An antimicrobial polypeptide for treating acne, its preparation method and application

Abstract

This invention discloses an antimicrobial peptide for treating acne, its preparation method, and its application, belonging to the field of biopeptides. The amino acid sequence of the antimicrobial peptide is shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5. This invention provides an antimicrobial peptide for treating acne, which exhibits good antimicrobial activity against Propionibacterium acnes, and also demonstrates good stability and low hemolytic activity, showing promise as a novel drug for treating acne.

Description

Technical Field

[0001] This invention belongs to the field of biopeptide drugs, specifically relating to an antibacterial polypeptide for treating acne, its preparation method, and its application. Background Technology

[0002] Acne, commonly known as "pimples" or "blackheads," is a common chronic inflammatory disease of the hair follicles and sebaceous glands, typically characterized by skin lesions such as comedones, papules, pustules, nodules, and cysts. Acne is a multifactorial disease involving sebaceous gland hyperplasia, excessive keratinization of the hair follicle, and colonization by Propionibacterium acnes as an opportunistic pathogen. Inhibiting the proliferation of Propionibacterium acnes is one of the core strategies for treating acne. However, long-term use of antibiotics can easily lead to drug resistance in Propionibacterium acnes, resulting in a gradual decline in treatment effectiveness and potentially causing adverse reactions such as intestinal flora imbalance and secondary infections. Benzoyl peroxide, on the other hand, may cause discomfort such as dry skin, peeling, and erythema, and some patients have poor tolerance to it.

[0003] Antimicrobial peptides are a class of small molecule polypeptides with antimicrobial activity that are induced to be produced in organisms. They are widely found in bacteria, fungi, plants, animals, and humans. Antimicrobial peptides possess broad-spectrum antimicrobial activity, exhibiting direct and potent antimicrobial activity against a wide range of microorganisms, including Gram-negative and Gram-positive bacteria, fungi, and viruses. They achieve their antimicrobial effect by rapidly disrupting bacterial cell membranes and causing leakage of contents, independent of bacterial metabolic pathways, thus reducing the likelihood of inducing drug resistance. Furthermore, some antimicrobial peptides also possess multiple biological functions such as immunomodulation and anti-inflammation, enabling them to intervene in the pathogenesis of acne from multiple perspectives. Therefore, antimicrobial peptides hold promise as a new treatment option for acne, providing safer and more effective treatment solutions for acne patients. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, this invention provides an antimicrobial peptide for treating acne. This peptide exhibits good antimicrobial activity against Propionibacterium acnes, and also has good stability and low hemolytic activity, making it a promising new drug for treating acne.

[0005] This invention also provides a method for preparing and applying antimicrobial peptides.

[0006] Technical solution: In order to achieve the above objective, the present invention provides an antibacterial polypeptide for treating acne, wherein the amino acid sequence of the antibacterial peptide is shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5.

[0007] The present invention discloses a method for preparing antimicrobial peptides for treating acne, wherein the peptides are designed based on the core structure-activity relationship of antimicrobial peptides to obtain SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, and the antimicrobial peptides are synthesized using a solid-phase peptide synthesis method.

[0008] The application of the antimicrobial polypeptide for treating acne described in this invention in the preparation of drugs for treating pathogenic bacterial infections.

[0009] The anti-pathogenic bacterial infection drug is an anti-pathogenic bacterial infection drug.

[0010] The pathogenic bacteria is Propionibacterium acnes.

[0011] The antimicrobial infection drug composition of the present invention comprises the aforementioned antimicrobial polypeptide and its pharmaceutically acceptable carrier.

[0012] The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

[0013] The application of the anti-pathogenic bacterial infection drug composition described in this invention in the preparation of anti-pathogenic bacterial infection drugs.

[0014] The anti-pathogenic bacterial infection drug is a drug for pathogenic bacteria infection.

[0015] The pathogenic bacteria is Propionibacterium acnes.

