A deuterated antimicrobial peptide, compositions and uses thereof

Abstract

The application discloses a kind of deuterated antibacterial peptide and its composition and application, belong to biological medicine field.The application is formed by introducing deuterated 2-amino butyric acid to form cyclic structure, and the affinity of antibacterial peptide to bacterial membrane is greatly improved compared with traditional drug (polymyxin B, D50) by combining with deuterium modification, and the selectivity of mammalian membrane is more than 884 times higher than that of control 884;The minimum bacteriostatic concentration of gram-negative bacteria in vitro is as low as 0.015 μg / mL, which is 67 times higher than polymyxin B, 33-67 times higher than D50 control peptide, about 17 times higher than non-deuterated antibacterial peptide 5, realizes efficient bacteriostasis at very low concentration;And human kidney cell toxicity is very low, and biological safety is better.In various infection models, its therapeutic effect is significantly better than the above three kinds of controls, and can be used for preparing anti-infection drugs, to provide "high efficiency and low toxicity" new scheme for clinical anti-infection treatment, and is suitable for the prevention and treatment of various infections.

Description

Technical Field

[0001] This invention relates to polypeptides and their applications, specifically to a deuterated antimicrobial peptide, its composition, and its applications. Background Technology

[0002] Bacterial drug resistance is a growing problem, particularly among Gram-negative bacteria such as *Escherichia coli*, *Klebsiella pneumoniae*, *Acinetobacter baumannii*, and *Pseudomonas aeruginosa*. These bacteria utilize biological mechanisms such as horizontal gene transfer and efflux pumps to build complex drug resistance networks, posing significant technical challenges to clinical treatment. Carbapenem-resistant *Klebsiella pneumoniae*, in particular, poses a significant threat to public health due to its high mortality rate.

[0003] Polymyxin, a drug for treating infections caused by drug-resistant Gram-negative bacteria, works by specifically binding to lipid A of the bacterial outer membrane lipopolysaccharide (LPS). However, significant technical limitations exist in its practical application: Firstly, resistant strains modify the LPS with phosphate ethanolamine, a modification that drastically reduces the affinity between the drug and bacteria and activates the efflux system, leading to rapid development of resistance. This raises the technical issue of the interaction between bacterial resistance mechanisms and drug action mechanisms, making it difficult to sustain the effectiveness of polymyxin in treating drug-resistant bacteria. Secondly, during polymyxin treatment, patients are prone to irreversible renal tubular damage, along with adverse reactions such as neurotoxicity and ototoxicity. From a therapeutic index perspective, its therapeutic index is low, failing to meet the clinical requirements for drug safety and efficacy. This highlights the limitations of polymyxin in the technical integration of drug development and clinical application.

[0004] Antimicrobial peptides, as natural amphiphilic molecules, exert their antibacterial effects by disrupting bacterial cell membranes. Technically, they exhibit higher killing efficiency against drug-resistant bacteria than traditional antibiotics and maintain good activity even at high temperatures. Furthermore, due to their diverse target sites, they can reduce the probability of bacterial resistance development from the perspective of the underlying mechanisms of bacterial resistance, providing a new technological approach to solving the antibiotic resistance problem. However, some publicly disclosed 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 limits their further application in the clinical translation process. Therefore, developing novel antimicrobial peptides that combine high antimicrobial activity, low toxicity, and high biocompatibility, based on clinical treatment needs and the technical goals of antimicrobial drug development, is of paramount importance for overcoming the clinical treatment dilemma caused by drug-resistant Gram-negative bacteria and meeting the technical and application-level needs of clinical treatment. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a deuterated antimicrobial peptide and its pharmaceutical composition, in order to provide more options for combating microbial infections, especially infections caused by drug-resistant Gram-negative bacteria.

[0006] Technical solution: The antimicrobial peptide of the present invention has a specific structure selected from any one of the following (I)-(IV):

[0007] (1) As shown in equation (I):

[0008] ;

[0009] (2) As shown in equation (II):

[0010] ;

[0011] (3) As shown in equation (Ⅲ):

[0012] ;

[0013] (4) As shown in equation (Ⅳ):

[0014] .

[0015] The pharmaceutical composition contains the antimicrobial peptide or its pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug as an active ingredient, with or without pharmaceutically acceptable excipients.

[0016] The pharmaceutical composition wherein the pharmaceutically acceptable excipients include one or more of the following: excipients, diluents, lubricants, flow aids, wetting agents, emulsifiers, and pH buffers.

[0017] 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, nebulizing solutions, film-forming agents, implants, orally disintegrating tablets, oral instant films, sponges, and capsules.

[0018] The use of the antimicrobial peptide or the pharmaceutical composition described herein in the preparation of medicaments for the prevention and / or control of microbial infections.

[0019] In the aforementioned application, the microbial infection is a bacterial infection.

[0020] In the aforementioned application, the bacteria are Gram-negative bacteria.

