Antimicrobial peptide and use thereof

Abstract

Provided are an antimicrobial peptide or a pharmaceutical composition thereof, and the use thereof. The antimicrobial peptide is a new efficient antimicrobial peptide, has the characteristics of small molecular weight, broad antibacterial spectrum, high efficiency, no cytotoxicity, no hemolytic property, high stability, low susceptibility to developing drug resistance, etc., and can kill microorganisms including clinically common drug-resistant strains, bacteria, and fungi. Moreover, the antimicrobial peptide has the advantages of simple structure, easy large-scale synthesis and low cost, and the antimicrobial peptide is expected to replace antibiotics to become a safe, green and efficient ideal antimicrobial agent, and has very wide application prospects in the fields of the preparation of antimicrobial drugs, antimicrobial preparations, antimicrobial products, etc.

Description

An antimicrobial peptide and its application Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to an antimicrobial peptide and an application thereof, in particular to a novel antimicrobial peptide and an application of the antimicrobial peptide in controlling microbial infection. Background Art

[0002] Antibiotics play a significant role in the prevention, control, and treatment of human diseases. Following the clinical introduction of penicillin in the 1940s, infectious diseases became widely and effectively treated. However, with the widespread and long-term use of antibiotics, many pathogens have continuously mutated and acquired resistance genes, leading to the widespread emergence of diverse drug-resistant strains, such as methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Escherichia coli (CREC), vancomycin-resistant Enterococci (VRE), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and carbapenem-resistant Klebsiella pneumoniae (CRKP). These strains can develop resistance to antibiotics through a variety of mechanisms, including the production of inactivating enzymes, alteration of antibiotic target sites, and the use of efflux pumps to pump drugs out of the cell. These resistant strains spread rapidly and are difficult to cure. The emergence of drug-resistant strains poses significant challenges to the treatment of infectious diseases and has made infectious diseases one of the top ten public health threats worldwide. However, the development of new antibiotics is far less rapid than the emergence of drug-resistant strains, and the resistance mechanisms of these newer drug-resistant strains are also more complex. Therefore, it is urgent to develop new, effective and less resistant antimicrobial drugs to solve the problem of antibiotic resistance.

[0003] Antimicrobial peptides (AMPs) are an important class of polypeptides and their derivatives with antimicrobial activity, exhibiting excellent inhibitory activity against a wide range of microorganisms, including bacteria, fungi, and viruses. In addition to their antimicrobial properties, AMPs also hold broad research and development potential in aquaculture, livestock and poultry feed, food preservation, cosmetics, oral care, and animal and plant genetic engineering. Compared to chemical drugs, AMPs offer advantages such as high antimicrobial activity, broad antimicrobial spectrum activity, excellent thermal stability, and low toxicity. Furthermore, compared to traditional single-target antibiotics, AMPs' membrane-targeted antimicrobial mechanism enables them to act simultaneously on multiple targets, including the cell membrane, thereby maintaining short-term stability of cell membrane properties and making it less likely for bacteria to develop resistance. Their non-membrane-targeting mechanism involves intracellular translocation of AMPs, where they participate in the inhibition of key biological processes such as DNA, RNA, and protein synthesis. The unique antimicrobial mechanism of AMPs holds great promise and potential for research and development in the treatment of infectious diseases, potentially replacing antibiotics as a new generation of antimicrobial drugs.

[0004] Antimicrobial peptides come from a wide range of sources, and most of the natural antimicrobial peptides discovered so far are purified from organisms such as animals, plants, and bacteria. Although there are many types of known natural antimicrobial peptides, they generally have problems such as large molecular weight, low content, poor stability, and high separation and purification costs. In addition, many antimicrobial peptides have weak antimicrobial activity and may even cause toxic side effects such as hemolysis on cells. In addition, most naturally purified antimicrobial peptides are composed of L-amino acids, which are unstable in the body and easily degraded by proteases. For example, the antimicrobial peptide Cbf-K isolated from the venom of Bungarus bungarus 16 Composed of thirty L-amino acids, antimicrobial peptides have poor stability in serum and are easily degraded and inactivated by proteases. Compared to L-amino acids, antimicrobial peptides composed of D-amino acids generally exhibit greater proteolytic and metabolic stability. Therefore, the introduction of D-amino acids into antimicrobial peptides can disrupt their amphipathic helical structure, thereby reducing potential side effects such as hemolytic activity and cytotoxicity.

[0005] Summary of the invention:

[0006] Purpose of the invention: To address the problems currently existing in the treatment of infectious diseases, the present invention proposes a strategy of using D-type antimicrobial peptides to replace antibiotics. The aim is to provide a novel antimicrobial peptide by introducing D-type amino acids instead of L-type amino acids, thereby solving the common clinical drug resistance problem of current antibiotics and the easy degradation, low activity and toxic side effects of natural antimicrobial peptides. This is of great significance for the research and development of new and effective anti-infective drugs.

[0007] Therefore, the technical problem to be solved by the present invention is to provide a novel antimicrobial peptide having broad-spectrum antimicrobial activity, low toxicity, high efficiency, high stability, and low resistance to drug resistance.

