Antimicrobial peptide liquid composition and formulation thereof

Abstract

To provide an antimicrobial peptide liquid composition, and to provide a formulation thereof.SOLUTION: Provided is an antimicrobial peptide composition comprising an antimicrobial peptide, at least one stabilizer, and a buffer system, the mass concentration of the antimicrobial peptide in the composition being 0.1 per mille to 10 per mille, the buffer system being a phosphate buffer system or an acetate buffer system, and the amino acid sequence of the antimicrobial peptide being: KWKSFLKTFaAbKTVLHTALKAISS. The antimicrobial peptide composition is a topical broad-spectrum anti-infection drug, suitable for treating various primary skin infections caused by pathogens, particularly resistant bacteria, as well as secondary skin infections such as eczema-complicated infections and ulcer-complicated infections, including stubborn infectious diseases such as diabetic foot disease, burn wound infections, and bedsore infections, and has potential for a wide range of future applications.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, and in particular to antimicrobial peptide liquid compositions and their formulations. [Background technology]

[0002] Antibacterial drugs mainly include antibiotics and synthetic antibacterial drugs. Since the 1920s and 1930s, the discovery and application of antibiotics has saved countless lives. Antibiotics are substances produced by bacteria or other microorganisms during their life processes, and have the effect of inhibiting or killing pathogenic microorganisms such as bacteria, spirochetes, mycoplasma, and chlamydia. Commercially available antibiotics mainly include beta-lactams, macrolides, polysaccharides (vancomycin, teicoplanin), aminoglycosides, tetracyclines, chloramphenicols, and lipopeptides (daptomycin).

[0003] While humanity once benefited from antibiotics, the world now must focus on solving the problem of the emergence and spread of resistant bacteria, a result of the overuse of antibiotics. As more and more "super bacteria" are discovered, the lineup of resistant bacteria becomes increasingly stronger, due to the phenomenon of antibiotic insensitivity caused by super bacteria, and in addition to "super bacteria," there are also "super super bacteria."

[0004] The development of antibiotic alternatives has become a hot topic in the biopharmaceutical field. Among these, antimicrobial peptides have become a "star of hope" for antibiotic alternatives because they possess broad-spectrum and highly effective bactericidal activity against bacteria and are less likely to develop drug resistance. The cell membrane is the primary target of antimicrobial peptides, and it has been reported that aggregation of antimicrobial peptide molecules in the cell membrane increases membrane permeability and causes a loss of membrane barrier function. For microorganisms to develop drug resistance to antimicrobial peptides, substantial changes to the lipid components of the microbial cell membrane are required.

[0005] Antimicrobial peptides are small polypeptides with biological activity that are induced and produced within living organisms. They have a molecular weight of approximately 2000-7000 and consist of 20-60 amino acid residues. Depending on their origin, antimicrobial peptides can be divided into plant antimicrobial peptides, animal antimicrobial peptides, bacterial antimicrobial peptides (also known as bacteriocins, which include cationic and neutral peptides and can be secreted by both gram-positive and gram-negative bacteria), and human-derived antimicrobial peptides.

[0006] In November 2013, a research group led by Zhang Yun, a researcher at the Kunming Institute of Zoology, Chinese Academy of Sciences, discovered that natural antibacterial peptides have selective immune activation and regulation functions and have good preventive and therapeutic effects against sepsis.

[0007] Other literature has reported that human-derived antimicrobial peptides have broad-spectrum antimicrobial activity, can regulate wound inflammation, and promote angiogenesis and regeneration of epithelial tissue at the wound site. During the human wound healing process, antimicrobial peptides are one of the important signaling molecules of the endogenous immune system, interacting with various cells and growth factors to coordinate the achievement of a balanced state and achieve wound repair.