[0021] In the aforementioned application, the Gram-negative bacteria are one or more of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

[0022] The application described herein refers to one or more of the following microbial infections: 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.

[0023] The aforementioned applications include microbial infections caused by Gram-negative bacteria, such as 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.

[0024] Preferred "pharmaceuticalally 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.

[0025] 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.

[0026] The core innovations of this invention are as follows:

[0027] I. Conformational Innovation: Breaking Through Traditional Peptide Mechanisms of Action

[0028] Novel cyclic structure design: The core, through the introduction of deuterated 2-aminobutyric acid, forms a cyclic conformation different from the linear structure of polymyxin B and the D50 control peptide. This enhances the 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) and extremely strong selectivity for mammalian membranes (more than 884 times higher than the control polymyxin B and control peptide D50). This demonstrates that it can precisely bind to bacterial membranes to exert antibacterial effects while avoiding damage to mammalian membranes. This "targeting bacteria and protecting the host" characteristic makes antimicrobial peptides show greater application potential than traditional polymyxin B and control peptide D50 in the development of novel antimicrobial drugs—the 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 deuteration modification: antimicrobial peptides 1-4 showed an approximately 17-fold increase in MIC values ​​against four types of bacteria compared to non-deuterated antimicrobial peptide 5.

[0029] Value: Conformational innovation breaks through the drug resistance barrier of traditional antibiotics at the molecular level, providing a new paradigm for the development of "low-toxicity and high-efficiency" antimicrobial peptides.

[0030] II. Antibacterial activity: Highly effective in eliminating drug-resistant bacteria with significant dose-dependent effects.

[0031] Breakthrough in in vitro antibacterial efficacy: The minimum inhibitory concentration (MIC) against 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 that of colistin B (MIC=1 μg / mL), 33-67 times higher than that of the D50 control peptide, and about 17 times higher than that of non-deuterated antimicrobial peptide 5, achieving "highly effective antibacterial efficacy at extremely low concentrations".

[0032] Significant advantages in in vivo efficacy: In a mouse thigh infection model, the antimicrobial peptide showed a more prominent in vivo antimicrobial effect compared with polymyxin B, D50 control peptide and non-deuterated antimicrobial peptide 5, demonstrating that the antimicrobial peptide has better antimicrobial efficacy than the control drug at the same dose, and exhibits a clear dose-dependent effect (the antimicrobial effect increases linearly with increasing dose).

[0033] Value: It solves the problem of "high mortality and difficulty in eradication" of multidrug-resistant bacteria in clinical practice, and provides new treatment options for superbug infections such as carbapenem-resistant bacteria.

[0034] III. Safety Optimization: Low Toxicity Breaks Through the Bottleneck of Traditional Antibiotics

[0035] Nephrotoxicity was significantly reduced: the antimicrobial peptide was more toxic to the proximal tubular epithelial cells of HK-2 human kidneys than colistin B (IC50). 50 =1) Safety improved by 87 times compared to the D50 control peptide (IC50). 50=5) is about 17 times higher than that of non-deuterated antimicrobial peptide 5, and about 5.8 times higher than that of polymyxin B and control peptide D50, avoiding the clinical dilemma of "high toxicity and low efficacy".

[0036] Selective targeting of bacterial membranes: MST experiments confirmed that antimicrobial peptides 1-4 exhibit extremely 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) and extremely strong selectivity for mammalian membranes (more than 884 times higher than the control polymyxin B and control peptide D50), demonstrating that they can precisely bind to bacterial membranes to exert antimicrobial effects while avoiding damage to mammalian membranes.

[0037] Value: Breaking through the limitations of traditional antibiotics' "narrow therapeutic window," it provides a safe option for patients requiring long-term medication or those with immunodeficiency.

[0038] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The antimicrobial peptide is a new type of highly efficient and broad-spectrum antimicrobial peptide. Antimicrobial peptides 1-4 have shown 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 it 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-toxicity 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. It is expected to reduce adverse reactions such as nephrotoxicity that occur during traditional antibiotic treatment. (3) The antimicrobial peptide, through its mechanism of action of destroying the bacterial cell membrane, is not likely to induce bacterial resistance, thus making up for the shortcomings of traditional antibiotics in terms of efficacy decline due to resistance problems, and providing 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 and low synthesis cost, and has shown excellent therapeutic effects in various infection models. It has great potential to be developed into a new type of anti-infective drug and is expected to be applied to the prevention and treatment of various infections such as respiratory system, urinary system, skin and soft tissue, bringing breakthrough progress to the treatment of multidrug resistant bacterial infections in clinical practice. It is expected to replace antibiotics as a safe, green and efficient ideal antimicrobial agent. Attached Figure Description

[0039] Figure 1 The HPLC chromatogram of antimicrobial peptide 1 is shown.

