Antibacterial peptide, pharmaceutical composition and application

Abstract

The invention discloses an antibacterial peptide, a pharmaceutical composition and application, and belongs to the field of polypeptide medicines.The antibacterial peptide forms a novel annular conformation by introducing n-leucine and 2-aminobutyric acid to construct a parent nucleus structure. The affinity of the antibacterial peptide to the main component phosphatidylglycerol of a bacterial cell membrane is remarkably improved, is improved by 31 times or more compared with polymyxin B and is improved by 16 times or more compared with D50 peptide, and the selectivity of the antibacterial peptide to the main component phosphatidylcholine of a mammalian cell membrane is improved by hundreds of times or more compared with that of a control peptide. The polymyxin B peptide has extremely low toxicity to HK-2 kidney cells, and the biological safety of the polymyxin B peptide is obviously superior to that of polymyxin B and D50 peptide. The minimum inhibitory concentration of the antibacterial peptide to gram-negative bacteria is as low as 0.03 mu g / mL, is about 33 times higher than that of polymyxin B, and is 17-33 times higher than that of D50 peptide. The treatment effect of the antibacterial peptide is obviously superior to that of polymyxin B and D50 peptide in various infection models, and the antibacterial peptide is expected to be developed into a novel anti-infection drug and is used for preventing and treating various infections.

Description

Technical Field

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

[0002] Antibiotics, as a key tool in combating bacterial invasion, have seen a dramatic increase in usage in recent years, leading to a more severe problem of bacterial resistance. Gram-negative bacteria such as Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa have formed complex and difficult-to-break resistance networks through various resistance strategies, including horizontal gene transfer and efflux pump mechanisms, posing a significant challenge to clinical treatment. In particular, carbapenem-resistant Klebsiella pneumoniae maintains a persistently high mortality rate, posing a major threat to public health security.

[0003] Polymyxin, as an important drug for treating infections caused by drug-resistant Gram-negative bacteria, faces a dilemma in clinical application: on the one hand, drug-resistant strains modify lipopolysaccharides with phosphoethanolamine, which significantly reduces the affinity of polymyxin for bacteria and activates the efflux system, leading to the rapid emergence of drug resistance; on the other hand, a considerable proportion of patients treated with polymyxin experience irreversible renal tubular damage, and may also experience adverse reactions such as neurotoxicity and ototoxicity. Its therapeutic index is far below the standard of ideal drugs, making it difficult to meet the actual needs of clinical treatment.

[0004] Against this challenging backdrop, antimicrobial peptides are gradually demonstrating unique advantages and potential. These naturally occurring amphiphilic molecules disrupt bacterial cell membranes through specific mechanisms of action, exhibiting significantly higher killing efficiency against drug-resistant bacteria than traditional antibiotics, and maintaining good activity even at higher temperatures. More importantly, the diversity of their target sites greatly reduces the probability of bacterial resistance, providing a new approach to solving the antibiotic resistance problem. However, some publicly disclosed antimicrobial peptides, such as the antimicrobial active peptide D50 reported in patent CN106232617A, which is one of the most potent antimicrobial peptides discovered to date, still suffer from insufficient activity against Gram-negative bacteria and high cytotoxicity in practical applications, limiting their clinical translation. Summary of the Invention

[0005] Purpose of the invention: The primary objective of this invention is to provide a novel antimicrobial peptide that possesses high bacterial cell membrane affinity, high antimicrobial activity, and high biocompatibility; another objective is to provide a pharmaceutical composition containing the antimicrobial peptide and its application.

[0006] Technical solution: The antimicrobial peptide of the present invention has the following characteristics:

[0007] (1) The sequence [(S)-4-amino-3-(3-ethyl-5-(trifluoromethyl)phenyl)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-) is shown in Formula (I):

[0008] Formula (I);

[0009] (2) The sequence shown is [(S)-4-amino-3-(3-isobutylphenyl)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 (II):

[0010] Formula (II);

[0011] (3) The sequence shown is [(S)-4-amino-3-(3-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 (III):

[0012] Formula (Ⅲ);

[0013] (4) The sequence shown is [(S)-4-amino-3-(3-chloro-5-methylphenyl)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 (Ⅳ):

[0014] Formula (Ⅳ);

[0015] (5) The structure of the sequence [(S)-4-amino-3-(3-chloro-5-fluorophenyl)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-) is shown in formula (V):

[0016] Formula (V).

[0017] 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.

[0018] 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.

[0019] 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.

[0020] The application of the antimicrobial peptides or pharmaceutical compositions described in this invention in the preparation of medicaments for treating microbial infections.

[0021] Preferably, the drug dosage form includes injections, oral preparations, or topical preparations, and the topical preparations include eye drops or lotions, wherein 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.

[0022] 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.

[0023] An antimicrobial agent comprising the said antimicrobial peptide or the said pharmaceutical composition.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Beneficial effects: Compared with the prior art, the antimicrobial peptides of the present invention have the following significant advantages:

