Antibacterial peptides or peptide derivatives, substituents, and their compositions, production methods, and applications

Abstract

The present application provides antimicrobial peptides or peptide derivatives and substitutions comprising at least one of the following amino acid sequences I and II, as well as compositions, production methods and applications thereof. [C1] JPEG2023542830000015.jpg19170where, X a1 , B a1 , U1, Z a1 , B a2 , X a2 , B a3 , Z a2 , B a4 , X a3 , X b1 , B b1 , C a1 , Z b1 , B b2 , X b2 , B b3 , Z b2 , B b4 , X b3 , C a2 are each independently selected from natural amino acids and / or unnatural amino acids, wherein the antimicrobial peptide or peptide derivative provided herein can bind to the lipid structure of cell walls / membranes, damage their physical and chemical properties, destroy microbial walls, and even kill microorganisms and tumor cells, and also have the effects of wound disinfection and anti-infection.

Description

Technical Field

[0001] The present application belongs to the technical field of peptides or peptide derivatives, and particularly relates to antibacterial peptides or peptide derivatives, substituents, and their compositions, manufacturing methods, and applications.

Background Art

[0002] In nature, antibacterial peptides are widely present in living organisms such as bacteria, viruses, fungi, insects, amphibians, animals, and plants. Antibacterial peptides have a very significant effect on killing bacteria, fungi, parasites, and viruses. With the massive use of antibiotics, microorganisms have developed drug resistance, which has driven researchers to conduct large-scale research and development on antibacterial peptides. After endogenous antibacterial peptides in the human body such as defensins were discovered and approved by the FDA (Food and Drug Administration) as drugs for anti-infection, anti-inflammation, and treatment of other injuries, and after it was further discovered that antibacterial peptides have the effect of stimulating the body's innate and adaptive immunity, antibacterial peptides have increasingly shown potential therapeutic advantages for several diseases. In addition to medical drugs, antibacterial peptides are also widely applied in many aspects such as food preservation, poultry and livestock industries, fishery industries, and daily medical health promotion, hygiene and beauty, cleaning and disinfection, and prevention and control of agricultural pests and diseases.

[0003] For example, acne caused by Propionibacterium is caused by the excessive growth of the microbial colony on the skin of normal humans, which is a Gram-positive bacterium, in the hair follicles of the sebaceous glands. Acne caused by Propionibacterium is one of the most common diseases that affect human skin, and about one million Chinese people are affected by this disease. Acne caused by Propionibacterium has different pathological manifestations, such as spines, papules, nodules, cysts, and follicular sebaceous gland inflammation, etc. Antibiotics are often used in the conventional treatment of acne caused by Propionibacterium. For example, oral administration of oxytetracycline, topical application of erythromycin, clindamycin, etc. can be mentioned. However, such treatments will soon lead to antibiotic resistance. In addition, clinically, benzoyl peroxide and 5% aminophenyl sulfone gel are adopted for the treatment of acne caused by Propionibacterium. These two kinds of drugs have low efficacy at low concentrations and large toxicity and side effects at high concentrations.

[0004] As another example, Pseudomonas aeruginosa is due to the invasive infection of Gram-negative bacteria into the human body and animal bodies, which can infect the respiratory, urinary, digestive, central nervous systems of the living body, as well as the blood, heart and skeletal systems. When Pseudomonas aeruginosa infects the eyes and ears, it often causes bacterial keratitis, scleral abscess, endophthalmitis, neonatal ophthalmia and otitis media in children in adults. In particular, Pseudomonas aeruginosa / bacterial keratitis increases the infection rate of using a pupilloscope, and in the United States, about 25,000 pupilloscope wearers encounter infections caused by this bacterium every year. According to the statistics of the Centers for Disease Control and Prevention in the United States, 4 out of every 1000 inpatients may encounter infections of Pseudomonas aeruginosa. Although Pseudomonas aeruginosa is treatable, with the use of antibiotics, the drug resistance of this microorganism improves, rapidly reducing the efficacy of treatment, and the dosing combination and treatment means must be changed. According to the data, the drug resistance of Pseudomonas aeruginosa isolated from intensive care units in the United States to ciprofloxacin is 51.6%, to piperacillin / tazobactam is 31.4%, to imipenem is 38%, and to ceftazidime is 23.6%. The drug resistance of Pseudomonas aeruginosa isolated from European intensive care units to aminoglycosides can reach 37-70%, to ceftazidime is 57%, to piperacillin / tazobactam is 53%, to ciprofloxacin is 56%, and to imipenem is 52%. Therefore, the treatment and control of serious drug-resistant infections caused by Pseudomonas aeruginosa become very important.

[0005] Typical antibacterial peptides reported currently are generally composed of 10-100 amino acid residues, contain many basic amino acids and are generally amphiphilic.

[0006] The host defense peptide PGLa extracted from the secretions of the Ranidae family has been reported to have excellent killing power against Helicobacter pylori (Gram-negative bacteria). There is also the natural antibacterial peptide TP4 isolated from fish, and furthermore, the natural antibacterial peptide LL-37 isolated from the human body also has excellent killing power against Helicobacter pylori. These findings indicate that antibacterial peptides have a particularly high therapeutic effect against Helicobacter pylori, and can be added to daily necessities as bacteriostatic additives, which is advantageous for protecting us from Helicobacter pylori. However, conventional antibacterial peptides have the defects of weak antibacterial activity and most do not have broad-spectrum antibacterial activity.

[0007] In addition, the types of antibacterial peptides isolated from animals and plants are diverse, widely distributed, and have been found in organisms such as insects, fish, mammals, amphibians, and plants. There is still no systematic research on the applicability of antibacterial peptides from different origins. In particular, plant antibacterial peptides are particularly unstable and are easily hydrolyzed by proteolytic enzymes, thereby significantly reducing their antibacterial activity.

Summary of the Invention

Problems to be Solved by the Invention

[0008] The object of the present application is to provide an antibacterial peptide or peptide derivative, a synthesis method, a composition, and an application, aiming to solve the problems that the antibacterial peptides in the prior art have weak antibacterial activity, are unstable, and do not have broad-spectrum antibacterial activity.

Means for Solving the Problems

[0009] To achieve the above object of the present application, the technical solution adopted by the present application is as follows.