[0016] Based on the core structure-activity relationship of antimicrobial peptides, this invention designs the following peptides:

[0017] H01 (KWKLFKKIKKFLHKELKF-NH2) (SEQ ID NO.1)

[0018] H05(KWKLFKKIKKFLHKELKR)(SEQ ID NO.2)

[0019] H11(KWKLFKKIKKFLHKELKI)(SEQ ID NO.3)

[0020] H12 (KWKLFKKIKKFLHKELKR-NH2) (SEQ ID NO.4)

[0021] H13 (KWKLFKKIKKFLHKELKI-NH2) (SEQ ID NO.5)

[0022] The peptide was designed and modified based on the core structure-activity relationship of antimicrobial peptides, specifically P18 (KWKLFKKIPKFLHLAKKF-NH2). The specific design basis is as follows:

[0023] (1) Replace proline: Replace the rigid Pro with lysine (Lys) to reduce the rigidity of the peptide chain while increasing its net positive charge, thereby reducing its hemolytic activity in mammals and enhancing its membrane binding capacity.

[0024] (1) Enhance cationicity: Introduce arginine (Arg) and lysine (Lys) residues on the polar side of the peptide chain to enhance the electrostatic interaction strength with the bacterial cell membrane and strengthen the membrane binding ability;

[0025] (2) Optimize amphiphilicity: By constructing hydrophobic surfaces through the site-specific distribution of hydrophobic residues such as leucine (Leu) and isoleucine (Ile), the efficiency of membrane insertion and disruption is improved;

[0026] (3) Reduce toxicity: Avoid excessive accumulation of hydrophobic residues, introduce non-hydrophobic amino acids into the hydrophobic surface to destroy the integrity of the hydrophobic surface and reduce hemolytic effects on mammalian cells;

[0027] (4) Improve stability: The C-terminus of the peptide chain is modified by amidation to neutralize the negative charge at the C-terminus and stabilize the α-helix structure.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0029] 1. The antibacterial polypeptide prepared in this invention has excellent antibacterial effect against Propionibacterium acnes.

[0030] 2. The antibacterial polypeptide prepared by this invention has both antibacterial activity and good stability.

[0031] 3. The antibacterial polypeptide prepared by this invention can be used in the preparation of anti-acne drugs and has excellent in vitro antibacterial activity; it can be used as an excellent alternative or adjuvant drug to existing antibiotics.

[0032] 4. The antibacterial peptides of this invention are designed and prepared in a simple and convenient manner, with novel design and readily available raw materials, and can be industrially produced and applied. Attached Figure Description

[0033] Figure 1 This is the HPLC chromatogram of the antibacterial polypeptide H05 of the present invention.

[0034] Figure 2 This is the HPLC chromatogram of the antibacterial polypeptide H11 of the present invention.

[0035] Figure 3 This is the HPLC chromatogram of the antibacterial polypeptide H12 of the present invention.

[0036] Figure 4 This is the HPLC chromatogram of the antibacterial polypeptide H13 of the present invention.

[0037] Figure 5 This is the HPLC chromatogram of the antibacterial polypeptide H01 of the present invention.

[0038] Figure 6 This is the mass spectrum of the antibacterial polypeptide H05 of the present invention.

[0039] Figure 7 This is the mass spectrum of the antibacterial polypeptide H11 of the present invention.

[0040] Figure 8 This is the mass spectrum of the antibacterial polypeptide H12 of the present invention.

[0041] Figure 9 This is the mass spectrum of the antibacterial polypeptide H13 of the present invention.

[0042] Figure 10 This is the mass spectrum of the antibacterial polypeptide H01 of the present invention. Detailed Implementation

[0043] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0044] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0045] The polypeptides designed in this invention can be synthesized directly by a biotechnology company, or they can be synthesized using existing polypeptide synthesis methods.