[0029] 1. This antimicrobial peptide, by introducing leucine and 2-aminobutyric acid to construct a core structure, forms a novel cyclic conformation distinct from polymyxin B and D50 peptide, significantly enhancing its affinity for phosphatidylglycerol, a major component of bacterial cell membranes, by more than 31 times compared to polymyxin B and more than 16 times compared to D50 peptide; simultaneously, its selectivity for phosphatidylcholine, a major component of mammalian cell membranes, is more than 100 times higher than the control peptide; 2. Against HK-2 human renal proximal tubular epithelial cells, this antimicrobial peptide exhibits extremely low toxicity compared to polymyxin B and D50 peptide, significantly improving biosafety; 3. The minimum inhibitory concentration (MIC) of this antimicrobial peptide against Gram-negative bacteria is as low as 0.03. Compared to polymyxin B, which is commonly used in clinical practice, this antimicrobial peptide has an antibacterial efficacy that is approximately 33 times higher than that of polymyxin B. Compared to the D50 control peptide, its antimicrobial activity is enhanced by approximately 17-33 times. Moreover, in various infection models, the therapeutic effect of this antimicrobial peptide is significantly better than that of polymyxin B and D50 peptide, showing great potential for its development into a novel anti-infective drug. It is expected to be applied to the prevention and treatment of various infections of the respiratory system, urinary system, skin and soft tissue, bringing breakthrough progress to the treatment of multidrug-resistant bacterial infections in clinical practice, and thus becoming a safe, green, and highly effective ideal antimicrobial agent. Attached Figure Description

[0030] Figure 1 The high-performance liquid chromatogram of antimicrobial peptide 1 prepared in Example 1;

[0031] Figure 2 The high-performance liquid chromatogram of antimicrobial peptide 2 prepared in Example 1;

[0032] Figure 3 The high-performance liquid chromatogram of antimicrobial peptide 3 prepared in Example 1;

[0033] Figure 4 The high-performance liquid chromatogram of antimicrobial peptide 4 prepared in Example 1;

[0034] Figure 5 The high-performance liquid chromatogram of antimicrobial peptide 5 prepared in Example 1;

[0035] Figure 6 This is a statistical graph showing the in vitro renal cytotoxicity assay results of antimicrobial peptides 1-5. Detailed Implementation

[0036] The technical solution of the present invention will be further described below.

[0037] Example 1: Preparation and purification of antimicrobial peptides

[0038] The sequence of antimicrobial peptide 1 is: [(S)-4-amino-3-(3-ethyl-5-(trifluoromethyl)phenyl)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-);

[0039] The sequence of antimicrobial peptide 2 is: [(S)-4-amino-3-(3-isobutylphenyl)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-);

[0040] The sequence of antimicrobial peptide 3 is: [(S)-4-amino-3-(3-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-);

[0041] The sequence of antimicrobial peptide 4 is: [(S)-4-amino-3-(3-chloro-5-methylphenyl)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-);

[0042] The sequence of antimicrobial peptide 5 is: [(S)-4-amino-3-(3-chloro-5-fluorophenyl)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-);

[0043] 1. Preparation and purification of antimicrobial peptide 1

[0044] (1) Preparation of 4-((tert-butoxycarbonyl)amino)-3-(3-ethyl-5-(trifluoromethyl)-phenyl)-butyric acid

[0045] A mixture of 10 g of (E)-3-(3-ethyl-5-(trifluoromethyl)phenyl)acrylic acid, 100 mL of methanol, and 4 mL of concentrated sulfuric acid was stirred at room temperature for 20 hours. The mixture was evaporated to dryness, and the residue was partitioned between dichloromethane (DCM) and water. The aqueous phase was separated and then extracted with dichloromethane. The organic extracts were combined, dried over magnesium sulfate, filtered, and evaporated to dryness to give 10.19 g of a white solid.

[0046] A mixture of 32 mL nitromethane and 8.5 mL 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added and stirred at room temperature for 20 hours. The mixture was evaporated to dryness, and the residue was partitioned between 0.5 M hydrochloric acid aqueous solution and diethyl ether. The aqueous phase was separated and extracted with diethyl ether. The organic extracts were combined, washed with brine, dried over magnesium sulfate, filtered, and evaporated to dryness. The residue was purified on silica gel and eluted with hexane and ethyl acetate (0-100%). Evaporation to dryness gave 9.93 g of a yellow oil.

[0047] 20.1 g of zinc powder was added to 90 mL of acetic acid solution containing a yellow oily substance stirred at 0 °C. The mixture was heated to room temperature and stirred for 19 hours. The mixture was evaporated to dryness, and the residue was partitioned between NaHCO3 solution and ethyl acetate. The mixture was then filtered through diatomaceous earth to separate the aqueous and organic phases. The aqueous phase was then extracted with ethyl acetate. The organic extracts were combined, washed with brine, dried over magnesium sulfate, filtered, and evaporated to dryness to give 4.80 g of an orange oily substance.

[0048] The mixture was dissolved in 100 mL of dichloromethane and treated with 5.28 g of di-tert-butyl dicarbonate. The mixture was stirred at room temperature for 18 hours. The mixture was evaporated to dryness, and the residue was purified on silica gel by elution with 0-100% ethyl acetate in hexane. Evaporation to dryness gave 2.59 g of the desired milky white solid product.

[0049] A mixture of 2.59 g of a milky white solid product, 546 mg of lithium hydroxide, 40 mL of 1,4-dioxane, and 40 mL of water was stirred at room temperature for 64 hours. The mixture was evaporated to dryness. The residue was dissolved in water, neutralized with 1 M HCl solution, and extracted twice with ethyl acetate. The organic extracts were combined, washed with brine, dried over magnesium sulfate, filtered, and evaporated to dryness to give 2.51 g of a yellow oily product of 4-((tert-butoxycarbonyl)amino)-3-(3-ethyl-5-(trifluoromethyl)-phenyl)-butyric acid.

[0050] 4-((tert-Butoxycarbonyl)amino)-3-(3-ethyl-5-(trifluoromethyl)-phenyl)butyric acid was purified by high performance liquid chromatography (HPLC), and the fraction was distilled under reduced pressure at a bath temperature of 40 °C to obtain the desired rapidly separated enantiomer, namely the title compound.

