Deuterated antibacterial peptide as well as composition and application thereof

Abstract

The invention discloses a deuterated antibacterial peptide as well as a composition and application thereof, and belongs to the field of biological medicines. By introducing deuterated 2-aminobutyric acid to form an annular structure and combining deuterated modification, the affinity of the antibacterial peptide to a bacterial membrane is greatly improved compared with that of traditional drugs (polymyxin B and D50), and the selectivity of the antibacterial peptide to a mammalian membrane is 884 times or above higher than that of a control; the minimum inhibitory concentration to gram-negative bacteria in vitro is as low as 0.015 mu g / mL, which is increased by 67 times compared with polymyxin B, is increased by 33-67 times compared with D50 control peptide, and is increased by about 17 times compared with non-deuterated antibacterial peptide 5, so that extremely-low-concentration efficient bacteriostasis is realized; the toxicity to human kidney cells is extremely low, and the biological safety is better. In a plurality of infection models, the treatment effect of the compound is obviously better than that of the three control substances, and the compound can be used for preparing anti-infection drugs, provides a new scheme of high efficiency and low toxicity for clinical anti-infection treatment, and is suitable for prevention and treatment of a plurality of infections.

Description

Technical Field

[0001] The present invention relates to polypeptides and applications thereof, and in particular to a deuterated antimicrobial peptide and a composition and application thereof. Background Art

[0002] The current problem of bacterial resistance is becoming increasingly serious, especially among Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. These bacteria, through biological mechanisms such as horizontal gene transfer and efflux pumps, have built complex resistance networks, posing significant technical challenges to clinical treatment. Carbapenem-resistant Klebsiella pneumoniae, with its high mortality rate, poses a significant threat to public health.

[0003] Polymyxin, a drug used to treat drug-resistant Gram-negative bacterial infections, works by specifically binding to lipopolysaccharide (LPS) lipid A on the bacterial outer membrane. However, in actual applications, there are obvious technical flaws: on the one hand, drug-resistant strains will modify LPS with phosphoethanolamine, which will significantly reduce the affinity between the drug and bacteria and activate the efflux system, causing the rapid emergence of drug resistance. This involves technical issues related to the interaction between the bacterial resistance mechanism and the drug action mechanism, making it difficult to sustain the effectiveness of polymyxin in the treatment of drug-resistant bacteria. On the other hand, during the treatment with polymyxin, patients are prone to irreversible renal tubular damage, accompanied by adverse reactions such as neurotoxicity and ototoxicity. From the perspective of the technical indicators of the drug therapeutic index, its therapeutic index is low, making it difficult to meet the clinical treatment requirements for drug safety and efficacy. This highlights the limitations of polymyxin in the technical connection between drug research and development and clinical application.

[0004] As natural amphiphilic molecules, antimicrobial peptides exert their antibacterial effects by disrupting bacterial cell membranes. Their technical properties are superior to those of traditional antibiotics in killing drug-resistant bacteria and they maintain good activity even at high temperatures. Furthermore, due to their diverse targets, they can reduce the probability of bacterial resistance from the perspective of the technical principles of bacterial resistance, providing a new technical approach to addressing the problem of antibiotic resistance. However, some currently available antimicrobial peptides, such as the optimal antimicrobial peptide D50 reported in patent CN106232617A, suffer from insufficient activity against Gram-negative bacteria and high cytotoxicity in practical applications. This has limited their further application in the clinical translation process. Therefore, based on clinical treatment needs and the technical goals of antimicrobial drug development, the development of novel antimicrobial peptides with high antimicrobial activity, low toxicity, and high biosafety is crucial for overcoming the clinical treatment dilemma caused by drug-resistant Gram-negative bacteria and meeting the technical and application needs of clinical treatment. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a deuterated antimicrobial peptide and a pharmaceutical composition thereof, in order to provide more options for antimicrobial infections, especially drug-resistant Gram-negative bacterial infections.

[0006] Technical solution: The antimicrobial peptide of the present invention has a specific structure selected from any one of the following (I)-(IV): (1) As shown in formula (I): ; (2) As shown in formula (II): ; (3) As shown in formula (III): ; (4) As shown in formula (IV): .

[0007] The pharmaceutical composition contains the antimicrobial peptide or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof as an active ingredient, with or without the addition of pharmaceutically acceptable excipients.

[0008] The pharmaceutical composition, the pharmaceutically acceptable excipients include one or more of excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, and pH buffer substances.

[0009] The dosage forms of the pharmaceutical composition include tablets, capsules, granules, oral liquids, syrups, powders, chewable tablets, effervescent tablets, sustained-release tablets, pellets, injections, powder injections, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, atomized solutions, film coatings, implants, orally disintegrating tablets, oral fast-dissolving films, sponges, and capsules.

[0010] Use of the antimicrobial peptide or the pharmaceutical composition in preparing a drug for preventing and / or controlling microbial infection.

[0011] In the application, the microbial infection is a bacterial infection.

[0012] In the application, the bacteria are Gram-negative bacteria.

[0013] In the application, the Gram-negative bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0014] In the application, the microbial infection is one or more of 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.

[0015] The application, the microbial infection includes urinary tract infection, pneumonia, burn infection, peritonitis, cholecystitis, pyelonephritis, sepsis, hospital-acquired pneumonia, trauma infection, 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.

[0016] Preferably, "pharmaceutically acceptable excipients" are substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit / risk ratio, and also include various excipients and diluents, which may contain liquids such as water, saline, glycerol and ethanol, or auxiliary substances such as lubricants, glidants, wetting agents, emulsifiers, and pH buffering substances.

[0017] Preferably, the pharmaceutical dosage form includes an injection, an oral agent or an external use agent, and the external use agent includes an eye drop or a lotion, wherein the dosage range of the antimicrobial peptide in the dosage form is: injection 0.001-1000 mg / kg; oral agent 0.001-1000 mg / kg; external use agent 1 / 10000-30% / vial; eye drops 1 / 10000-30% / vial; lotion 1 / 100000-20‰ / vial.

[0018] The core innovations of the present invention are as follows: 1. Conformational innovation: breaking through the traditional peptide mechanism of action

[0019] A novel cyclic structure design: The introduction of deuterated 2-aminobutyric acid into the core creates a cyclic conformation distinct from the linear structure of polymyxin B and the control peptide D50. This enhances their ultra-high affinity for bacterial membranes (140-280-fold compared to polymyxin B, 75-150-fold compared to D50, and 21-43-fold compared to the non-deuterated antimicrobial peptide 5) and their strong selectivity for mammalian membranes (over 884-fold compared to the control peptides polymyxin B and D50). This demonstrates their ability to precisely bind to bacterial membranes to exert their antibacterial effects while avoiding damage to mammalian membranes. This "bacterial targeting, host protection" property gives these antimicrobial peptides greater potential for application in the development of new antimicrobial drugs than traditional polymyxin B and the control peptide D50. This dual breakthrough in affinity and selectivity is the core advantage of antimicrobial peptides 1-4 that distinguishes them from traditional peptides. In addition, the unique advantage of deuterated modification: the MIC values ​​of antimicrobial peptides 1-4 against the four bacteria were increased by about 17 times compared with the non-deuterated antimicrobial peptide 5.