[0046] Example 1

[0047] Preparation of antimicrobial peptides:

[0048] The peptides were designed based on the core structure-activity relationship of antimicrobial peptides, and the specific design basis is as follows:

[0049] (1) Enhance cationicity: Introducing arginine (Arg) residues into the polar side of the peptide chain enhances the electrostatic interaction strength with the bacterial cell membrane (which is negatively charged), thereby strengthening membrane binding capacity;

[0050] (2) Optimize amphiphilicity: By constructing hydrophobic surfaces through the site-specific distribution of hydrophobic residues such as leucine (Leu), isoleucine (Ile), and aromatic amino acids, the efficiency of membrane insertion and disruption is improved;

[0051] (3) Reduce toxicity: Avoid excessive accumulation of hydrophobic residues, introduce non-hydrophobic amino acids into the hydrophobic surface to destroy the integrity of the hydrophobic surface and reduce hemolytic effects on mammalian cells;

[0052] (4) Improved stability: Amide modification of the C-terminus of the peptide chain neutralizes the negative charge at the C-terminus and stabilizes the α-helix structure. Solid-phase synthesis of peptides:

[0053] Peptide synthesis: Peptide synthesis proceeded sequentially from the C-terminus to the N-terminus. Fmoc-Phe-Rink Resin (for amidated peptides, Fmoc-Phe-Rink Amide MBHA / AM Resin) was soaked in dichloromethane for 15 min until the resin swelled. The dichloromethane was then removed. A 1:4 volume ratio of hexahydropyridine / DMF solution (10 mL per gram of resin) was added, and the mixture was agitated with nitrogen. The reaction was repeated twice, for 5 min and 15 min respectively. After the reaction, the resin was washed 6 times with DMF. 20-40 peptides were then mixed with 2-3 drops each of colorimetric reagents A, B, and C (A: ninhydrin / anhydrous ethanol; B: pyridine; C: phenol / anhydrous ethanol). The mixture was heated at 100°C for 3 min. The solution and resin turned blue, indicating the removal of the amino group protection. Excess Fmoc-Val-OH and HOBT (twice the molar amount of the reaction mixture) were added, dissolved in 10 mL DMF per gram of resin, and then DIC and Collidine (twice the molar amount of the reaction mixture) were added. The mixture was agitated with nitrogen and reacted for 1 h. After the reaction, the resin was washed 6 times with DMF. The condensation reaction was repeated to sequentially ligate each Fmoc-protected amino acid to complete the synthesis of the linear sequence. The resin was soaked in dichloromethane and diethyl ether and then dried. TFA was added, and the mixture was reacted in a constant-temperature shaker at 110 r / min and 25℃ for 2 h. The resin was filtered off, and anhydrous diethyl ether was added to the filtrate. After centrifugation, the solid was obtained, washed with anhydrous diethyl ether, centrifuged again, and the process was repeated several times before drying to obtain the crude polypeptide.

[0054] Polypeptide purification: A certain amount of crude product was weighed, and an appropriate amount of acetonitrile was added. The mixture was sonicated until clear, and large particulate impurities were removed using a filter. Simultaneously, the sample was collected in fractions using a preparative liquid chromatograph. Gradient analysis was performed using an analytical chromatograph, and samples reaching the desired purity were retained. Finally, the samples were freeze-dried.

[0055] Results of purity determination (HPLC method) and mass spectrometry analysis of peptides: After synthesis, the peptides were purified to obtain the finished product, which was identified by high performance liquid chromatography and mass spectrometry.

[0056] Liquid chromatography analysis conditions: C18 column (4.6 × 250 mm, 5 µm); mobile phase A was acetonitrile solution containing 0.1% trifluoroacetic acid, and mobile phase B was purified water containing 0.1% TFA. Detection wavelength was 220 nm; flow rate was 1.0 mL / min; injection volume was 20 µl, with gradient elution.

[0057] The amino acid sequences of the polypeptides of this invention are as shown in SEQ ID NO. 1-5, with a purity greater than 95%, and their HPLC and MS results are as follows: Figure 1-5 and Figure 6-10 As shown, it matches the theoretical value.