[0008] CN10111256A is an antimicrobial peptide NA L and D-NA L Disclosed is the amino acid sequence Ac-Lys-Trp-Lys-Ser-Phe-Leu-Lys-Thr-Phe-Lys-Ser-Ala-Ala-Lys-Thr-Val-Leu-His-Thr-Ala-Leu-Lys-Ala-Ile-Ser-Ser-NH2, which has antimicrobial activity, a desirable level of hemolytic activity and a broad spectrum therapeutic index against Gram-positive and Gram-negative bacteria and other microorganisms having lipid bilayer-forming cellular or structural components.

[0009] The main problem with protein drug formulations is poor drug stability. To improve the stability of protein drugs, it is easy to imagine that preparing protein drugs into sterile lyophilized powders would enhance the stability of protein drugs. For liquid compositions, stability can be enhanced by adding additives (stabilizers) such as polyols (e.g., sorbitol, mannitol, glycerin, and propylene glycol), sugars (e.g., lactose, colloids, dextrin, glucose, and trehalose), amino acids (e.g., glycine, serine, glutamic acid, and lysine), salts (e.g., citrate, acetate, and phosphate), and surfactants. However, lyophilized formulations have the disadvantage of complex formulation and processing, making it extremely valuable to provide stable liquid protein formulations. Summary of the Invention [Means for solving the problem]

[0010] The present invention aims to provide an antimicrobial peptide liquid composition in the hope of solving the increasingly serious problem of drug resistance of bacteria and the suffering caused to many patients due to stubborn infections.

[0011] The antimicrobial peptide liquid composition provided by the present invention comprises an antimicrobial peptide, at least one stabilizer, and a buffer system, wherein the mass concentration of the antimicrobial peptide in the composition is 0.1‰-10‰, the mass concentration of the stabilizer is 0.5%-5%, and the buffer system is a phosphate buffer system or an acetate buffer system.

[0012] The antimicrobial peptide is a polypeptide of the following general formula: Segment A-IA-II-Segment B, I is selected from any of the following amino acid residues: L-leucine, D-leucine, L-valine, D-valine, L-alanine, D-alanine, glycine, L-serine, D-serine, L-lysine and D-lysine (L- and D- are the optical isomers L, A, S, V and K, and G); II is selected from any of the following amino acid residues: L-leucine, D-leucine, L-valine, D-valine, L-alanine, D-alanine, glycine, L-serine, D-serine, L-lysine and D-lysine (L- and D- are the optical isomers L, A, S, V and K, and G); Segment A has the sequence: KWKSFLKTFK, as shown in SEQ ID No: 1; Segment B has the sequence: KTVLHTALKAISS as shown in SEQ ID No: 2; A represents an alanine residue, In the general formula, the direction is from the N-terminal to the C-terminal.

[0013] Preferably, the antimicrobial peptide is NA L and D-NA L is.

[0014] The above NA L has the sequence of SEQ ID No: 3, which is: KWKSFLKTFKSAAKTVLHTALKAISS, The D-NA L has the sequence SEQ ID No: 4, which is: KWKSFLKTFKSAAKTVLHTALKAISS (all amino acids are in the D-configuration except for A at position 13, which is in the L-configuration).

[0015] In the names of antimicrobial peptides, the capital letter D- (non-subscript) indicates that the antimicrobial peptide is composed entirely of D-amino acids, except at specific positions (e.g., D-NA L is composed entirely of D-amino acids, except that this antimicrobial peptide contains L-alanine substitutions at the center of the nonpolar surface, which is represented by a capital N).

[0016] Preferably, the concentration of the antimicrobial peptide is 0.5‰ to 6‰, more preferably 1‰ to 4‰, and even more preferably 1‰ to 2‰, and specifically may be 0.2‰, 0.5‰, 1‰, or 2‰.

[0017] Preferably, the buffer system is a disodium hydrogen phosphate-citric acid buffer system, wherein the ion concentration of the buffer system in the composition is 0.01M-0.1M, preferably the ion concentration is 0.01M-0.02M, and more preferably the ion concentration is 0.015M.

[0018] Preferably, the pH value of the composition is between 3.5 and 5.5, specifically 3.5, 4.5 or 5.5.