[0020] Value: Conformational innovation breaks through the resistance barrier of traditional antibiotics at the molecular level, providing a new paradigm for the development of "low-toxic and high-efficiency" antimicrobial peptides.

[0021] 2. Antibacterial activity: Highly effective in eliminating drug-resistant bacteria with significant dose dependence

[0022] Breakthrough in in vitro antibacterial efficacy: The minimum inhibitory concentration (MIC) for four Gram-negative bacteria, including Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, is as low as 0.015 μg / mL, which is 67 times higher than colistin B (MIC=1 μg / mL), 33-67 times higher than the D50 control peptide, and about 17 times higher than the non-deuterated antimicrobial peptide 5, achieving "high-efficiency antibacterial effect at extremely low concentrations."

[0023] Significant in vivo efficacy advantage: In the mouse thigh infection model, the antimicrobial peptide showed a more prominent in vivo antibacterial effect compared with polymyxin B, D50 control peptide and non-deuterated antimicrobial peptide 5, reflecting that the antimicrobial peptide has better antibacterial efficacy than the control drug at the same dose, and shows a clear dose-dependence (the antibacterial effect increases linearly with increasing dose).

[0024] Value: Solve the clinical problem of "high mortality rate and difficulty in elimination" of multidrug-resistant bacteria, and provide new treatment options for super bacteria infections such as carbapenem-resistant bacteria.

[0025] 3. Safety Optimization: Low Toxicity Breaks Through the Bottleneck of Traditional Antibiotics

[0026] Nephrotoxicity is significantly reduced: antimicrobial peptides are more toxic to HK-2 human renal proximal tubular epithelial cells than colistin B (IC 50 =1) with an 87-fold increase in safety compared to the D50 control peptide (IC 50=5) increased by about 17 times, and increased by about 5.8 times compared with the non-deuterated antimicrobial peptide 5, avoiding the clinical dilemma of "high toxicity and low efficiency" of polymyxin B and the control peptide D50.

[0027] Selective targeting of bacterial membranes: MST experiments have confirmed that antimicrobial peptides 1-4 have ultra-high affinity for bacterial membranes (140-280 times higher than polymyxin B, 75-150 times higher than D50, and 21-43 times higher than non-deuterated antimicrobial peptide 5), as well as extremely strong selectivity for mammalian membranes (more than 884 times higher than the controls polymyxin B and control peptide D50), proving that they can not only precisely bind to bacterial membranes to exert antibacterial effects, but also avoid damage to mammalian membranes.

[0028] Value: It breaks through the limitation of “narrow therapeutic window” of traditional antibiotics and provides a safe option for patients who take long-term medication or have immune deficiency.

[0029] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: (1) The antimicrobial peptide is a new type of highly effective and broad-spectrum antimicrobial peptide. Antimicrobial peptides 1-4 show excellent antimicrobial activity against a variety of Gram-negative bacteria such as Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. In vitro and in vivo experiments have confirmed that its antimicrobial efficacy is significantly better than that of polymyxin B, D50 peptide and non-deuterated antimicrobial peptide 5, and has good dose dependence, providing a new and effective means for the treatment of multidrug-resistant Gram-negative bacterial infections; (2) The antimicrobial peptide is a type of low-toxic and safe antimicrobial peptide. The antimicrobial peptide has extremely low toxicity to HK-2 cells, and its safety is significantly improved compared with polymyxin B and D50 control peptides, which is expected to reduce adverse reactions such as nephrotoxicity that occur during traditional antibiotic treatment. , improving the patient's tolerance and treatment safety; (3) The antimicrobial peptide destroys the bacterial cell membrane and is not easy to induce bacterial resistance, which makes up for the deficiency of traditional antibiotics in which the efficacy is reduced due to the problem of drug resistance, and provides a new idea and direction for solving the global antibiotic resistance crisis; (4) The antimicrobial peptide of the present invention has the advantages of good biocompatibility, high stability, low synthesis cost, etc., and has shown excellent therapeutic effects in a variety of infection models. It has great potential to be developed into a new type of anti-infective drug and is expected to be used in the prevention and treatment of various infections such as the respiratory system, urinary system, skin and soft tissue, bringing breakthrough progress to the treatment of clinical multidrug-resistant bacterial infections, and is expected to replace antibiotics and become a safe, green and efficient ideal antimicrobial agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the HPLC spectrum of antimicrobial peptide 1; Figure 2 is the HPLC spectrum of antimicrobial peptide 2; Figure 3 is the HPLC spectrum of antimicrobial peptide 3; Figure 4 is the HPLC spectrum of antimicrobial peptide 4; Figure 5 The results of in vitro renal cell toxicity assay of antimicrobial peptides are shown. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below.

[0032] The polypeptide compound in the embodiment of the present invention, antimicrobial peptide 1 is: Antimicrobial peptide 2 is:

[0033] Antimicrobial peptide 3 is:

[0034] Antimicrobial peptide 4 is:

[0035] Example 1: Preparation of antimicrobial peptides 1-4 1. Preparation and purification of antimicrobial peptide 1 (1) Preparation of 4-((tert-Butyloxycarbonyl)amino)-3-(3-isopropylphenyl)-butyric acid A mixture of 10 g of (E)-3-(3-isopropylphenyl)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 extracted with dichloromethane. The combined organic extracts were dried over magnesium sulfate, filtered, and evaporated to dryness to yield 10.19 g of a white solid.

[0036] A mixture of 32 mL of nitromethane and 8.5 mL of 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 aqueous hydrochloric acid and diethyl ether. The aqueous phase was separated and extracted with diethyl ether. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered, and evaporated to dryness. The residue was purified on silica gel, eluting with hexane and ethyl acetate (0-100%). Evaporation to dryness yielded 9.93 g of a yellow oil.

[0037] To a solution of the yellow oil in 90 mL of acetic acid, stirred at 0°C, was added 20.1 g of zinc powder. The mixture was warmed to room temperature and stirred for 19 hours. The mixture was evaporated to dryness, and the residue was partitioned between a NaHCO3 solution and ethyl acetate. The mixture was then filtered through celite to separate the aqueous and organic phases. The aqueous phase was further extracted with ethyl acetate. The combined organic extracts were washed with brine, dried over magnesium sulfate, filtered, and evaporated to dryness to yield 4.80 g of an orange oil.

[0038] The orange oil obtained in the previous step 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, eluting with 0-100% ethyl acetate in hexane. Evaporation to dryness gave 2.59 g of the desired product as an off-white solid.