[0058] The details are as follows:

[0059] H01 (KWKLFKKIKKFLHKELKF-NH2) (SEQ ID NO.1)

[0060] H05(KWKLFKKIKKFLHKELKR)(SEQ ID NO.2)

[0061] H11(KWKLFKKIKKFLHKELKI)(SEQ ID NO.3)

[0062] H12 (KWKLFKKIKKFLHKELKR-NH2) (SEQ ID NO.4)

[0063] H13 (KWKLFKKIKKFLHKELKI-NH2) (SEQ ID NO.5).

[0064] Example 2

[0065] The determination of the MIC of peptides and clindamycin in this invention

[0066] The Propionibacterium acnes strain used in this experiment was the ATCC6919 standard strain.

[0067] Experimental methods:

[0068] 1. Preparation of culture medium

[0069] Take 38.5g of BHI culture medium, add it to 1000mL of distilled water, heat to boiling to dissolve, and dispense into individual containers.

[0070] 2. Preparation and sterilization of experimental equipment

[0071] Place the BHI culture medium, matching pipette tips, pipette slots, and test tubes together in an autoclave for sterilization at 121°C for 15 minutes. Before use, the clean bench and operating room must be sterilized with ultraviolet light for at least 30 minutes.

[0072] 3. Preparation of antimicrobial peptide mother liquor

[0073] Weigh an appropriate amount of polypeptide, dissolve it in physiological saline to prepare a stock solution of 256 μg / mL, filter it through a 0.22 μm aqueous filter to sterilize it, dispense it into individual containers, and store it at 4℃ for later use.

[0074] 4. Preparation of bacterial suspension

[0075] Remove the bacterial culture stored in glycerol from the -80℃ freezer. Streak a small amount of the bacterial suspension onto a Columbia blood agar plate (four zones), seal it in an anaerobic bag, and incubate at 37℃ for 72 hours to activate it. Transfer a single activated colony to 4 mL of BHI medium and incubate for another 48 hours. Then, centrifuge the bacterial suspension to remove the medium, dilute with physiological saline to a turbidity of 0.5, and then further dilute with medium 100 times (to approximately 10^6 colonies). 6 (CFU / mL) for later use.

[0076] 5. Sample dilution and bacterial addition

[0077] Add 100 μL of BHI broth to each well of a 96-well plate. Then, dilute the sample twofold by adding 100 μL of sample to the first well of each of the three rows (A, B, and C). Use a pipette to thoroughly mix the sample with the broth (at least three times). Then, add 100 μL of the diluted bacterial culture to the second well and mix thoroughly again. Repeat this process until the last well. Finally, add 100 μL of the diluted bacterial culture to each well to achieve a final colony count of 5 × 10⁻⁶. 5 CFU, repeat three times (A / B / C rows of samples).

[0078] Meanwhile, a growth control (bacteria only) was set up in column 10 of the same plate, and a blank control (culture medium only) was set up in column 11.

[0079] 6. Observation Results

[0080] The 96-well plates were sealed with anaerobic bags and incubated at 37°C for 72 hours. The results were observed, and the lowest concentration of samples in which no bacteria were visible to the naked eye was defined as the MIC. The experimental results are shown in Table 1.

[0081] Table 1. MICs of antimicrobial peptides and clindamycin against Propionibacterium acnes

[0082]

[0083] As shown in Table 1, the peptides of this invention exhibit good antibacterial activity. Omiganan is an antibacterial peptide already in clinical use that can inhibit Propionibacterium acnes, with a MIC value of 4 μg / mL. The peptides of this invention all have MIC values ​​of 4 μg / mL or lower against Propionibacterium acnes, demonstrating inhibitory activity comparable to or even better than Omiganan. This indicates that the peptides of this invention have a good bactericidal effect against Propionibacterium acnes. Furthermore, while clindamycin has a low MIC against the standard strain ATCC 6919 of Propionibacterium acnes, long-term use of clindamycin can cause some side effects. The MIC of P18 is around 2 μg / mL, and the inhibitory activity of the peptides of this invention is comparable to or better than that of clindamycin, with significantly lower hemolytic activity (see Example 3 for details).