[0051] High-performance liquid chromatograph: Agilent 1200; Column: Phenomenex Hyperclone C18 BDS, 5 μm, 4.6 mm × 150 mm; Detection wavelength: 210, 254 nm; Mobile phase A: water / acetonitrile (90 / 10, v / v), containing 0.15% TFA; Mobile phase B: acetonitrile / water (90 / 10, v / v), containing 0.15% TFA; Flow rate: 1 mL / min; Injection volume: 20 μL; Elution gradient: 0 min, 100% mobile phase A; 20 min, 40% mobile phase A, 60% mobile phase B; 21 min, 100% mobile phase B; 23 min, 100% mobile phase B; 23.5 min, 100% mobile phase A; 25 min, 100% mobile phase A.

[0052] (2) Cyclic peptides were prepared using the standard Fmoc solid-phase peptide synthesis method in an automated peptide synthesizer. 10 g of CTC-Resin with a degree of substitution of 0.5 ± 0.1 mmol / g was added to the peptide reactor, along with 2 g of Fmoc-Thr(tBu)-OH and 10 mL of N,N-diisopropylethylamine (DIEA). The mixture was placed on a shaker and gently shaken for 2 hours. The reaction solution was then removed and the mixture was washed twice with DMF. After removing the Fmoc protection, the mixture was washed twice with N,N-dimethylformamide (DMF), once with dichloromethane (DCM), and twice with DMF.

[0053] (3) Add equal amounts of amino acids in sequence for reaction, including 2.22g Fmoc-Dab(Boc)-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.78g Fmoc-D-Nle-OH, 1.63g Fmoc-Abu-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.72g Fmoc-Dab-OH, 2.15g Fmoc-Dap(Boc)-OH, 2g Fmoc-Thr(tBu)-OH, 2.22g Fmoc-Dab(Boc)-OH, and 1.89g 4-[(tert-butoxycarbonyl)amino]-3-(3-ethyl-5-(trifluoromethyl)phenyl)butyric acid. For each peptide addition, 10 mL of N,N-diisopropylethylamine (DIEA) was added. The mixture was placed on a shaker and gently shaken for 2 hours. The reaction solution was then removed, and the mixture was washed twice with DMF. Fmoc deprotection was performed using a 20% piperidine N,N-dimethylformamide solution for 0.5 hours.

[0054] (4) After the reaction was completed, the resin was washed twice with DMF, once with DCM, and twice with DMF. The washed resin was then washed twice with methanol, and then the resin was removed twice with 200 mL of trifluoroethanol / dichloromethane at a volume ratio of 1:2, with each reaction being stirred for 2 hours. The resin was filtered off, the reaction solution was collected, and evaporated to dryness to obtain 6.8 g of (S)-4-amino-3-(3-ethyl-5-(trifluoromethyl)phenyl)butyryl-Thr(tBu)-Dap(Boc)-Dab-Dab(Boc)-D-Nle-Abu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH.

[0055] (5) Dissolve 6.8 g of (S)-4-amino-3-(3-ethyl-5-(trifluoromethyl)phenyl)butyryl-Thr(tBu)-Dap(Boc)-Dab-Dab(Boc)-D-Nle-Abu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH in 20 mL of DMF solution, add 0.5 g of 1-hydroxybenzotriazole (HOBT) and 0.5 mL of N, N'-Diisopropylcarbodiimide (DIC) was reacted at room temperature with stirring at 400 rpm for 2 hours. The reaction solution was then poured into 200 mL of ice water and magnetically stirred at 400 rpm for 2 hours. After filtration to obtain a solid, the solid was washed twice with 50 mL of water, and the filter cake was dried using an oil pump to obtain 5.4 g of white solid (S)-4-amino-3-(3-ethyl-5-(trifluoromethyl)phenyl)butyryl-Thr(tBu)-Dap(Boc)-Cyclo(Dab-Dab(Boc)-D-Nle-Abu-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH).

[0056] (6) Dissolve 5.4g of the aforementioned product in 60mL of trifluoroacetic acid / water / triisopropylsilane (volume ratio 8:1:1), stir at 15℃ and 400rpm for 3 hours, add dropwise to 500mL of ice-cold ether solution, centrifuge the precipitate, wash three times with ether, dry under reduced pressure to obtain 2.2g of white solid (S)-4-amino-3-(3-ethyl-5-(trifluoromethyl)phenyl)butyryl-Thr-Dap-Cyclo(Dab-Dab-D-Nle-Abu-Dab-Dab-Thr-OH).

[0057] (7) The aforementioned product was dissolved in 10 ml of 10% acetonitrile-water solution, filtered, and purified by high performance liquid chromatography (HPLC) to obtain 1.1 g of antimicrobial peptide 1 with an HPLC purity of 98.49% (see [link to HPLC]). Figure 1 The chromatographic conditions are as follows:

[0058] High-performance liquid chromatograph: Agilent 1200; Column: Phenomenex Hyperclone BDS C18, 5 μm, 4.6 mm × 150 mm; Detection wavelength: 210 nm; Mobile phase A: water / acetonitrile (90 / 10, v / v), containing 0.15% TFA; Mobile phase B: acetonitrile / water (90 / 10, v / v), containing 0.15% TFA; Flow rate: 1 mL / min; Elution gradient: 0 min, 100% mobile phase A; 20 min, 40% mobile phase A, 60% mobile phase B; 21 min, 0% mobile phase A, 100% mobile phase B; 23 min, 0% mobile phase A, 100% mobile phase B; 23.5 min, 100% mobile phase A; 25 min, 100% mobile phase A.

