH buffers.

[0020] Preferably, the dosage forms of the pharmaceutical composition include tablets, capsules, granules, oral liquids, syrups, powders, chewable tablets, effervescent tablets, sustained-release tablets, microcapsules, injections, powder for injection, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, dressings, nebulizing solutions, film-forming agents, implants, transdermal patches, microemulsions, liposomes, nanoparticles, orally disintegrating tablets, oral instant films, sponges, and capsules.

[0021] The application of the antimicrobial peptides or pharmaceutical compositions described in this invention in the preparation of medicaments for treating microbial infections.

[0022] Preferably, the drug dosage form includes injections, oral preparations, or topical preparations, wherein the topical preparations include eye drops or lotions, and the dosage range of the antimicrobial peptide in the dosage form is: injections 0.001-1000 mg / kg; oral preparations 0.001-1000 mg / kg; topical preparations 1 / 10000-30% / vial; eye drops 1 / 10000-30% / vial; lotions 1 / 100000-20‰ / vial.

[0023] Preferably, the application is the use of the antimicrobial peptide or the pharmaceutical composition in the preparation of a medicament for the prevention and / or control of respiratory tract infections, soft tissue infections, gastrointestinal tract infections, urinary tract infections, bloodstream infections, meningitis, bone and joint infections, eye infections, and ear infections caused by Gram-negative bacteria.

[0024] An antimicrobial agent comprising the said antimicrobial peptide or the said pharmaceutical composition.

[0025] The use of the antimicrobial peptides or pharmaceutical compositions described in this invention in the preparation of medicaments for the prevention and / or control of microbial infections.

[0026] Preferably, the microbial infection is a Gram-negative bacterial infection, and the Gram-negative bacteria are any one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0027] Preferably, the microbial infection is a respiratory system infection, urinary system infection, musculoskeletal system infection, skin and soft tissue infection, systemic infection type, circulatory system infection, digestive system infection, nervous system infection, endocrine system infection, or reproductive system infection caused by Gram-negative bacteria.

[0028] Preferably, the Gram-negative bacterial infections include urinary tract infections, pneumonia, burn infections, peritonitis, cholecystitis, pyelonephritis, sepsis, hospital-acquired pneumonia, trauma infections, neonatal meningitis, necrotizing fasciitis, liver abscess, osteomyelitis, suppurative arthritis, endocarditis, keratitis, otitis media, sinusitis, cellulitis, ventilator-associated pneumonia, melioidosis, chancroid, Legionnaires' disease, gastritis, peptic ulcer, and cholangitis caused by Gram-negative bacteria.

[0029] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The antimicrobial peptide is a novel and highly efficient antimicrobial peptide with good biocompatibility and high stability; 2. The antimicrobial peptide has a simple structure, is easy to synthesize on a large scale, and has low synthesis cost; 3. The antimicrobial peptide has highly efficient and broad-spectrum antimicrobial properties, and its performance is superior to polymyxin B and control peptide D65 in infections caused by a variety of bacteria. Moreover, it is not prone to drug resistance and is expected to replace antibiotics as a safe, green, and highly efficient ideal antimicrobial agent. Detailed Implementation

[0030] The technical solution of the present invention will be further described below.

[0031] The polypeptide compound in the embodiments of the present invention, the antimicrobial peptide 1 sequence is: [(S)-4-amino-3-(3-(trifluoromethyl)phenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-ortholeucine-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-);

[0032] The sequence of antimicrobial peptide 2 is: [(S)-4-amino-3-(3-(trifluoromethyl)phenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-[2-aminobutyric acid]-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-);

[0033] The sequence of antimicrobial peptide 3 is: [(S)-4-amino-3-(3-(trifluoromethyl)phenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-n-valine-[2,4-diaminobutyric acid]-[D-phenylalanine]-valine-[2,4-diaminobutyric acid]-[D-phenylalanine]-valine-[D-phenylalanine] ...

[0034] [2,4-Diaminobutyric acid]-threonine-);

[0035] The sequence of antimicrobial peptide 4 is: [(S)-4-amino-3-(4-chlorophenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-ortholeucine-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-);

[0036] The sequence of antimicrobial peptide 5 is: [(S)-4-amino-3-(4-chlorophenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-leucine]-[2-aminobutyric acid]-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-);

[0037] The peptides designed in this invention were all directly synthesized by Sangon Biotech.