[0084] Example 3

[0085] Hemolytic activity experiment of the polypeptide of the present invention

[0086] Experimental methods:

[0087] Fresh red blood cells (RBCs) were collected from ICR mice and humans. RBCs were washed at least three times with 0.01 mM PBS buffer until no color was visible in the supernatant. The RBCs were then diluted with PBS to obtain a 4.0% (v / v) RBC solution. 150 μL of RBCs were mixed with an equal volume of antimicrobial peptide to achieve final peptide concentrations of 256, 128, 64, 32, and 16 μg / mL, and incubated at 37°C for 1 hour. The mixture was then centrifuged (1500 × g, 5 min) to collect the supernatant. 150 μL of the supernatant was transferred to a 96-well plate. A mixture of 2% Triton X-100 solution and an equal volume of 4.0% RBC solution served as a positive control, and a mixture of PBS and an equal volume of 4.0% RBC solution served as a negative control. The absorbance (OD) of hemoglobin was measured at 570 nm using a microplate reader. 570 The formula is as follows:

[0088] Hemolysis rate (%) = [(A-A0) / (A1-A0)] × 100.

[0089] A represents the absorbance of the polypeptide. A0 represents the absorbance of the PBS group, and A1 represents the absorbance of the Triton X-100 group.

[0090] Three independent replicate experiments were conducted. The hemolytic activity of H01, H05, H11, H12, and H13 against mouse and human erythrocytes was evaluated. The experimental results are shown in Tables 2 and 3. According to the results, at a concentration of 128 μg / mL, the hemolytic activity of the peptides of this invention against mouse erythrocytes was low, while at 256 μg / mL, the hemolytic activity against human erythrocytes was even lower, far lower than that of its template peptide P18, and there was no hemolytic toxicity within the effective dose range of the drug.

[0091] Table 2. Hemolysis rate (%) of antimicrobial peptides on mouse erythrocytes

[0092]

[0093] Table 3. Hemolysis rate (%) of antimicrobial peptides on human erythrocytes

[0094]

[0095] Example 4

[0096] Thermostability Experiment of Peptides in this Invention

[0097] Experimental methods

[0098] The peptide dissolved in physiological saline was incubated at 25°C and 60°C, and samples were taken from the solution at 12, 24, 36, 48 h and 14 days, for a total of six times. The MIC of the incubated sample solution against Propionibacterium acnes was determined using the experimental method of Example 1. The experimental results are shown in Table 4. According to the results, the MIC of the sample solution against Propionibacterium acnes remained unchanged after incubation at 25°C for 14 days; after incubation at 60°C for 14 days, the MIC of H11 against Propionibacterium acnes remained unchanged, while the MICs of H5, H12, and H13 against Propionibacterium acnes only decreased to 4 μg / mL. This indicates that the peptide of the present invention has good thermal stability.

[0099] Table 4. MIC (μg / mL) of peptides against Propionibacterium acnes after incubation at 25 and 60℃.

[0100]

[0101] Example 5

[0102] This invention relates to a polypeptide eye irritation test and a chicken embryo chorioallantoic membrane test.

[0103] Experimental methods:

[0104] Fertilized chicken embryos of breeds such as White Laihang chickens, weighing 50g-60g, were selected, with SPF embryos preferred. The embryo quality met relevant standards. Incubation temperature was 37.5℃ ± 0.5℃, relative humidity 55%-70%, and egg turning frequency 3-6 times / hour. On day 8 of incubation, eggs were candled using an egg candler. Unfertilized and inactive embryos were discarded, and embryos with well-developed blood vessels were selected. The location of the air cell was marked on the eggshell surface. Six embryos were used per group. 0.5 and 1 mg / mL H13 polypeptide solutions were prepared using physiological saline as test samples.