[0019] The stabilizer described in the present invention can be at least one selected from polyols (e.g., sorbitol, mannitol, glycerin, propylene glycol, etc.), sugars (e.g., lactose, colloids, dextrin, glucose, trehalose, etc.), amino acids (e.g., glycine, serine, glutamic acid, lysine, etc.), salts (e.g., citrate, acetate, phosphate, etc.), surfactants, etc., and can not only stabilize the antimicrobial peptide but also maintain the antibacterial activity of the antimicrobial peptide.

[0020] Preferably, the stabilizer is mannitol, and the concentration of mannitol in the antimicrobial peptide liquid composition is 0.5% to 5%, preferably 1% to 2%, more preferably 1%, specifically, for example, 0.5%, 1%, 1.25%, 1.5%, or 5%.

[0021] Stability tests have demonstrated that the antimicrobial peptide composition provided by the present invention is stable for at least 24 months (at 4°C).

[0022] The antimicrobial peptide composition provided by the present invention is mainly suitable for the treatment of skin infectious diseases. Compared with gels, creams, etc., spray-formed drugs can effectively avoid direct contact with the skin wound, act directly on the lesion site, reduce systemic toxic reactions, and have good stability and easy absorption, improving the safety and adaptability of clinical drugs and more meeting clinical needs.

[0023] Therefore, the formulation of the composition provided by the present invention is a liquid formulation, and its dosage form may be a spray, solution, gel, emulsion, sol, drop, syrup, suspension, oral liquid, cleanser or liniment, etc., and is preferably a spray.

[0024] The present invention further provides a method for preparing the above antimicrobial peptide liquid composition.

[0025] Furthermore, the method for preparing the topical compositions of the present invention is simple; all that is required is to select the raw materials and auxiliary agents necessary for each composition and prepare them according to the conventional preparation methods for each dosage form disclosed in the prior art.

[0026] The method for preparing an antimicrobial peptide liquid composition provided by the present invention comprises the following steps: 1) Measure out 70 to 90% of the volume of water for injection, add the substances that form the buffer system to each, and stir until dissolved. Further, add the stabilizer to the above solution and stir until completely dissolved. 2) The antimicrobial peptide is weighed and added to the solution in step 1), stirred to dissolve, and then made up to the full volume with water for injection.

[0027] The method further comprises filtering the antimicrobial peptide liquid composition through a micropore filter film having a size of 0.22 microns.

[0028] According to one embodiment of the present invention, a specific method for preparing a spray formulation is disclosed, in which the antimicrobial peptide liquid composition is prepared using the above method, then filled into a spray bottle at 5 ml per bottle, and the spray pump is tightly tightened to obtain an antimicrobial peptide spray.

[0029] The antibacterial peptide used in the present invention is a novel broad-spectrum anti-infection drug that achieves bactericidal effects by destroying bacterial cell membranes. It has unique pharmacological effects and strong bactericidal effects, so unlike conventional antibiotics, it is less likely to develop drug resistance and there is almost no cross-drug resistance phenomenon compared to conventional antibiotics, making it a very effective route and method for solving the problem of drug resistance in pathogenic bacteria.

[0030] The main problem with protein drug formulations is poor drug stability. To improve the stability of protein drugs, it is easy to consider formulating them into sterile lyophilized powders to enhance their stability. For liquid compositions, stability can be enhanced by adding additives (stabilizers) such as polyols (e.g., sorbitol, mannitol, glycerin, and propylene glycol), sugars (e.g., lactose, colloids, dextrin, glucose, and trehalose), amino acids (e.g., glycine, serine, glutamic acid, and lysine), salts (e.g., citrate, acetate, and phosphate), and surfactants. The stability of liquid protein compositions is undoubtedly more difficult to ensure than that of lyophilized powders. The present invention focuses on the stability of antimicrobial peptide liquid compositions.

[0031] The research team of the present invention discovered that mannitol can significantly increase the stability of antimicrobial peptides compared to other stabilizers (e.g., glycerin, lactose, glycine).