[0040] Figure 2 The HPLC chromatogram of antimicrobial peptide 2;

[0041] Figure 3The HPLC chromatogram of antimicrobial peptide 3;

[0042] Figure 4 The HPLC chromatogram of antimicrobial peptide 4;

[0043] Figure 5 This is the result of an in vitro renal cytotoxicity assay for the antimicrobial peptide. Detailed Implementation

[0044] The technical solution of the present invention will be further described below.

[0045] The polypeptide compound in this embodiment of the invention, antimicrobial peptide 1, is:

[0046]

[0047] Antimicrobial peptide 2 is:

[0048]

[0049] Antimicrobial peptide 3 is:

[0050]

[0051] Antimicrobial peptide 4 is:

[0052]

[0053] Example 1: Preparation method of antimicrobial peptides 1-4

[0054] 1. Preparation and purification of antimicrobial peptide 1

[0055] (1) Preparation of 4-((tert-butoxycarbonyl)amino)-3-(3-isopropylphenyl)-butyric acid

[0056] A mixture of 10 g (E)-3-(3-isopropylphenyl)acrylic acid, 100 mL methanol, and 4 mL 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.

[0057] 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.

[0058] 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.

[0059] The orange oily substance 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 by elution with 0-100% ethyl acetate in hexane. Evaporation to dryness gave 2.59 g of the desired milky white solid product.

[0060] 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-isopropylphenyl)-butyric acid.

[0061] 4-((tert-Butoxycarbonyl)amino)-3-(3-isopropylphenyl)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.

[0062] 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.

[0063] (2) Cyclic peptides were prepared using an automated peptide synthesizer and 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 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.

[0064] (3) Add equal amounts of amino acids in sequence for reaction, including 2.22g Fmoc-Dab(Boc)-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.65g Fmoc-Abu-OH-D3, 1.95g Fmoc-D-Phe-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-isopropylphenyl)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.

[0065] (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-isopropylphenyl)butyryl-Thr(tBu)-Dap(Boc)-Dab-Dab(Boc)-D-Phe-Abu-D3-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH;

[0066] (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 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, 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-isopropylphenyl)butyryl-Thr(tBu)-Dap(Boc)-Cyclo(Dab-Dab(Boc)-D-Phe-Abu-D3-Dab(Boc)-Dab(Boc)-Thr(tBu)-OH).

[0067] (6) Dissolve 5.4g of the product obtained in the previous step 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-isopropylphenyl)butyryl-Thr-Dap-Cyclo(Dab-Dab-D-Phe-Abu-D3-Dab-Dab-Thr-OH).

[0068] (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:

[0069] 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.

[0070] 2. Preparation and purification of antimicrobial peptide 2

[0071] 4-((tert-Butoxycarbonyl)amino)-3-(3-chlorophenyl)-butyric acid was prepared using 10 g (E)-3-(3-chlorophenyl)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). In step (3), 2.22g of Fmoc-Dab(Boc)-OH, 2.22g of Fmoc-Dab(Boc)-OH, 1.65g of Fmoc-Abu-OH-D3, 1.78g of Fmoc-D-Leu-OH, 2.22g of Fmoc-Dab(Boc)-OH, 1.72g of Fmoc-Dab-OH, 2.15g of Fmoc-Dap(Boc)-OH, 2g of Fmoc-Thr(tBu)-OH, 2.22g of Fmoc-Dab(Boc)-OH, and 1.89g of 4-[(tert-butoxycarbonyl)amino]-3-(3-chlorophenyl)butyric 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 ).

[0072] 3. Preparation and purification of antimicrobial peptide 3

[0073] 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 98.39% (see...). Figure 3 ).

[0074] 4. Preparation and purification of antimicrobial peptide 4

[0075] 4-((tert-butoxycarbonyl)amino)-3-(3,5-dichlorophenyl)-butyric acid was prepared as described in step (1) above using 10 g (E)-3-(3,5-dichlorophenyl)acrylic acid, 100 mL methanol and 4 mL concentrated sulfuric acid as raw materials. The reactions of nitration, reduction, Boc protection, hydrolysis, peptide coupling and deprotection are carried out sequentially in steps (2) to (7). In step (3), 2.22g Fmoc-Dab(Boc)-OH, 2.22g Fmoc-Dab(Boc)-OH, 1.65g Fmoc-Abu-OH-D3, 1.78g Fmoc-D-Nle-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-(3,5-dichlorophenyl)butyric acid are added. The crude product was purified by HPLC to obtain antimicrobial peptide 4, with an HPLC purity of 98.12 (see...). Figure 4 ).

[0076] Combination Figure 1-4 The HPLC chromatograms show that the peak area of ​​each antimicrobial peptide exceeds 98% (98.56% for antimicrobial peptide 1, 98.32% for antimicrobial peptide 2, 98.39% for antimicrobial peptide 3, and 98.12% for antimicrobial peptide 4), indicating that the prepared antimicrobial peptides 1-4 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 1-4 And as shown in Table 1:

[0077]

[0078] Example 2: Microthermophoresis (MST) to determine the interaction between antimicrobial peptides and lipids

[0079] In this study, polymyxin B, D50 peptide, and non-deuterated antimicrobial peptide 5 were used as control peptides. They were prepared and synthesized by Shanghai Jier Biochemical Co., Ltd. (purity >96%) and could be used without further purification.