[0010] In a first aspect, the present application provides an antibacterial peptide or peptide derivative containing at least one of the following amino acid sequences.

Chemical formula

[0011] The antibacterial peptide or peptide derivative according to the present application can specifically bind to the lipid structure of the cell wall / membrane, destroy the microbial wall, and further kill microorganisms and cancer cells.

[0012] In a second aspect, there is provided a variant of the antibacterial peptide or peptide derivative, in which at least one amino acid of the amino acid sequence I or / and the amino acid sequence II contained in the antibacterial peptide or peptide derivative of the present application is substituted.

[0013] The variant provided by the present application can ensure the activity of the antibacterial peptide or peptide derivative, is included in the content of the present application, and by substituting the amino acids on the antibacterial peptide or peptide derivative, the antibacterial activity of the antibacterial peptide or peptide derivative can be improved and its broad-spectrum antibacterial activity can be increased.

[0014] In a third aspect, the present application provides an antibacterial peptide or peptide derivative composition comprising at least the antibacterial peptide or peptide derivative described in any one of the above paragraphs and at least one pharmaceutically acceptable carrier.

[0015] Regarding the antibacterial composition provided by the third aspect of the present application, the antibacterial peptide or peptide derivative provided by the present application can be mixed with most solvents to produce a composition.

[0016] In a fourth aspect, the present application provides a method for manufacturing an antibacterial composition, including the step of mixing an antibacterial peptide or peptide derivative with a solvent.

[0017] According to the method for manufacturing the antibacterial composition of the present application, on the one hand, the antibacterial peptide or peptide derivative can stably exist in most solvents, and the antibacterial activity of the antibacterial peptide or peptide derivative can be preserved by mixing the solvent with the antibacterial peptide or peptide derivative. On the other hand, the stability of the antibacterial peptide or peptide derivative against peptidase or proteolysis is ensured.

[0018] In a fifth aspect, the present application provides the application of the antibacterial peptide or peptide derivative composition of the present application in packaging, the end of food processing, clothing, medical supplies, medical devices, personal hygiene products, disinfectants, detergents, anti-infective drugs, anti-inflammatory drugs, and drugs for inhibiting the unlimited proliferation of cells.

[0019] The application of the antibacterial peptide or peptide derivative composition of the present application has a wide range of uses mainly because the antibacterial peptide or peptide derivative can kill microorganisms, kill tumor cells, and perform anticoagulant and anti-inflammatory functions, and mainly has four advantages.

[0020] First, the antibacterial peptide or peptide derivative can be combined with or incorporated into packaging, clothing, medical supplies, medical devices, and personal hygiene product materials to endow them with antibacterial functions or serve as preservatives for other materials that are easily decomposed by microorganisms.

[0021] Second, the antibacterial peptide or peptide derivative can be manufactured into disinfectants, detergents, anti-infective drugs, and anti-inflammatory drugs to play the roles of sterilization, anti-infection, and anti-inflammation.

[0022] Third, the antimicrobial peptide or peptide derivative composition is manufactured into a coating, applied in food processing and at the very end, and can reduce the microorganisms on the surface at the very end of food processing, thereby reducing the risk of food poisoning.

[0023] Fourth, it can kill cells and limit proliferation, and is used for anti-tumor treatment, local cosmetic callus and wart removal, etc.

Brief Description of the Drawings

[0024]

Figure 1

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Figure 9

Modes for Carrying Out the Invention

[0025] To make the technical problems, technical solutions and beneficial effects to be solved by this application clearer, the following will refer to the embodiments to explain this application in more detail. It should be understood that the specific embodiments described herein are only for explaining this application and do not limit this application.

[0026] In this application, the term "and / or" describes the relationship of related objects and indicates that there may be three types of relationships. For example, A and / or B can indicate the cases where A exists alone, A and B exist simultaneously, and B exists alone. Here, A and B may be singular or plural. The character " / " generally indicates that the related objects before and after are in an "or" relationship.

[0027] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following items (pieces)" or similar expressions mean any combination of these items, including any combination of single items (pieces) or multiple items (pieces). For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all represent a, b, c, a-b (that is, a and b), a-c, b-c, or a-b-c, where a, b, and c may be single or plural respectively.

[0028] It should be understood that in various embodiments of this application, the magnitude of the numbers of the above processes does not mean the order of execution before and after. Some or all of the steps can be executed in parallel or before and after. The execution order of each process should be determined by its function and inherent logic, and the implementation process of the embodiments of this application is not limited in any way.

[0029] The terms used in the embodiments of the present application are for the purpose of describing specific embodiments only and are not intended to limit the present application. In the context, unless clearly indicated otherwise, the singular forms "a kind", "the above", and "the" used in the embodiments of the present application and the appended claims are also intended to include the plural forms.

[0030] The weights of the related components of the embodiments of the present application mentioned in the specification not only refer to the specific contents of each component, but also can represent the proportional relationship of the weights among the components. Therefore, whether it is enlarged or reduced proportionally according to the contents of the related components described in the specification of the embodiments of the present application, it is within the scope disclosed in the specification of the embodiments of the present application. Specifically, the masses described in the specification of the embodiments of the present application may be mass units known in the chemical industry field such as μg, mg, g, kg, etc.

[0031] First, the terms "first" and "second" are only used to explain the object, for example, to distinguish substances from each other, and it cannot be understood that they indicate or imply relative importance or implicitly indicate the quantity of the indicated technical features. For example, without departing from the scope of the embodiments of the present application, the 1XX may be called the 2XX, and similarly, the 2XX may be called the 1XX. Thus, the features limited by "first" and "second" can explicitly or implicitly include one or more of such features.

[0032] In a first aspect, the embodiments of the present application provide an antibacterial peptide or a peptide derivative, and the general representation of its amino acid sequence is as follows.