[0059] 2. Preparation and purification of antimicrobial peptide 2

[0060] 4-((tert-Butoxycarbonyl)amino)-3-(3-isobutylphenyl)-butyric acid was prepared using 10 g (E)-3-(3-isobutylphenyl)acrylic acid, 100 mL methanol, and 4 mL concentrated sulfuric acid as raw materials, as described in step (1) above. Nitrification, reduction, Boc protection, hydrolysis, peptide coupling, and deprotection reactions were carried out sequentially as described in steps (2) to (7). The crude product was purified by HPLC to obtain antimicrobial peptide 2, with an HPLC purity of 98.35% (see [link to HPLC]). Figure 2 ).

[0061] 3. Preparation and purification of antimicrobial peptide 3

[0062] Using 10 g (E)-3-(3-chlorophenyl)acrylic acid, 100 mL methanol, and 4 mL concentrated sulfuric acid as raw materials, 4-((tert-butoxycarbonyl)amino)-3-(3-chlorophenyl)-butyric acid was prepared as described in step (1) above. Then, nitration, reduction, Boc protection, hydrolysis, peptide coupling, and deprotection reactions were carried out sequentially as described in steps (2) to (7). The crude product was purified by HPLC to obtain antimicrobial peptide 3 with an HPLC purity of 98.48% (see...). Figure 3 ).

[0063] 4. Preparation and purification of antimicrobial peptide 4

[0064] 4-((tert-Butoxycarbonyl)amino)-3-(3-chloro-5-methylphenyl)butyric acid was prepared using 10 g (E)-3-(3-chloro-5-methylphenyl)acrylic acid, 100 mL methanol, and 4 mL concentrated sulfuric acid as raw materials, as described in step (1) above. Nitration, reduction, Boc protection, hydrolysis, peptide coupling, and deprotection reactions were carried out sequentially as described in steps (2) to (7). The crude product was purified by HPLC to obtain antimicrobial peptide 4, with an HPLC purity of 98.11% (see [link to HPLC]). Figure 4 ).

[0065] 5. Preparation and purification of antimicrobial peptide 5

[0066] 4-((tert-Butoxycarbonyl)amino)-3-(3-chloro-5-fluorophenyl)butyric acid was prepared using 10 g (E)-3-(3-chloro-5-fluorophenyl)acrylic acid, 100 mL methanol, and 4 mL concentrated sulfuric acid as raw materials, as described in step (1) above. Nitrification, reduction, Boc protection, hydrolysis, peptide coupling, and deprotection reactions were carried out sequentially as described in steps (2) to (7). The crude product was purified by HPLC to obtain antimicrobial peptide 5 with an HPLC purity of 98.23% (see [link to HPLC]). Figure 5 ).

[0067] Combination Figure 1-5 The HPLC chromatograms show that the peak area of ​​each antimicrobial peptide exceeds 98% (98.49% for antimicrobial peptide 1, 98.35% for antimicrobial peptide 2, 98.48% for antimicrobial peptide 3, 98.11% for antimicrobial peptide 4, and 98.23% for antimicrobial peptide 5), indicating that the prepared antimicrobial peptides 1-5 have high purity. The peptides prepared above were identified and analyzed using an Agilent 1200 tandem with an AB SCIEX API3200, and the results are as follows... Figure 1-5 And as shown in Table 1:

[0068] Table 1. Basic chemical parameters, HPLC retention time, and mass-to-charge ratio (m / z) of antimicrobial peptides.

[0069]

[0070] Example 2: Microthermophoresis (MST) for determining the interaction between antimicrobial peptides and lipids

[0071] In this study, polymyxin B and D50 peptide were used as control peptides, prepared and synthesized by Shanghai Jier Biochemical Co., Ltd. (purity >96%), and could be used without further purification. 1,2-Dimyristoylphosphatidylglycerol (DMPG, Avanti Polar Lipids, catalog number 840445) or 1,2-dimyristoylphosphatidylcholine (DMPC, Avanti Polar Lipids, catalog number 850345) were weighed into flasks, and a chloroform-methanol mixture with a volume ratio of 3:1 was added to fully dissolve and obtain a clear lipid solution. After drying the lipid solution under a nitrogen stream, it was placed in a vacuum environment overnight to obtain DMPG and DMPC lipid films. Phosphate buffer with a pH of 7.4 and a concentration of 10 mmol / L was added for hydration to bring the final concentration of DMPG or DMPC lipids to 100 μmol / L. After shaking on a horizontal shaker at room temperature for 1 h, the above system was sonicated at 150 W for 0.5 h using a Sonics Vibra-Cell sonic cell disruptor to obtain DMPG or DMPC liposomes. The particle size of the prepared liposomes was determined using dynamic light scattering with a Zetasizer Nano ZS90 instrument from Malvern Instruments Ltd., UK. The results showed that the particle sizes of DMPG and DMPC liposomes were 101±8 nm and 380±11 nm, respectively. Antimicrobial peptides 1-5, polymyxin B, or D50 peptide were dissolved in PBS at pH 7.4 to prepare 200 nM solutions. A series of dilutions of liposomes with concentrations of 100, 50, 25, 12.5, 6.25, 3.125, 1.563, 0.781, 0.391, 0.195, 0.098, 0.049, 0.024, 0.012, 0.006, and 0.003 μmol / L were prepared. The antimicrobial peptide solutions and the liposome dilutions were thoroughly mixed at a volume ratio of 1:1 and injected into standard glass capillaries. Detection was performed using a NanoTemper Monolith NT.115 molecular interaction analyzer. The MST experimental parameters were set as follows: LED power (heating solution) 50%, MST power (blue light excitation) 20%, on / off time 30 seconds and 5 seconds respectively, and experimental temperature controlled at 37℃. The NanoTemper Monolith NT.115 was used for the analysis. Data analysis using analysis software yielded the affinity (K) of antimicrobial peptides 1-5, polymyxin B, and D50 peptide for DMPG and DMPC. d (μM), the results are shown in Table 2 below.