[0032] The research team of the present invention discovered that an acidic pH buffer system can make antibacterial peptides more stable, and that buffer systems such as phosphate buffer systems (e.g., disodium hydrogen phosphate + citric acid, or disodium hydrogen phosphate + sodium dihydrogen phosphate, etc.), acetate buffer systems (e.g., sodium acetate + phosphoric acid, or sodium acetate + acetic acid, etc.), and citrate buffer systems (e.g., sodium citrate + acetic acid, or sodium citrate + citric acid, etc.) can be considered. In terms of combining the stability and bacteriostatic tests of antibacterial peptides, phosphate buffer systems and acetate buffer systems (including disodium hydrogen phosphate, citric acid, sodium acetate, and glacial acetic acid) are preferred, and a buffer system consisting of disodium hydrogen phosphate and citric acid is more preferred.

[0033] In the composition of the present invention, the ion concentration of the buffer system also has a certain effect on the stability of the antimicrobial peptide, and the inventors' research has revealed that the ion concentration of the buffer system is 0.01M to 0.1M, preferably the ion concentration is 0.01M to 0.02M, and more preferably the ion concentration is 0.015M.

[0034] The antimicrobial peptide spray of the present invention exhibits good bioavailability and safety in use, can reduce irritation to the skin and / or wound surface, has high stability, can improve the microenvironment of the wound surface during long-term wound coverage and inhibit bacterial growth, can rapidly repair wounds, promote healing, and shorten wound healing time, and has broad potential in the field of clinical wound care applications.

[0035] The antimicrobial peptide composition of the present invention can be used to prepare topical broad-spectrum anti-infection drug preparations and can also be used to treat local infections or diseases caused by local infections. It is suitable for pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), erythromycin-resistant and -sensitive strains of Streptococcus pyogenes, and IPM-R and IPM-S strains of Pseudomonas aeruginosa, and has a wide range of potential applications, particularly in various primary skin infections caused by resistant bacteria, as well as secondary skin infections such as eczema-complicated infections and ulcer-complicated infections, and in stubborn infectious diseases such as diabetic foot lesions, burn wound infections, and bedsore infections.

[0036] The present invention further relates to topical broad-spectrum infection prevention products, the active ingredient of which comprises the antimicrobial peptide compositions provided by the present invention.

[0037] Illustratively, the product may be a drug or a drug formulation.

[0038] The present invention further provides a method for localized infection prevention, comprising the steps of: administering an antimicrobial peptide composition described in the present invention to a recipient animal for localized infection prevention.

[0039] The present invention further provides a method for treating a disease caused by a local infection, comprising the steps of: administering to a recipient animal an antimicrobial peptide composition described in the present invention to treat a disease caused by a local infection.

[0040] In the present invention, the animal may be a mammal, for example, a human, and the animal may also be the infected animal other than a mammal, for example, a mouse.

[0041] The above infections are caused by at least one pathogenic bacterium selected from the group consisting of methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, erythromycin-resistant strains of Streptococcus pyogenes, erythromycin-sensitive strains of Streptococcus pyogenes, and IPM-R and IPM-S strains of Pseudomonas aeruginosa. The above infections include various primary skin infections caused by resistant bacteria, and secondary skin infections such as eczema-complicated infections and ulcer-complicated infections, as well as at least one stubborn infectious disease such as diabetic foot lesions, burn wound infections, and pressure ulcer infections. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention will be further described below with reference to specific examples, but the scope of the present invention is not limited to these examples. The above methods are conventional methods unless otherwise specified. The above raw materials can be obtained from public commercial sources unless otherwise specified.

[0043] The sequence of the antimicrobial peptide used in the following examples (having the sequence of SEQ ID No: 3) can be artificially synthesized. [Example]

[0044] Example 1: Screening of stabilizers for antimicrobial peptide compositions Mannitol, glycerin, lactose, and glycine were selected as stabilizers for antimicrobial peptides, and aqueous solutions of the antimicrobial peptide compositions (antimicrobial peptide concentration 2‰) were prepared. The stability of the antimicrobial peptide compositions was examined, and the measurement indicators included properties, related substances (maximum single impurity and total impurities), and content.