Chemical formula

[0033] In the examples, based on the above examples, in order to improve the antibacterial activity of the antibacterial peptide or peptide derivative, X a1 , X a2 , X a3 , X b1 , X b2 , X b3 are each independently selected from residues with a positive charge, or / and B a1 , B a2 , B a3 , B a4 , B b1 , B b2 , B b3 , B b4 are each independently selected from residues with a hydrophobic side chain, or / and U1 is one selected from Gly (G-glycine), Pro (P-proline), Cys (C-cysteine), Cys(R), where R represents a protecting group for the Cys disulfide bond, or / and Z a1 , Z a2 , Z b1 , Z b2 are each independently selected from non-polar residues, or / and C a1 , C a2 are each independently one selected from Cys (C-cysteine), Cys(R). In amino acid sequence I, X a1 or X a3 represents the N-terminus, and X a1 or X a3 represents the C-terminus. In amino acid sequence II, X b1and / or B2 represents the N-terminus, and C a2 represents the C-terminus. Since the antibacterial peptides and peptide derivatives of the present application contain residues of hydrophobic side chains, residues of positive charges, or residues of non-polarity, the antibacterial peptide or peptide derivative chain provided in this example can form a typical amphipathic helical structure. Due to the charge and amphipathic helical structure of the antibacterial peptide or peptide derivative, it can bind to the lipid structure of the cell wall / membrane, destroy the microbial wall or cell membrane, and further kill microorganisms and tumor cells.

[0034] In the examples, the general representation of its amino acid sequence is as follows.

Chemical formula

[0035] In the examples, the general representation of its amino acid sequence is as follows.

Chemical formula

[0036] In the examples, the general representation of the amino acid sequence is as follows.

Chemical formula

[0037] In the examples, the general representation of the amino acid sequence is as follows.

Chemical formula

[0038] In the examples, based on the above examples, to improve the stability of the antibacterial peptide or peptide derivative, X a1 X a2 X a3 X b1 X b2 X b3 B b4 are each independently one, two, or three selected from Arg (R-arginine), His (H-histidine), Lys (K-lysine), Orn (ornithine), Har (homoarginine), Dab (2,4-diaminopropionic acid), and non-natural amino acids. On the one hand, Arg (R-arginine), His (H-histidine), Lys (K-lysine), Orn (ornithine), Har (homoarginine), Dab (2,4-diaminopropionic acid), and non-natural amino acids are residues with positive charges. Looking at the overall structure, they can form an amphipathic helical structure together with other residues, and can increase the antibacterial activity of the antibacterial peptide or peptide derivative. On the other hand, if the residues with positive charges in the antibacterial peptide chain or peptide derivative are too long, the charge of the antibacterial peptide or peptide derivative will become unbalanced and it will be prone to instability. Therefore, to ensure the stability of the peptide or peptide derivative against peptidase or proteolysis, the number of residues with positive charges is one, two, or three, or / and B a1 B a2 B a3, B a4 , B b1 , B b2 , B b3 are each independently two, three, or four selected from Ala (A - alanine), Val (V - valine), Ile (I - isoleucine), Leu (L - leucine), Met (M - methionine), and non - natural amino acids. Ala (A - alanine), Val (V - valine), Ile (I - isoleucine), Leu (L - leucine), Met (M - methionine), and non - natural amino acids have hydrophobic side chains. The hydrophobic side chains can balance the water solubility and charge property of the peptide chain. That is, if the hydrophobic side chain residues in the peptide chain or peptide derivative are too long, the peptide chain is likely to become unstable. Therefore, in order to ensure the stability of the peptide chain against peptidase or proteolysis, the number of hydrophobic side chain residues is one of the situations of two, three, or four, or / and Z a1 , Z a2 , Z b1 , Z b2 are each independently one selected from Asn (N - asparagine), Gln (Q - glutamine), Ser (S - serine), Thr (T - threonine), and non - natural amino acids, which are selected from non - polar residues and can balance the water solubility and charge property of the peptide chain. That is, if the non - polar residues in the peptide chain or peptide derivative are too long, the peptide chain is likely to become unstable. Therefore, in order to ensure the stability of the antibacterial peptide or peptide derivative against peptidase or proteolysis, the number of non - polar residues is one of the situations of two, three, or four, or / and X a1 and B a1 , X a2 and B a2 , X a3 and B a3 , X b1 and B b1 , X b2 and B b2 , X b3 and B b3 , X b4 and B b4Ensure that the sum of the number of residues is 3, 4, 5, 6, or 7, preferably 3 or 4. For the activity of the antibacterial peptide or peptide derivative of the present application, the longer the peptide chain length, the better. However, considering the synthesis cost and manufacturing cost, and to ensure the stability of the peptide chain, X a1 and B a1 、X a2 and B a2 、X a3 and B a3 、X b1 and B b1 、X b2 and B b2 、X b3 and B b3 、X b4 and B b4 The preferred number of the sum of the number of residues is 3 and 4. The amino acid sequence and / or some amino acid sequences of the antibacterial peptide of the present application are derived from 8 to 25 amino acids of the virus.

[0039] In the examples, the general representation of the amino acid sequence is as follows.

Chemical formula

[0040] In the examples, the general representation of its amino acid sequence is as follows.

Chemical formula

[0041] In the examples, the general representation of its amino acid sequence is as follows.

Chemical formula

[0042] In some embodiments, the Cys disulfide bond contains acetamidomethyl (Acm), methyl methane thiosulfonate, or other Cys protecting groups, and Cys(R) represents a protecting group containing a Cys disulfide bond, where C a2 is the terminal group of the antibacterial peptide or peptide derivative, and C a1It is located at an intermediate position of the antibacterial peptide or peptide derivative, and thus can form a cyclic structure inside the molecule of the antibacterial peptide or peptide derivative, thereby improving the stability of the antibacterial peptide and extending the bactericidal time of the antibacterial peptide. The antibacterial peptide or peptide derivative has higher antibacterial activity.

[0043] In some embodiments, the amino acid sequence I and / or II includes that at least one amino acid has been modified. In actual applications, the stability and antibacterial activity of the antibacterial peptide or peptide derivative are very important. By modifying the amino acids on the antibacterial peptide or peptide derivative of the present application, the performance of the peptide chain can be changed, the broad-spectrum property of the antibacterial peptide or peptide derivative can be improved, the stability of the antibacterial peptide or peptide derivative against peptidase or proteolysis can be improved, and its broad-spectrum antibacterial activity can be increased.