[0072] Table 2 clearly presents the differences in affinity of each test sample for DMPG (a mimic of phosphatidylglycerol, a major component of bacterial cell membranes) and DMPC (a mimic of phosphatidylcholine, a major component of mammalian cell membranes). Polymyxin B showed an affinity of 2.8 μM for DMPG and 9.1 μM for DMPC, with a membrane selectivity of 3.3. The control peptide D50 showed an affinity of 1.5 μM for DMPG and 17.0 μM for DMPC, with a membrane selectivity of 11.3. Antimicrobial peptides 1-5 exhibited superior performance, with antimicrobial peptide 1 showing an affinity of 0.07 μM for DMPG, antimicrobial peptide 2 0.05 μM, antimicrobial peptide 3 0.06 μM, antimicrobial peptide 4 0.09 μM, and antimicrobial peptide 5 0.08 μM. Furthermore, all of them showed an affinity for DMPC greater than 200 μM and a membrane selectivity greater than 2000. Antimicrobial peptides 1-5 exhibited an affinity for DMPG that was more than 31 times greater than that of colistin B and more than 16 times greater than that of D50 peptide. This result demonstrates that antimicrobial peptides 1-5 exhibit extremely strong affinity for bacterial cell membrane phospholipids, while showing very low affinity for mammalian cell membrane phospholipids. This implies that they can act on bacterial cell membranes with high selectivity, exerting antimicrobial effects while minimizing the potential risk of damage to host cells. This provides important experimental evidence and theoretical support for the development of novel, highly effective, and low-toxicity antimicrobial drugs.

[0073] Table 2. Affinity of antimicrobial peptides 1-5, polymyxin B, and control peptide D50 to DMPG and DMPC

[0074]

[0075] Example 3: Antibacterial activity experiment of antimicrobial peptides

[0076] According to the Clinical and Laboratory Standards Institute (CLSI) document M07-A10, namely the standard operating procedure for the aerobic dilution method for antimicrobial susceptibility testing, the minimum inhibitory concentration (MIC) of antimicrobial peptides 1-5 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 method.

[0077] (1) Fresh Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC13822, Pseudomonas aeruginosa ATCC27853, and Acinetobacter baumannii NCTC13424 colonies were cultured on Mueller-Hinton (MH) agar plates for 18 hours under aerobic conditions at 35℃. Bacterial suspensions were prepared using sterile physiological saline and calibrated to 0.5 McFarland turbidity by turbidimetric method.

[0078] (2) In a sterile 96-well microplate, the antimicrobial peptides 1-5, D50 peptide or polymyxin B were subjected to double serial dilutions using cationic-adjusted MH broth to form antimicrobial solutions with concentration gradients of 0.0075, 0.015, 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.

[0079] (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;

[0080] (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.

[0081] 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.

[0082] The results are shown in Table 3. The minimum inhibitory concentrations (MICs) of antimicrobial peptides 1-5 against *Escherichia coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii* were all 0.03 μg / mL, exhibiting extremely strong antimicrobial activity. In contrast, polymyxin B had an MIC of 1 μg / mL. Experimental data show that compared to the clinically commonly used polymyxin B, the antimicrobial efficacy of antimicrobial peptides 1-5 is approximately 33 times higher; compared to the D50 control peptide, the antimicrobial activity of antimicrobial peptides 1-5 is approximately 17-33 times stronger. These experimental results indicate that antimicrobial peptides 1-5 can effectively inhibit the growth of Gram-negative bacteria (*Escherichia coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii*) at extremely low concentrations (0.03 μg / mL). Their antimicrobial efficacy is significantly superior to existing clinical drugs polymyxin B and the control peptide D50, demonstrating in vitro antimicrobial efficacy far exceeding that of traditional antibiotics, and has potential application value in the treatment of Gram-negative bacterial infections.

[0083] Table 3. Minimum Inhibitory Concentration (MIC) of Antimicrobial Peptides against Bacteria

[0084]

[0085] Example 4: In vitro renal cytotoxicity assay of antimicrobial peptides

[0086] Cytotoxicity was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole bromide (MTT) colorimetric method.

[0087] (1) HK-2 human kidney 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. They were then seeded in 96-well plates at a density of 2.5×10³ cells / well and pre-cultured for 24 hours until the cells were completely adhered.

[0088] (2) Add serially diluted antimicrobial peptides 1-5, D50 peptide or polymyxin B to adherent cell wells, with final concentration gradients of 0.0075, 0.015, 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;

[0089] (3) After removing the drug-containing culture medium, 10 μL of 5 mg / mL MTT-PBS solution was mixed with 100 μL of DMEM culture medium. 110 μL of MTT working solution was added to each well and incubated in the dark for 3 hours to induce the formation of formazan crystals.

[0090] (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 570 nm.

[0091] (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 using a nonlinear regression model. 50 ), defined as the concentration of a compound that reduces cell viability by 50% compared to the negative control.

[0092] The results are as follows Figure 6 As shown, antimicrobial peptides 1-5 exhibit extremely low toxicity to proximal tubular epithelial cells of HK-2 human kidneys. Compared to the commonly used clinical drug polymyxin B, antimicrobial peptides 1-5 demonstrate at least a 60-fold improvement in safety, and compared to the D50 control peptide, its safety is at least a 12-fold improvement. These experimental results indicate that antimicrobial peptides 1-5, while exerting their antibacterial effect, can minimize the risk of damage to normal cells (such as proximal tubular epithelial cells of HK-2 human kidneys). Their safety is significantly superior to existing clinical drugs polymyxin B and the control peptide D50, providing a technical solution for antimicrobial therapy that combines highly effective antibacterial activity with low cytotoxicity.