[0045] Related substance measurement methods: In accordance with the high-performance liquid chromatography method (Chinese Pharmacopoeia, 2015, Part II, Appendix VD), octadecylsilane-bonded silica gel was used as the packing material. 20 μL of the solution was injected into a liquid chromatography system, and the chromatogram was recorded. Related substances and limits: known impurities A, B, C, and D and unknown single impurities. Each impurity must not exceed 1.0%. The maximum single impurity is calculated by summing the maximum value of all single impurities. Total impurities include the sum of impurities A, B, C, D, and other unknown single impurities. The total impurities must not exceed 3.0%.

[0046] In the table below: antimicrobial peptide (2‰) + mannitol (1%) composition, Antimicrobial peptide (2‰) + glycerin (1%) composition, Antimicrobial peptide (2‰) + lactose (1%) composition, Antimicrobial peptide (2‰) + glycine (1%) composition. [Table 1] [Table 2]

[0047] The stability of the antimicrobial peptide composition in Tables 1 and 2 shows that mannitol (1%), as a stabilizer for the antimicrobial peptide, ensured that the antimicrobial peptide content of the antimicrobial peptide composition solution remained at 97% or more after 36 months (at 4°C).

[0048] Considering the stability in different mannitol systems with different antimicrobial peptide concentrations, we further considered the stability in different mannitol (0.5%–5%) systems with different antimicrobial peptide concentrations (2‰ and 0.2‰), and the results are shown in Table 3 . [Table 3]

[0049] As shown in Table 3, when the antimicrobial peptide concentration is 2‰ and the mannitol concentration in the composition system is 0.5%, the antimicrobial peptide content of the antimicrobial peptide composition solution after 6 months (at 25°C) is 97.55%. When the mannitol concentration in the composition system reaches 1% or more, the antimicrobial peptide content of the antimicrobial peptide composition solution after 6 months (at 25°C) can be maintained at 98% or more. Therefore, a mannitol concentration of 1% is preferred.

[0050] Example 2: Consideration of buffer systems and buffer system molarities for antimicrobial peptide compositions The buffer system of the antimicrobial peptide composition is considered according to the combinations in the table below, and in each buffer system of the antimicrobial peptide composition in the table below, the mass concentration of the antimicrobial peptide is 4‰. [Table 4] [Table 5]

[0051] As can be seen from Table 5, the selection of a buffer system of disodium hydrogen phosphate and citric acid ensures that the antimicrobial peptide content of the antimicrobial peptide composition solution remains at 98% or higher after 3 months (at 25°C). Therefore, the buffer system of disodium hydrogen phosphate and citric acid is the preferred buffer system.

[0052] For the preferred disodium hydrogen phosphate and citrate buffer system, the influence of the buffer system ion concentration (0.01 M to 0.1 M) on the stability of the antimicrobial peptide was considered, and the results are shown in Table 6. [Table 6]

[0053] From Table 6, the results of the investigation into the stability of the antimicrobial peptide composition solution depending on the buffer ion concentration show that an ion concentration of approximately 0.015 M can ensure that the antimicrobial peptide content of the antimicrobial peptide composition solution remains at 98% after 3 months (at 25°C).

[0054] Example 3: Consideration of pH value of antimicrobial peptide composition Mannitol (1%) was selected for stability, and disodium hydrogen phosphate and citric acid were used as a buffer system (0.015M) to prepare antimicrobial peptide (2‰) composition solutions at different pH values ​​(3.0, 3.5, 4.5, 5.5, 6.5). The stability of the antimicrobial peptide composition under different pH value conditions was examined, and the results are shown in Table 7.