[0044] In the embodiments, the modification includes phosphorylation, halogenation, acetylation, cyclization, and an endpoint capping reaction. Here, the cyclization includes at least one of cyclization formed by a disulfide bond, head-to-tail cyclization, and cyclization of side groups in the internal structure. The capping reaction of the amino acid endpoint includes at least one of C-terminal amidation, N-terminal acetylation, and N-terminal lipidation. By performing a modification treatment such as cyclization on the amino acid, the stability of the peptide chain can be improved, and by performing a phosphorylation treatment on the amino acid, for example, the antibacterial activity of the antibacterial peptide or peptide derivative can be improved.

[0045] In some embodiments, the amino acid sequence I and / or II contains at least one motif, is always connected to a predetermined amino acid endpoint sequence (N-terminus and / or C-terminus), and may be directly inserted in the middle of a predetermined amino acid sequence. Generally, it is not an amino acid sequence endpoint independently. The connection between the motif and the peptide chain or the insertion between amino acids can change the performance of the peptide chain when the motif is inserted into the antibacterial peptide or peptide derivative, and can increase the antibacterial activity of the peptide chain and the stability of the antibacterial peptide or peptide derivative against peptidase or proteolysis.

[0046] In some embodiments, the antibacterial peptide or peptide derivative is a multimer formed by including the amino acid sequence I and / or the amino acid sequence II. Here, at least one of a dimer and a tetramer is in a general multimer state. The antibacterial peptide monomer and / or multimer is conjugated with at least one drug or antibody to form an antibacterial drug, mainly including small molecule chemically synthesized drugs, macromolecular antibodies, and macromolecular drugs synthesized by biological recombinant technology. The multimer formed by the antibacterial peptide or peptide derivative of the present application is conjugated with a drug or antibody molecule to form an antibacterial drug and can be used for the modification of drugs or antibodies.

[0047] In some embodiments, the antibacterial peptide or peptide derivative contains the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 39.

[0048] In some embodiments, it is a nanostructure of an antibacterial peptide or peptide derivative. A single peptide chain and its peptide derivative, or a plurality or multiple peptide chains and peptide derivatives can all form nanostructures. Here, the general nanostructure is at least one selected from micelles, vesicles, nanotubes, and nanobelts. The peptide chain and its peptide derivative form a nanostructure, which helps the peptide chain and its peptide derivative to exist stably in the solution and to be transmitted in the solution.

[0049] In a second aspect, the present application provides a substitution of an antibacterial peptide or a peptide derivative, and obtaining a substitution by substituting an amino acid in any of the amino acid sequences I or II in the above paragraphs is also included in the content of the present application. For example, substituting an L-amino acid with a corresponding D-amino acid or an unnatural amino acid can be mentioned. By substituting the amino acid on the antibacterial peptide or peptide derivative, the antibacterial activity of the antibacterial peptide or peptide derivative can be improved, and its broad-spectrum antibacterial activity can be increased.

[0050] In a third aspect, the present application provides an antibacterial peptide or peptide derivative composition comprising an antibacterial peptide or peptide derivative and / or a substitution and at least one pharmaceutically acceptable carrier and / or solvent. The antibacterial peptide or peptide derivative provided by the present application can combine with a pharmaceutically acceptable carrier for most drugs to form a pharmaceutical composition. Similarly, it can be mixed with most solvents to be manufactured into a composition.

[0051] In some embodiments, further comprising at least one of an excipient, an isotonic agent, an absorption retardant or a base polymer, and by mixing with these substances, the antibacterial peptide or peptide derivative can be manufactured into various antibacterial drugs or antibacterial solvents.

[0052] In some embodiments, the excipient is at least one selected from water, salts, phosphates, glucose, glycerin, ethanol, and these are all common medical reagents. The antibacterial peptide or derivative can be mixed with these reagents, which is helpful for the popularization and use of the antibacterial peptides and derivatives of the present application.

[0053] In some embodiments, the isotonic agent is selected from sugars, polyols and sodium chloride. The isotonic agent can retain the peptide activity, and by mixing the antibacterial peptide and derivative with the isotonic agent, the antibacterial peptide or peptide derivative is more likely to bind to the living body.

[0054] In some embodiments, the polyol is selected from mannitol and sorbitol. By mixing the polyol with an antibacterial peptide or peptide derivative, the polyol can retain the activity of the antibacterial peptide or derivative, and the antibacterial peptide or derivative is more likely to bind to the living body.

[0055] Here, the pharmaceutically acceptable carrier is selected from a minimum amount of auxiliary substances, the pharmaceutically acceptable carrier is selected from wetting agents, emulsifiers, preservatives, and buffers. The pharmaceutically acceptable carrier and the antibacterial peptide or peptide derivative form a composition, which can retain the activity of the antibacterial peptide or derivative, and the antibacterial peptide or derivative is more likely to bind to the living body.

[0056] In an embodiment, the antibacterial peptide or peptide derivative binds to a base polymer and is prepared in an aqueous or non-aqueous solution, and these aqueous or non-aqueous solutions can be applied to the surface of an object to form an antibacterial protective layer.

[0057] Also, the above substances may be composed of various forms or various components, including but not limited to extracts, sheets, films, laminates, knitted fabrics, woven fabrics, non-woven fabrics, fibers, filaments, threads, particles, coatings, and / or foams. This application also includes various molded products made of the above substances and antibacterial peptides, including but not limited to injection molding, extrusion molding, blow molding, thermoforming, solution coating, blow film, weaving, knitting, and textile products.

[0058] In a fourth aspect, this application provides a method for producing an antibacterial peptide or peptide derivative composition, which includes the step of mixing and treating the antibacterial peptide or peptide derivative with the solvent.

[0059] In an embodiment, when preparing a mixture of an antibacterial peptide or peptide derivative and a base polymer, to ensure sufficient mixing, the mixing temperature is controlled in the range of -10 to 150 °C, and the mixing time is controlled in the range of 0.1 to 5760 min. Here, the mixing temperature is in the range of 25 to 80 °C, and the mixing time is in the range of 1 to 1440 min, which has a better mixing effect.

[0060] In some embodiments, it further includes pretreating a pharmaceutically acceptable carrier, excipient, solvent, dispersion medium, isotonic agent, absorption delaying agent or base polymer, wherein the pretreatment includes but is not limited to at least one of oxidation, reduction, hydrolysis, plasma or radiation, and the pretreatment can improve the environment of the antimicrobial peptide or peptide derivative, and further improve the stability of the antimicrobial peptide or peptide derivative against protease or proteolysis.

