Use of antibacterial peptide humamp_23 in preparation of drugs for treating burn infection

Abstract

The application belongs to the technical field of biological medicine, and discloses application of antibacterial peptide humAMP_23 in preparation of a medicine for treating burn infection, and is used for promoting healing of a burn wound surface. It is found that local application of the antibacterial peptide humAMP_23 can effectively reduce inflammatory reaction and mortality. It is proved by cell experiments that humAMP_23 has no cytotoxicity and good biocompatibility. The application further provides a pharmaceutical composition containing the antibacterial peptide humAMP_23 and a pharmaceutically acceptable local external carrier. The pharmaceutical composition can be prepared into various forms such as gels, creams, ointments, sprays, hydrocolloid dressings or fibrin glue, is convenient to use in medicine, has the advantages of convenient use, high local drug concentration, remarkable effect of promoting wound healing, and is suitable for local administration of burn infection wound, especially burn infection wound caused by Staphylococcus aureus or Pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of the antimicrobial peptide humAMP_23 in the preparation of drugs for treating burn infections. Background Technology

[0002] Burns are a common type of trauma in clinical practice. After a burn, the skin barrier function is lost, and the wound is rich in necrotic tissue and exudate, making it highly susceptible to colonization and infection by pathogenic microorganisms. Burn infections, especially those caused by opportunistic pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, are one of the core causes of delayed wound healing, spread of inflammation, sepsis, and patient death.

[0003] Currently, the main clinical treatments for burn infections include systemic antibiotic administration and topical antibacterial agents, such as silver sulfadiazine cream. However, the effectiveness of conventional anti-infective therapy is significantly reduced due to bacterial resistance. Furthermore, simply controlling the infection is often insufficient to actively promote tissue repair, reduce excessive inflammatory responses, or lower mortality rates from systemic complications. Therefore, there is a need to develop novel agents or treatments that combine anti-infection, healing-promoting, inflammation-regulating, and survival-enhancing effects, while also possessing good biocompatibility and low susceptibility to inducing drug resistance.

[0004] Antimicrobial peptides, as effector molecules in the innate immune system, possess broad-spectrum antimicrobial activity. Their mechanism of action is primarily based on physical membrane disruption, which differs from the targets of traditional antibiotics, making it less likely to induce bacterial resistance. In recent years, antimicrobial peptides have become a hot topic in anti-infective drug development. However, some reported antimicrobial peptides are derived from amphibians, insects, or are chemically synthesized, exhibiting poor in vivo stability, potential immunogenicity, or cytotoxicity, limiting their clinical translation. In contrast, endogenous human antimicrobial peptides, derived from the body's own proteins, theoretically possess a lower risk of immune rejection and higher biocompatibility.

[0005] Although some human endogenous antimicrobial peptides have been reported to have antimicrobial effects, in the specific pathological environment of burn infection, how to simultaneously inhibit common pathogens in burn wounds, promote wound epithelialization, and reduce local and systemic inflammatory responses still needs further research and development.

[0006] On the other hand, not all antimicrobial peptides are suitable for formulation with excipients to create products suitable for medical treatment of burn infections. Some antimicrobial peptides have a high proportion of hydrophobic amino acids, which makes them prone to aggregation in aqueous environments, resulting in poor solubility and stability in topical preparations such as gels, creams, or sprays. Other antimicrobial peptides lack flexible structural motifs and have excessively rigid molecules, making them prone to precipitation or loss of activity during storage or when combined with excipients. This makes it difficult to truly transform existing antimicrobial peptides into topical drugs suitable for the treatment of burn infections. Summary of the Invention

[0007] The purpose of this invention is to provide a novel use for the antimicrobial peptide humAMP_23, as well as specific products and treatment methods for achieving this use, in order to address the aforementioned challenges faced in the treatment of burn infections in the prior art.

[0008] To achieve the above objectives, the present invention provides the following solution:

[0009] This invention provides the use of the antimicrobial peptide humAMP_23 in the preparation of a medicament for treating burn infections.

[0010] Among them, the amino acid sequence of the antimicrobial peptide humAMP_23 is RRRKKKGGPLRALSSKTW, which was disclosed in Chinese invention patent application CN2025117620688 on February 24, 2026. It is a human endogenous antimicrobial peptide predicted and verified from the human proteome through deep learning methods.