[0039] A mixture of 2.59 g of the off-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 combined organic extracts were washed with brine, dried over magnesium sulfate, filtered, and evaporated to dryness to yield 2.51 g of 4-((tert-butoxycarbonyl)amino)-3-(3-isopropylphenyl)-butanoic acid as a yellow oil.

[0040] 4-((tert-Butyloxycarbonyl)amino)-3-(3-isopropylphenyl)butanoic acid was purified by high performance liquid chromatography (HPLC) and the fractions were evaporated under reduced pressure at a bath temperature of 40°C to give the desired rapidly separated enantiomer, the title compound.

[0041] High-performance liquid chromatography: Agilent 1200; chromatographic column: Phenomenex Hyperclone C18 BDS, 5 μm, 4.6 mm × 150 mm; detection wavelengths: 210 and 254 nm; mobile phase A: water / acetonitrile (90 / 10, volume ratio), containing 0.15% TFA; mobile phase B: acetonitrile / water (90 / 10, volume ratio), 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.

[0042] (2) Cyclic peptides were prepared using an automatic peptide synthesizer using the standard Fmoc solid-phase peptide synthesis method. 10 g of CTC-Resin with a degree of substitution of 0.5 ± 0.1 mmol / g was added to a peptide reactor, followed by 2 g of Fmoc-Thr(tBu)-OH and 10 mL of N,N-diisopropylethylamine (DIEA). The mixture was placed on an oscillator and gently shaken for 2 hours. The reaction solution was then removed and 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. (3) Equal amounts of amino acids were added in sequence for reaction, including 2.22 g Fmoc-Dab(Boc)-OH, 2.22 g Fmoc-Dab(Boc)-OH, 1.65 g Fmoc-Abu-OH-D3, 1.95 g Fmoc-D-Phe-OH, 2.22 g Fmoc-Dab(Boc)-OH, 1.72 g Fmoc-Dab-OH, 2.15 g Fmoc-Dap(Boc)-OH, 2 g Fmoc-Thr(tBu)-OH, 2.22 g Fmoc-Dab(Boc)-OH, and 1.89 g 4-[(tert-butyloxycarbonyl)amino]-3-(3-isopropylphenyl)butanoic acid. For each peptide segment, 10 mL of N,N-diisopropylethylamine (DIEA) was added and the mixture was gently shaken on a shaker for 2 hours. The reaction solution was then aspirated and washed twice with DMF. Fmoc deprotection was performed using a 20% piperidine solution in N,N-dimethylformamide for 0.5 hours.

[0043] (4) After the reaction, 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 removed twice with 200 mL of trifluoroethanol / dichloromethane (volume ratio 1:2). The stirring time for each reaction was 2 hours. The resin was filtered off, the reaction solution was collected, and dried to obtain 6.8 g of (S)-4-amino-3-(3-isopropylphenyl)butyryl-Thr(tBu)-Dap(Boc)-Dab-Dab(Boc)-D-Phe-Abu-D3-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH. (5) Dissolve 6.8 g of (S)-4-amino-3-(3-isopropylphenyl)butyryl-Thr(tBu)-Dap(Boc)-Dab-Dab(Boc)-D-Phe-Abu-D3-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 stirred at 400 rpm at room temperature for 2 hours. The reaction solution was poured into 200 mL of ice water and stirred with a magnetic stirrer at 400 rpm for 2 hours. The solid was filtered and rinsed twice with 50 mL of water. The filter cake was drained with an oil pump to obtain 5.4 g of a white solid (S)-4-amino-3-(3-isopropylphenyl)butyryl-Thr(tBu)-Dap(Boc)-Cyclo(Dab-Dab(Boc)-D-Phe-Abu-D3-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH). (6) 5.4 g of the product obtained in the previous step was dissolved in 60 mL of trifluoroacetic acid / water / triisopropylsilane (volume ratio 8:1:1) solution, stirred at 15 °C and 400 rpm for 3 hours, and then added dropwise to 500 mL of icy ether solution. The precipitate was centrifuged, washed three times with ether, and dried under reduced pressure to obtain 2.2 g of white solid (S)-4-amino-3-(3-isopropylphenyl)butyryl-Thr-Dap-Cyclo (Dab-Dab-D-Phe-Abu-D3-Dab-Dab-Thr-OH).

[0044] (7) The product obtained in the previous step 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 98.56% (see Figure 1 ). The chromatographic conditions are as follows: High-performance liquid chromatography: Agilent 1200; chromatographic column: Phenomenex Hyperclone BDS C18, 5 μm, 4.6 mm×150 mm; detection wavelength: 210 nm; mobile phase A: water / acetonitrile (90 / 10, volume ratio) containing 0.15% TFA; mobile phase B: acetonitrile / water (90 / 10, volume ratio) 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.

[0045] 2. Preparation and purification of antimicrobial peptide 2 4-((tert-Butyloxycarbonyl)amino)-3-(3-chlorophenyl)-butyric acid was prepared as described in step (1) using 10 g of (E)-3-(3-chlorophenyl)acrylic acid, 100 mL of methanol, and 4 mL of concentrated sulfuric acid. Nitration, reduction, Boc protection, hydrolysis, peptide coupling, and deprotection were performed sequentially as described in steps (2) to (7). In step (3), 2.22 g Fmoc-Dab(Boc)-OH, 2.22 g Fmoc-Dab(Boc)-OH, 1.65 g Fmoc-Abu-OH-D3, 1.78 g Fmoc-D-Leu-OH, 2.22 g Fmoc-Dab(Boc)-OH, 1.72 g Fmoc-Dab-OH, 2.15 g Fmoc-Dap(Boc)-OH, 2 g Fmoc-Thr(tBu)-OH, 2.22 g Fmoc-Dab(Boc)-OH, and 1.89 g 4-[(tert-butyloxycarbonyl)amino]-3-(3-chlorophenyl)butanoic acid were added. The crude product was purified by HPLC to obtain antimicrobial peptide 2 with an HPLC purity of 98.32% (see Figure 2 ).

[0046] 3. Preparation and purification of antimicrobial peptide 3 4-((tert-Butoxycarbonyl)amino)-3-(3-chlorophenyl)-butyric acid was prepared as described in step (1) using 10 g (E)-3-(3-chlorophenyl)acrylic acid, 100 mL methanol, and 4 mL concentrated sulfuric acid as raw materials. Nitration, reduction, Boc protection, hydrolysis, peptide coupling, and deprotection were performed in sequence as described in steps (2) to (7). The crude product was purified by HPLC to obtain antimicrobial peptide 3 with an HPLC purity of 98.39% (see Figure 3 ).