[0061] In some embodiments, the effective concentration range of the mixture of the antimicrobial peptide or peptide derivative and the above substances is between 1 μg and 20 gwt%, or between a concentration of 5 pmol and 2 mol. In order to store the antimicrobial peptide or peptide derivative composition, it is necessary to control the temperature in the range of -20 to 25 °C to ensure the antibacterial activity of the antimicrobial peptide or peptide derivative. Also, when the temperature is controlled in the range of -4 to 4 °C, the biological activity characteristics of the antimicrobial peptide can be ensured only at lower temperatures.

[0062] In a fifth aspect, the present application provides the application of any of the following antimicrobial peptide or peptide derivative compositions.

[0063] It is a packaging, the end of food processing, clothing, medical supplies, medical devices, personal hygiene products, disinfectants, detergents, anti-infective drugs, anti-inflammatory drugs, and drugs for inhibiting the unlimited proliferation of cells. The antimicrobial peptide or peptide derivative composition can be widely used as a drug or non-drug for humans, animals (including wild animals, livestock, companion animals), plants (including crops), aquatic and aquaculture animals, poultry farming animals, etc. Hereinafter, the concept definitions of humans and animals and plants refer to the concept definitions including the humans, animals (including wild animals, livestock, partner animals), plants (including crops), aquatic and aquaculture animals, poultry farming animals, etc. mentioned above, and the same applies hereinafter.

[0064] In some embodiments, in the application of the compositions described in the above paragraphs to packaging, the antimicrobial peptide or peptide derivative can be bound to or incorporated into the packaging material, or used as a preservative for other materials that are easily decomposed by microorganisms. Here, the packaging components include, but are not limited to, packaging films, pads, absorbent pads, trays, container parts, caps, covers, adhesives, applicators, etc. for meat products. Such packaging can be in any packaging form suitable for special applications, such as aluminum tanks, cases, bottles, glass cans, bags, cosmetic packaging, sealed tubes, etc. The above packaging includes, but is not limited to, packaging molded articles manufactured by processes such as injection molding, extrusion molding, blow molding, thermoforming, solution coating, blow film, etc.

[0065] Here, the packaging products are further applied to the packaging of prescription / non-prescription drugs and health promotion and hygiene products, such as bottles, tips, applicators, caps, etc. manufactured for capsules, tablets, solutions, emulsifiers, detergents, powders, shampoos, hair care products, deodorants, antiperspirants, etc. The packaging products are further applied to the packaging of applicators such as lipsticks, lip creams, and makeup setting oils, eye makeup packaging such as mascaras, eyeliners, and eyeshadows, spray powders, bath powders, blushers, foundations, and lotions. This applicator is used on different parts and surfaces of the organism, and such applications significantly reduce or eliminate the growth of bacteria on the human body surface. It also includes further other forms of packaging configurations, such as drinking bottle necks for containing liquids, solutions or suspensions, replaceable covers, non-replaceable covers, food or drug dispensing systems, food and beverage transportation systems, baby feeding bottles, covers, nipples, etc. The packaging also includes the use of gel droplet dispersants or droplet injectors.

[0066] In some embodiments, it is used for food processing and end - use applications of the compositions described in the above paragraph, or for temporarily or permanently preparing coatings on food surfaces. For example, food additives, food processing equipment, food conveyor belt assemblies and components that replace antibiotics, assemblies and components of different equipment for mixing, grinding, crushing, tumbling, granulating, and pressing food, and equipment assemblies and components for cutting and slicing food. If the surface of the above - mentioned equipment is a metallic substance, it is necessary to apply a layer of a polymer having functionality to the metal surface, and the polymer is a polymer containing the antibacterial peptide or peptide derivative described in the present application. The antibacterial peptide or peptide derivative composition is prepared for coating and applied at the food processing and end - use, which can reduce the microorganisms on the end - surface of food processing and reduce the risk of food poisoning.

[0067] In some embodiments, it is an application of the compositions described in the above paragraph in clothing. Such clothing has bactericidal and bacteriostatic functions, and examples include swimsuits, underwear, shoe components (such as knitted or non - knitted liners or shoe pads), sports protection pads, children's clothing, etc. The present application also includes protective medical clothing or isolation supplies, such as protective clothing, masks, gloves, slippers, boots, head covers or curtains, etc.

[0068] In some embodiments, there is an application of the composition described in the above paragraph in medical supplies and devices, where the antimicrobial peptide or peptide derivative can be combined with or incorporated into the medical supplies and materials, or an antimicrobial peptide or peptide derivative coating can be further applied to the surface of the medical device, or manufactured by spraying to sterilize and disinfect the medical supplies and devices. Here, the medical supplies and devices include human and animal implants, such as bandages, adhesives, gauze, gauze pads, syringe stents, peripatetic or central venous cannulas composed of polyurethane or silicone rubber, urethral fistula ports, orthopedic correctors, correction needles, pacemaker leads, defibrillator leads, external ear shunts, vascular stents, plastic implants, otolaryngological implants, implantable pumps, hernia patches, and related dishes, screws, blood bags, external blood pumps, infusion systems, cardiopulmonary devices, dialysis devices, artificial skin, artificial hearts, ventricular assist devices, hearing aids, vascular grafts, pacemaker components, hip implants, knee implants, dental implants, etc.

[0069] In some embodiments, there is an application of the composition described in the above paragraph in personal hygiene products, where the antimicrobial peptide or peptide derivative can be combined with or incorporated into the personal hygiene product materials, and further the personal hygiene products have a bactericidal and bacteriostatic effect, such as diapers, incontinence pads, sanitary napkins, sports pads, sanitary tampons, and the accessory devices for applying them. In terms of health promotion products, antimicrobial wet tissues, wet tissues for infants, personal wiping wet tissues, cosmetic wet tissues, diapers, medical wet tissues (such as wet tissues or cotton pads containing antibiotics, acne treatment drugs, hemorrhoid treatment drugs, antipruritic drugs, anti-inflammatory drugs, and preservatives, etc.) are included.