[0011] Furthermore, the drug is used to promote the healing of burn wounds.

[0012] Furthermore, the drug is used to reduce the content of inflammatory factors and alleviate local or systemic inflammatory responses caused by burn wound infection.

[0013] The inflammatory factors include IL-10, IL-1β, IL-6, and TNF-α.

[0014] Furthermore, the drug is used to improve survival rates after burn infection.

[0015] Furthermore, the burn infection is caused by Gram-positive and / or Gram-negative bacteria.

[0016] Furthermore, the Gram-positive bacteria include Staphylococcus aureus, and the Gram-negative bacteria include Pseudomonas aeruginosa.

[0017] The present invention also provides a pharmaceutical composition for any of the foregoing applications, the composition comprising the antimicrobial peptide humAMP_23 and a pharmaceutically acceptable topical carrier.

[0018] Furthermore, the pharmaceutical composition also contains at least one additional active ingredient selected from the group consisting of antibiotics, growth factors, analgesics, anti-inflammatory agents, or immunomodulators.

[0019] Furthermore, the dosage form of the pharmaceutical composition is a gel, cream, ointment, or spray.

[0020] The present invention also provides the use of the pharmaceutical composition in the preparation of medical dressings or medical bioadhesives.

[0021] Preferably, the medical dressing is a hydrocolloid dressing; the medical bioadhesive is fibrin glue.

[0022] Due to the adoption of the above technical solution, the beneficial effects of the present invention are as follows:

[0023] This invention has discovered that topical application of the antimicrobial peptide humAMP_23 can not only effectively inhibit the infection of common pathogens in burn wounds, such as Staphylococcus aureus and Pseudomonas aeruginosa, but also significantly promote burn wound healing and reduce local and systemic inflammatory responses. This overcomes the shortcomings of existing treatments that can only control infection and cannot simultaneously address tissue repair and systemic prognosis.

[0024] The antimicrobial peptide humAMP_23 used in this invention is a human endogenous antimicrobial peptide. Unlike traditional antibiotics, it is not easy to induce bacterial resistance and maintains good antimicrobial effects against drug-resistant strains. Cell experiments have confirmed that humAMP_23 is non-cytotoxic, has excellent biocompatibility, and can be safely used in sensitive tissues such as burn wounds.

[0025] The antimicrobial peptide humAMP_23 used in this invention has high hydrophilicity and a flexible structural motif, making it difficult to aggregate in an aqueous environment. It is well-compatible with excipients in various topical dosage forms such as gels, creams, ointments, and sprays, and has high storage stability, enabling uniform drug delivery. This overcomes the problem that some existing antimicrobial peptides are difficult to formulate into stable topical preparations due to their strong hydrophobicity or excessive molecular rigidity.

[0026] Furthermore, the topical drug compositions such as creams and gels containing the antimicrobial peptide humAMP_23 provided by this invention have diverse dosage forms, which are convenient for flexible selection according to wound characteristics and clinical needs. This not only improves treatment adaptability and patient compliance, but also provides a practical and feasible formulation basis for the precise and personalized management of burn infections. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below:

[0028] Figure 1 This is a graph showing the effect of humAMP_23 on cell viability in Experiment Example 1 of the present invention;

[0029] Figure 2 Survival curves of mice in each group in the burn infection model in Experiment Example 2 of the present invention;

[0030] Figure 3This is a macroscopic characterization result of wound healing in mice in Experiment Example 2 of the present invention. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Unless otherwise specified, all materials, reagents, etc. used in the following embodiments of the present invention are commercially available.

[0037] Example 1

[0038] This embodiment describes the preparation of a drug for treating burn infections. The drug uses the antimicrobial peptide humAMP_23 as its active ingredient, and its preparation method is as follows:

[0039] (1) Swelling and pretreatment of resin

[0040] Weigh 0.1 mmol equivalent of Rink Amide MBHA resin into a solid-phase synthesis column, add 10 mL of dichloromethane solution, and shake to swell at room temperature for 30 min. After removing the dichloromethane, wash the resin three times with 10 mL of N,N-dimethylformamide.

[0041] (2) Removal of Fmoc protecting groups

[0042] Add 5 mL of N,N-dimethylformamide solution containing 20% ​​piperidine (v / v) to the reaction column and shake at room temperature for 15 min to remove the Fmoc protecting group from the resin surface. Drain the reaction solution and wash the resin 5 times with 10 mL of N,N-dimethylformamide to ensure complete removal of piperidine.