[0047] 4. Preparation and purification of antimicrobial peptide 4 4-((tert-Butyloxycarbonyl)amino)-3-(3,5-dichlorophenyl)-butyric acid was prepared as described in step (1) using 10 g of (E)-3-(3,5-dichlorophenyl)acrylic acid, 100 mL of methanol, and 4 mL of concentrated sulfuric acid. As in steps (2) to (7), nitration, reduction, Boc protection, hydrolysis, peptide coupling and deprotection reactions were carried out in sequence, wherein 2.22 g Fmoc-Dab(Boc)-OH, 2.22 g Fmoc-Dab(Boc)-OH, 1.65 g Fmoc-Abu-OH-D3, 1.78 g Fmoc-D-Nle-OH, 2.22 g Fmoc-Dab(Boc)-OH, 1.72 g Fmoc-Dab-OH, 2.15 g Fmoc-Dap(Boc)-OH, 2 g Fmoc-Thr(tBu)-OH, 2.22 g Fmoc-Dab(Boc)-OH, and 1.89 g 4-[(tert-butyloxycarbonyl)amino]-3-(3,5-dichlorophenyl)butanoic acid were added in step (3). The crude product was purified by HPLC to obtain antimicrobial peptide 4 with an HPLC purity of 98.12 (see Figure 4 ).

[0048] Combine Figure 1-4 The HPLC chromatogram shows that the main peak area of ​​each antimicrobial peptide exceeds 98% (antimicrobial peptide 1 is 98.56%, antimicrobial peptide 2 is 98.32%, antimicrobial peptide 3 is 98.39%, and antimicrobial peptide 4 is 98.12%), indicating that the prepared antimicrobial peptides 1-4 have high purity. The peptides prepared above were identified and analyzed using an Agilent 1200 in conjunction with an AB SCIEX API3200. The results are as follows Figure 1-4 As shown in Table 1:

[0049] Example 2: Microthermophoresis (MST) determination of the interaction between antimicrobial peptides and lipids In this study, polymyxin B, D50 peptide, and non-deuterated antimicrobial peptide 5 were prepared and synthesized by Shanghai Jier Biochemical Co., Ltd. (purity >96%) and used without further purification.

[0050]

[0051] 1,2-Dimyristoylphosphatidylglycerol (DMPG, Avanti Polar Lipids, Product No. 840445) or 1,2-Dimyristoylphosphatidylcholine (DMPC, Avanti Polar Lipids, Product No. 850345) was weighed into a flask and fully dissolved in a 3:1 volume ratio of chloroform-methanol mixture to obtain a clear lipid solution. The lipid solution was dried under a nitrogen stream and then placed in a vacuum overnight to obtain a DMPG or DMPC lipid film. 10 mmol / L phosphate buffer (pH 7.4) was added for hydration to a final DMPG or DMPC lipid concentration of 100 μmol / L. After shaking on a horizontal shaker at room temperature for 1 hour, the system was sonicated at 150 W for 0.5 hour using a Sonics Vibra-Cell ultrasonic cell disruptor to prepare DMPG or DMPC liposomes. The particle size of the prepared liposomes was measured using a Zetasizer Nano ZS90 instrument from Malvern Instruments Ltd., UK, using dynamic light scattering. 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 or polymyxin B or D50 peptide were dissolved in PBS (pH 7.4) to prepare 200 nM solutions. Serial dilutions of liposomes were prepared 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. The antimicrobial peptide solutions and serial liposome dilutions were thoroughly mixed at a volume ratio of 1:1 and injected into standard glass capillaries. The samples were detected using a NanoTemper Monolith NT.115 molecular interaction instrument. The MST experimental parameters were set as follows: LED power (heating solution) was 50%, MST power (blue light excitation) was 20%, on and off times were set to 30 s and 5 s, respectively, and the experimental temperature was controlled at 37°C. The affinity of antimicrobial peptides 1-5, polymyxin B and D50 peptide to DMPG and DMPC (K d , μM), and the results are shown in Table 2 below.

[0052] The results, shown in Table 2, demonstrate that antimicrobial peptides 1-4 exhibit exceptionally high affinity for bacterial membrane DMPG (140-280-fold greater than polymyxin B, 75-150-fold greater than D50, and 21-43-fold greater than the non-deuterated antimicrobial peptide 5), as well as exceptional selectivity for mammalian membrane DMPC (over 884-fold greater than the control peptides polymyxin B and D50). This demonstrates their ability to precisely bind to bacterial membranes to exert their antimicrobial effects while avoiding damage to mammalian membranes. This "bacterial targeting, host protection" property gives antimicrobial peptides greater potential for application in the development of novel antimicrobial drugs than traditional polymyxin B, the control peptide D50, and the non-deuterated antimicrobial peptide 5. This dual breakthrough in affinity and selectivity is the core advantage of antimicrobial peptides 1-4 over traditional peptides.

[0053]

[0054] Example 3: Antibacterial activity experiment of antimicrobial peptides According to the CLSI (Clinical Laboratory Standards Institute) M07-A10 standard operating procedure, the minimum inhibitory concentration (MIC) of antimicrobial peptides 1-5 against four common clinical Gram-negative bacteria, including Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC43816, Pseudomonas aeruginosa ATCC27853, and Acinetobacter baumannii NCTC13424, was determined by the microserial two-fold dilution method.

[0055] According to the synthesis method disclosed in CN106232617A, a D50 control peptide was synthesized for comparison.

[0056] (1) Fresh Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC43816, Pseudomonas aeruginosa ATCC27853, and Acinetobacter baumannii NCTC13424 colonies cultured on Mueller-Hinton (MH) agar plates at 35°C for 18 hours were used to prepare bacterial suspensions using sterile saline and calibrated to a McFarland turbidity of 0.5 by turbidimetry. (2) In a sterile 96-well microplate, a double gradient dilution of the antimicrobial peptides 1-5, D50 control peptide, or polymyxin B was performed using cationic MH broth to form an antimicrobial solution with a concentration gradient 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 DMSO concentration was 0.1%, and 150 μL of gradient dilution solution was pre-placed in each well. (3) Quantitatively inoculate 20 μL of standardized bacterial suspension into each well to make the final volume of the system 170 μL / well, and set up two parallel replicate wells for each test concentration; (4) The microplate was then placed in a 35°C incubator for 20 hours. The lowest drug concentration that completely inhibited visible microbial growth was determined by visual observation, with the disappearance of turbidity serving as the endpoint.

[0057] For compounds that were independently repeated five times or more, the median of the experimental results was taken as the final MIC value, and the concentration unit was uniformly expressed in μg / mL.