[0070] In some embodiments, it is an application in an article that directly contacts the oral cavity of the composition described in the above paragraph. The antimicrobial peptide or peptide derivative is non-toxic to the human body and can be disinfected. Therefore, it can directly contact the oral cavity and achieve the purpose of oral sterilization and bacteriostasis. Here, the articles that directly contact the oral cavity include preventing skin infections in infants, such as baby bottles, nipples, dental instruments, stretch tapes, dentures, cups, water cups, toothpaste, and tooth hardening toys. Applications mainly aimed at preventing microbial infections in infant articles are also within this scope of protection, such as baby bottles, infant books, plastic scissors, toys, diaper pails, and containers for holding clean wet tissues.

[0071] In some embodiments, it is the use of the composition described in the above paragraph in household items. In order to prevent bacteria from entering through the mouth, the antimicrobial peptide or peptide derivative can be combined with or mixed into household item materials. Here, household items include telephones, mobile phones, fiber fillings, beddings, window treatments, carpet floor cleaning treatments, foam pads on the back of foot pads or carpet pads, interior decorations (such as foam pads), non-woven drying papers, papers containing softeners, automotive wiping wet tissues, household cleaning wet tissues, table wet tissues, bath curtains, bath curtain fabrics, towels, face towels, rags, mops, tablecloths, walls, and countertops, etc.

[0072] In some embodiments, it is an application in a disinfectant of the composition described in the above paragraph. In order to avoid cross-infection, the antimicrobial peptide or peptide derivative of the present application can be prepared into a disinfectant. The environments where the disinfectant is used include air disinfection in closed spaces, airplanes, trains, cinemas, theaters, etc. For example, disinfection of medical endoscopes, etc. can be mentioned. The antimicrobial peptide or antimicrobial peptide derivative can reduce or prevent the formation of biofilms on the surface of independent membranes. These independent membranes include pervaporation membranes, dialysis membranes, reverse osmosis membranes, ultrafiltration membranes, microfiltration membranes, etc.

[0073] In some embodiments, it is an application of the composition described in the above paragraph in a detergent, such that such a detergent has a bactericidal effect and needs to be washed with water after use. Preferably, it is washed with deionized water. Optionally, the washed article can be dried, and the drying methods can include ambient air drying, oven drying, and forced air drying. The drying temperature is from 50°C to 120°C, preferably the drying temperature is from 50°C to 100°C, and the drying time is from about 15 min to 24 h.

[0074] Also, the treatment of all the above articles with the antimicrobial peptide or antimicrobial peptide composition includes the entire process before industrial production, after industrial production, and during industrial production. For example, when manufacturing an antibacterial shower curtain, first the antimicrobial peptide is combined with a base polymer, and then the curtain is treated with the mixture. The manufacturing process includes at least one of injection molding, extrusion molding, blow molding, thermoforming, solution coating, and blown film, but is not limited thereto.

[0075] In some embodiments, it is an application of the composition described in the above paragraph in an anti-infective agent and an anti-inflammatory agent, which sterilizes to prevent infection, and the infection includes those caused by bacteria, viruses, and fungi. For example, it includes those caused by yeast, pathogens, gram-negative bacteria and / or gram-positive bacteria, single-celled or multi-celled organisms, and may be caused by symbiotic or non-symbiotic organisms, pathogenic organisms, colonies or non-colony bacteria. Specifically, the antimicrobial peptide or antimicrobial peptide derivative can treat or reduce skin infections caused by bacteria, viruses, and fungi, and such infections can be any part related to the human or animal integumentary system. For example, the epidermis, dermis, subcutaneous tissue, one or more hair follicles, one or more sebaceous glands, or other sites related to the skin. In other words, the above infections are related to the skin, and further, the infection may be related to a part of vision or a part of hearing. For example, it may be a part of the ear, such as the tympanic cavity or a part of the hearing system including the tympanic cavity.

[0076] In the examples, the anti-infective agent is used to treat infections caused by one or more kinds of bacteria in humans or animals, and the antibacterial peptide can treat the excessive increase of one or more kinds of symbiotic organisms, and such symbiotic organisms may be non-pathogens or symbiotic organisms beneficial to the human body or animals. Some of these symbiotic organisms may be derived from staphylococci, mycobacteria, and propionic acid bacteria. Also, other bacterial infections may be derived from known pathogens, such as the genus Pseudomonas.

[0077] In the examples, the anti-infective agent can be used to treat infections caused by non-bacterial microorganisms, such as treating infections caused by one or more kinds of fungi (such as yeast). For example, the antibacterial peptide can treat infections caused by Malassezia. Malassezia is a symbiotic organism, and its excessive growth causes dandruff, seborrheic dermatitis, tinea, folliculitis, etc., and for its treatment, growth control, removal of related inflammation, and control of secondary infections are always adopted. The growth of Malassezia is stubborn and difficult to treat, and the treatment cycle is long and requires the combined action of many drugs. Therefore, the drugs used in the current market are not only highly toxic and have large side effects but also extremely expensive. The antibacterial peptide of the present application can treat infections caused by fungi such as Malassezia within an effective treatment concentration range.

[0078] In the examples, the anti-inflammatory agent is used to treat inflammation caused by infection, non-infection, and physical trauma, and the antibacterial peptide or antibacterial peptide derivative has been shown to have the effects of reducing, alleviating, and treating inflammation in experiments. For example, in cells, bacteria, and animals, the above-mentioned combined peptide has been shown to reduce the release of inflammatory factors, such as cytokinesis, chemokines, and their analogs.

[0079] Moreover, such inflammation is a physiologically defined inflammatory reaction, including erythema, swelling, induction of one or more inflammatory cytokines at the cellular and molecular transcription and translation levels, induction of one or more cell signaling pathways related to inflammation, induction of receptors present on the cell membrane, cell infiltration present in vascularized tissues, and other manifestation symptoms related to inflammation within the technical scope of this field.

[0080] In some embodiments, the application of the composition described in the above paragraph in a cell growth inhibition agent, an anti-tumor effect, where the non-infinite growth cells (In Vitro within the cell line and In Vivo in vivo) include cells constructed by humans, cells in a diseased state, such as cancer cells, etc. Such non-immortal cells also include primary cultured cells, such as keratinocytes, microvascular endothelial cells, corneal epithelial cells, and dermal fibroblasts. The antibacterial peptide composition results in a decrease in cell membrane receptor - pattern-recognition receptors for specific bacterial binding. As a result of the above reactions, animals and parts of animals show an obvious anti-inflammatory effect, for example, tissue swelling decreases or disappears, and cell infiltration decreases or disappears, etc.