[0038] Example 1: Antibacterial activity experiment of antimicrobial peptides

[0039] According to the CLSI (Clinical Laboratory Standards Institute) M07-A10 standard operating procedure, the minimum inhibitory concentration (MIC) of antimicrobial peptides 1-8 against four clinically common Gram-negative bacteria, including Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC13822, Pseudomonas aeruginosa ATCC27853, and Acinetobacter baumannii NCTC13424, was determined using the micro-dilution serial two-fold method.

[0040] D65 peptide was synthesized according to the synthesis method disclosed in CN106232617A as a comparison.

[0041] (1) Fresh Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC13822, Pseudomonas aeruginosa ATCC27853, and Acinetobacter baumannii NCTC13424 colonies that have been cultured on Mueller-Hinton (MH) agar plates at 35℃ for 18 hours were collected and bacterial suspensions were prepared using sterile physiological saline and calibrated to 0.5 McFarland turbidity by turbidimetric method.

[0042] (2) In a sterile 96-well microplate, the antimicrobial peptides 1-5, D65 peptide or polymyxin B were subjected to double serial dilutions in cationic-adjusted MH broth to form antimicrobial solutions with concentration gradients of 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32 and 64 μg / mL. The final concentration of DMSO was 0.1%. 150 μL of gradient dilution solution was pre-placed in each well.

[0043] (3) Inoculate each well with 20 μL of standardized bacterial suspension to make the final volume of the system 170 μL / well, and set up 2 parallel replicate wells for each test concentration;

[0044] (4) The microplate was then placed in a 35°C incubator and incubated for 20 hours. The minimum drug concentration required to completely inhibit visible microbial growth was determined by visual observation, with the disappearance of turbidity as the endpoint.

[0045] For compounds that have undergone five or more independent replicate experiments, the median of the results from each experiment is taken as the final MIC value, and the concentration unit is uniformly expressed in μg / mL.

[0046] The results are shown in Table 2. The minimum inhibitory concentrations (MICs) of antimicrobial peptides 1-5 against bacteria were significantly better than those of polymyxin B and the control peptide D65, and their antimicrobial activity was more prominent.

[0047] Table 2. Minimum Inhibitory Concentration (MIC) of Antimicrobial Peptides against Bacteria

[0048]

[0049] Example 3: In vitro renal cytotoxicity assay of antimicrobial peptides

[0050] Cytotoxicity was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole bromide (MTT) colorimetric method.

[0051] (1) HK-2 human renal proximal tubular epithelial cells (CRL-2190) were seeded in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2 until the logarithmic growth phase. The cells were then inoculated at a concentration of 2.5 × 10⁻⁶ cells / year. 3 Cells were seeded at a density per well in 96-well plates and pre-cultured for 24 hours until the cells were fully adhered.

[0052] (2) Add serially diluted antimicrobial peptides 1-5, D65 peptide or polymyxin B to adherent cell wells, with final concentration gradients of 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 μg / mL, and continue to expose and culture for 48 hours;

[0053] (3) After removing the drug-containing culture medium, add 110 μL of freshly prepared MTT working solution to each well and incubate in the dark for 3 hours to induce formazan crystal formation.

[0054] Each 110 μL of MTT working solution is prepared by mixing 10 μL of 5 mg / mL MTT-PBS solution with 100 μL of LMEM culture medium.

[0055] (4) After removing the MTT solution, add 100 μL of formazan solution containing 40% N,N-dimethylformamide, 16% sodium dodecyl sulfate and 2% glacial acetic acid to each well, shake for 10 minutes to fully dissolve the crystals, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at a wavelength of 570 nm.

[0056] (5) The absorbance of wells treated with 0.25% DMSO was used as the negative control (100% cell viability), and the wells treated with polymyxin B were used as the positive control (0% cell viability). The half-maximal inhibitory concentration (IC50) was calculated by nonlinear regression model and defined as the concentration of the compound that reduces cell viability by 50% compared with the negative control.

[0057] The results are shown in Table 3. In the safety assessment of HK-2 human renal proximal tubular epithelial cells, antimicrobial peptides 1-5 showed better safety compared with polymyxin B and control peptide D65, and their toxicity to renal cells was significantly lower.