[0093] Example 5: In vivo efficacy evaluation of antimicrobial peptides

[0094] 1. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Escherichia coli ATCC25922

[0095] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in independent 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:

[0096] Treatment groups: 0.4 mg / kg polymyxin B, 1.6 mg / kg polymyxin B, and 3.2 mg / kg polymyxin B.

[0097] Treatment groups with 0.4 mg / kg D50 control peptide, 1.6 mg / kg D50 control peptide, and 3.2 mg / kg D50 control peptide.

[0098] Treatment groups: 0.4 mg / kg antimicrobial peptide 1, 1.6 mg / kg antimicrobial peptide 1, and 3.2 mg / kg antimicrobial peptide 1.

[0099] Treatment groups: 0.4 mg / kg antimicrobial peptide 2, 1.6 mg / kg antimicrobial peptide 2, and 3.2 mg / kg antimicrobial peptide 2.

[0100] Treatment groups: 0.4 mg / kg antimicrobial peptide 3, 1.6 mg / kg antimicrobial peptide 3, and 3.2 mg / kg antimicrobial peptide 3.

[0101] Treatment groups: 0.4 mg / kg antimicrobial peptide 4, 1.6 mg / kg antimicrobial peptide 4, and 3.2 mg / kg antimicrobial peptide 4.

[0102] Treatment groups: 0.4 mg / kg antimicrobial peptide 5, 1.6 mg / kg antimicrobial peptide 5, and 3.2 mg / kg antimicrobial peptide 5.

[0103] And a blank control group;

[0104] (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.

[0105] (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, D50 peptide, or antimicrobial peptide were administered according to the above groupings, while the blank control group received physiological saline.

[0106] (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 (CFU / g), the results are shown in Table 4.

[0107] Table 4. Logarithmic difference of antimicrobial peptides 1-5 in reducing the bacterial load of Escherichia coli ATCC25922 infection in mouse thigh.

[0108]

[0109] 2. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Klebsiella pneumoniae ATCC43816

[0110] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in independent 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:

[0111] Treatment groups: 0.4 mg / kg polymyxin B, 1.6 mg / kg polymyxin B, and 3.2 mg / kg polymyxin B.

[0112] Treatment groups with 0.4 mg / kg D50 control peptide, 1.6 mg / kg D50 control peptide, and 3.2 mg / kg D50 control peptide.

[0113] Treatment groups: 0.4 mg / kg antimicrobial peptide 1, 1.6 mg / kg antimicrobial peptide 1, and 3.2 mg / kg antimicrobial peptide 1.

[0114] Treatment groups: 0.4 mg / kg antimicrobial peptide 2, 1.6 mg / kg antimicrobial peptide 2, and 3.2 mg / kg antimicrobial peptide 2.

[0115] Treatment groups: 0.4 mg / kg antimicrobial peptide 3, 1.6 mg / kg antimicrobial peptide 3, and 3.2 mg / kg antimicrobial peptide 3.

[0116] Treatment groups: 0.4 mg / kg antimicrobial peptide 4, 1.6 mg / kg antimicrobial peptide 4, and 3.2 mg / kg antimicrobial peptide 4.

[0117] Treatment groups: 0.4 mg / kg antimicrobial peptide 5, 1.6 mg / kg antimicrobial peptide 5, and 3.2 mg / kg antimicrobial peptide 5.

[0118] And a blank control group;

[0119] (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.

[0120] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5 CFU Klebsiella pneumoniae ATCC43816 standard strain suspension was administered via tail vein injection at 2, 6, and 10 hours post-infection. Polymyxin B, D50 peptide, or antimicrobial peptide were administered according to the above groupings, while the blank control group received physiological saline.

[0121] (4) Sixteen hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, each thigh tissue sample was homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto CLED agar and incubated at 37°C for 24 hours. Bacterial load was determined by CFU counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated (log...). 10 (CFU / g), the results are shown in Table 5.

[0122] Table 5. Logarithmic difference in the reduction of bacterial load of Klebsiella pneumoniae ATCC43816 infection in mouse thigh by antimicrobial peptides 1-5

[0123]

[0124] 3. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Acinetobacter baumannii NCTC13424

[0125] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in independent 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:

[0126] Treatment groups: 0.4 mg / kg polymyxin B, 1.6 mg / kg polymyxin B, and 3.2 mg / kg polymyxin B.

[0127] Treatment groups with 0.4 mg / kg D50 control peptide, 1.6 mg / kg D50 control peptide, and 3.2 mg / kg D50 control peptide.

[0128] Treatment groups: 0.4 mg / kg antimicrobial peptide 1, 1.6 mg / kg antimicrobial peptide 1, and 3.2 mg / kg antimicrobial peptide 1.

[0129] Treatment groups: 0.4 mg / kg antimicrobial peptide 2, 1.6 mg / kg antimicrobial peptide 2, and 3.2 mg / kg antimicrobial peptide 2.

[0130] Treatment groups: 0.4 mg / kg antimicrobial peptide 3, 1.6 mg / kg antimicrobial peptide 3, and 3.2 mg / kg antimicrobial peptide 3.

[0131] Treatment groups: 0.4 mg / kg antimicrobial peptide 4, 1.6 mg / kg antimicrobial peptide 4, and 3.2 mg / kg antimicrobial peptide 4.

[0132] Treatment groups: 0.4 mg / kg antimicrobial peptide 5, 1.6 mg / kg antimicrobial peptide 5, and 3.2 mg / kg antimicrobial peptide 5.

[0133] And a blank control group;

[0134] (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.

[0135] (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, D50 peptide, or antimicrobial peptide were administered according to the above groupings, while the blank control group received physiological saline.