[0080]

[0081] 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.

[0082] The results, as shown in Table 2, demonstrate that antimicrobial peptides 1-4 exhibit extremely high affinity for bacterial membrane DMPG (140-280 times higher than polymyxin B, 75-150 times higher than D50, and 21-43 times higher than non-deuterated antimicrobial peptide 5), and extremely strong selectivity for mammalian membrane DMPC (more than 884 times higher than the control polymyxin B and control peptide D50). This proves that they can precisely bind to bacterial membranes to exert antimicrobial effects while avoiding damage to mammalian membranes. This "targeting bacteria and protecting the host" characteristic makes antimicrobial peptides more promising for the development of novel antimicrobial drugs than traditional polymyxin B, control peptide D50, and non-deuterated antimicrobial peptide 5. The dual breakthroughs in affinity and selectivity are the core advantages that distinguish antimicrobial peptides 1-4 from traditional peptides.

[0083]

[0084] Example 3: Antibacterial activity experiment of antimicrobial peptides

[0085] 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 clinically common Gram-negative bacteria, including Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC43816, Pseudomonas aeruginosa ATCC27853, and Acinetobacter baumannii NCTC13424, was determined using the micro-dilution serial two-fold method.

[0086] According to the synthesis method disclosed in CN106232617A, a D50 control peptide was synthesized as a comparison.

[0087] (1) Fresh Escherichia coli ATCC25922, Klebsiella pneumoniae ATCC43816, 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.

[0088] (2) In sterile 96-well microplates, cationic-adjusted MH broth was used to perform double-dilute serial dilutions of the antimicrobial peptides 1-5, D50 control peptides, or polymyxin B 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%, and 150 μL of gradient dilution was pre-placed in each well.

[0089] (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;

[0090] (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.

[0091] 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.

[0092] The results are shown in Table 3. In the determination of the minimum inhibitory concentration (MIC) against *Escherichia coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, and *Acinetobacter baumannii*, the MIC values ​​of antimicrobial peptides 1-4 were all 0.015 μg / mL, while the MIC value of polymyxin B was 1 μg / mL. Experimental data show that antimicrobial peptides 1-4 increased the MIC values ​​against all four bacteria by approximately 67 times compared to polymyxin B; compared to the D50 control peptide, they increased by approximately 33 times against *Escherichia coli*, *Klebsiella pneumoniae*, and *Pseudomonas aeruginosa*, and by approximately 67 times against *Acinetobacter baumannii*; and compared to non-deuterated antimicrobial peptide 5, the MIC values ​​of antimicrobial peptides 1-4 increased by approximately 17 times against all four bacteria. This indicates that antimicrobial peptides can effectively inhibit the growth of Gram-negative bacteria at extremely low concentrations, exhibiting in vitro antibacterial efficacy far exceeding that of traditional antibiotics. With this characteristic, antimicrobial peptides 1-4 are expected to reduce the risk of potential toxic side effects by lowering the dosage, laying a key foundation for the development of novel anti-infective agents. They have potential for in-depth exploration in the clinical translation process and may bring innovative breakthroughs to the existing pattern of anti-infective therapy, providing new directions for solving clinical problems related to bacterial infections.

[0093]

[0094] Example 4: In vitro renal cytotoxicity assay of antimicrobial peptides

[0095] Cytotoxicity was evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazole bromide (MTT) colorimetric method.

[0096] (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.

[0097] (2) Add serially diluted antimicrobial peptides 1-5, D50 control peptides, 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, and 128 μg / mL, and continue to expose and culture for 48 hours;

[0098] (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 the formation of formazan crystals.

[0099] Each 110 μL of MTT working solution is prepared by mixing 10 μL of 5 mg / mL MTT-PBS solution with 100 μL of DMEM culture medium.

[0100] (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.

[0101] (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.

[0102] The results are as follows Figure 5 As shown, antimicrobial peptides 1-4 exhibit significant advantages: On the one hand, they show extremely low toxicity to proximal tubular epithelial cells of HK-2 human kidneys, with a safety profile at least 87 times higher than that of commonly used polymyxin B, approximately 17 times higher than that of the D50 control peptide, and approximately 5.8 times higher than that of non-deuterated antimicrobial peptide 5. When exerting their antibacterial effects, they minimize the risk of damage to normal cells, solving the clinical dilemma of "high toxicity and low efficacy" of polymyxin B and potentially becoming an ideal candidate drug to replace it. On the other hand, this low cytotoxicity provides a stronger safety foundation for their use in anti-infective therapy, strongly supporting the development of novel and safe anti-infective agents. It 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 towards a safer and more effective direction.