[0058] Table 3. Cytotoxicity of antimicrobial peptides 1-5 against HK-2.

[0059]

[0060]

[0061] Example 4: In vivo efficacy evaluation of antimicrobial peptides

[0062] 1. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Escherichia coli ATCC25922

[0063] (1) CD-1 mice were used in the experiment, with 10 mice per group (n=10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0064] Treatment groups: 0.5 mg / kg polymyxin B, 2 mg / kg polymyxin B, and 3.5 mg / kg polymyxin B.

[0065] 0.5 mg / kg D65 peptide treatment group, 2 mg / kg D65 peptide treatment group, 3.5 mg / kg D65 peptide treatment group,

[0066] Treatment groups: 0.5 mg / kg antimicrobial peptide 1, 2 mg / kg antimicrobial peptide 1, and 3.5 mg / kg antimicrobial peptide 1.

[0067] Treatment groups: 0.5 mg / kg antimicrobial peptide 2, 2 mg / kg antimicrobial peptide 2, and 3.5 mg / kg antimicrobial peptide 2.

[0068] Treatment groups: 0.5 mg / kg antimicrobial peptide 3, 2 mg / kg antimicrobial peptide 3, and 3.5 mg / kg antimicrobial peptide 3.

[0069] Treatment groups included 0.5 mg / kg antimicrobial peptide 4, 2 mg / kg antimicrobial peptide 4, 3.5 mg / kg antimicrobial peptide 4, 0.5 mg / kg antimicrobial peptide 5, 2 mg / kg antimicrobial peptide 5, 3.5 mg / kg antimicrobial peptide 5, and a blank control group.

[0070] (2) On the 4th day and the 1st day before the experiment, all mice were injected intraperitoneally with cyclophosphamide twice. The first dose was 150 mg / kg and the second dose was 100 mg / kg to induce a persistent neutrophilia.

[0071] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5 CFU Escherichia coli ATCC25922 standard strain suspension was administered via tail vein injection at 1, 3.5, and 6 hours post-infection. Polymyxin B (PMB), D65 peptide, or antimicrobial peptide were administered according to the above grouping. The blank control group was given physiological saline.

[0072] (4) Nine hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, individual thigh tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony-forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 The results (CFU / g) are shown in Table 4.

[0073] Table 4. Logarithmic differences in the reduction of Escherichia coli ATCC25922 bacterial load in mouse thighs by antimicrobial peptides 1-5

[0074]

[0075]

[0076] 2. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Klebsiella pneumoniae ATCC13822

[0077] (1) CD-1 mice were used in the experiment, with 10 mice per group (n=10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0078] Treatment groups: 1 mg / kg polymyxin B, 2 mg / kg polymyxin B, and 4 mg / kg polymyxin B.

[0079] 1 mg / kg D65 peptide treatment group, 2 mg / kg D65 peptide treatment group, 4 mg / kg D65 peptide treatment group

[0080] Treatment groups: 1 mg / kg antimicrobial peptide 1, 2 mg / kg antimicrobial peptide 1, and 4 mg / kg antimicrobial peptide 1.

[0081] Treatment groups: 1 mg / kg antimicrobial peptide 2, 2 mg / kg antimicrobial peptide 2, and 4 mg / kg antimicrobial peptide 2.

[0082] 1 mg / kg antimicrobial peptide 3 treatment group, 2 mg / kg antimicrobial peptide 3 treatment group, 4 mg / kg antimicrobial peptide 3 treatment group,

[0083] 1 mg / kg antimicrobial peptide 4 treatment group, 2 mg / kg antimicrobial peptide 4 treatment group, 4 mg / kg antimicrobial peptide 4 treatment group

[0084] The treatment groups included 1 mg / kg antimicrobial peptide 5, 2 mg / kg antimicrobial peptide 5, 4 mg / kg antimicrobial peptide 5, and a blank control group.

[0085] (2) On the 4th day and the 1st day before the experiment, all mice were injected intraperitoneally with cyclophosphamide twice. The first dose was 150 mg / kg and the second dose was 100 mg / kg to induce a persistent neutrophilia.

[0086] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5 CFU Klebsiella pneumoniae ATCC13822 standard strain suspension was administered via tail vein injection at 2, 6, and 10 hours post-infection. Polymyxin B (PMB), D65 peptide, or antimicrobial peptide were administered according to the above grouping. The blank control group was given physiological saline.