[0058] The results are shown in Table 3. In the minimum inhibitory concentration (MIC) assays against Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, antimicrobial peptides 1-4 all had MIC values ​​of 0.015 μg / mL, compared to 1 μg / mL for polymyxin B. The experimental data showed that antimicrobial peptides 1-4 increased their MIC values ​​by approximately 67-fold compared to polymyxin B against all four bacteria. Compared to the D50 control peptide, antimicrobial peptides 1-4 increased their MIC values ​​by approximately 33-fold against Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, and by approximately 67-fold against Acinetobacter baumannii. Furthermore, antimicrobial peptides 1-4 increased their MIC values ​​by approximately 17-fold compared to the non-deuterated antimicrobial peptide 5 against all four bacteria. This demonstrates that antimicrobial peptides can effectively inhibit the growth of Gram-negative bacteria at extremely low concentrations, demonstrating in vitro antibacterial potency far exceeding that of traditional antibiotics. With this characteristic, antimicrobial peptides 1-4 are expected to reduce the dosage and the risk of potential toxic and side effects in their application, thus laying a key foundation for the development of new anti-infective preparations. They have potential worthy of further exploration in the process of clinical transformation, and may bring innovative breakthroughs to the existing pattern of anti-infective treatment and provide new exploration directions for solving clinical problems related to bacterial infections.

[0059] Example 4: In vitro renal cell toxicity assay of antimicrobial peptides The cytotoxicity was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) colorimetric assay.

[0060] (1) HK-2 human renal proximal tubule epithelial cells CRL-2190 were inoculated in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2 until the logarithmic growth phase. They were 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 attached. (2) Add serially diluted antimicrobial peptides 1-5 to the adherent cell wells, D50 control peptide, or polymyxin B to a final concentration gradient of 0.0075, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / mL, and continue exposure and incubation for 48 hours; (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. Among them, each 110 μL of MTT working solution was obtained by mixing 10 μL of 5 mg / mL MTT-PBS solution with 100 μL of DMEM culture medium; (4) After aspirating 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 and shake for 10 minutes to fully dissolve the crystals. Measure the absorbance at a wavelength of 570 nm using a microplate reader. (5) The absorbance value of the wells treated with 0.25% DMSO was used as the negative control (100% cell viability) and the wells treated with polymyxin B was used as the positive control (0% cell viability). The half-maximal inhibitory concentration (IC 50 ), defined as the concentration of compound that reduces cell viability by 50% compared with the negative control.

[0061] The results are as follows Figure 5 As shown in the results, antimicrobial peptides 1-4 exhibit significant advantages: on the one hand, they exhibit extremely low toxicity to HK-2 human renal proximal tubular epithelial cells, and their safety is at least 87 times higher than that of the commonly used polymyxin B in clinical practice, and about 17 times higher than that of the D50 control peptide. Compared with the non-deuterated antimicrobial peptide 5, their safety is about 5.8 times higher. When exerting their antimicrobial effects, they can minimize the risk of damage to the body's normal cells, solving the clinical dilemma of "high toxicity and low efficiency" of polymyxin B, and are expected to become ideal candidate drugs to replace polymyxin B. On the other hand, this low cytotoxicity characteristic lays a solid foundation for its better safety in anti-infection treatment, strongly supports the development of new and safe anti-infective preparations, and is expected to reduce adverse reactions caused by drug toxicity in clinical applications, improve the safety and tolerability of treatment, and promote the development of antimicrobial drugs in a safer and more effective direction.

[0062] Example 5: In vivo efficacy evaluation of antimicrobial peptides 1. Efficacy of antimicrobial peptides in the neutropenic mouse thigh model infected with Escherichia coli ATCC25922 (1) CD-1 mice were selected for the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages. The room temperature was maintained at 24 ± 2°C during the experiment. All mice had free access to food and water. The mice were randomly assigned to: 0.4 mg / kg polymyxin B treatment group, 1.6 mg / kg polymyxin B treatment group, 3.2 mg / kg polymyxin B treatment group, 0.4 mg / kg D50 control peptide treatment group, 1.6 mg / kg D50 control peptide treatment group, 3.2 mg / kg D50 control peptide treatment group, 0.4 mg / kg antimicrobial peptide 1 treatment group, 1.6 mg / kg antimicrobial peptide 1 treatment group, 3.2 mg / kg antimicrobial peptide 1 treatment group, 0.4 mg / kg antimicrobial peptide 2 treatment group, 1.6 mg / kg antimicrobial peptide 2 treatment group, 3.2 mg / kg antimicrobial peptide 2 treatment group, 0.4 mg / kg antimicrobial peptide 3-treatment group, 1.6 mg / kg antimicrobial peptide 3-treatment group, 3.2 mg / kg antimicrobial peptide 3-treatment group, 0.4 mg / kg antimicrobial peptide 4 treatment group, 1.6 mg / kg antimicrobial peptide 4 treatment group, 3.2 mg / kg antimicrobial peptide 4 treatment group, 0.4 mg / kg antimicrobial peptide 5-treated group, 1.6 mg / kg antimicrobial peptide 5-treated group, 3.2 mg / kg antimicrobial peptide 5-treated group, and a blank control group; (2) On the 4th and 1st day before the experiment, all mice were intraperitoneally injected with cyclophosphamide twice, with the first dose being 150 mg / kg and the second dose being 100 mg / kg, to induce a persistent neutrophil deficiency state; (3) All mice were injected with 1×10 5 CFU Escherichia coli ATCC25922 standard strain suspension was injected into the tail vein 1, 3.5, and 6 hours after infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide were given according to the above grouping. The blank control group was given normal saline. (4) After 9 hours of infection, the bilateral thigh tissues of all mice were collected aseptically. In an ice-water bath, a single thigh tissue sample was added with 9 mL of PBS per gram of sample, mixed with PBS, and homogenized. 1 mL of thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and cultured at 37°C for 24 hours. The bacterial load was determined by colony forming unit (CFU) counting. The logarithm of the bacterial load reduction between each treatment group and the control group was calculated (log 10 CFU / g), the results are shown in Table 4.

[0063] The results, shown in Table 4, show that antimicrobial peptides 1-4 demonstrated significant bacterial load reduction in a mouse thigh infection model with E. coli ATCC25922. Compared to polymyxin B, each dose group demonstrated a 9- to 18-fold improvement in bacterial load reduction; compared to the D50 control peptide, the improvement was 7- to 14-fold; and compared to the non-deuterated antimicrobial peptide 5, the improvement was approximately 7- to 10-fold. The logarithmic difference in load reduction increased with increasing dose, demonstrating a clear dose-effect relationship. Low doses can effectively eliminate bacteria, providing strong support for the development of novel, highly effective anti-E. coli agents and possessing high potential for clinical translation.