[0103] Example 5: In vivo efficacy evaluation of antimicrobial peptides

[0104] 1. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Escherichia coli ATCC25922

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

[0106] Treatment groups: 0.4 mg / kg polymyxin B, 1.6 mg / kg polymyxin B, and 3.2 mg / kg polymyxin B.

[0107] 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.

[0108] Treatment groups: 0.4 mg / kg antimicrobial peptide 1, 1.6 mg / kg antimicrobial peptide 1, and 3.2 mg / kg antimicrobial peptide 1.

[0109] Treatment groups: 0.4 mg / kg antimicrobial peptide 2, 1.6 mg / kg antimicrobial peptide 2, and 3.2 mg / kg antimicrobial peptide 2.

[0110] Treatment groups: 0.4 mg / kg antimicrobial peptide 3, 1.6 mg / kg antimicrobial peptide 3, and 3.2 mg / kg antimicrobial peptide 3.

[0111] Treatment groups: 0.4 mg / kg antimicrobial peptide 4, 1.6 mg / kg antimicrobial peptide 4, and 3.2 mg / kg antimicrobial peptide 4.

[0112] Treatment groups: 0.4 mg / kg antimicrobial peptide 5, 1.6 mg / kg antimicrobial peptide 5, and 3.2 mg / kg antimicrobial peptide 5.

[0113] And a blank control group;

[0114] (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.

[0115] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5 CFU Escherichia coli ATCC25922 standard strain suspension was administered via tail vein injection at 1, 3.5, and 6 hours post-infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide were administered according to the above grouping, while the blank control group was given physiological saline.

[0116] (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.

[0117] The results are shown in Table 4. Antimicrobial peptides 1-4 demonstrated a significant ability to reduce bacterial load in the mouse Escherichia coli ATCC25922 infection model. Compared with polymyxin B, the bacterial load reduction efficacy was 9-18 times higher in each dose group; 7-14 times higher than the D50 control peptide; and approximately 7-10 times higher than the non-deuterated antimicrobial peptide 5. The logarithmic difference in load reduction increased with increasing dose, demonstrating a clear dose-response relationship. Low doses can effectively clear bacteria, providing strong support for the development of novel and highly effective anti-Escherichia coli agents and possessing high clinical translational potential.

[0118]

[0119] 2. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Klebsiella pneumoniae ATCC43816

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

[0121] Treatment groups: 0.4 mg / kg polymyxin B, 1.6 mg / kg polymyxin B, and 3.2 mg / kg polymyxin B.

[0122] 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.

[0123] Treatment groups: 0.4 mg / kg antimicrobial peptide 1, 1.6 mg / kg antimicrobial peptide 1, and 3.2 mg / kg antimicrobial peptide 1.

[0124] Treatment groups: 0.4 mg / kg antimicrobial peptide 2, 1.6 mg / kg antimicrobial peptide 2, and 3.2 mg / kg antimicrobial peptide 2.

[0125] Treatment groups: 0.4 mg / kg antimicrobial peptide 3, 1.6 mg / kg antimicrobial peptide 3, and 3.2 mg / kg antimicrobial peptide 3.

[0126] Treatment groups: 0.4 mg / kg antimicrobial peptide 4, 1.6 mg / kg antimicrobial peptide 4, and 3.2 mg / kg antimicrobial peptide 4.

[0127] Treatment groups: 0.4 mg / kg antimicrobial peptide 5, 1.6 mg / kg antimicrobial peptide 5, and 3.2 mg / kg antimicrobial peptide 5.

[0128] And a blank control group;

[0129] (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.

[0130] (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 (PMB) or D50 control peptide or antimicrobial peptide were administered according to the above grouping, while the blank control group was given physiological saline.

[0131] (4) Sixteen hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, individual thigh tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony-forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 (CFU / g), the results are shown in Table 5.

[0132] The results are shown in Table 5. Antimicrobial peptides 1-4 demonstrated a significant ability to reduce bacterial load in the mouse Klebsiella pneumoniae ATCC43816 infection model. Compared with polymyxin B, the bacterial load reduction efficacy was 6-11 times higher in each dose group; 3-8 times higher than the D50 control peptide; and approximately 3.6-7 times higher than the non-deuterated antimicrobial peptide 5. With gradual increase in dosage, the logarithmic difference in bacterial load reduction showed a relatively stable upward trend, reflecting the dose-response relationship. Notably, significant antimicrobial effects were already observed at lower doses.

[0133]

[0134] 3. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Acinetobacter baumannii NCTC13424

[0135] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0136] Treatment groups: 0.4 mg / kg polymyxin B, 1.6 mg / kg polymyxin B, and 3.2 mg / kg polymyxin B.

[0137] 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.

[0138] Treatment groups: 0.4 mg / kg antimicrobial peptide 1, 1.6 mg / kg antimicrobial peptide 1, and 3.2 mg / kg antimicrobial peptide 1.