[0081] In some embodiments, the above inflammatory cytokines include cytokines related to inflammation, including but not limited to tumor necrosis factor, interleukin 8, interleukin 1, and interleukin 6. Also, the intracellular signaling pathways include signaling pathways related to inflammation, including but not limited to NFkB, AP-1. "Cell membrane-related receptors" include but are not limited to pattern recognition receptors, the TLR receptor family (including TLR-2 and TLR4).

[0082] In an embodiment, the therapeutic concentration and treatment time of the antibacterial peptide depend on many factors, such as the disease state, age, gender, weight, and individual physical condition. The antibacterial peptide is applied to stem cell therapy, and its effective concentration is greater than 0.01 ug / ml.

[0083] In an embodiment, the effective concentration is 0.2 ug / ml or more.

[0084] In the examples, the anti-infective agent, anti-inflammatory agent, and cell growth inhibition agent include at least one of a liquid, semi-solidified liquid, cream-like solid, ointment, and gel. For example, the topical dosage form can be used on the skin, hair, and other topical parts. Further, under certain conditions, the topical dosage form can be used for one or both eyes and can also be prepared as eye drops.

[0085] In the examples, the antibacterial peptide or antibacterial peptide derivative may be prepared in an intravenous dosage form or an intratympanic / intertympanic administration dosage form, and the solvent may be a sterilized isotonic aqueous solution.

[0086] Also, the antibacterial peptide dosage form used in the medicine is consistent with the administration route, and the administration route includes gastrointestinal administration, intravenous injection, subcutaneous, intradermal, oral, intranasal (inhalation), vaginal, anal, epidermal, mucosal administration, intertympanic, intratympanic, rectal administration, and other acceptable administration methods. The antibacterial peptide preparation is consistent with the administration route according to the pharmaceutical components. For example, it is administered to humans and / or animals, such as livestock and companion animals, etc., by administration methods such as intravenous injection, subcutaneous, intramuscular, ganglionic, oral, nasal, inner, inner ear, and subepidermal, etc., and can also be applied to aquatic animals, poultry, etc.

[0087] Also, the definition of the amino acid represented by one alphabet in the application is consistent with the international amino acid code and the representative sequence standard (IUPAC-IYUB) and is as shown in Table 1.

[0088]

Table 1

Examples

[0089] The following will be described with reference to specific examples. Examples 1 - 39 Examples 1-39 were synthesized by standard internationally known polypeptide solid-phase synthesis, solution-phase synthesis, or recombinant biosynthesis, and the synthesis results are as shown in the amino acid sequence listing shown in Table 2.

[0090] [Table 2-1] [Table 2-2]

[0091] Performance Test and Result Analysis (1) Escherichia coli Inhibition Experiment An Escherichia coli inhibition experiment was conducted on 39 antibacterial peptides in Polypeptide Synthesis Examples 1-39. 30 μl of an equal amount of Escherichia coli liquid was taken, and 30 μl of various antibacterial peptides (concentration 200 μg / ml) was added. The control group used 30 μl of PBS isotonic solution and reacted at room temperature for 15 min. Then, it was applied to an agarose plate. After uniform application, it was cultured at 37°C for 12 h, and the number of Escherichia coli on each plate was counted and recorded. The experimental results are as shown in Figure 1. According to the experimental results, antibacterial peptides SEQ ID NO:1 to SEQ ID NO:39 all have an inhibitory effect on Escherichia coli, and among them, the effect of SEQ ID NO:8 is the most significant. To make the experimental results clearer, six experimental samples were selected for control analysis. As shown in Figure 2, A is the control tube, consisting of 251 Escherichia coli clones. B is SEQ ID NO:5, consisting of 236 Escherichia coli clones. D is SEQ ID NO:6, consisting of 226 Escherichia coli clones. F is SEQ ID NO:7, consisting of 216 Escherichia coli clones. C is SEQ ID NO:8, consisting of 1 Escherichia coli clone. E is SEQ ID NO:35, consisting of 95. It has been shown that multiple polypeptides have the effect of killing Gram-negative bacteria. Also, it has been shown that there is 1 of SEQ ID NO:8, and the amino acid sequence of the optimal antibacterial peptide is SEQ ID NO:8, and the MIC of Escherichia coli is 32 ug / ml.

[0092] (2) Inhibitory Ring Experiment on Staphylococcus aureus Take a liquid of clinically isolated wild Staphylococcus aureus (Staphylococcus aureus is a Gram-positive bacterium) and spread it on an agarose plate. Place it in an Oxford cup for inoculation, and add 200 ul of the antibacterial peptide of SEQ ID NO:8 (the concentrations are 1 / 4: 375 ug / ml, 1 / 16: 93.7 ug / ml, 1 / 64: 23.34 ug / ml respectively). Incubate at 37°C for 18 h and measure the size of the bacteriostatic ring. The experimental results are as follows. 1 / 4: The bacteriostatic ring is 3.95 cm. 1 / 16: The bacteriostatic ring is 3.5 cm. 1 / 64: The bacteriostatic ring is 3.1 cm. Refer to Figure 3. According to the experimental results, as the concentration of the antibacterial peptide decreases, the bacteriostatic ring becomes smaller. Also, compared with Escherichia coli, the antibacterial peptide is more sensitive to Staphylococcus aureus, and the antibacterial peptide of the present application has excellent anti-Gram-positive bacterium properties.