[0008] The technical problem that the present invention also aims to solve is to provide applications of antimicrobial peptides in multiple fields such as pharmaceutical production, livestock and poultry farming, aquaculture, fruit and vegetable preservation, production of disinfection and antiseptic products, additive production, agricultural production, and production of health products (food, nursing products, health products, feed and cosmetics, etc.).

[0009] Technical solution: In order to solve the above technical problems, the present invention provides an antimicrobial peptide, which comprises the following common amino acid formula: Ac-Arg-Leu-Leu-BZ-Leu-ZB-NH2, or Ac-Arg-Leu-Leu-BZ-Leu-ZB-Arg-NH2, or Ac-Arg-Arg-Leu-Leu-BZ-Leu-ZB-Arg-Arg-NH2, wherein Ac represents that the amino terminus of the polypeptide is acetylated, NH2 represents that the carboxyl terminus of the polypeptide is amidated, B is selected from an aromatic amino acid residue, and one or two Zs are selected from an arginine residue or a lysine residue.

[0010] The aromatic amino acid residues refer to amino acid residues containing aromatic rings in their molecular structures, mainly including 3-(2-naphthyl)-alanine residues, phenylalanine residues and phenylalanine derivative residues.

[0011] Wherein, one or more Arg residues in the sequence of the antimicrobial peptide are replaced by lysine residues.

[0012] Wherein, one or more Leu residues in the sequence of the antimicrobial peptide are substituted by a norleucine residue, a norvaline residue, a homoleucine residue, an alanine residue or a valine residue.

[0013] The antimicrobial peptide includes all L-type and all D-type enantiomers; or any one or more amino acids of the antimicrobial peptide are substituted with L-type or D-type amino acids.

[0014] Wherein, polyethylene glycol is connected to the amino terminal, carboxyl terminal or side chain amino group of lysine residue of the antimicrobial peptide.

[0015] The molecular weight of the polyethylene glycol is 200 to 4000. Preferably, the polyethylene glycol includes polyethylene glycol 200, polyethylene glycol 500, polyethylene glycol 1000, polyethylene glycol 2000 or polyethylene glycol 4000.

[0016] The present invention also includes a pharmaceutical composition comprising the antimicrobial peptide.

[0017] The present invention also includes the use of the antimicrobial peptide or its pharmaceutical composition in the preparation of a drug for preventing and / or controlling microbial infection.

[0018] The preferred dosage range of the antimicrobial peptide preparation of the present invention is 0.001-1000 mg by weight.

[0019] The drug comprises at least one antimicrobial peptide as described above, or a pharmaceutically acceptable salt, ester, solvate, hydrate, or prodrug thereof, and at least one pharmaceutically acceptable carrier, excipient, vehicle, diluent, buffer, adjuvant, auxiliary agent, or vehicle. A "pharmaceutically acceptable" excipient is one that is suitable for administration to humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. The term "pharmaceutically acceptable carrier" refers to a vehicle used for administering a therapeutic agent, including various excipients and diluents. The term refers to pharmaceutical carriers that are not themselves essential active ingredients and are not excessively toxic upon administration. Suitable carriers are well known to those of ordinary skill in the art. A comprehensive description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991). Pharmaceutically acceptable carriers in the composition may contain liquids such as water, saline, glycerol, and ethanol. In addition, these carriers may also contain auxiliary substances, such as lubricants, glidants, wetting agents or emulsifiers, pH buffer substances, etc.

[0020] The term "pharmaceutically acceptable excipient" refers to any suitable pharmaceutically acceptable adjuvant, carrier, diluent, preservative, etc. used in pharmaceutical formulations. For illustrative purposes only, known adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysophosphatidylcholine, complex polyols, polyanions, peptides, oil emulsions, hydrocarbon emulsions, keyhole limpet hemocyanin, etc. Known carriers include, but are not limited to, sterile liquids such as water, physiological saline, oil, or mixtures of water and oil, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Known diluents include, but are not limited to, water, saline, glucose, ethanol, glycerol, and the like. Known preservatives include, but are not limited to, thimerosal and EDTA, etc. The selection of pharmaceutically acceptable excipients can be accomplished using techniques known in the art. Those skilled in the art can select appropriate pharmaceutically acceptable excipients based on existing technology according to the desired polypeptide pharmaceutical dosage form. For example, for the preparation of oral liquid preparations (including but not limited to suspensions, microemulsions, or multiple emulsions), the selected excipients may include, for example, water, oils, alcohols, flavoring agents, preservatives, colorants, etc. For another example, for the preparation of oral solid preparations (including but not limited to powders, powder aerosols, capsules, or tablets), the selected excipients may include, for example, starch, sugars, diluents, granulating agents, lubricants, binders, disintegrants, etc. Furthermore, if desired, the polypeptide drugs of the present invention may also be prepared as sugar-coated or enteric-coated preparations, or as controlled-release preparations.

