An antimicrobial polypeptide, a method for preparing a gel containing it, and its application.

Abstract

This invention relates to an antimicrobial peptide, a gel containing the same, and a method for preparing the same. The amino acid sequence of the antimicrobial peptide is shown in SEQ ID No. 1.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and in particular to an antimicrobial peptide that can be used to treat bacterial vaginosis. Background Technology

[0002] The vagina, as an open cavity, is home to a large number of flora, and the normal flora has a protective effect on the vagina. If the balance of the vaginal flora is disrupted, such as by excessive proliferation of anaerobic bacteria, bacterial vaginosis can easily occur. Bacterial vaginosis is one of the most common vaginal infections, accounting for nearly half of all vaginal infection cases. Patients may experience symptoms such as vaginal itching and abnormal vaginal discharge. Patients with bacterial vaginosis need timely treatment; otherwise, the risk of miscarriage, postpartum endometritis, and other complications increases. Currently, metronidazole and other antibiotics are the first-line treatment for bacterial vaginosis, but long-term use can lead to the formation of drug-resistant strains. Antimicrobial peptides are a class of small molecule polypeptides widely found in animals, plants, insects, and humans. They are genetically encoded and have specific spatial structures, with molecular weights ranging from approximately 2000 to 7000 Da, composed of 20 to 60 amino acid residues. They possess good antimicrobial activity and are a class of promising biological macromolecules that can replace traditional antibiotics. They are widely available, have excellent antibacterial properties and are not prone to inducing bacterial resistance, but natural antimicrobial peptides have certain limitations, including low antibacterial activity, poor stability and high hemolytic activity. Summary of the Invention

[0003] One aspect of the present invention provides an antimicrobial peptide, the amino acid sequence of which is shown in SEQ ID No. 1.

[0004] The second invention provides a composition containing the antimicrobial peptide of claim 1.

[0005] In one embodiment, the composition further includes a gelling agent, a humectant, a pH adjuster, and water.

[0006] In one specific embodiment, the antimicrobial peptide comprises 0.5 to 2.0 parts by weight, the gelling agent comprises 1 to 2 parts by weight, the moisturizer comprises 10 to 20 parts by weight, and the sterile water comprises 500 to 700 parts by weight.

[0007] In one specific embodiment, the gelling agent is selected from carbomer.

[0008] In one embodiment, the gelling agent is selected from at least one of carbomer 980, carbomer 934, and carbomer 941.

[0009] In one embodiment, the moisturizer is selected from at least one of glycerin, propylene glycol, ethanol, sorbitol, and polyethylene glycol.

[0010] In one specific embodiment, the pH adjuster is selected from triethanolamine and / or sodium hydroxide.

[0011] In one embodiment, the pH value of the composition is 4.7 to 7.3.

[0012] In one embodiment, the composition further includes a preservative.

[0013] In one specific embodiment, the preservative is methylparaben.

[0014] In one specific embodiment, the amount of preservative used is 1 to 2 parts by weight.

[0015] The third invention relates to the antimicrobial peptide of the first invention and the composition of any one of the second inventions of the present invention in the use of a medicament for preparing an antimicrobial peptide against at least one of Staphylococcus spp., Streptococcus spp., Enterococcus spp., Micrococcus spp., Moraxella spp., Corynebacterium spp., Klebsiella spp., Enterobacter spp., Serratia spp., Proteus spp., Pseudomonas spp., Morganella morganii, Haemophilus spp., Xanthomonas spp., Acinetobacter spp. and Propionibacterium spp.

[0016] In one specific embodiment, the application is in the preparation of a medicament for use against at least one of Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa.

[0017] In one specific embodiment, the application is in the preparation of a medicament for treating at least one of bacterial vaginosis.

[0018] The beneficial effects of this invention are:

[0019] The antibacterial peptide carbomer gel prepared by this invention has excellent antibacterial activity, low hemolysis rate, easy gel application, good comfort, does not interfere with the normal physiological function of the skin, has a certain water retention effect, and can also promote transdermal absorption of drugs, resulting in better drug delivery effect. It can be used in clinical treatment of bacterial vaginosis. Detailed Implementation

[0020] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0021] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0022] The amino acid sequence of the antimicrobial peptide of the present invention is shown in SEQ ID No. 1. It was synthesized by Gir Chemical and the content of the synthesized antimicrobial peptide is 97.7%.

[0023] Both the standard nutrient agar medium and the nutrient broth medium were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0024] PBS (pH 7.2) was purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0025] Bacillus subtilis CMCC(B) 63501, Staphylococcus aureus CMCC(B) 26003, Escherichia coli CMCC(B) 44102, and Pseudomonas aeruginosa CMCC(B) 10104 were all purchased from Beijing Sanyao Technology Development Co., Ltd.