[0064]

[0065] 2. Efficacy of antimicrobial peptides in the neutropenic mouse thigh model infected with Klebsiella pneumoniae ATCC43816 (1) CD-1 mice were selected for the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages. The room temperature was maintained at 24 ± 2°C during the experiment. All mice had free access to food and water. The mice were randomly assigned to: 0.4 mg / kg polymyxin B treatment group, 1.6 mg / kg polymyxin B treatment group, 3.2 mg / kg polymyxin B treatment group, 0.4 mg / kg D50 control peptide treatment group, 1.6 mg / kg D50 control peptide treatment group, 3.2 mg / kg D50 control peptide treatment group, 0.4 mg / kg antimicrobial peptide 1 treatment group, 1.6 mg / kg antimicrobial peptide 1 treatment group, 3.2 mg / kg antimicrobial peptide 1 treatment group, 0.4 mg / kg antimicrobial peptide 2 treatment group, 1.6 mg / kg antimicrobial peptide 2 treatment group, 3.2 mg / kg antimicrobial peptide 2 treatment group, 0.4 mg / kg antimicrobial peptide 3-treatment group, 1.6 mg / kg antimicrobial peptide 3-treatment group, 3.2 mg / kg antimicrobial peptide 3-treatment group, 0.4 mg / kg antimicrobial peptide 4 treatment group, 1.6 mg / kg antimicrobial peptide 4 treatment group, 3.2 mg / kg antimicrobial peptide 4 treatment group, 0.4 mg / kg antimicrobial peptide 5-treated group, 1.6 mg / kg antimicrobial peptide 5-treated group, 3.2 mg / kg antimicrobial peptide 5-treated group, and a blank control group; (2) On the 4th and 1st day before the experiment, all mice were intraperitoneally injected with cyclophosphamide twice, with the first dose being 150 mg / kg and the second dose being 100 mg / kg, to induce a persistent neutrophil deficiency state; (3) All mice were injected with 1×10 5 CFU Klebsiella pneumoniae ATCC43816 standard strain suspension was injected into the tail vein 2, 6, and 10 hours after infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide were given according to the above grouping. The blank control group was given normal saline. (4) After 16 hours of infection, the bilateral thigh tissues of all mice were collected aseptically. In an ice-water bath, a single thigh tissue sample was added with 9 mL of PBS per gram of sample, mixed with PBS, and homogenized. 1 mL of thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and cultured at 37°C for 24 hours. The bacterial load was determined by colony forming unit (CFU) counting. The logarithm of the bacterial load reduction between each treatment group and the control group was calculated (log 10 CFU / g), the results are shown in Table 5.

[0066] The results, shown in Table 5, show that antimicrobial peptides 1-4 demonstrated significant bacterial load reduction in a mouse thigh Klebsiella pneumoniae ATCC43816 infection model. Compared with polymyxin B, each dose group demonstrated a 6- to 11-fold increase in bacterial load reduction; compared with the D50 control peptide, the increase was 3- to 8-fold; and compared with the non-deuterated antimicrobial peptide 5, the increase was approximately 3.6- to 7-fold. With increasing doses, the logarithmic difference in bacterial load reduction showed a relatively stable upward trend, demonstrating a dose-response relationship. Notably, significant antimicrobial efficacy was demonstrated even at relatively low doses.

[0067]

[0068] 3. Efficacy of antimicrobial peptides in a neutropenic mouse thigh model infected with Acinetobacter baumannii NCTC13424 (1) CD-1 mice were selected for the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages. The room temperature was maintained at 24 ± 2°C during the experiment. All mice had free access to food and water. The mice were randomly assigned to: 0.4 mg / kg polymyxin B treatment group, 1.6 mg / kg polymyxin B treatment group, 3.2 mg / kg polymyxin B treatment group, 0.4 mg / kg D50 control peptide treatment group, 1.6 mg / kg D50 control peptide treatment group, 3.2 mg / kg D50 control peptide treatment group, 0.4 mg / kg antimicrobial peptide 1 treatment group, 1.6 mg / kg antimicrobial peptide 1 treatment group, 3.2 mg / kg antimicrobial peptide 1 treatment group, 0.4 mg / kg antimicrobial peptide 2 treatment group, 1.6 mg / kg antimicrobial peptide 2 treatment group, 3.2 mg / kg antimicrobial peptide 2 treatment group, 0.4 mg / kg antimicrobial peptide 3-treatment group, 1.6 mg / kg antimicrobial peptide 3-treatment group, 3.2 mg / kg antimicrobial peptide 3-treatment group, 0.4 mg / kg antimicrobial peptide 4 treatment group, 1.6 mg / kg antimicrobial peptide 4 treatment group, 3.2 mg / kg antimicrobial peptide 4 treatment group, 0.4 mg / kg antimicrobial peptide 5-treated group, 1.6 mg / kg antimicrobial peptide 5-treated group, 3.2 mg / kg antimicrobial peptide 5-treated group, and a blank control group; (2) On the 4th and 1st day before the experiment, all mice were intraperitoneally injected with cyclophosphamide twice, with the first dose being 150 mg / kg and the second dose being 100 mg / kg, to induce a persistent neutrophil deficiency state; (3) All mice were injected with 1×10 5 CFU suspension of Acinetobacter baumannii NCTC13424 standard strain was injected into the tail vein 2, 6, and 10 hours after infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide were given according to the above grouping. The blank control group was given normal saline. (4) After 16 hours of infection, the bilateral thigh tissues of all mice were collected aseptically. In an ice-water bath, a single thigh tissue sample was added with 9 mL of PBS per gram of sample, mixed with PBS, and homogenized. 1 mL of thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and cultured at 37°C for 24 hours. The bacterial load was determined by colony forming unit (CFU) counting. The logarithm of the bacterial load reduction between each treatment group and the control group was calculated (log 10 CFU / g), the results are shown in Table 6.

[0069] The results, shown in Table 6, show that antimicrobial peptides 1-4 demonstrated remarkable bacterial load reduction in the mouse thigh infection model of Acinetobacter baumannii NCTC13424. Compared to polymyxin B, each dose group demonstrated a 7.9- to 14.8-fold improvement in bacterial load reduction; compared to the D50 control peptide, the improvement was 5.5- to 7.7-fold; and compared to the non-deuterated antimicrobial peptide 5, the improvement was approximately 5- to 6-fold. With increasing doses, the logarithmic difference in bacterial load reduction steadily increased, initially demonstrating a dose-response relationship. Notably, significant antimicrobial effects were observed at relatively low doses, potentially providing a breakthrough solution for the treatment of Acinetobacter baumannii infections.