[0021] The dosage forms of the medicine may include injections, tablets, oral preparations, external preparations, eye drops, lotions, powders, granules, pills, capsules, caplets, pills, powders, elixirs, suspensions, mixtures, enemas, liniments, solutions, liquid preparations, rubber preparations, mucilages, infusions, extracts, gels, sachets, cachets, solutions, syrups, aqueous solutions, spirits, glycerin preparations, transdermal patches, film preparations, granules, smoke preparations, emulsions, transdermal patches, tinctures, penetrants, wine preparations, traditional Chinese medicine preparations, aerosols, creams, ear drops, ointments, oral films, gargles, lotions, pastes, sprays, foams, inhalants, gels, injections, dews, dry powder injections, enteric coatings, nanospheres, microspheres, powder aerosols, decoctions, ointments, pills, nasal sprays, nasal drops, Gargles, sublingual tablets, insufflations, suppositories, films, drops, liniments, nasal washes, eye washes, ear washes, intraocular inserts, eye masks, eye ointments, sprays, dry powder inhalers, powder inhalers, nebulizers, sticks, threads, strips, tooth drops, suspension drops, emulsions, sprinkles, paints, coatings, creams, patches, plasters, oral patches, implants, irrigants, tablets Preparations, cakes, biscuits, decoctions, plasters, teas, porridges, oil extracts, sols, fluid extracts, powder injections, water injections, sterile powders, sponges, microcapsules, nanocapsules, honey pills, water pills, water-honeyed pills, paste pills, concentrated pills, wax pills, sugar pills, coated tablets, enteric-coated tablets, effervescent tablets, chewable tablets, soluble tablets, dispersible tablets, sustained-release tablets, controlled-release tablets, orally disintegrating tablets, buccal pills, microemulsions or multiple emulsions.

[0022] Preferably, the pharmaceutical dosage form includes an injection, an oral agent or an external agent, and the external agent includes an eye drop or a lotion, wherein the dosage range of the antimicrobial peptide in the dosage form is: injection 0.001-1000 mg / kg; oral agent 0.001-1000 mg / kg; external agent 1 / 10000-30% / tube; eye drop 1 / 10000-30% / tube; lotion 1 / 100000-20‰ / tube

[0023] The microorganisms include one or more of bacteria, fungi, actinomycetes, archaea, cyanobacteria, mycoplasmas, chlamydia, rickettsiae, spirochetes, subviruses, viruses, protozoa or algae.

[0024] The application includes any of the following:

[0025] (1) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of anti-inflammatory and / or anti-tumor drugs;

[0026] (2) Application of the antimicrobial peptide or its pharmaceutical composition in aquaculture;

[0027] (3) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of oral care products;

[0028] (4) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of oral cleaning preparations;

[0029] (5) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of tooth cleaning products;

[0030] (6) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of tooth coatings;

[0031] (7) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of a drug for preventing and treating dental caries;

[0032] (8) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of a product for treating skin infections;

[0033] (9) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of intracellular sterilization products;

[0034] (10) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of a product for removing bacterial biofilm;

[0035] (11) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of disinfectant products;

[0036] (12) Use of the antimicrobial peptide or its pharmaceutical composition in preventing and / or treating wheat scab;

[0037] (13) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of antifungal and preservative agents;

[0038] (14) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of food preservatives;

[0039] (15) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of cosmetic preservatives;

[0040] (16) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of cosmetics;

[0041] (17) Use of the antimicrobial peptide or its pharmaceutical composition in livestock and poultry feed and / or feed additives;

[0042] (18) Use of the antimicrobial peptide or its pharmaceutical composition in the improvement of animal and plant varieties and / or animal and plant breeding.

[0043] (19) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of wound dressings;

[0044] (20) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of antimicrobial cling film;

[0045] (21) Application of the antimicrobial peptide or its pharmaceutical composition in preserving fruits and vegetables;

[0046] (22) Application of the antimicrobial peptide or its pharmaceutical composition in animal husbandry;

[0047] (23) Use of the antimicrobial peptide or its pharmaceutical composition in the preparation of antimicrobial agents, medicines, veterinary drugs, feeds, foods, daily chemicals, health products, cosmetics, bactericides, virucides, algaecides, sterilizers, feeds, additives, oral cleaning preparations, disinfectants, anti-inflammatory products, cleaning agents, preservatives, excipients, mildew inhibitors, algaecides, detergent aids, detergent compositions, cleaning agents or preservatives.

[0048] The antimicrobial peptides and pharmaceutical compositions thereof can be prepared into any medically available biological carrier or preparation form and administered to patients with infectious diseases.

[0049] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the antimicrobial peptides described in the present invention are a new type of highly effective antimicrobial peptides, which have the characteristics of small molecular weight, broad antibacterial spectrum, high efficiency, non-cytotoxicity, non-hemolytic activity, high stability, and low resistance to drug resistance. They can kill microorganisms including common clinically resistant strains, bacteria, and fungi, and they have a simple structure, are easy to synthesize on a large scale, and are low in cost. They are expected to replace antibiotics and become a safe, green, and highly effective ideal antimicrobial agent. They have broad application prospects in the preparation of antimicrobial drugs, antimicrobial preparations, and antimicrobial products. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG1 shows the cytotoxicity test results of the antimicrobial peptides LV-1 to LV-30 of the present invention. DETAILED DESCRIPTION

[0051] Unless otherwise defined, the technical terms used in the following examples have the same meanings as commonly understood by those skilled in the art to which this invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are conventional biochemical reagents; the experimental methods described, unless otherwise specified, are conventional methods.

[0052] The present invention will be further described below with reference to specific embodiments.