[0093] (3) MIC Measurement of Pseudomonas aeruginosa (Pseudomonas aeruginosa ATCC 27853) Take the antibacterial peptide of SEQ ID NO:8 with concentrations of 1024 ug / ml, 512 ug / ml, 256 ug / ml, 128 ug / ml, 64 ug / ml, 32 ug / ml, 16 ug / ml, 8 ug / ml, 4 ug / ml, and 2 ug / m, set positive and negative controls, mix them with standard Pseudomonas aeruginosa respectively, then add them to 12 wells of a culture plate respectively, and number the 12 wells of the culture plate. Among them, as shown in Figure 4, 1 corresponds to 1024 ug / ml, 2 corresponds to 512 ug / ml, 3 corresponds to 256 ug / ml, 4 corresponds to 128 ug / ml, 5 corresponds to 64 ug / ml, 6 corresponds to 32 ug / ml, 7 corresponds to 16 ug / ml, 8 corresponds to 8 ug / ml, 9 corresponds to 4 ug / ml, 10 corresponds to 2 ug / m, 11 corresponds to the positive control, 12 corresponds to the negative control. After culturing at 37°C for 2 days, observe the turbidity degree of the culture plate. The experimental results are as shown in Figure 4. According to the experimental results, it is shown that the antibacterial peptide has a high inhibitory effect on Pseudomonas aeruginosa, and the MIC against Pseudomonas aeruginosa is 64 ug / ml.

[0094] (4) Inhibitory effect on oral bacteria Take the mouthwash for the elderly, divide it into 4 equal parts, mix each part with different amounts of 2 mg / ml of SEQ ID NO:8 for 30 min, and then uniformly apply it to a plate containing agarose sheep blood. Here, mix with A: PBS (control), B: 2 ul, C: 10 ul, and D: 20 ul. After anaerobic culture at 37°C for 4 days, observe the number of colonies on the plate. The experimental results are as shown in Figure 5. Specifically, there is growth of Candida albicans and anaerobic bacteria on the control plate, there is a small amount of bacterial growth on the antibacterial peptide plate of B: 2 ul, no growth of any oral bacteria is seen on the antibacterial peptide of C: 10 ul, and no growth of oral bacteria is seen on the bacteria of D: 20 ul either.

[0095] (5) Experiment to kill RNA virus with antibacterial peptide Transfect 293T cells with plasmid pNL4.3Δ, pVSV-G, p-enhancer and lipo2000, culture at 37°C for two days, then collect the culture supernatant and centrifuge to remove organelle impurities. Mix 500 μl of the cell supernatant with an equal volume of 200 μg / ml of SEQ ID NO:8 and react for 15 min. In the control group, an equal volume of PBS is used to replace SEQ ID NO:8. Remove the antimicrobial peptide by centrifugation method (Millipore Amicon Ultra-15) to remove the killing effect of the antimicrobial peptide on cells. Infect the sample to be measured with the 293T cell line, culture at 37°C for 12 h, recover the 293T cell line, and measure the difference in live virus of infectable cells by RT-PCR method. Here, the various experimental environments of the control group and the experimental group are the same, and the difference is that the amino acid of the SEQ ID NO:8 sequence of the present application is added to the experimental group. As a result, the copy of the active virus gene in the control group was 1.7×10 7 and the copy of the active virus in the antimicrobial peptide group of the experimental group was 5×10 5 It was shown that the antimicrobial peptide of the present application has the effect of killing obvious viruses.

[0096] (6) Experiment on killing tumor cells with antimicrobial peptides Culture K562 cells, react equal amounts of K562 cells with 10 ng, 20 ng, and 40 ng of SEQ ID NO:8 respectively, then measure apoptotic cells with a flow cytometry PI kit, and find that cell death is directly proportional to the antimicrobial peptide content, indicating that the antimicrobial peptide can effectively kill K562 cells.

[0097] (7) Experiment on killing leukemia lymphocytes with antimicrobial peptides Jurkat cell line: leukemia lymphocytes, 1 μg / m of the polypeptide of SEQ ID NO:10 in 20 μl for 10 5React with cells for 10 min, centrifuge at 1000 rpm for 3 min, and as shown in Figure 6, the experimental results show that leukemia lymphocytes aggregate into clumps and are accompanied by cell fragmentation.

[0098] (8) Experiment on killing leukemia cells with antibacterial peptides K562 cell line: leukemia cells, 20 μl of 1 μg / ml antibacterial peptide of SEQ ID NO: 17 was added to 10 5 React with cells for 10 min, centrifuge at 1000 rpm for 3 min, and as shown in Figure 7, the results show that leukemia cells aggregate into clumps.

[0099] (9) Experiment on killing liver cancer cells with antibacterial peptides HypG2 cell line: liver cancer cells, 20 μl of 1 μg / ml polypeptide of SEQ ID NO: 15 was added to 10 5 React with cells for 10 min, centrifuge at 1000 rpm for 3 min, and as shown in Figure 8, the results show that liver cancer cells aggregate into clumps and are crushed and deformed.

[0100] (10) Experiment on the effect of antibacterial peptides on red blood cells in blood Human blood red blood cells, 20 μl of 1 μg / ml antibacterial peptide of SEQ ID NO: 27 was reacted with 2% RBC for 10 min, centrifuged at 1000 rpm for 3 min, and the experimental results, as shown in Figure 9, show that human blood red blood cells aggregate into clumps and no obvious hemolysis is observed.

[0101] The above description is only a preferred embodiment of the present application and does not limit the present application. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present application should all be included within the protection scope of the present application.

Claims

1. An antibacterial peptide comprising at least one of the amino acid sequences shown from SEQ ID NO: 1 to SEQ ID NO:

39.

2. The antibacterial peptide according to Claim 1, wherein the amino acid sequence includes that at least one amino acid is modified.

3. The antibacterial peptide according to Claim 2, wherein the modification includes at least one of acetylation or an endpoint capping reaction.

4. The antibacterial peptide according to any one of Claims 1 to 3, wherein at least one of the antibacterial peptides is formed into a nanostructure.

5. The antibacterial peptide according to Claim 4, wherein the nanostructure includes at least one of micelles, vesicles, nanotubes, and nanobelts.

6. An antibacterial peptide composition comprising the antibacterial peptide according to any one of Claims 1 to 5, and at least one pharmaceutically acceptable carrier and / or solvent.

7. The antibacterial peptide composition according to Claim 6, further comprising at least one of an excipient, an isotonic agent, an absorption delaying agent, or a base polymer.

8. A method for producing the antibacterial peptide composition according to Claim 6, comprising the step of mixing the antibacterial peptide and the solvent.

9. Use of the antibacterial peptide composition according to Claim 7 in the manufacture of packaging, the end of food processing, clothing, medical supplies, medical devices, personal hygiene products, disinfectants, detergents, anti-infective drugs, anti-inflammatory drugs, and drugs for inhibiting the unlimited proliferation of cells.

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