[0139] Treatment groups: 0.4 mg / kg antimicrobial peptide 2, 1.6 mg / kg antimicrobial peptide 2, and 3.2 mg / kg antimicrobial peptide 2.

[0140] Treatment groups: 0.4 mg / kg antimicrobial peptide 3, 1.6 mg / kg antimicrobial peptide 3, and 3.2 mg / kg antimicrobial peptide 3.

[0141] Treatment groups: 0.4 mg / kg antimicrobial peptide 4, 1.6 mg / kg antimicrobial peptide 4, and 3.2 mg / kg antimicrobial peptide 4.

[0142] Treatment groups: 0.4 mg / kg antimicrobial peptide 5, 1.6 mg / kg antimicrobial peptide 5, and 3.2 mg / kg antimicrobial peptide 5.

[0143] And a blank control group;

[0144] (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.

[0145] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5CFU Acinetobacter baumannii NCTC13424 standard strain suspension was administered via tail vein injection at 2, 6, and 10 hours post-infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide were administered according to the above grouping, while the blank control group was given physiological saline.

[0146] (4) Sixteen hours after infection, bilateral thigh tissue was aseptically collected from all mice. Under ice-water bath conditions, individual thigh tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample. One mL of the thigh homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony-forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 (CFU / g), the results are shown in Table 6.

[0147] The results are shown in Table 6. Antimicrobial peptides 1-4 exhibited excellent bacterial load reduction capabilities in the mouse Acinetobacter baumannii NCTC13424 infection model of thigh baumannii. Compared with polymyxin B, the bacterial load reduction efficacy of each dose group was increased by 7.9-14.8 times; compared with the D50 control peptide, it was increased by 5.5-7.7 times; and compared with non-deuterated antimicrobial peptide 5, it was increased by approximately 5-6 times. With the gradual increase of the administered dose, the logarithmic difference in bacterial load reduction showed a steady upward trend, initially demonstrating a dose-effect correlation. Notably, significant antibacterial effects have been observed at relatively low doses, which may provide a breakthrough solution for the treatment of Acinetobacter baumannii infection.

[0148]

[0149] 4. Efficacy of antimicrobial peptides in the thigh model of neutropenia in mice infected with Pseudomonas aeruginosa ATCC27853

[0150] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0151] Treatment groups: 0.4 mg / kg polymyxin B, 1.6 mg / kg polymyxin B, and 3.2 mg / kg polymyxin B.

[0152] 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.

[0153] Treatment groups: 0.4 mg / kg antimicrobial peptide 1, 1.6 mg / kg antimicrobial peptide 1, and 3.2 mg / kg antimicrobial peptide 1.

[0154] Treatment groups: 0.4 mg / kg antimicrobial peptide 2, 1.6 mg / kg antimicrobial peptide 2, and 3.2 mg / kg antimicrobial peptide 2.

[0155] Treatment groups: 0.4 mg / kg antimicrobial peptide 3, 1.6 mg / kg antimicrobial peptide 3, and 3.2 mg / kg antimicrobial peptide 3.

[0156] Treatment groups: 0.4 mg / kg antimicrobial peptide 4, 1.6 mg / kg antimicrobial peptide 4, and 3.2 mg / kg antimicrobial peptide 4.

[0157] Treatment groups: 0.4 mg / kg antimicrobial peptide 5, 1.6 mg / kg antimicrobial peptide 5, and 3.2 mg / kg antimicrobial peptide 5.

[0158] And a blank control group;

[0159] (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.

[0160] (3) All mice were injected with 1×10⁻⁶ mmol / L into the bilateral thigh muscle tissue. 5 CFU suspension of Pseudomonas aeruginosa ATCC27853 standard strain was administered via tail vein injection at 1, 3.5, and 6 hours post-infection. Polymyxin B (PMB) or D50 control peptide or antimicrobial peptide were administered according to the above grouping, while the blank control group was given physiological saline.

[0161] (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 7.

[0162] The results are shown in Table 7. Antimicrobial peptides 1-4 demonstrated excellent bacterial load reduction capabilities in the mouse Pseudomonas aeruginosa ATCC27853 infection model. Compared with polymyxin B, the bacterial load reduction efficacy of each dose group was increased by 5-11.8 times; compared with the D50 control peptide, it was increased by 4-7 times; and compared with non-deuterated antimicrobial peptide 5, it was increased by approximately 3.9-6.6 times. With increasing dosage, the logarithmic difference in load reduction showed a stable upward trend, exhibiting a clear dose-response relationship, demonstrating highly effective antibacterial effects at low doses.

[0163]

[0164] 5. Efficacy of antimicrobial peptides in a mouse model of neutropenia infected with Acinetobacter baumannii NCTC13424

[0165] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0166] Treatment groups: 5 mg / kg polymyxin B, 7 mg / kg polymyxin B, and 14 mg / kg polymyxin B.