[0053] Example 1 Antimicrobial Peptide Sequence Information

[0054] As shown in Table 1, the first 30 antimicrobial peptides (LV-1 to LV-30) from sequence number 1 to sequence number 30 are all D-amino acids; the last 30 antimicrobial peptides (LV-31 to LV-60) from sequence number 31 to sequence number 60 are all L-amino acids. In Table 1, X represents a 3-(2-naphthyl)-alanine residue, X1 represents a 4-fluoro-phenylalanine residue, X2 represents a 3,4-difluorophenylalanine residue, X3 represents a 4-chlorophenylalanine residue, X4 represents a 3,4-dichlorophenylalanine residue, X5 represents a 4-trifluoromethyl-phenylalanine residue, X6 represents a 4-bromophenylalanine residue, X7 represents a 4-methyl-phenylalanine residue, X8 represents a norleucine residue, X9 represents a norvaline residue, and X10 represents a homoleucine residue. Ac represents acetylation modification of the amino terminus of the peptide, and NH2 represents amidation modification of the carboxyl terminus of the peptide. The phenylalanine derivative residues mainly include 4-fluoro-phenylalanine residues, 3,4-difluorophenylalanine residues, 4-chlorophenylalanine residues, 3,4-dichlorophenylalanine residues, 4-trifluoromethyl-phenylalanine residues, 4-bromophenylalanine residues and 4-methyl-phenylalanine residues. At the same time, for the 25 antimicrobial peptides (LV-1 to LV-25) with sequence numbers 1 to 25 and the 25 antimicrobial peptides (LV-31 to LV-55) with sequence numbers 31 to 55 in Table 1, their amino termini are acetylated and their carboxyl termini are amidated. The PEG200 of the antimicrobial peptides LV-26 and LV-56 is polyethylene glycol 200, and PEG200 is connected to the carboxyl termini of LV-26 and LV-56, respectively; the PEG500 of the antimicrobial peptides LV-27 and LV-57 is polyethylene glycol 500, and PEG500 is connected to the carboxyl termini of LV-27 and LV-57, respectively; the PEG1000 of LV-28 and LV-58 is polyethylene glycol 1000, and PEG1000 is connected to the carboxyl termini of LV-28 and LV-58, respectively; the PEG2000 of the antimicrobial peptides LV-29 and LV-59 is PEG2000, and PEG2000 is connected to the amino termini of LV-29 and LV-59, respectively; the PEG4000 of LV-30 and LV-60 is polyethylene glycol 4000, and PEG4000 is connected to the carboxyl termini of LV-30 and LV-60, respectively.

[0055] A summary of antimicrobial peptide sequence information is shown in Table 1. All 60 peptides (LV 1–60) share a common amino acid sequence motif: their first, second, sixth, eighth, tenth, and eleventh amino acids are positively charged arginine or lysine, their third, fourth, and seventh amino acids are hydrophobic, and their fifth and ninth amino acids are aromatic. Ten representative peptides (LV-1, LV-2, LV-6, LV-12, LV-17, LV-18, LV-19, LV-20, LV-25, and LV-27) exhibited significant in vivo efficacy at high, medium, and low doses.

[0056] Table 1 Summary of antimicrobial peptide sequence information

[0057] Example 2: Antibacterial activity experiment of antimicrobial peptides

[0058] 1. Antibacterial test of LV-1 to LV-60 against Staphylococcus aureus (NRS 384)

[0059] According to the National Center for Clinical Laboratory Standards (NCCLS) antimicrobial susceptibility testing operating standards, the classic micro-serial two-fold dilution method was used to determine the minimum inhibitory concentration of all 60 peptides (serial number: LV-1 to LV-60) against Staphylococcus aureus (NRS384). The experiment was repeated 5 times in parallel. The final concentration of the peptide solution was 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / ml. The minimum inhibitory concentration (MIC) of the antimicrobial peptide was the lowest drug concentration that completely inhibited bacterial growth in the well. The antimicrobial drug levofloxacin and the antimicrobial peptide Cbf-K 16 (Cbf-K 16 The sequence of KFFRKLKKSVKKRAKKFFKKPRVIGVSIPF was selected as a positive control to test the antibacterial effect of the polypeptide on Staphylococcus aureus (NRS 384). The results are shown in Table 2.

[0060] As shown in Table 2, the antimicrobial peptide Cbf-K 16 All 60 peptides tested (serial numbers: LV-1 to LV-60) showed better antibacterial effects against Staphylococcus aureus (NRS 384) (MIC≤8μg / ml) compared with levofloxacin (MIC=32μg / ml), among which 30 peptides (serial numbers: LV-1 to LV-30) showed the best antibacterial activity (MIC=1μg / ml), which was comparable to the antibacterial activity of the antibacterial drug levofloxacin.

[0061] Table 2 Experimental results of the minimum inhibitory concentration (MIC) (μg / ml) of all 60 peptides tested (serial number: LV-1 to LV-60) against Staphylococcus aureus (NRS 384)

[0062] 2. Antibacterial test of LV-1~LV-30 against 10 common sensitive strains and 6 fungi

[0063] According to the National Center for Clinical Laboratory Standards (NCCLS) antimicrobial susceptibility testing procedures, the minimum inhibitory concentrations (MICs) of 30 antimicrobial peptides (numbered LV-1 to LV-30) against 10 common clinically sensitive strains and 6 fungi were further determined using the classic serial two-fold microdilution method. The experiment was repeated five times in parallel, with final peptide solution concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / ml. The minimum inhibitory concentration (MIC) (μg / ml) of the antimicrobial peptides was determined to be the lowest concentration that completely inhibited bacterial or fungal growth within the wells. The results are shown in Tables 3 and 4.