[0055] At the same time, hydrochloric acid was selected as a pH adjuster to adjust the pH value of the antimicrobial peptide aqueous solution to 4.5. The stability of the antimicrobial peptide was examined and compared with that of an antimicrobial peptide composition solution buffered with disodium hydrogen phosphate and citric acid. The results are shown in Table 8. [Table 7] [Table 8]

[0056] Tables 7 and 8 show that the antimicrobial peptide composition can ensure that the antimicrobial peptide content of the antimicrobial peptide composition solution after 3 months (at 25°C) is 98% or more when the pH is between 3.5 and 5.5. When the pH of the antimicrobial peptide aqueous solution adjusted with hydrochloric acid is 4.5, the antimicrobial peptide content in the antimicrobial peptide aqueous solution after 3 months (at 25°C) decreased to 95% or less.

[0057] Example 4 Antimicrobial peptide spray composition (1) Prescription [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14]

[0058] (2) Preparation process (1) Take 70-90% of the prescribed amount of water for injection, add the prescribed amounts of disodium hydrogen phosphate and citric acid, and stir for 15 minutes until dissolved. Then add the prescribed amount of mannitol to the above solution and stir for 10 minutes until it is completely dissolved.

[0059] (2) Weigh out the prescribed amount of antimicrobial peptide and add it to the above solution, stir to dissolve, and add water for injection to the full volume.

[0060] (3) The mixture is filtered through a micro-pore filter film of 0.22 microns, and a sample is taken to measure the properties, content, pH value, etc. of the intermediate.

[0061] Fill a spray bottle with 5 ml per bottle and tighten the spray pump to obtain an antimicrobial peptide spray.

[0062] After filling, take an appropriate amount of product and inspect it according to the inspection methods of the quality standard items.

[0063] (3) Quality requirements 1) Related substances: In accordance with the high-performance liquid chromatography method (Chinese Pharmacopoeia 2015, Part II, Appendix VD), octadecylsilane-bonded silica gel was used as the packing material, and 20 μL of the solution was injected into a liquid chromatography device and the chromatogram was recorded. Related substances and limits: known impurities A, B, C, D and unknown single impurities, each of which must not exceed 1.0%. Total impurities include the sum of impurities A, B, C, D and other unknown single impurities, and the total impurities must not exceed 3.0%.

[0064] 2) pH: It should fall within the specified pH range of 3.5 to 5.5.

[0065] 3) Content: Measured by high performance liquid chromatography (Chinese Pharmacopoeia 2015, Part 4 General Provisions 0512). An appropriate amount of this product was precisely taken, dissolved in water, measured and diluted to make a solution of 0.25 mg per ml, which was used as the test substance solution. An accurate amount of 10 μl was taken and injected into the liquid chromatography device, and the chromatography was recorded. An appropriate amount of the antibacterial peptide control product was also taken and measured by the same method.

[0066] The stability of Formulation 1 and Formulation 6 was examined, and the results are shown in Table 9. [Table 15]

[0067] As can be seen from Table 9, both Formulation 1 and Formulation 6 can maintain the antimicrobial peptide content of 98% or more within 6 months at 25°C.

[0068] (4) In vitro bactericidal and antibacterial tests The antimicrobial peptide compositions (Formulations 2 and 4) were tested for their in vitro bactericidal and antibacterial effects against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant coagulase-negative Staphylococcus aureus (MRSCNS), and methoxycillin-susceptible coagulase-negative Staphylococcus aureus (MSSCNS). Vancomycin was used as a control. The results are shown in Tables 10 and 11.

[0069] Test strains: Standard strain: Staphylococcus aureus ATCC29213 (originating from the Clinical Laboratory Center of Jiangsu Provincial People's Hospital and preserved by the Nanjing Skin Research Institute). Clinical strains: MRSA, MSSA, MRSCNS, and MSSCNS strains were all derived from the Clinical Laboratory Center of Jiangsu Provincial People's Hospital and stored by the Nanjing Skin Research Institute. [Table 16] [Table 17]