[0136] (4) Sixteen hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, each thigh tissue sample was homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto CLED agar and incubated at 37°C for 24 hours. Bacterial load was determined by CFU counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated (log...). 10 (CFU / g), the results are shown in Table 6.

[0137] Table 6. Logarithmic difference in the reduction of bacterial load of Acinetobacter baumannii NCTC13424 infection in mouse thigh by antimicrobial peptides 1-5

[0138]

[0139] 4. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Pseudomonas aeruginosa ATCC27853

[0140] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in independent 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:

[0141] Treatment groups: 0.4 mg / kg polymyxin B, 1.6 mg / kg polymyxin B, and 3.2 mg / kg polymyxin B.

[0142] Treatment groups with 0.4 mg / kg D50 control peptide, 1.6 mg / kg D50 control peptide, and 3.2 mg / kg D50 control peptide.

[0143] Treatment groups: 0.4 mg / kg antimicrobial peptide 1, 1.6 mg / kg antimicrobial peptide 1, and 3.2 mg / kg antimicrobial peptide 1.

[0144] Treatment groups: 0.4 mg / kg antimicrobial peptide 2, 1.6 mg / kg antimicrobial peptide 2, and 3.2 mg / kg antimicrobial peptide 2.

[0145] Treatment groups: 0.4 mg / kg antimicrobial peptide 3, 1.6 mg / kg antimicrobial peptide 3, and 3.2 mg / kg antimicrobial peptide 3.

[0146] Treatment groups: 0.4 mg / kg antimicrobial peptide 4, 1.6 mg / kg antimicrobial peptide 4, and 3.2 mg / kg antimicrobial peptide 4.

[0147] Treatment groups: 0.4 mg / kg antimicrobial peptide 5, 1.6 mg / kg antimicrobial peptide 5, and 3.2 mg / kg antimicrobial peptide 5.

[0148] And a blank control group;

[0149] (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.

[0150] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5 CFU Pseudomonas aeruginosa ATCC27853 standard strain suspension was administered via tail vein injection at 1, 3.5, and 6 hours post-infection. Polymyxin B, D50 peptide, or antimicrobial peptide were administered according to the above groupings, while the blank control group received physiological saline.

[0151] (4) Nine hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, each thigh tissue sample was homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto CLED agar and incubated at 37°C for 24 hours. Bacterial load was determined by CFU counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated (log...). 10 (CFU / g), the results are shown in Table 7.

[0152] Table 7. Logarithmic differences in the reduction of bacterial load of Pseudomonas aeruginosa ATCC27853 infection in mouse thigh by antimicrobial peptides 1-5

[0153]

[0154] As shown in Tables 4-7, in a mouse model of neutropenia caused by Gram-negative bacterial infection, antimicrobial peptides 1-5 exhibited excellent antimicrobial activity at all tested doses. Their antimicrobial efficacy significantly increased with increasing dose, demonstrating a good dose-dependent relationship. Furthermore, under the same dosage conditions, the therapeutic effect of antimicrobial peptides 1-5 was significantly superior to the control: in the *E. coli* ATCC25922 infection model, compared with polymyxin B, the bacterial load reduction efficacy of each dose group was 6.8-12.0 times higher, and compared with the D50 control peptide, it was 5.4-7.3 times higher; in the *Klebsiella pneumoniae* ATCC43816 infection model, compared with polymyxin B, the bacterial load reduction efficacy of each dose group was 5.2-8.2 times higher, and compared with the D50 control peptide, it was 3.0-... In the Acinetobacter baumannii NCTC13424 infection model, compared with polymyxin B, the bacterial load reduction efficacy of each dose group was increased by 5.9-10.5 times, and compared with the D50 control peptide, it was increased by 4.1-5.6 times. In the Pseudomonas aeruginosa ATCC27853 infection model, compared with polymyxin B, the bacterial load reduction efficacy of each dose group was increased by 3.9-8.6 times, and compared with the D50 control peptide, it was increased by 3.2-5.4 times. The above experimental results show that antimicrobial peptides 1-5 exhibit significantly superior antimicrobial advantages over existing clinical drugs polymyxin B and control peptide D50 in the thigh model of neutropenia mice infected with Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa).

[0155] 5. Efficacy of antimicrobial peptides in a mouse model of neutropenia infected with Acinetobacter baumannii NCTC13424

[0156] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in independent 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:

[0157] Treatment groups: 5 mg / kg polymyxin B, 7 mg / kg polymyxin B, and 14 mg / kg polymyxin B.

[0158] 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group,

[0159] Treatment groups: 5 mg / kg antimicrobial peptide 1, 7 mg / kg antimicrobial peptide 1, and 14 mg / kg antimicrobial peptide 1.

[0160] Treatment groups: 5 mg / kg antimicrobial peptide 2, 7 mg / kg antimicrobial peptide 2, and 14 mg / kg antimicrobial peptide 2.

[0161] 5 mg / kg antimicrobial peptide 3 treatment group, 7 mg / kg antimicrobial peptide 3 treatment group, 14 mg / kg antimicrobial peptide 3 treatment group,

[0162] 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group,

[0163] Treatment groups: 5 mg / kg antimicrobial peptide 5, 7 mg / kg antimicrobial peptide 5, and 14 mg / kg antimicrobial peptide 5.

[0164] And a blank control group;

[0165] (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.

[0166] (3) Mice were intranasally inoculated with 1×10 7 CFU / lung lobe 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, with polymyxin B, D50 peptide, or antimicrobial peptide administered according to the above-described groupings. The blank control group received normal saline.

[0167] (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 CLED agar and incubated at 37°C for 24 hours. Bacterial load was determined by CFU counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated (log...). 10 (CFU / g), the results are shown in Table 8.