[0070]

[0071] 4. Efficacy of antimicrobial peptides in the neutropenic mouse thigh model infected with Pseudomonas aeruginosa ATCC27853 (1) CD-1 mice were selected for the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages. The room temperature was maintained at 24 ± 2°C during the experiment. All mice had free access to food and water. The mice were randomly assigned to: 0.4 mg / kg polymyxin B treatment group, 1.6 mg / kg polymyxin B treatment group, 3.2 mg / kg polymyxin B treatment group, 0.4 mg / kg D50 control peptide treatment group, 1.6 mg / kg D50 control peptide treatment group, 3.2 mg / kg D50 control peptide treatment group, 0.4 mg / kg antimicrobial peptide 1 treatment group, 1.6 mg / kg antimicrobial peptide 1 treatment group, 3.2 mg / kg antimicrobial peptide 1 treatment group, 0.4 mg / kg antimicrobial peptide 2 treatment group, 1.6 mg / kg antimicrobial peptide 2 treatment group, 3.2 mg / kg antimicrobial peptide 2 treatment group, 0.4 mg / kg antimicrobial peptide 3-treatment group, 1.6 mg / kg antimicrobial peptide 3-treatment group, 3.2 mg / kg antimicrobial peptide 3-treatment group, 0.4 mg / kg antimicrobial peptide 4 treatment group, 1.6 mg / kg antimicrobial peptide 4 treatment group, 3.2 mg / kg antimicrobial peptide 4 treatment group, 0.4 mg / kg antimicrobial peptide 5-treated group, 1.6 mg / kg antimicrobial peptide 5-treated group, 3.2 mg / kg antimicrobial peptide 5-treated group, and a blank control group; (2) On the 4th and 1st day before the experiment, all mice were intraperitoneally injected with cyclophosphamide twice, with the first dose being 150 mg / kg and the second dose being 100 mg / kg, to induce a persistent neutrophil deficiency state; (3) All mice were injected with 1×10 5 CFU Pseudomonas aeruginosa ATCC27853 standard strain suspension was injected into the tail vein 1, 3.5, and 6 hours after infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide were given according to the above grouping. The blank control group was given normal saline. (4) After 9 hours of infection, the bilateral thigh tissues of all mice were collected aseptically. In an ice-water bath, a single thigh tissue sample was added with 9 mL of PBS per gram of sample, mixed with PBS, and homogenized. 1 mL of thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and cultured at 37°C for 24 hours. The bacterial load was determined by colony forming unit (CFU) counting. The logarithm of the bacterial load reduction between each treatment group and the control group was calculated (log 10 CFU / g), the results are shown in Table 7.

[0072] The results, shown in Table 7, show that antimicrobial peptides 1-4 demonstrated excellent bacterial load reduction in a mouse thigh infection model with Pseudomonas aeruginosa ATCC27853. Compared to polymyxin B, each dose group demonstrated a 5- to 11.8-fold improvement in bacterial load reduction; compared to the D50 control peptide, the improvement was 4- to 7-fold; and compared to the non-deuterated antimicrobial peptide 5, the improvement was approximately 3.9- to 6.6-fold. The logarithmic difference in bacterial load reduction showed a steady upward trend with increasing dose, demonstrating a clear dose-effect relationship, demonstrating a highly effective antibacterial effect even at low doses.

[0073]

[0074] 5. Efficacy of antimicrobial peptides in a neutropenic mouse lung model infected with Acinetobacter baumannii NCTC13424 (1) CD-1 mice were selected for the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages. The room temperature was maintained at 24 ± 2°C during the experiment. All mice had free access to food and water. The mice were randomly assigned to: 5 mg / kg polymyxin B treatment group, 7 mg / kg polymyxin B treatment group, 14 mg / kg polymyxin B treatment group, 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group, 5 mg / kg antimicrobial peptide 1 treatment group, 7 mg / kg antimicrobial peptide 1 treatment group, 14 mg / kg antimicrobial peptide 1 treatment group, 5 mg / kg antimicrobial peptide 2 treatment group, 7 mg / kg antimicrobial peptide 2 treatment group, 14 mg / kg antimicrobial peptide 2 treatment group, 5 mg / kg antimicrobial peptide 3-treatment group, 7 mg / kg antimicrobial peptide 3-treatment group, 14 mg / kg antimicrobial peptide 3-treatment group, 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group, 5 mg / kg antimicrobial peptide 5-treatment group, 7 mg / kg antimicrobial peptide 5-treatment group, 14 mg / kg antimicrobial peptide 5-treatment group, and a blank control group; (2) On the 4th and 1st day before the experiment, all mice were intraperitoneally injected with cyclophosphamide twice, with the first dose being 200 mg / kg and the second dose being 150 mg / kg, to induce a persistent neutrophil deficiency state; (3) Mice were intranasally inoculated with 1×10 7 A standard suspension of Acinetobacter baumannii NCTC13424 was used to achieve precise bilateral lung lobe infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide was administered subcutaneously in the back of the neck 2, 6, and 10 hours after infection, according to the above groupings. A blank control group received normal saline. (4) After 16 hours of infection, the bilateral lung tissues of all mice were aseptically removed. In an ice-water bath, the lung tissue samples were mixed with PBS at a ratio of 9 mL per gram of sample and homogenized. 1 mL of lung tissue homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and cultured at 37°C for 24 hours. The bacterial load was determined by colony forming unit (CFU) counting. The logarithm of the bacterial load reduction between each treatment group and the control group (log 10 CFU / g), the results are shown in Table 8.

[0075] The results, shown in Table 8, show that antimicrobial peptides 1-4 demonstrated excellent bacterial load reduction in the mouse lung infection model of Acinetobacter baumannii NCTC13424. Compared to polymyxin B, each dose group demonstrated a 7.9- to 13.8-fold improvement in bacterial load reduction; compared to the D50 control peptide, the improvement was 6.5- to 9-fold; and compared to the non-deuterated antimicrobial peptide 5, the improvement was approximately 5.9- to 7.8-fold. The logarithmic difference in load reduction showed a steady upward trend with increasing dose, demonstrating a clear dose-response relationship. Low doses can achieve efficient bacterial clearance, potentially opening a new avenue for the treatment of pulmonary Acinetobacter baumannii infection.

[0076]

[0077] 6. Efficacy of antimicrobial peptides in a neutropenic mouse lung model infected with Pseudomonas aeruginosa ATCC27853 (1) CD-1 mice were selected for the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages. The room temperature was maintained at 24 ± 2°C during the experiment. All mice had free access to food and water. The mice were randomly assigned to: 5 mg / kg polymyxin B treatment group, 7 mg / kg polymyxin B treatment group, 14 mg / kg polymyxin B treatment group, 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group, 5 mg / kg antimicrobial peptide 1 treatment group, 7 mg / kg antimicrobial peptide 1 treatment group, 14 mg / kg antimicrobial peptide 1 treatment group, 5 mg / kg antimicrobial peptide 2 treatment group, 7 mg / kg antimicrobial peptide 2 treatment group, 14 mg / kg antimicrobial peptide 2 treatment group, 5 mg / kg antimicrobial peptide 3-treatment group, 7 mg / kg antimicrobial peptide 3-treatment group, 14 mg / kg antimicrobial peptide 3-treatment group, 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group, 5 mg / kg antimicrobial peptide 5-treatment group, 7 mg / kg antimicrobial peptide 5-treatment group, 14 mg / kg antimicrobial peptide 5-treatment group, and a blank control group; (2) On the 4th and 1st day before the experiment, all mice were intraperitoneally injected with cyclophosphamide twice, with the first dose being 200 mg / kg and the second dose being 150 mg / kg, to induce a persistent neutrophil deficiency state; (3) Mice were intranasally inoculated with 1×10 7 A standard suspension of Pseudomonas aeruginosa ATCC27853 with a CFU / lung lobe was used to achieve precise infection of both lung lobes. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide was administered subcutaneously in the back of the neck 2, 6, and 10 hours after infection, according to the above groupings. A blank control group received normal saline. (4) After 16 hours of infection, the bilateral lung tissues of all mice were aseptically removed. In an ice-water bath, the lung tissue samples were mixed with PBS at a ratio of 9 mL per gram of sample and homogenized. 1 mL of lung tissue homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and cultured at 37°C for 24 hours. The bacterial load was determined by colony forming unit (CFU) counting. The logarithm of the bacterial load reduction between each treatment group and the control group (log 10 CFU / g), the results are shown in Table 9.