[0064] From the experimental results in Tables 3 and 4, it can be seen that LV-1 to LV-30 showed strong antimicrobial activity against all 16 common clinical sensitive strains and fungi tested.

[0065] Table 3. Minimum inhibitory concentration (MIC) (μg / ml) of LV-1 to LV-30 against 10 common sensitive strains

[0066] Table 4. Experimental results of minimum inhibitory concentration (MIC) (μg / ml) of LV-1 to LV-30 against common fungi

[0067] 3. Antibacterial experiments of peptides LV-1 to LV-30 against five common clinical drug-resistant strains

[0068] According to the National Center for Clinical Laboratory Standards (NCCLS) antimicrobial susceptibility testing procedures, the minimum inhibitory concentrations (MICs) of peptides LV-1 to LV-30 against five common clinically resistant strains were further determined using the classic serial two-fold microdilution method. The experiment was repeated five times with final peptide concentrations of 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / ml. The minimum inhibitory concentration (MIC) of the antimicrobial peptide was defined as the lowest concentration that completely inhibited bacterial growth within the wells. The resistant strains tested included methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Escherichia coli (CREC), vancomycin-resistant Enterococci (VRE), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and carbapenem-resistant Klebsiella pneumoniae (CRKP). The results are shown in Table 5.

[0069] As shown in Table 5, antimicrobial peptides LV-1 to LV-30 exhibited good antimicrobial activity (MIC ≤ 8 μg / ml) against all five common drug-resistant strains tested. The antimicrobial experiments in Tables 2, 3, 4, and 5 demonstrate that the peptides LV-1 to LV-30 described herein possess highly effective and broad-spectrum antimicrobial activity against microorganisms including clinically common bacteria, fungi, and drug-resistant strains.

[0070] Table 5 shows the minimum inhibitory concentration (MIC) (μg / ml) of LV-1 to LV-30 against common clinical drug-resistant strains.

[0071] Example 3 Hemolytic Activity Detection of Antimicrobial Peptides

[0072] Fresh rabbit blood was centrifuged at 500 g for 5 min, the supernatant was removed, and the red blood cells were repeatedly washed with PBS (0.01 M, pH 7.4) until the supernatant was clear. The red blood cells were resuspended to 10 ml and diluted 25 times with PBS (0.01 M, pH 7.4). The red blood cells were about 2 × 10 8 RBC / ml, prepare red blood cell suspension. Antimicrobial peptides were serially diluted with physiological saline (0.85%, w / v, NaCl) according to the serial dilution method. The final concentrations of the peptide solution were 1.95, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μg / ml. 100 μL of antimicrobial peptide solution was added to a 96-well plate, with 5 replicates for each concentration. Then, approximately 2×10 red blood cells were added to each well. 8100 μl of rabbit blood (RBC / ml) was incubated at 37°C for 1 hour and centrifuged at 500 g for 5 minutes. 100 μl of the supernatant was transferred to a new 96-well plate and the OD value was measured at 541 nm using a microplate reader. Triton X-100 (10 mg / ml as a starting concentration, followed by two-fold serial dilutions) was used as a positive control. Hemolysis rate was calculated using the formula: [OD541 of the experimental group - OD541 of the negative control group] / [OD541 of the positive control group - OD541 of the negative control group] × 100%. Specific hemolysis test results are shown in Table 6. Minimum hemolytic concentration (MHC) is the lowest drug concentration that causes 10% hemolysis.

[0073] As shown in Table 6, at concentrations far above the minimum inhibitory concentration (MIC), all the antimicrobial peptides tested (LV-1 to LV-60) showed no obvious hemolysis on rabbit red blood cells, indicating that they have good safety.

[0074] Table 6 Minimum hemolytic concentration (MHC) of peptides for red blood cell hemolysis

[0075] Example 4 Cytotoxicity Assay of Antimicrobial Peptides

[0076] HEK293T cells (from ATCC) were cultured in DMEM containing 10% fetal bovine serum in an incubator at 37°C and 5% CO2. Cells in the logarithmic phase were collected and adherent cells were treated with 0.25% trypsin to adjust the cell suspension concentration to 5 × 10 4 Cells were cultured at 37°C in a 5% CO2 incubator for 24 hours, followed by aspiration of the culture medium. After washing twice with PBS, 100 μL of an antimicrobial peptide solution (final concentration of 128 μg / mL) was added to each well, with five replicates per well. The 96-well plate was then placed in a cell culture incubator for 48 hours. 20 μL of a 5 mg / mL MTT solution was added to each well, and the culture was continued for 4 hours. The culture medium in the 96-well plate was carefully aspirated, and 150 μL of DMSO was added to each well. The plates were shaken carefully to fully dissolve the crystals, and the absorbance (OD) of each well was measured at 570 nm using a microplate reader. Cell survival rate (%) was calculated as follows: (OD value of the experimental group - OD value of the blank control group) / (OD value of the control group - OD value of the blank control group) × 100%.