[0043] (3) Amino acid coupling reaction

[0044] Take 0.4 mmol of Fmoc-amino acid monomer, 4 mmol of N,N-diisopropylcarbodiimide, and 4 mmol of Oxyma Pure, dissolve them in an appropriate amount of N,N-dimethylformamide, activate at room temperature for 3 min, and then add the activated solution to the reaction column and mix with the resin. Continue shaking and react at room temperature for 60 min. After the reaction is complete, drain the coupling solution and wash the resin three times with N,N-dimethylformamide. After each round of coupling reaction, the resin can be sampled and tested using ninhydrin or bromophenol blue detection methods to ensure complete coupling. If the test is positive, a second coupling reaction is required.

[0045] (4) Cyclic linking of amino acids

[0046] Repeat steps (2) and (3), and sequentially link each Fmoc-protected amino acid according to the amino acid sequence from the C-terminus to the N-terminus of the antimicrobial peptide humAMP_23 RRRKKKGGPLRALSSKTW.

[0047] (5) Final deprotection and resin cutting

[0048] After all amino acid sequences were assembled, the final Fmoc removal was performed. The resin was then washed with dichloromethane and dried under vacuum.

[0049] The cutting mixture was prepared according to a volume ratio of trifluoroacetic acid, triisopropylsilane, and water of 95:2.5:2.5. The dried resin was then added to 10 mL of the cooled cutting mixture, and the mixture was shaken and reacted at room temperature for 3 hours. After the reaction was complete, the mixture was filtered using a sintered glass funnel, and the filtrate containing the crude peptide was collected and precipitated in cold diethyl ether. The white peptide precipitate was collected by centrifugation at 5000 rpm for 10 minutes at 4°C.

[0050] (6) Purification

[0051] The peptide precipitate was first washed three times with cold anhydrous ether, collected by centrifugation, and then placed in a fume hood to allow the residual ether to evaporate. Finally, it was vacuum dried to obtain the crude antimicrobial peptide humAMP_23.

[0052] The crude peptide was then dissolved in a mixed solution of acetonitrile and water containing 0.1% trifluoroacetic acid, and purified using semi-preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic conditions were as follows: a C18 column, 4.6 × 250 mm, with a particle size of 5 μm; mobile phase A was a 0.1% trifluoroacetic acid aqueous solution, and mobile phase B was a 0.1% trifluoroacetic acid acetonitrile solution; the gradient was set as follows: 15% B to 45% B for 30 min, 45% B to 100% B for 3 min, 100% B for 5 min, 100% B to 15% B for 2 min, and 15% B for 10 min; the detection wavelength was 214 nm. The fraction corresponding to the main peak was collected, and after HPLC analysis showing a purity greater than 90%, the fractions were combined and lyophilized to obtain a purified antimicrobial peptide, humAMP_23, as a white powder, which is a drug for treating burn infections.

[0053] Example 2

[0054] This embodiment provides a method for preparing an antimicrobial peptide humAMP_23 cream, used to prepare a pharmaceutical composition comprising the antimicrobial peptide humAMP_23 and a pharmaceutically acceptable topical carrier, wherein the dosage form is a cream. The specific preparation method is as follows:

[0055] Based on a total mass of 100g, the cream base is formulated as follows: the oil phase contains 15% stearic acid, 12% liquid paraffin and 8% petrolatum; the aqueous phase contains 10% glycerin and 48% purified water; the emulsification system uses 4% Span 80 and 2% glyceryl monostearate; and the preservative is 0.1% propylparaben.

[0056] In preparation, the oil phase component, oil-soluble preservative, and aqueous phase component were first heated to 75°C and 70°C respectively and stirred until homogeneous. Then, the preheated aqueous phase was slowly added dropwise to the oil phase under continuous stirring at 300-400 rpm / min to form a primary emulsion. The emulsion was then allowed to cool naturally under stirring. Once the emulsion temperature dropped below 40°C, the humAMP_23 antimicrobial peptide solution, pre-dissolved in phosphate buffer, was slowly added and gently stirred at 150-200 rpm / min until uniformly dispersed, finally yielding a homogeneous W / O type antimicrobial peptide cream.