[0026] Example 1

[0027] Antibacterial activity test

[0028] Add 100 μL of nutrient broth to each well of a 96-well plate. Then, add 100 μL of an antimicrobial peptide aqueous solution (i.e., a mixture of antimicrobial peptide and water) at a concentration of 2048 μg / mL to the first row of the first column. Mix the nutrient broth and antimicrobial peptide aqueous solution in the well thoroughly. Take 100 μL from the first row of the first column and add it to the second row of the first column. Mix thoroughly, then take 100 μL from the second row of the first column and add it to the next row. Repeat this process until 100 μL is taken from the eighth row of the first column and discarded. Then, dilute Staphylococcus aureus CMCC(B) 26003 to 10... 4 Up to 10 5 CFU / mL, 100 μL of bacterial culture was inoculated into each well of a 96-well plate. At this point, the final concentration of the antimicrobial peptide in the first row of the first column was 1024 μg / mL, and the final concentration in the eighth row of the first column was 4 μg / mL. Columns 2 and 3 were prepared as replicates of column 1. Columns 4, 5, and 6 were prepared using purified water as negative controls. Columns 7, 8, and 9 were prepared as positive controls with ampicillin at a final concentration of 1 μg / mL, following the method in column 1. The 96-well plates were incubated overnight at 37°C, and the OD was measured using a microplate reader. 620 The value of . Plot the antimicrobial peptide concentration on the x-axis and OD value on the y-axis. 620 Plot a curve with the values ​​on the ordinate. The concentration corresponding to the first measurement point to the right of the inflection point of the curve is the minimum inhibitory concentration (MIC).

[0029] The determination of minimum inhibitory concentration (MIC) for Bacillus subtilis CMCC(B) 63501, Escherichia coli CMCC(B) 44102, and Pseudomonas aeruginosa CMCC(B) 10104 was the same as the determination of activity against Staphylococcus aureus CMCC(B) 26003.

[0030] The results of the minimum inhibitory concentration (MIC) of the antimicrobial peptides against four types of bacteria are shown in Table 1.

[0031] Table 1. Antimicrobial peptide antibacterial results

[0032]

[0033] As shown in Table 1, antimicrobial peptides at concentrations above 8 μg / mL have good inhibitory effects on both Gram-positive and Gram-negative bacteria.

[0034] Example 2

[0035] In vitro hemolysis rate detection

[0036] The blood sample used for detecting the hemolysis rate of animal erythrocytes by antimicrobial peptides was defibrinated sheep blood.

[0037] The method for detecting the hemolytic activity of antimicrobial peptides was as follows: Red blood cells were washed with PBS buffer (pH 7.2, purchased from Beijing Solarbio Science & Technology Co., Ltd.), centrifuged (1500 rpm, 15 min), and the supernatant was discarded. A 0.9 wt% sodium chloride aqueous solution (92:8 volume ratio) was mixed with the discarded red blood cell solution to prepare an 8% red blood cell suspension. 100 μL of the 8% red blood cell suspension was added to each well of a 96-well plate. Then, an antimicrobial peptide aqueous solution (prepared from antimicrobial peptide and purified water) was added to the first to third columns, resulting in the following final concentrations of antimicrobial peptide in the wells of the first column (rows 1-8): 2048 μg / mL; 1024 μg / mL; 512 μg / mL; 256 μg / mL; 128 μg / mL; 64 μg / mL; 32 μg / mL; 16 μg / mL. The second and third columns were replicates of the first column. PBS buffer was used as the negative control, and 0.1% was used as the positive control. (v / v) Triton X-100 solution. Incubate at 37°C for 1 h, centrifuge at 1500 rpm for 5 min, transfer 100 μl of supernatant to a new 96-well plate, and measure OD using a microplate reader. 414 Value. According to OD 414 The hemolysis rate of the antimicrobial peptide was calculated, and the results are shown in Table 2.

[0038] Method for calculating the hemolysis rate of antimicrobial peptides: (OD of detection well) 414 - Negative control well OD 414 ) / (Positive control well OD 414 - Negative control well OD 414 )×100%.

[0039] Table 2 Results of Antimicrobial Peptide Hemolysis and Blood Activation Detection

[0040]

[0041] The lower the hemolysis rate of an antimicrobial peptide, the lower its hemolytic toxicity. Table 2 shows that the hemolysis rate of the antimicrobial peptide is very low, especially below 2048 μg / mL, where no significant hemolysis occurred.

[0042] Example 3

[0043] Carbomer, glycerol, and triethanolamine were selected, and an orthogonal experiment with 3 levels and 3 factors (see Table 3) was designed to obtain 9 formulations of antimicrobial peptide carbomer gel, from sample 1 to sample 9, as detailed in Table 4.