[0087] (4) Sixteen hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, individual thigh tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony-forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 The results (CFU / g) are shown in Table 5.

[0088] Table 5. Logarithmic difference of antimicrobial peptides 1-5 in reducing bacterial load of Klebsiella pneumoniae ATCC13822 infection in mouse thigh.

[0089]

[0090] 3. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Acinetobacter baumannii NCTC13424

[0091] (1) CD-1 mice were used in the experiment, with 10 mice per group (n=10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0092] Treatment groups: 1 mg / kg polymyxin B, 2 mg / kg polymyxin B, and 4 mg / kg polymyxin B.

[0093] 1 mg / kg D65 peptide treatment group, 2 mg / kg D65 peptide treatment group, 4 mg / kg D65 peptide treatment group

[0094] Treatment groups: 1 mg / kg antimicrobial peptide 1, 2 mg / kg antimicrobial peptide 1, and 4 mg / kg antimicrobial peptide 1.

[0095] Treatment groups: 1 mg / kg antimicrobial peptide 2, 2 mg / kg antimicrobial peptide 2, and 4 mg / kg antimicrobial peptide 2.

[0096] 1 mg / kg antimicrobial peptide 3 treatment group, 2 mg / kg antimicrobial peptide 3 treatment group, 4 mg / kg antimicrobial peptide 3 treatment group,

[0097] 1 mg / kg antimicrobial peptide 4 treatment group, 2 mg / kg antimicrobial peptide 4 treatment group, 4 mg / kg antimicrobial peptide 4 treatment group

[0098] The treatment groups included 1 mg / kg antimicrobial peptide 5, 2 mg / kg antimicrobial peptide 5, 4 mg / kg antimicrobial peptide 5, and a blank control group.

[0099] (2) On the 4th day and the 1st day before the experiment, all mice were injected intraperitoneally with cyclophosphamide twice. The first dose was 150 mg / kg and the second dose was 100 mg / kg to induce a persistent neutrophilia.

[0100] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5 CFU Acinetobacter baumannii NCTC13424 standard strain suspension was administered via tail vein injection at 2, 6, and 10 hours post-infection. Polymyxin B (PMB), D65 peptide, or antimicrobial peptide were administered according to the above groupings, while the blank control group received physiological saline.

[0101] (4) Sixteen hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, individual thigh tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony-forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 The results (CFU / g) are shown in Table 6.

[0102] Table 6. Logarithmic difference in the reduction of bacterial load of Acinetobacter baumannii NCTC13424 infection in mouse thigh by antimicrobial peptides 1-5

[0103]

[0104] 4. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Pseudomonas aeruginosa ATCC27853

[0105] (1) CD-1 mice were used in the experiment, with 10 mice per group (n=10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0106] Treatment groups: 8 mg / kg polymyxin B, 15 mg / kg polymyxin B, and 20 mg / kg polymyxin B.

[0107] 8 mg / kg D65 peptide treatment group, 15 mg / kg D65 peptide treatment group, 20 mg / kg D65 peptide treatment group,

[0108] Treatment groups with 8 mg / kg antimicrobial peptide 1, 15 mg / kg antimicrobial peptide 1, and 20 mg / kg antimicrobial peptide 1 were identified.

[0109] Treatment groups with 8 mg / kg antimicrobial peptide 2, 15 mg / kg antimicrobial peptide 2, and 20 mg / kg antimicrobial peptide 2 were identified.

[0110] Treatment groups: 8 mg / kg antimicrobial peptide 3, 15 mg / kg antimicrobial peptide 3, and 20 mg / kg antimicrobial peptide 3.

[0111] The treatment groups included 8 mg / kg antimicrobial peptide 4, 15 mg / kg antimicrobial peptide 4, 20 mg / kg antimicrobial peptide 4, 8 mg / kg antimicrobial peptide 5, 15 mg / kg antimicrobial peptide 5, 20 mg / kg antimicrobial peptide 5, and a blank control group.

[0112] (2) On the 4th day and the 1st day before the experiment, all mice were injected intraperitoneally with cyclophosphamide twice. The first dose was 150 mg / kg and the second dose was 100 mg / kg to induce a persistent neutrophilia.