[0057] Creams with final concentrations of 500 μg / mL and 250 μg / mL were obtained by uniformly dispersing 5 mg or 2.5 mg of humAMP_23 antimicrobial peptide in 10 g of cream matrix, respectively. All formulations were sealed after preparation and stored at 4°C protected from light.

[0058] Example 3

[0059] This embodiment provides a method for preparing an antimicrobial peptide humAMP_23 gel. This gel formulation can be used to prepare medical dressings or medical bioadhesives. The specific preparation method is as follows:

[0060] A chitosan-acetic acid solution was prepared by magnetically stirring overnight until completely dissolved, with a chitosan concentration of 2% (w / v) and an acetic acid volume fraction of 1%. A 1% (w / v) sodium tripolyphosphate aqueous solution was also prepared. Under stirring, the sodium tripolyphosphate solution was slowly added dropwise to the chitosan-acetic acid solution at a volume ratio of 5:1 until a homogeneous sol was formed. The sol was poured into a mold and frozen at -20°C for 12 hours, followed by freeze-drying at -50°C for 24 hours to obtain a porous chitosan sponge-like gel. The freeze-dried gel was hydrated by soaking in a pH 6.0 phosphate buffer solution, with the buffer solution changed several times to balance the pH and remove residual acetic acid. A 250 μg / mL humAMP_23 antimicrobial peptide solution was prepared using a pH 6.0 phosphate buffer solution. The equilibrated, moistened chitosan gel was immersed in this antimicrobial peptide solution and slowly agitated at 4°C for 24 hours to allow adsorption. After adsorption is complete, the loaded gel is removed, and the surface peptides that are not firmly adsorbed are quickly rinsed with a small amount of phosphate buffer at pH 7.4. Finally, the gel is immersed in phosphate buffer at pH 7.4 for equilibration, thus obtaining an antimicrobial peptide humAMP_23 gel.

[0061] Effect Experiment Example 1

[0062] This experiment evaluated the cytotoxicity of the antimicrobial peptide humAMP_23 to various cell lines by examining its effect on cell viability, as detailed below:

[0063] (1) Cell culture: Mouse normal hepatocytes AML12, human normal colonic epithelial cells NCM460, human liver cancer cells HepG2, mouse colon cancer cells CT26, human gastric cancer cells AGS and HGC-27, mouse T lymphocyte leukemia cells AKR, and mouse T lymphocyte cell line CT1 were placed in a 37°C, 5% carbon dioxide incubator and cultured in DMEM, RPMI-1640 or DMEM / F12 medium containing 10% fetal bovine serum.

[0064] (2) Cell seeding and drug administration: Each cell line in the logarithmic growth phase was seeded at 4 × 10⁶ cells per well. 4Cells were seeded at a density of [number] cells per well in 96-well plates and pre-cultured for 24 hours to ensure complete adherence. For suspension cells AKR and CT1, drug treatment was administered immediately after seeding. Once adherent cells had adhered, the culture medium was removed and replaced with fresh complete medium containing 250 µg / mL humAMP_23. Wells containing only the culture medium served as a blank control, and wells containing both cells and culture medium but not the antimicrobial peptide served as a negative control (Control). Cells and antimicrobial peptide were co-cultured in an incubator for 24 hours.

[0065] (3) Cell viability detection: After culture, 10 µL of CCK-8 solution was added to each well, and the 96-well plate was incubated in the dark for 2 hours. The absorbance of each well was then measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability percentage equals the difference between the absorbance of the sample well and the absorbance of the blank well, divided by the difference between the absorbance of the negative control well and the absorbance of the blank well, multiplied by 100%. Data are expressed as mean ± standard deviation. Results are as follows: Figure 1 .

[0066] Depend on Figure 1 It can be seen that, compared with the negative control, the cell lines showed good cell survival, indicating that the antimicrobial peptide humAMP_23 prepared in this invention is non-cytotoxic and suitable for drug development.

[0067] Effect Experiment Example 2

[0068] This experimental example is used to evaluate the effects of the antimicrobial peptide humAMP_23 prepared in Examples 1-3 on the survival rate, wound healing, and inflammatory factors in mice with burn infection.