[0078] The results, shown in Table 9, show that antimicrobial peptides 1-4 demonstrated significant bacterial load reduction in a mouse lung infection model with Pseudomonas aeruginosa ATCC27853. Compared with polymyxin B, each dose group demonstrated a 5.9- to 11.8-fold improvement in bacterial load reduction; compared with the D50 control peptide, the improvement was 5.5- to 8.4-fold; and compared with the non-deuterated antimicrobial peptide 5, the improvement was approximately 5- to 6.6-fold. With increasing doses, the logarithmic difference in bacterial load reduction increased in a regular pattern, demonstrating a clear dose-response relationship. Highly effective bacterial clearance was achieved at low doses, providing an important basis for the development of novel agents for combating Pseudomonas aeruginosa infection.

[0079]

[0080] 7. Efficacy of antimicrobial peptides in a neutropenic mouse lung model infected with Klebsiella pneumoniae ATCC43816 (1) CD-1 mice were selected for the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages. The room temperature was maintained at 24 ± 2°C during the experiment. All mice had free access to food and water. The mice were randomly assigned to: 5 mg / kg polymyxin B treatment group, 7 mg / kg polymyxin B treatment group, 14 mg / kg polymyxin B treatment group, 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group, 5 mg / kg antimicrobial peptide 1 treatment group, 7 mg / kg antimicrobial peptide 1 treatment group, 14 mg / kg antimicrobial peptide 1 treatment group, 5 mg / kg antimicrobial peptide 2 treatment group, 7 mg / kg antimicrobial peptide 2 treatment group, 14 mg / kg antimicrobial peptide 2 treatment group, 5 mg / kg antimicrobial peptide 3-treatment group, 7 mg / kg antimicrobial peptide 3-treatment group, 14 mg / kg antimicrobial peptide 3-treatment group, 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group, 5 mg / kg antimicrobial peptide 5-treatment group, 7 mg / kg antimicrobial peptide 5-treatment group, 14 mg / kg antimicrobial peptide 5-treatment group, and a blank control group; (2) On the 4th and 1st day before the experiment, all mice were intraperitoneally injected with cyclophosphamide twice, with the first dose being 200 mg / kg and the second dose being 150 mg / kg, to induce a persistent neutrophil deficiency state; (3) Mice were intranasally inoculated with 1×107 A standard suspension of Klebsiella pneumoniae ATCC43816 with a CFU / lobe was used to precisely infect both lung lobes. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide was administered subcutaneously in the back of the neck 2, 6, and 10 hours after infection, according to the above groupings. A blank control group received normal saline. (4) After 16 hours of infection, the bilateral lung tissues of all mice were aseptically removed. In an ice-water bath, the lung tissue samples were mixed with PBS at a ratio of 9 mL per gram of sample and homogenized. 1 mL of lung tissue homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and cultured at 37°C for 24 hours. The bacterial load was determined by colony forming unit (CFU) counting. The logarithm of the bacterial load reduction between each treatment group and the control group (log 10 CFU / g), the results are shown in Table 10.

[0081] The results are shown in Table 10. Antimicrobial peptides 1-4 demonstrated excellent ability to reduce bacterial load in the Klebsiella pneumoniae ATCC43816 infection model in mouse lungs. Compared with polymyxin B, the efficacy of reducing bacterial load in each dose group increased by 7-14 times; compared with the D50 control peptide, it increased by 5-8 times; and compared with the non-deuterated antimicrobial peptide 5, it increased by about 4.8-7 times. As the dosage gradually increased, the logarithmic difference of the bacterial load reduction showed a stable increasing trend, which clearly reflected the correlation between dose and effect. It is worth noting that a more significant bacterial clearance effect has been observed under lower dose conditions, and the antibacterial effect is outstanding.

[0082]

[0083] According to the experimental data in Tables 8-10, in the neutropenic mouse lung model infected with Gram-negative bacteria, antimicrobial peptides 1-4 showed clear antibacterial activity across the entire dose range, and their antibacterial efficacy increased linearly with increasing dose, showing a typical dose-dependent characteristic. More importantly, under the same dose conditions, antimicrobial peptides 1-4 showed dual advantages over the clinical first-line drug polymyxin B and the control peptide D50: on the one hand, the in vivo antibacterial efficacy against Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae was significantly improved, and the bacterial clearance effect could be achieved at a low dose; on the other hand, the human renal proximal tubular epithelial cell toxicity experiment showed that its HK-2 cell IC 50 The value is 87 times higher than that of colistin B, which breaks through the clinical bottleneck of "high toxicity and low efficiency" of traditional antibiotics from a mechanistic perspective. As a new type of polypeptide molecule with both high antibacterial activity and renal safety, it shows clear clinical transformation value in the treatment of multidrug-resistant Gram-negative bacterial infections.

[0084] In the specific embodiments of the present invention, the Chinese meanings of the English abbreviations used in the application documents are shown in the table: .

Claims

1. An antimicrobial peptide, characterized in that The specific structure is selected from any one of the following (I)-(IV): (1) As shown in formula (I): ； (2) As shown in formula (II): ； (3) As shown in formula (III): ； (4) As shown in formula (IV): 。 2. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the antimicrobial peptide according to claim 1 or a pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug thereof as an active ingredient, with or without the addition of pharmaceutically acceptable excipients.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutically acceptable excipients include one or more of excipients, diluents, lubricants, glidants, wetting agents, emulsifiers, and pH buffer substances.

4. 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, chewable tablets, effervescent tablets, sustained-release tablets, pellets, injections, powder injections, infusions, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, atomized solutions, film coatings, implants, orally disintegrating tablets, oral fast-dissolving films, sponges, and capsules.

5. Use of the antimicrobial peptide according to claim 1 or the pharmaceutical composition according to any one of claims 2 to 4 in the preparation of a medicament for preventing and / or controlling microbial infection.

6. The use according to claim 5, characterized in that The microbial infection is a bacterial infection.

7. The use according to claim 6, characterized in that The bacteria are Gram-negative bacteria.

8. The use according to claim 7, characterized in that The Gram-negative bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

9. The use according to claim 5, characterized in that The microbial infection is one or more of 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.

10. The use according to claim 7, characterized in that The microbial 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.

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