[0113] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5 CFU suspension of Pseudomonas aeruginosa ATCC27853 standard strain was administered via tail vein injection at 1, 3.5, and 6 hours post-infection. Polymyxin B (PMB), D65 peptide, or antimicrobial peptide were administered according to the above groupings, while the blank control group received physiological saline.

[0114] (4) Nine hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, individual thigh tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony-forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated.10 The results (CFU / g) are shown in Table 7.

[0115] Table 7. Logarithmic difference of antimicrobial peptides 1-5 in reducing bacterial load of Pseudomonas aeruginosa ATCC27853 infection in mouse thigh.

[0116]

[0117]

[0118] As shown in Table 4-7, in a mouse model of neutropenia infected with Gram-negative bacteria, antimicrobial peptides 1-5 exhibited antimicrobial activity at all tested doses. Their antimicrobial efficacy significantly increased with increasing dose, demonstrating a favorable dose-dependent relationship. Notably, at the same dose, antimicrobial peptides 1-5 showed significantly better therapeutic effects than polymyxin B (more than 3 times) and D65 (more than 2 times), demonstrating a stronger antimicrobial advantage.

[0119] 5. Efficacy of antimicrobial peptides in a mouse model of neutropenia infected with Acinetobacter baumannii NCTC13424

[0120] (1) CD-1 mice were used in the experiment, with 10 mice per group (n=10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0121] Treatment groups: 15 mg / kg polymyxin B, 20 mg / kg polymyxin B, and 30 mg / kg polymyxin B.

[0122] 15 mg / kg D65 peptide treatment group, 20 mg / kg D65 peptide treatment group, 30 mg / kg D65 peptide treatment group

[0123] Treatment groups: 15 mg / kg antimicrobial peptide 1, 20 mg / kg antimicrobial peptide 1, and 30 mg / kg antimicrobial peptide 1.

[0124] Treatment groups: 15 mg / kg antimicrobial peptide 2, 20 mg / kg antimicrobial peptide 2, and 30 mg / kg antimicrobial peptide 2.

[0125] Treatment groups: 15 mg / kg antimicrobial peptide 3, 20 mg / kg antimicrobial peptide 3, and 30 mg / kg antimicrobial peptide 3.

[0126] The treatment groups included 15 mg / kg antimicrobial peptide 4, 20 mg / kg antimicrobial peptide 4, 30 mg / kg antimicrobial peptide 4, 15 mg / kg antimicrobial peptide 5, 20 mg / kg antimicrobial peptide 5, 30 mg / kg antimicrobial peptide 5, and a blank control group.

[0127] (2) On the 4th day and the 1st day before the experiment, all mice were injected intraperitoneally with cyclophosphamide twice. The first dose was 200 mg / kg and the second dose was 150 mg / kg to induce a persistent neutrophilia.

[0128] (3) Mice were intranasally inoculated with 1×10 7 CFU / lung Acinetobacter baumannii NCTC13424 standard bacterial suspension was used to achieve precise bilateral lung lobe infection. The bacteria were administered subcutaneously via the neck and back at 2, 6, and 10 hours post-infection, and were given polymyxin B (PMB), D65 peptide, or antimicrobial peptide according to the above-described groupings. The blank control group received normal saline.

[0129] (4) Sixteen hours after infection, bilateral lung tissue was aseptically harvested from all mice. Under ice-water bath conditions, the lung tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the lung tissue homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony-forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 The results (CFU / g) are shown in Table 8.

[0130] Table 8. Logarithmic difference in the reduction of bacterial load of Acinetobacter baumannii NCTC13424 infection in mouse lungs by antimicrobial peptides 1-5

[0131]

[0132] 6. Efficacy of antimicrobial peptides in a mouse model of neutropenia induced by Pseudomonas aeruginosa ATCC27853 infection

[0133] (1) CD-1 mice were used in the experiment, with 10 mice per group (n=10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0134] Treatment groups: 15 mg / kg polymyxin B, 20 mg / kg polymyxin B, and 40 mg / kg polymyxin B.

[0135] 15 mg / kg D65 peptide treatment group, 20 mg / kg D65 peptide treatment group, 40 mg / kg D65 peptide treatment group

[0136] Treatment groups: 15 mg / kg antimicrobial peptide 1, 20 mg / kg antimicrobial peptide 1, and 40 mg / kg antimicrobial peptide 1.