[0069] (1) Establishment of a burn infection model: Mice were anesthetized, and their backs were shaved and the hair was removed. A preheated copper block contact method was used for modeling. The copper block was 1.5 cm in diameter and heated to 95°C in a metal bath. It was then vertically and without pressure applied to the lifted skin folds on the back for 9 seconds to form a standard full-thickness burn wound. Immediately after the procedure, hemostasis was achieved by gentle pressure with sterile gauze, and preheated saline was injected subcutaneously at a dose of 20 mL / kg. Within 30 minutes of modeling, 10 µL of a solution containing 1×10⁻⁶ g of saline was inoculated into the wound. 8 CFU suspensions of Staphylococcus aureus and Pseudomonas aeruginosa were spread evenly with a sterile swab.

[0070] (2) Animal grouping and drug treatment: Mice were randomly grouped and treatment began 1 hour after bacterial inoculation. The wound was cleaned with sterile saline every day, followed by application of the appropriate preparation at a dose of 50 mg / cm² once a day for 17 days. During the treatment period, mice were housed individually with free access to food and water, and their condition, mortality rate, and wound healing were observed.

[0071] The groups were as follows: the sham surgery group (Sham) had infection but no burns; the model group (Model) had burns with infection; the solvent group (Vehicle) had burns with infection and was given blank cream; the positive control group (SSD) had burns with infection and was given silver sulfadiazine ointment; the low-dose group (250 μg / mL humAMP_23) had burns with infection and was given cream containing 250 μg / mL humAMP_23; and the high-dose group (500 μg / mL humAMP_23) had burns with infection and was given cream containing 500 μg / mL humAMP_23.

[0072] (3) Detection indicators: After drug administration, mouse serum was collected, and the levels of IL-10, IL-1β, IL-6, and TNF-α in the serum were detected. Survival curves for each group of mice were also recorded, such as... Figure 2 As shown; and representative photos of the wound were taken on days 1, 7, 13, and 17, as shown. Figure 3 As shown in Table 1, the macroscopic characteristics of wound healing were observed, and the wound area and inflammatory factor content were calculated.

[0073] Table 1. Effects of humAMP_23 on wound healing and inflammatory factors in a mouse model of burn infection.

[0074]

[0075] Note: All results in the table are Mean ± SEM.

[0076] Experimental results show that the antimicrobial peptide humAMP_23 has good biocompatibility. Several drugs containing the antimicrobial peptide humAMP_23 prepared in this invention can effectively reduce the content of inflammatory factors, alleviate inflammatory response, and significantly promote the healing of burn wounds, thereby reducing the mortality rate of animals with burn infections.

[0077] In other embodiments, a medicament for treating burn infections is prepared from the white powder of the antimicrobial peptide humAMP_23, which is a gel, cream, ointment, spray, hydrocolloid dressing, or fibrin glue made by taking the white powder of the antimicrobial peptide humAMP_23 with pharmaceutically acceptable excipients, additional active ingredients, and a topical carrier.

[0078] The additional active ingredient is at least one of antibiotics, growth factors, analgesics, anti-inflammatory agents, or immunomodulators.

[0079] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of antimicrobial peptide humAMP_23 in the preparation of drugs for treating burn infections.

2. The application according to claim 1, characterized in that, The drug is used to promote the healing of burn wounds.

3. The application according to claim 1, characterized in that, The drug is used to reduce the content of inflammatory factors and alleviate local or systemic inflammatory responses caused by burn wound infection.

4. The application according to claim 1, characterized in that, The drug is used to improve survival rates after burn infection.

5. The application according to any one of claims 1 to 4, characterized in that, The burn infection was caused by Gram-positive and / or Gram-negative bacteria.

6. The application according to claim 5, characterized in that, The Gram-positive bacteria include Staphylococcus aureus, and the Gram-negative bacteria include Pseudomonas aeruginosa.

7. A pharmaceutical composition for use in any one of claims 1 to 6, characterized in that, It contains the antimicrobial peptide humAMP_23 and a pharmaceutically acceptable topical carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, It also contains at least one additional active ingredient selected from the group consisting of antibiotics, growth factors, analgesics, anti-inflammatory agents, or immunomodulators.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that, Its dosage form is gel, cream, ointment or spray.

10. The use of the pharmaceutical composition according to any one of claims 7 to 9 in the preparation of medical dressings or medical bioadhesives.

Images