[0070] (5) In vivo drug efficacy test As infectious bacteria, we selected Staphylococcus aureus and Streptococcus pyogenes, which are clinically the most common in localized skin bacterial infections, and Pseudomonas aeruginosa, which is clinically the most common in stubborn infections. By infecting mice with localized or mildly lesional skin infections, respectively, we developed a localized skin infection model with clinical strains of MRSA (methicillin-resistant Staphylococcus aureus), MSSA (methicillin-sensitive Staphylococcus aureus), erythromycin-resistant and -sensitive strains of Streptococcus pyogenes, and IPM-R and IPM-S strains of Pseudomonas aeruginosa. [1~3] The efficacy of the antimicrobial peptide spray against local skin infections caused by the above bacterial strains was quantitatively evaluated using 0.5% ofloxacin gel as a control and a solvent group (1% mannitol, 0.015 M disodium hydrogen phosphate and citric acid buffer, pH 4.5) as a negative control. The results are shown in Tables 12 to 14.

[0071] All test strains originated from the Clinical Laboratory Center of Jiangsu Provincial People's Hospital and were stored by the Nanjing Skin Research Institute, and the strain codes were generated by the Nanjing Skin Research Institute.

[0072] Antimicrobial peptide sprays containing 1‰ (Formulation 1), 0.5‰ (Formulation 4), and 0.2‰ (Formulation 6) showed significant inhibitory effects against local skin infections in mice caused by Staphylococcus aureus (MRSA and MSSA) and Streptococcus pyogenes (erythromycin-susceptible and -resistant strains). The 1‰ antimicrobial peptide spray also showed inhibitory effects against local skin infections in mice caused by Pseudomonas aeruginosa (IPM-R and IPM-S). (Inhibition rate calculation for the antimicrobial peptide group = [1 - (bacterial solution concentration in homogenate of the treatment group / bacterial solution concentration in homogenate of the vehicle group)] × 100%. Inhibition rate calculation for the ofloxacin gel group = [1 - (bacterial solution concentration in homogenate of the treatment group / bacterial solution concentration in homogenate of the model group)] × 100%). Statistical analysis of the post-treatment inhibitory rates revealed that the dose of antimicrobial peptide was significantly lower than that ofloxacin to achieve the same therapeutic effect. [Table 18] [Table 19] [Table 20] [Table 21] [Table 22]

[0073] References: 1. Clinical Trial Guidelines for Antibiotics for Uncomplicated and Complicated Skin Infections, published by the US FDA in July 1998, translated by the National Center for Drug Evaluation and Research (NCRRE) in 2009 2. Xu Shuyun et al. Pharmacological Testing Methodology. People's Health Publishing House, 2006,11, 3rd edition 3. Summary of Guiding Principles for Preclinical Research of New Drugs (Western Drugs), Pharmaceutical Affairs Bureau, Ministry of Health of the People's Republic of China, July 1993