[0167] 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group,

[0168] Treatment groups: 5 mg / kg antimicrobial peptide 1, 7 mg / kg antimicrobial peptide 1, and 14 mg / kg antimicrobial peptide 1.

[0169] Treatment groups: 5 mg / kg antimicrobial peptide 2, 7 mg / kg antimicrobial peptide 2, and 14 mg / kg antimicrobial peptide 2.

[0170] 5 mg / kg antimicrobial peptide 3 treatment group, 7 mg / kg antimicrobial peptide 3 treatment group, 14 mg / kg antimicrobial peptide 3 treatment group,

[0171] 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group,

[0172] Treatment groups: 5 mg / kg antimicrobial peptide 5, 7 mg / kg antimicrobial peptide 5, and 14 mg / kg antimicrobial peptide 5.

[0173] And a blank control group;

[0174] (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.

[0175] (3) Mice were intranasally inoculated with 1×10 7 CFU / lung Acinetobacter baumannii NCTC13424 standard bacterial suspension was used to achieve precise bilateral lung lobe infection. Injections were administered subcutaneously into the neck and back at 2, 6, and 10 hours post-infection. Patients were given polymyxin B (PMB), D50 control peptide, or antimicrobial peptide according to the above-described groupings. The blank control group received normal saline.

[0176] (4) Sixteen hours after infection, bilateral lung tissue was aseptically harvested from all mice. The lung tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample under ice-water bath conditions. One mL of the lung tissue homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 (CFU / g), the results are shown in Table 8.

[0177] The results are shown in Table 8. Antimicrobial peptides 1-4 demonstrated excellent bacterial load reduction capabilities in a mouse model of Acinetobacter baumannii NCTC13424 lung infection. Compared with polymyxin B, the bacterial load reduction efficacy was increased by 7.9–13.8 times in each dose group; by 6.5–9 times compared with the D50 control peptide; and by approximately 5.9–7.8 times compared with non-deuterated antimicrobial peptide 5. With increasing dosage, the logarithmic difference in load reduction showed a stable upward trend, exhibiting a clear dose-response relationship. Highly efficient bacterial clearance can be achieved with low doses, potentially opening a new avenue for the treatment of Acinetobacter baumannii lung infection.

[0178]

[0179] 6. Efficacy of antimicrobial peptides in a mouse model of neutropenia induced by Pseudomonas aeruginosa ATCC27853 infection

[0180] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0181] Treatment groups: 5 mg / kg polymyxin B, 7 mg / kg polymyxin B, and 14 mg / kg polymyxin B.

[0182] 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group,

[0183] Treatment groups: 5 mg / kg antimicrobial peptide 1, 7 mg / kg antimicrobial peptide 1, and 14 mg / kg antimicrobial peptide 1.

[0184] Treatment groups: 5 mg / kg antimicrobial peptide 2, 7 mg / kg antimicrobial peptide 2, and 14 mg / kg antimicrobial peptide 2.

[0185] 5 mg / kg antimicrobial peptide 3 treatment group, 7 mg / kg antimicrobial peptide 3 treatment group, 14 mg / kg antimicrobial peptide 3 treatment group,

[0186] 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group,

[0187] Treatment groups: 5 mg / kg antimicrobial peptide 5, 7 mg / kg antimicrobial peptide 5, and 14 mg / kg antimicrobial peptide 5.

[0188] And a blank control group;

[0189] (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.

[0190] (3) Mice were intranasally inoculated with 1×10 7 CFU / lung Pseudomonas aeruginosa ATCC27853 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 (PMB) or D50 control peptide or antimicrobial peptide administered according to the above-described groupings. The blank control group received normal saline.

[0191] (4) Sixteen hours after infection, bilateral lung tissue was aseptically harvested from all mice. The lung tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample under ice-water bath conditions. One mL of the lung tissue homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 (CFU / g), the results are shown in Table 9.

[0192] The results are shown in Table 9. Antimicrobial peptides 1-4 demonstrated a significant ability to reduce bacterial load in a mouse model of Pseudomonas aeruginosa ATCC27853 lung infection. Compared with polymyxin B, the bacterial load reduction efficacy was increased by 5.9-11.8 times in each dose group; by 5.5-8.4 times compared with the D50 control peptide; and by approximately 5-6.6 times compared with non-deuterated antimicrobial peptide 5. With gradually increasing doses, the logarithmic difference in bacterial load reduction showed a regular increasing trend, clearly demonstrating a dose-response relationship. The ability to achieve highly efficient bacterial clearance at low dose levels provides important evidence for the development of novel anti-Pseudomonas aeruginosa infection agents.

[0193]

[0194] 7. Efficacy of antimicrobial peptides in a mouse model of neutropenia caused by Klebsiella pneumoniae ATCC43816 infection.