[0137] Treatment groups: 15 mg / kg antimicrobial peptide 2, 20 mg / kg antimicrobial peptide 2, and 40 mg / kg antimicrobial peptide 2.

[0138] Treatment groups: 15 mg / kg antimicrobial peptide 3, 20 mg / kg antimicrobial peptide 3, and 40 mg / kg antimicrobial peptide 3.

[0139] The treatment groups included 15 mg / kg antimicrobial peptide 4, 20 mg / kg antimicrobial peptide 4, 40 mg / kg antimicrobial peptide 4, 15 mg / kg antimicrobial peptide 5, 20 mg / kg antimicrobial peptide 5, 40 mg / kg antimicrobial peptide 5, and a blank control group.

[0140] (2) On the 4th day and the 1st day before the experiment, all mice were injected intraperitoneally with cyclophosphamide twice. The first dose was 200 mg / kg and the second dose was 150 mg / kg to induce a persistent neutrophilia.

[0141] (3) Mice were intranasally inoculated with 1×10 7 CFU / lung lobe Pseudomonas aeruginosa ATCC27853 standard bacterial suspension was used to achieve precise bilateral lung lobe infection. The drugs were administered subcutaneously via the neck and back at 2, 6, and 10 hours post-infection, and were given polymyxin B (PMB), D65 peptide, or antimicrobial peptide according to the above-described groupings. The blank control group received normal saline.

[0142] (4) Sixteen hours after infection, bilateral lung tissue was aseptically harvested from all mice. Under ice-water bath conditions, the lung tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the lung tissue homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony-forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 The results (CFU / g) are shown in Table 9.

[0143] Table 9. Logarithmic differences in the reduction of bacterial load of Pseudomonas aeruginosa ATCC27853 infection in mouse lungs by antimicrobial peptides 1-5.

[0144]

[0145] According to the data in Tables 8-9, in a mouse model of enlarged lungs due to neutropenia caused by Gram-negative bacterial infection, antimicrobial peptides 1-5 exhibited antimicrobial activity at all tested doses, and the antimicrobial effect was positively correlated with the dose, demonstrating excellent dose-dependency. Importantly, at the same dose, the therapeutic effect of antimicrobial peptides 1-5 was significantly better than that of polymyxin B by more than 3 times and better than that of D65 by more than 2 times, showing a stronger antimicrobial advantage.

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide has the following characteristics: (1) The structure of the sequence [(S)-4-amino-3-(4-chlorophenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-phenylalanine]-ortholeucine-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-) is shown in Formula (IV): Formula (IV); (2) The sequence shown is [(S)-4-amino-3-(4-chlorophenyl)butyryl]-threonine-[2,3-diaminopropionic acid]-cyclo-([2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-[D-leucine]-[2-aminobutyric acid]-[2,4-diaminobutyric acid]-[2,4-diaminobutyric acid]-threonine-), and the structure is shown in formula (V): Formula (V).

2. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the antimicrobial peptide as described in claim 1 or a pharmaceutically acceptable salt thereof as the active ingredient.

3. The pharmaceutical composition according to claim 2, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients.

4. The pharmaceutical composition according to claim 3, characterized in that, The pharmaceutically acceptable excipients include excipients selected from one or more of diluents, lubricants, flow aids, wetting agents, emulsifiers, or pH buffers.

5. The pharmaceutical composition according to claim 2, characterized in that, The dosage forms of the pharmaceutical composition include tablets, capsules, granules, oral liquids, syrups, powders, microcapsules, injections, powder injections, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, dressings, nebulizing solutions, film-forming agents, implants, microemulsions, liposomes, nanoparticles, oral instant films, and sponges.

6. The use of the antimicrobial peptide of claim 1 or the pharmaceutical composition of any one of claims 2-5 in the preparation of a medicament for the prevention and / or control of microbial infections, wherein the microorganism is selected from one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

7. The application according to claim 6, characterized in that, The microbial infection is any one or more of the following: respiratory system infection, urinary system infection, musculoskeletal system infection, skin and soft tissue infection, systemic infection type, circulatory system infection, digestive system infection, nervous system infection, and reproductive system infection.

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