[0168] Table 8. Logarithmic difference between antimicrobial peptides 1-5 and the reduction of bacterial load of Acinetobacter baumannii NCTC13424 in mouse lungs.

[0169]

[0170] 6. Efficacy of antimicrobial peptides in a mouse model of neutropenia induced by Pseudomonas aeruginosa ATCC27853 infection

[0171] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in independent 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:

[0172] Treatment groups: 5 mg / kg polymyxin B, 7 mg / kg polymyxin B, and 14 mg / kg polymyxin B.

[0173] 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group,

[0174] Treatment groups: 5 mg / kg antimicrobial peptide 1, 7 mg / kg antimicrobial peptide 1, and 14 mg / kg antimicrobial peptide 1.

[0175] Treatment groups: 5 mg / kg antimicrobial peptide 2, 7 mg / kg antimicrobial peptide 2, and 14 mg / kg antimicrobial peptide 2.

[0176] 5 mg / kg antimicrobial peptide 3 treatment group, 7 mg / kg antimicrobial peptide 3 treatment group, 14 mg / kg antimicrobial peptide 3 treatment group,

[0177] 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group,

[0178] Treatment groups: 5 mg / kg antimicrobial peptide 5, 7 mg / kg antimicrobial peptide 5, and 14 mg / kg antimicrobial peptide 5.

[0179] And a blank control group;

[0180] (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.

[0181] (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, with polymyxin B, D50 peptide, or antimicrobial peptide administered according to the above-described groupings. The blank control group received normal saline.

[0182] (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 CLED agar and incubated at 37°C for 24 hours. Bacterial load was determined by CFU counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated (log...). 10 (CFU / g), the results are shown in Table 9.

[0183] Table 9. Logarithmic differences in the reduction of bacterial load of Pseudomonas aeruginosa ATCC27853 infection in mouse lungs by antimicrobial peptides 1-5

[0184]

[0185] 7. Efficacy of antimicrobial peptides in a mouse model of neutropenia caused by Klebsiella pneumoniae ATCC43816 infection.

[0186] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in independent 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:

[0187] Treatment groups: 5 mg / kg polymyxin B, 7 mg / kg polymyxin B, and 14 mg / kg polymyxin B.

[0188] 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group,

[0189] Treatment groups: 5 mg / kg antimicrobial peptide 1, 7 mg / kg antimicrobial peptide 1, and 14 mg / kg antimicrobial peptide 1.

[0190] Treatment groups: 5 mg / kg antimicrobial peptide 2, 7 mg / kg antimicrobial peptide 2, and 14 mg / kg antimicrobial peptide 2.

[0191] 5 mg / kg antimicrobial peptide 3 treatment group, 7 mg / kg antimicrobial peptide 3 treatment group, 14 mg / kg antimicrobial peptide 3 treatment group,

[0192] 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group,

[0193] Treatment groups: 5 mg / kg antimicrobial peptide 5, 7 mg / kg antimicrobial peptide 5, and 14 mg / kg antimicrobial peptide 5.

[0194] And a blank control group;

[0195] (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.

[0196] (3) Mice were intranasally inoculated with 1×10 7 CFU / lung lobe Klebsiella pneumoniae ATCC43816 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, D50 peptide, or antimicrobial peptide according to the above-described groupings. The blank control group received normal saline.

[0197] (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 CLED agar and incubated at 37°C for 24 hours. Bacterial load was determined by CFU counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated (log...). 10 (CFU / g), the results are shown in Table 10.

[0198] Table 10. Logarithmic difference of antimicrobial peptides 1-5 in reducing bacterial load of Klebsiella pneumoniae ATCC43816 infection in mice.

[0199]

[0200] According to the experimental data shown in Table 8-10, in a mouse lung model of neutropenia infected with Gram-negative bacteria, antimicrobial peptides 1-5 showed clear antimicrobial activity across the entire dose range. Their antimicrobial efficacy increased linearly with increasing dose, exhibiting typical dose-dependent characteristics. Most importantly, at the same dosage, antimicrobial peptides 1-5 showed significant advantages over first-line clinical drugs polymyxin B and control peptide D50: In the Acinetobacter baumannii NCTC13424 infection model, compared with polymyxin B, the efficacy of each dose group in reducing bacterial load was increased by approximately 7.3-11.5 times, and compared with the D50 control peptide, it was increased by approximately 5.7-7.8 times; in the Pseudomonas aeruginosa ATCC27853 infection model, compared with polymyxin B, the efficacy of each dose group in reducing bacterial load was increased by approximately 5.2-9.2 times, and compared with the D50 control peptide, it was increased by 4.8-6.6 times; in the Klebsiella pneumoniae ATCC43816 infection model, compared with polymyxin B, the efficacy of each dose group in reducing bacterial load was increased by approximately 6.2-11.5 times, and compared with the D50 control peptide, it was increased by approximately 4.4-6.6 times. The above experimental results show that antimicrobial peptides 1-5 have demonstrated clear clinical translational value in the treatment of infections caused by multidrug-resistant Gram-negative bacteria (including Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae). Their antimicrobial activity is significantly superior to that of existing first-line clinical drugs polymyxin B and control peptide D50, providing a new technical solution for the treatment of multidrug-resistant bacterial infections.

[0201] A table of abbreviations used in the embodiments (English-Chinese translation):

[0202] .

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide has the following characteristics: The sequence [(S)-4-amino-3-(3-chloro-5-fluorophenyl)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-) 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, ester, solvate, or hydrate thereof, as an 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 any one or more of the following: excipients, 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 compositions 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.

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 preventing and / or controlling infections by any one or more of the following microorganisms: 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, endocrine system infection, and reproductive system infection.

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