[0195] (1) CD-1 mice were used in the experiment, with 10 mice per group (n = 10). The mice were housed in individually ventilated cages, and the room temperature was maintained at 24±2℃ during the experiment. All mice had free access to food and water. The mice were randomly assigned to:

[0196] Treatment groups: 5 mg / kg polymyxin B, 7 mg / kg polymyxin B, and 14 mg / kg polymyxin B.

[0197] 5 mg / kg D50 control peptide treatment group, 7 mg / kg D50 control peptide treatment group, 14 mg / kg D50 control peptide treatment group,

[0198] Treatment groups: 5 mg / kg antimicrobial peptide 1, 7 mg / kg antimicrobial peptide 1, and 14 mg / kg antimicrobial peptide 1.

[0199] Treatment groups: 5 mg / kg antimicrobial peptide 2, 7 mg / kg antimicrobial peptide 2, and 14 mg / kg antimicrobial peptide 2.

[0200] 5 mg / kg antimicrobial peptide 3 treatment group, 7 mg / kg antimicrobial peptide 3 treatment group, 14 mg / kg antimicrobial peptide 3 treatment group,

[0201] 5 mg / kg antimicrobial peptide 4 treatment group, 7 mg / kg antimicrobial peptide 4 treatment group, 14 mg / kg antimicrobial peptide 4 treatment group,

[0202] Treatment groups: 5 mg / kg antimicrobial peptide 5, 7 mg / kg antimicrobial peptide 5, and 14 mg / kg antimicrobial peptide 5.

[0203] And a blank control group;

[0204] (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.

[0205] (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, with polymyxin B (PMB) or D50 control peptide or antimicrobial peptide administered according to the above-described groupings. The blank control group received normal saline.

[0206] (4) Sixteen hours after infection, bilateral lung tissue was aseptically harvested from all mice. The lung tissue samples were homogenized with PBS at a ratio of 9 mL per gram of sample under ice-water bath conditions. One mL of the lung tissue homogenate was quantitatively inoculated onto cystine lactose electrolyte deficient (CLED) agar and incubated at 37°C for 24 hours. Bacterial load was determined by colony forming unit (CFU) counting. The logarithmic decrease in bacterial load between each treatment group and the control group was calculated. 10 (CFU / g), the results are shown in Table 10.

[0207] The results are shown in Table 10. Antimicrobial peptides 1-4 demonstrated excellent bacterial load reduction capabilities in a mouse model of Klebsiella pneumoniae ATCC43816 infection. Compared with polymyxin B, the bacterial load reduction efficacy was increased by 7-14 times in each dose group; 5-8 times compared with the D50 control peptide; and approximately 4.8-7 times compared with non-deuterated antimicrobial peptide 5. With gradual increases in dosage, the logarithmic difference in bacterial load reduction showed a stable increasing trend, clearly demonstrating the dose-effect correlation. Notably, significant bacterial clearance effects were observed at lower doses, indicating outstanding antimicrobial efficacy.

[0208]

[0209] Based on the experimental data in Table 8-10, in a mouse model of neutropenia infected with Gram-negative bacteria, antimicrobial peptides 1-4 exhibited clear antimicrobial activity across the entire dose range. Their antibacterial efficacy increased linearly with increasing dose, exhibiting a typical dose-dependent characteristic. Importantly, under the same dose conditions, antimicrobial peptides 1-4 showed dual advantages over the first-line clinical drug polymyxin B and the control peptide D50: firstly, significantly enhanced in vivo antimicrobial efficacy against Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, achieving bacterial clearance at low doses; secondly, human renal proximal tubular epithelial cell cytotoxicity assays showed that their IC50 in HK-2 cells...50 The value is 87 times higher than that of colistin B, which breaks through the clinical bottleneck of "high toxicity and low efficacy" of traditional antibiotics from a mechanistic perspective. As a novel polypeptide molecule that combines high antibacterial activity and renal safety, it has shown clear clinical translational value in the treatment of multidrug-resistant Gram-negative bacterial infections.

[0210] In specific embodiments of the present invention, the Chinese meanings of the English abbreviations used in the application documents are shown in the table below:

[0211] .

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 equation (I): ； (2) As shown in equation (II): ； (3) As shown in equation (Ⅲ): ； (4) As shown in equation (Ⅳ): 。 2. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the antimicrobial peptide as described in claim 1 or a pharmaceutically acceptable salt thereof as the active ingredient, with or without pharmaceutically acceptable excipients.

3. The pharmaceutical composition according to claim 2, characterized in that, Pharmaceutically acceptable excipients include one or more of the following: diluents, lubricants, flow aids, wetting agents, emulsifiers, and pH buffers.

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, microcapsules, injections, powder for injection, suspensions, ointments, creams, gels, sprays, eye drops, ear drops, nasal drops, patches, lotions, suppositories, films, and implants.

5. The use of the antimicrobial peptide of claim 1 or the pharmaceutical composition of any one of claims 2-4 in the preparation of a medicament for preventing and / or controlling infections by one or more of the following microorganisms: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa.

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