[0077] The results of the cytotoxicity test are shown in Figure 1. The antimicrobial peptides LV-1 to LV-30 described in the present invention had no significant effect on the survival rate of HEK293T cells at a high concentration of 128 μg / ml, indicating that the antimicrobial peptides have no significant toxic side effects on mammalian immune cells, can well distinguish between bacterial cells and mammalian cells, have high safety, and have broad application prospects in the preparation of antimicrobial drugs, antimicrobial preparations, and antimicrobial products. Example 5 Determination of proteolytic stability of antimicrobial peptides

[0078] Trypsin (250 U / mg, pH=8.0) and antimicrobial peptides (LV-1 to LV-30) were added to a reaction buffer (0.2 M sodium phosphate, 1 mM CaCl2, pH 7.5) at a ratio of 1:10 (w / w) and incubated at 37°C for 3 h. According to the same experimental method in Example 2, the peptide solution and Staphylococcus aureus (NRS 384) bacterial solution were added to a sterile 96-well cell culture plate, with 5 replicates for each concentration. The final concentration range of the peptide was 0.125, 0.25, 0.5, 1, 2, 4, 8, 16, 32, 64, and 128 μg / ml. The cells were incubated in a 37°C constant temperature incubator for 16 to 24 h until visible turbidity appeared in the negative control wells. The MIC values ​​of the antimicrobial peptides after trypsin treatment were observed.

[0079] The results are shown in Table 7. The MIC values ​​of the antimicrobial peptides LV-1 to LV-30 of the present invention did not change after being treated with trypsin. However, the positive control L-type antimicrobial peptide Cbf-K 16 After treatment with trypsin, all of them lost their antibacterial activity, indicating that the peptide bonds formed by D-type amino acids have stronger enzyme resistance than L-type amino acids, which significantly improves the clinical application value of antimicrobial peptides.

[0080] Table 7 Protease stability assay of antimicrobial peptides

[0081] Example 6 Determination of Serum Stability of Antimicrobial Peptides LV-1 to LV-30

[0082] 30 μL of antimicrobial peptide (LV-1 to LV-30) solution (10 mg / mL stock solution) was added to human serum (300 μL) for incubation. After incubation at 37°C for 12 h, samples were taken and the residual amount of antimicrobial peptide in human serum was determined by reverse-phase high-performance liquid chromatography.

[0083] The results are shown in Table 8. After incubation of antimicrobial peptides LV-1 to LV-30 in human serum for 12 hours, the residual amounts of antimicrobial peptides were all above 97%, indicating that antimicrobial peptides LV-1 to LV-30 showed good stability in human serum.

[0084] Table 8 Residual amounts of antimicrobial peptides in human serum

[0085] Example 7 Treatment trial of antimicrobial peptides LV-1 to LV-30 external preparations on mouse back skin infection

[0086] This study used female and male ICR mice, half male and half female, randomly divided by weight into 10 groups each and housed separately. The mice were divided into a blank group, a model group, a positive control methicillin group, and a topical group of antimicrobial peptides LV-1 to LV-30. A suspension of methicillin-sensitive Staphylococcus aureus (NRS 384) and methicillin-resistant Staphylococcus aureus (MRSA) was prepared and adjusted to a concentration of 5 × 10 6 First, the hair on the back of the mouse was shaved with a razor, and a wound was created on the back surface with a biopsy punch and infected with methicillin-sensitive Staphylococcus aureus or methicillin-resistant Staphylococcus aureus (0.1 ml, 5×10 6 CFU / ml) to establish an infection model. Except for the blank and model groups, all other groups received a 0.1 ml application of a different topical agent to the wound site twice daily for four consecutive days. The day after the final administration, aseptically sampled the infected skin from each group of animals, and the inhibition rate was calculated. The results are shown in Table 9.

[0087] Table 9 shows that, compared with the methicillin group, the high, medium, and low dose groups of antimicrobial peptides LV-1 to LV-30 not only achieved inhibition rates of over 95% against sensitive Staphylococcus aureus, but also achieved inhibition rates of over 85% against methicillin-resistant Staphylococcus aureus, significantly superior to the methicillin group. This indicates that the topical antimicrobial peptides LV-1 to LV-30 have a significant inhibitory effect on Staphylococcus aureus in skin wound infections.

[0088] Table 9 Effects of antimicrobial peptides LV-1 to LV-30 topical preparations on skin infections (n=10)

[0089] Example 8 Treatment Test of Antimicrobial Peptides LV-1 to LV-30 Injections on Peritonitis Infection in Mice

[0090] This study used female and male ICR mice, half male and half female, randomly divided by weight into 10 groups each and housed separately. The mice were divided into a blank control group, a negative control group, a positive control group containing levofloxacin, and groups containing antimicrobial peptides LV-1 to LV-30. A suspension of Pseudomonas aeruginosa ATCC 27853 was prepared and its concentration was adjusted to 1 × 10 9 CFU / ml, set aside. 9 CFU / ml) were injected into the peritoneal cavity of mice to establish a model. Immediately after successful model establishment, dosing was performed according to body weight. Except for the blank control group and the infection-negative control group, all other groups received a different injection via the tail vein twice daily for three consecutive days and were observed for 14 days. Mice in each group were observed for mortality and survival time. The effects of antimicrobial peptides LV-1 to LV-30 injections on the survival of mice infected with Pseudomonas aeruginosa are shown in Table 10.