[0105] The experiment began on day 9. The air cell portion of the eggshell was carefully removed using dental forceps. A few drops of physiological saline were applied to the eggshell membrane surface to thoroughly moisten it. After pouring out the saline solution, the eggshell membrane was carefully removed with forceps, ensuring the exposed allantoic membrane remained intact without any damage. 0.3 ml of the polypeptide solution was applied directly to the chorioallantoic membrane (CAM). The solution was spread as widely as possible, ensuring coverage of at least 50%. After 3 minutes of application, the CAM surface was gently rinsed with double-distilled water, completing the rinsing within 30 seconds. The liquid was then poured out, and the degree of change in each toxic effect was immediately observed under a stereomicroscope and scored (ES).

[0106] Results observation:

[0107] Bleeding: Blood flows out from the blood vessels or capillaries of the CAM. Bleeding is scored as 0, 1, 2, or 3 points respectively, based on no bleeding, mild bleeding, moderate bleeding, and severe bleeding.

[0108] Coagulation: refers to the denaturation of proteins inside and outside blood vessels, manifested as thrombosis, swelling of the vessel wall, appearance of coagulation points inside and outside blood vessels, and milky opacity outside the blood vessels. It is scored as 0, 1, 2, and 3 points respectively based on no coagulation, mild coagulation, moderate coagulation, and severe coagulation.

[0109] Vascular ablation: refers to the ablation of blood vessels on the CAM membrane. A score of 0, 1, 2, and 3 is assigned based on whether there is no vascular ablation, mild vascular ablation, moderate vascular ablation, or severe vascular ablation, respectively.

[0110] Results calculation and evaluation:

[0111] The average ES score was calculated using the following formula, and the stimulus classification was determined according to Table 5. The experimental results are shown in Table 6. When H13 at concentrations of 0.5 and 1 mg / mL was directly applied to the chorioallantoic membrane of chicken embryos, changes in chorioallantoic membrane toxicity indicators (such as hemorrhage, coagulation, and vascularization) were observed. The ES values ​​were 6 and 11, respectively, both less than 12. This indicates that H13 has low toxicity to the chorioallantoic membrane beyond the recommended dosage range, demonstrating that H13 has no or mild ocular irritation, thus confirming the safety of H13 as a topical preparation for ocular use.

[0112]

[0113] Table 5. Results Evaluation

[0114]

[0115] Table 6 Evaluation of H13 eye irritation results

[0116]

Claims

1. An antibacterial polypeptide for treating acne, characterized in that, The amino acid sequence of the antimicrobial peptide is shown in any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO.

5.

2. A method for preparing the antibacterial polypeptide for treating acne according to claim 1, characterized in that, The peptides were designed based on the core structure-activity relationship of antimicrobial peptides to obtain SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, and SEQ ID NO. 5, and the antimicrobial peptides were synthesized using a solid-phase peptide synthesis method.

3. The use of the antimicrobial polypeptide for treating acne as described in claim 1 in the preparation of drugs for treating pathogenic bacterial infections.

4. The application according to claim 3, characterized in that, The anti-pathogenic bacterial infection drug is an anti-pathogenic bacterial infection drug.

5. The application according to claim 4, characterized in that, The pathogenic bacteria is Propionibacterium acnes.

6. A drug composition for treating pathogenic bacterial infections, characterized in that, It includes the antimicrobial polypeptide of claim 1 and its pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition is a capsule, powder, tablet, granule, pill, injection, syrup, oral liquid, inhaler, ointment, suppository or patch.

8. The use of the antimicrobial drug composition of claim 6 in the preparation of an antimicrobial drug.

9. The application according to claim 8, characterized in that, The anti-pathogenic bacterial infection drug is a drug for pathogenic bacteria infection.

10. The application according to claim 9, characterized in that, The pathogenic bacteria is Propionibacterium acnes.

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