[0044] Table 3. Orthogonal Design Table with 3 Levels and 3 Factors

[0045]

[0046] Table 4. Antimicrobial peptide carbomer gel obtained through orthogonal experiments.

[0047]

[0048] The preparation steps of the antimicrobial peptide carbomer gel are as follows:

[0049] Step 1: Weigh out carbomer and glycerin according to Table 4.

[0050] Step 2: Take 500 g of sterile water, sprinkle in carbomer 980 powder while stirring at high speed, and continue stirring until the powder is completely dispersed in the sterile water. Add triethanolamine while stirring at low speed to adjust the pH to the value in Table 4 to form a gel matrix.

[0051] Step 3: Add 1.0 g of antimicrobial peptide to 100 g of sterile water, mix well, add glycerin, stir and mix well, and then gradually add it to the gel matrix to make antimicrobial peptide carbomer gel.

[0052] Step 4: Fill and dispense the antimicrobial peptide carbomer gel into vaginal applicators at a rate of 3ml / vial, and then package it.

[0053] The nine samples obtained were subjected to performance tests at 25±2°C to assess product appearance, visible foreign matter, viscosity, pH, and antibacterial activity. The specific test methods are as follows:

[0054] (1) Appearance: Visually observe whether the antimicrobial peptide carbomer gel is colorless to light yellow gel with uniform color under an illumination of 3000 to 5000 lx. The test results are shown in Table 5.

[0055] (2) Visible foreign matter: According to the visible foreign matter test method on page 0904 of Part IV of the 2020 edition of the Chinese Pharmacopoeia: the antimicrobial peptide carbomer gel dispensed in the vaginal applicator was visually inspected under an illuminance of 1000 to 1500 lx. Result judgment: No obvious visible foreign matter such as metal shavings, glass shavings, fibers with a length exceeding 2 mm, or lumps with a maximum particle size exceeding 2 mm should be detected in the antimicrobial peptide carbomer gel. The test results are shown in Table 5.

[0056] (3) Viscosity: The viscosity was measured using a digital viscometer according to the method specified in the 2020 edition of the Chinese Pharmacopoeia. The average value of three measurements was taken for the same sample. The test results are shown in Table 5.

[0057] (4) pH value: The pH value was measured directly using a pH meter. The average value of three measurements was taken for the same sample. The test results are shown in Table 5.

[0058] (5) Antibacterial rate test: The test was conducted according to the People's Republic of China Health Industry Standard WS / T 650-2019, the method for testing antibacterial rate, as follows:

[0059] Staphylococcus aureus CMCC(B) 26003 was inoculated onto ordinary nutrient agar medium and cultured for 24 hours. The culture was then washed with PBS buffer and diluted to approximately 5.0 × 10⁻⁶ ppm. 5 CFU / mL up to 4.5 × 10⁻⁶ 6 CFU / mL bacterial suspension was prepared to obtain the test bacterial suspension. 5.0 mL of antimicrobial peptide carbomer gel was added to a sterile test tube and incubated in a 20℃±1℃ water bath for 5 min. Then, 0.1 mL of the test bacterial suspension was added, and the mixture was quickly mixed to obtain the test sample. Timing was started immediately. Staphylococcus aureus in the test sample was allowed to interact with the antimicrobial peptide solution for 10 min. 1.0 mL of the test sample was then inoculated into two Petri dishes. If subsequent colony counting was impossible due to excessive density, a 10-fold serial dilution with PBS buffer was performed. Then, the same procedure was repeated, inoculating 1.0 mL into two Petri dishes. Ordinary nutrient agar medium was then poured into the Petri dish containing the test sample and mixed thoroughly. The mixture was incubated at 36℃±1℃ for 48 h, and colony counting was performed. The bacterial content in the test sample was determined based on the colony count. The experiment was repeated three times. Simultaneously, a parallel experiment was performed using PBS buffer instead of the antimicrobial peptide carbomer gel as a positive control. Based on colony counting, the bacterial count in the positive control was calculated to be 1.0 × 10⁻⁶. 4 CFU / mL up to 9.0 × 10⁻⁶ 4 The concentration was between CFU / mL. PBS buffer and liquid nutrient medium from the same batch, without the added bacterial suspension, were used as negative controls. The inhibition rate was calculated using the following formula:

[0060]

[0061] In the formula:

[0062] X—Antibacterial rate, %

[0063] A0—Bacterial content of the positive control, in CFU / mL;

[0064] A1—Bacterial content of the test sample, in CFU / mL;

[0065] The method for calculating the activity level difference is as follows:

[0066] Activity level difference =

[0067] The results of the above tests are shown in Table 5.