[0091] As shown in Table 10, all 10 mice in the negative control group died within 2 days after modeling (10 / 10). The mean survival days of mice in the antimicrobial peptide groups (LV-1 to LV-30) were significantly different from those in the negative control group (**P < 0.01, ***P < 0.001, or ****P < 0.0001), indicating that the antimicrobial peptide LV-1 to LV-30 groups all had good anti-infection effects. Although the levofloxacin control group effectively prolonged the survival time of infected mice, the survival time of infected mice in the antimicrobial peptide LV-1 to LV-30 groups was significantly longer than that in the levofloxacin group. Therefore, it can be seen that the antimicrobial peptide LV-1 to LV-30 injections have a good in vivo therapeutic effect on mice infected with Pseudomonas aeruginosa. In addition, 10 representative peptides (including LV-1, LV-2, LV-6, LV-12, LV-17, LV-18, LV-19, LV-20, LV-25 and LV-27) were selected from all 30 antimicrobial peptides with a common motif structure to conduct high-, medium- and low-dose group efficacy experiments on mouse peritonitis infection. As shown in Table 10, there was a very significant difference in the average survival days of mice in the high-, medium- and low-dose groups of these 10 peptides compared with the negative control group (**P < 0.01, ***P < 0.001 or ****P < 0.0001). The anti-infection effects of their high-, medium- and low-dose groups were significantly better than those of the levofloxacin group.

[0092] Table 10 Effects of antimicrobial peptides LV-1 to LV-30 injections on the survival time of mice infected with Pseudomonas aeruginosa (n=10)

[0093] Note: Compared with the negative control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

[0094] Example 8 Therapeutic Test of Oral Formulations of Antimicrobial Peptides LV-1 to LV-30 on Animals with Systemic Bacterial Infections

[0095] This study used female and male ICR mice, half male and half female, randomly divided by weight into 10 groups each and housed in separate cages. The mice were divided into blank control, negative control, positive control levofloxacin group, and antimicrobial peptide LV-1 to LV-30 groups. A suspension of Escherichia coli ATCC 25922 was prepared and its concentration was adjusted to 1×10 7 CFU / ml, set aside. 7 CFU / ml) were injected into the peritoneal cavity of mice to establish a model. Immediately after successful model establishment, dosing was performed based on body weight. Except for the blank control group and the infection-negative control group, all other groups received different compounds orally three times daily for seven consecutive days, followed by observation for 14 days. Mice in each group were observed for mortality, and survival was recorded. The effects of oral formulations of antimicrobial peptides LV-1 to LV-30 on the survival of mice infected with E. coli are shown in Table 11.

[0096] As shown in Table 11, all 10 mice in the negative control group died within 5 days after modeling (10 / 10). There was a significant difference in the average survival days between the antimicrobial peptide LV-1 to LV-30 groups and the negative control group (**P < 0.01, ***P < 0.001, or ****P < 0.0001), indicating that the antimicrobial peptide LV-1 to LV-30 groups all had a good anti-infection effect. Although the levofloxacin control group effectively prolonged the survival time of infected mice, the survival time of infected mice in the antimicrobial peptide LV-1 to LV-30 groups was significantly longer than that in the levofloxacin group. Therefore, it can be seen that the antimicrobial peptide LV-1 to LV-30 injection has a good in vivo therapeutic effect on mice infected with Escherichia coli. In addition, among all 30 antimicrobial peptides (LV-1 to LV-30) with a common motif structure, 10 representative peptides (including LV-1, LV-2, LV-6, LV-12, LV-17, LV-18, LV-19, LV-20, LV-25 and LV-27) showed significant effects in the high, medium and low dose groups of mouse systemic infection efficacy experiments. As shown in Table 11, there was a very significant difference in the average survival days of mice in the high, medium and low dose groups of these 10 peptides compared with the negative control group (**P < 0.01, ***P < 0.001 or ****P < 0.0001), and their anti-infection effects in the high, medium and low dose groups were significantly better than those in the levofloxacin group.

[0097] Table 11 Effect of oral preparations of antimicrobial peptides LV-1 to LV-30 on the survival time of mice infected with Escherichia coli (n = 10)

[0098] Note: Compared with the negative control group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001

[0099] As can be seen from the above examples, the antimicrobial peptides described in the present invention are a class of highly effective antimicrobial peptides. They have the characteristics of small molecular weight, broad antimicrobial spectrum, high efficiency, non-cytotoxicity, non-hemolytic activity, high stability, and low resistance to drug resistance. They can kill microorganisms including common clinically drug-resistant strains, bacteria, and fungi. In addition, they have a simple structure, are easy to synthesize on a large scale, and are low in cost. They are expected to replace antibiotics as a safe, green, and highly effective ideal antimicrobial agent. They have broad application prospects in the preparation of antimicrobial drugs, antimicrobial preparations, and antimicrobial products.

Claims

1. An antimicrobial peptide, characterized in that, The antimicrobial peptide or its pharmaceutical composition comprises the following common amino acid general formula: Ac-Arg-Leu-Leu-B-Z-Leu-Z-B-NH2, or Ac-Arg-Leu-Leu-B-Z-Leu-Z-B-Arg-NH2, or Ac-Arg-Arg-Leu-Leu-B-Z-Leu-Z-B-Arg-Arg-NH2, wherein Ac represents acetylation modification at the amino terminus of the polypeptide, NH2 represents amidation modification at the carboxyl terminus of the polypeptide, B is selected from aromatic amino acid residues, and one or two Zs are selected from arginine residues or lysine residues.