[0074] Industrial Applicability The antimicrobial peptide composition of the present invention can be used to prepare topical broad-spectrum anti-infection drug formulations, which are suitable for use against pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive Staphylococcus aureus (MSSA), erythromycin-resistant and -sensitive strains of Streptococcus pyogenes, and IPM-R and IPM-S strains of Pseudomonas aeruginosa. In particular, it has a wide range of potential applications in the treatment of various primary skin infections caused by resistant bacteria, as well as secondary skin infections such as eczema-complicated infections and ulcer-complicated infections, and stubborn infectious diseases such as diabetic foot lesions, burn wound infections, and bedsore infections. In certain embodiments, for example, the following items are provided: (Item 1) an antimicrobial peptide, at least one stabilizer, and a buffer system, wherein the mass concentration of the antimicrobial peptide in the composition is 0.1‰ to 10‰, and the buffer system is a phosphate buffer system or an acetate buffer system; The antimicrobial peptide is a polypeptide of the following general formula: Segment A1A-n-Segment B, I is selected from any of the following amino acid residues: L-leucine, D-leucine, L-valine, D-valine, L-alanine, D-alanine, glycine, L-serine, D-serine, L-lysine and D-lysine; II is selected from any of the following amino acid residues: L-leucine, D-leucine, L-valine, D-valine, L-alanine, D-alanine, glycine, L-serine, D-serine, L-lysine and D-lysine; Segment A has the sequence: KWKSFLKTFK, as shown in SEQ ID No: 1; Segment B has the sequence: KTVLHTALKAISS as shown in SEQ ID No: 2; A represents an alanine residue, The antimicrobial peptide liquid composition, wherein the general formula is from the N-terminus to the C-terminus. (Item 2) 2. The composition according to item 1, wherein the mass concentration of the antimicrobial peptide is 0.5‰ to 6‰, preferably 1‰ to 4‰, and more preferably 1‰ to 2‰. (Item 3) 3. The composition according to item 1 or 2, wherein the buffer system is a disodium hydrogen phosphate-citric acid buffer system, and the ion concentration of the buffer system in the composition is 0.01 M to 0.1 M, preferably 0.01 M to 0.02 M, and more preferably 0.015 M. (Item 4) The antimicrobial peptide is NA L or D-NA, The above NA L has the sequence of SEQ ID No: 3, the amino acid sequence of which is: KWKSFLKTFKSAAKTVLHTALKAISS, The D-NA L has the sequence of SEQ ID No: 4, the amino acid sequence of which is: 4. The composition according to any one of items 1 to 3, wherein all amino acids are in the D-configuration except for A at position 13, which is in the L-configuration. (Item 5) the stabilizer is selected from at least one of polyols, amino acids, salts, and surfactants; Preferably, the stabilizer is mannitol, and the mass concentration of mannitol in the composition is 0.5% to 5%, preferably 1% to 2%, more preferably 1%. (Item 6) 6. The composition according to any one of items 1 to 5, wherein the pH value of the composition is 3.5 to 5.5. (Item 7) 7. The composition according to any one of items 1 to 6, wherein the composition is in the form of a liquid preparation, which may be in the form of a spray, solution, gel or emulsion, sol, infusion, syrup, suspension, oral liquid, cleanser, or liniment, and is preferably in the form of a spray. (Item 8) 8. Use of the composition according to any one of items 1 to 7 in the manufacture of a topical broad-spectrum infection prevention product. (Item 9) 8. Use of the composition according to any one of items 1 to 7 in the treatment of a local infection and / or a disease caused by a local infection. (Item 10) said product is a medicine, and said infection is caused by at least one pathogen among methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, Streptococcus pyogenes erythromycin-resistant strains, Streptococcus pyogenes erythromycin-sensitive strains, and Pseudomonas aeruginosa IPM-R and IPM-S strains; 10. The use according to item 8 or 9, wherein the infections include various primary skin infections caused by resistant bacteria, and secondary skin infections such as infections complicated with eczema and infections complicated with ulcers, and at least one of stubborn infectious diseases such as diabetic foot, burn wound infection, and bedsore infection. (Item 11) 8. A topical broad-spectrum infection prevention product, the active ingredient of which comprises the composition according to any one of items 1 to 7. (Item 12) the product is a drug; the infection is caused by at least one of the following pathogens: methicillin-resistant Staphylococcus aureus, methicillin-sensitive Staphylococcus aureus, erythromycin-resistant strains of Streptococcus pyogenes, erythromycin-sensitive strains of Streptococcus pyogenes, and IPM-R and IPM-S strains of Pseudomonas aeruginosa; The product according to item 11, characterized in that the infections include various primary skin infections caused by resistant bacteria, and secondary skin infections such as infections complicated with eczema and infections complicated with ulcers, and also includes at least one of stubborn infectious diseases such as diabetic foot disease, burn wound infections, and bedsore infections. (Item 13) A method for preventing local infection, comprising administering to a recipient animal the composition according to any one of items 1 to 7 or the product according to item 11 or 12 for preventing local infection. (Item 14) A method for treating a disease caused by a local infection, comprising administering to a recipient animal a composition according to any one of items 1 to 7 or a product according to item 11 or 12, for treating a disease caused by a local infection. A method for treating diseases caused by

Claims

[Claim 1] The invention described in the specification.

Images