2. The antimicrobial peptide according to claim 1, characterized in that, The aromatic amino acid residues mainly include 3-(2-naphthyl)-alanine residue, phenylalanine residue or other phenylalanine derivative residues.

3. The antimicrobial peptide according to claim 1, characterized in that, One or more Arg residues in the sequence of the antimicrobial peptide are replaced by lysine residues.

4. The antimicrobial peptide according to claim 1, wherein One or more Leu residues in the sequence of the antimicrobial peptide are replaced by norleucine residue, norvaline residue, homoleucine residue, alanine residue or valine residue.

5. The antimicrobial peptide according to any one of claims 1 to 4, characterized in that, The antimicrobial peptide includes all L–type and all D–type enantiomers; or any one or more amino acids of the antimicrobial peptide are replaced by L–type or D–type amino acids.

6. The antimicrobial peptide according to any one of claims 1 to 4, characterized in that, Polyethylene glycol is linked to the amino terminus, carboxyl terminus or the side-chain amino group of the lysine residue of the antimicrobial peptide.

7. The antimicrobial peptide according to claim 6, wherein The molecular weight of the polyethylene glycol is 200 to 4000.

8. The antimicrobial peptide according to claim 1, wherein The sequence of the antimicrobial peptide is shown in the following table: , in Table 1, X represents 3-(2-naphthyl)-alanine residue, X1 represents 4-fluoro-phenylalanine residue, X2 represents 3,4-difluorophenylalanine residue, X3 represents 4-chlorophenylalanine residue, X4 represents 3,4-dichlorophenylalanine residue, X5 represents 4-trifluoromethyl-phenylalanine residue, X6 represents 4-bromophenylalanine residue, X7 represents 4-methyl-phenylalanine residue, X8 represents norleucine residue, X9 represents norvaline residue, X10 represents homoleucine residue, Ac represents acetylation modification at the amino terminus of the polypeptide, NH2 represents amidation modification at the carboxyl terminus of the polypeptide; phenylalanine derivative residues mainly include 4-fluoro-phenylalanine residue, 3,4-difluorophenylalanine residue, 4-chlorophenylalanine residue, 3,4-dichlorophenylalanine residue, 4-trifluoromethyl-phenylalanine residue, 4-bromophenylalanine residue or 4-methyl-phenylalanine residue.

9. A pharmaceutical composition, characterized in that, It includes the antimicrobial peptide according to any one of claims 1 to 8.

10. Use of the antimicrobial peptide according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of a drug for preventing and / or controlling microbial infections.

11. The application according to claim 10, characterized in that, The drug comprises at least one antimicrobial peptide according to any one of claims 1 to 7 or its pharmaceutically acceptable salt, ester, solvate, hydrate or prodrug, and at least one pharmaceutically acceptable carrier, excipient, vehicle, diluent, buffer, adjuvant, coadjuvant or medium.

12. The application according to claim 10, wherein The dosage form of the drug includes injection, oral preparation or topical preparation.

13. The application according to claim 12, wherein The oral preparations include tablets, oral liquids, capsules, granules or traditional Chinese medicine preparations, and the external preparations include ointments, gels, suppositories or aerosols.

14. The application according to any one of claims 10 to 13, characterized in that, The applications include any one of the following: (1) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of anti-inflammatory and / or anti-tumor drugs; (2) The application of the antimicrobial peptide or its pharmaceutical composition in aquaculture; (3) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of oral care products; (4) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of oral cleansing preparations; (5) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of tooth cleaning products; (6) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of dental coatings; (7) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of drugs for preventing and treating dental caries; (8) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of products for treating skin infections; (9) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of intracellular bactericidal products; (10) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of products for removing bacterial biofilms; (11) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of disinfection products; (12) The application of the antimicrobial peptide or its pharmaceutical composition in the prevention and / or treatment of Fusarium head blight of wheat; (13) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of mildew-proof and preservative agents; (14) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of food preservatives; (15) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of cosmetic preservatives; (16) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of cosmetics; (17) The application of the antimicrobial peptide or its pharmaceutical composition in livestock and poultry feeds and / or feed additives; (18) The application of the antimicrobial peptide or its pharmaceutical composition in the improvement of animal and plant varieties and / or animal and plant breeding; (19) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of wound dressings; (20) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of antimicrobial food wraps; (21) The application of the antimicrobial peptide or its pharmaceutical composition in the preservation of fruits and vegetables; (22) The application of the antimicrobial peptide or its pharmaceutical composition in livestock farming; (23) The application of the antimicrobial peptide or its pharmaceutical composition in the preparation of antimicrobial agents, pharmaceuticals, veterinary drugs, feeds, foods, daily chemical products, health products, cosmetics, bactericides, virucides, algaecides, sterilants, feeds, additives, oral cleansing preparations, disinfectants, anti-inflammatory products, cleaning agents, preservatives, excipients, mildew-proof agents, algaecide-proof agents, washing aids, washing compositions, cleaning agents or preservatives.

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