[0068] Table 5 Results of antimicrobial peptide carbomer gel test

[0069]

[0070] As shown in Table 5, samples 1 to 9 all meet the requirements. Among them, sample 5 is the best, which is the product with the best performance, achieved by adjusting the pH to 5.5 with triethanolamine, 15g of glycerol, and 1.5g of carbomer.

[0071] Example 4

[0072] Stability study of antimicrobial peptide carbomer gel

[0073] Samples 1 to 9 of the antimicrobial peptide carbomer gel prepared in Example 3 were placed in a test chamber at a temperature of 25±2℃, relative humidity of 60±10%, and light intensity of 200±10LX for 6 months. Samples were taken at the end of the 0th, 1st, 2nd, 3rd, and 6th months to investigate the stability of key items. The specific indicators investigated were appearance, visible foreign matter, viscosity, pH, and antimicrobial activity. The investigation method was the same as in Example 3.

[0074] The results are shown in Table 6.

[0075] Table 6. Results of stability study of antimicrobial peptide carbomer gel

[0076]

[0077]

[0078] As shown in Table 6, the stability test over 6 months showed no significant changes in the indicators of appearance, visible foreign matter, viscosity, pH, and antibacterial activity, indicating that the antibacterial peptide carbomer gel has good stability.

[0079] Example 5

[0080] Vaginal mucosal irritation test of carbomer gel

[0081] 1. Testing Basis: Section 2.3.5 of the "Disinfection Technical Specifications" (2002 Edition) – Vaginal Mucosal Irritation Test

[0082] 2. Sample preparation: The carbomer gel of sample 5 prepared in Example 3 was used.

[0083] 3. Test methods:

[0084] 3.1 Preparation of experimental animals: Healthy, newly-adult female white New Zealand rabbits of the same strain, weighing 2.0 kg to 2.5 kg, were selected. Before the experiment, the vaginal opening of each animal was carefully examined for any discharge, congestion, edema, or other injuries. The animals were divided into a stimulation group and a control group, with 3 rabbits in each group.

[0085] 3.2 Stimulation Method: Animals were immobilized, exposing the perineum and vaginal opening. A vaginal applicator filled with 3 mL of the test carbomer gel was moistened with the test solution and gently inserted 4-5 cm into the vagina. 3 mL of the test carbomer gel was slowly injected, and the applicator was then withdrawn, completing the stimulation. Control animals were treated with physiological saline in the same manner. 24 hours after stimulation, animals were euthanized using an air embolization method, and the intact vagina was removed. The vagina was longitudinally incised for observation and fixation. Tissue sections were prepared from three locations: both ends and the center of the vagina. After HE staining, histopathological examination was performed. Scoring and grading were conducted according to the relevant provisions in the "Disinfection Technical Specifications" (2002 edition), and the vaginal stimulation index was calculated.

[0086] The formula for calculating the vaginal stimulation index is as follows:

[0087] Stimulation index = Average score of the stimulation group - Average score of the control group

[0088] The results are shown in Table 7.

[0089] Table 7. Pathological Examination Record of Vaginal Mucosal Irritation Test in New Zealand Rabbits

[0090]

[0091] According to Table 7, the carbomer gel had a vaginal mucosal irritation score of 0.45 (<1) in New Zealand rabbits, indicating that the response intensity of the vaginal irritation test in New Zealand rabbits was non-irritating.

[0092] Example 6

[0093] The antimicrobial peptide carbomer gel formulation and preparation method differ from those of sample 5 in Example 3 in that carbomer 980 is replaced with carbomer 941.

[0094] The stability test was conducted in the same manner as in Example 3, and the results are shown in Table 8.

[0095] Example 7

[0096] The antimicrobial peptide carbomer gel formulation and preparation method differ from those of sample 5 in Example 3 in that carbomer 980 is replaced with carbomer 934.

[0097] The stability test was conducted in the same manner as in Example 3, and the results are shown in Table 8.

[0098] Example 8

[0099] The antimicrobial peptide carbomer gel formulation and preparation method differ from those of sample 5 in Example 3 in that glycerol is replaced with propylene glycol.

[0100] The stability test was conducted in the same manner as in Example 3, and the results are shown in Table 8.

[0101] Example

[0102] The antimicrobial peptide carbomer gel formulation and preparation method differ from those of sample 5 in Example 3 in that glycerol is replaced with ethanol.

[0103] The stability test was conducted in the same manner as in Example 3, and the results are shown in Table 8.

[0104] Example 10

[0105] The antimicrobial peptide carbomer gel formulation and preparation method differ from those of sample 5 in Example 3 in that glycerol is replaced with sorbitol.

[0106] The stability test was conducted in the same manner as in Example 3, and the results are shown in Table 8.

[0107] Example 11

[0108] The antimicrobial peptide carbomer gel formulation and preparation method differ from those of sample 5 in Example 3 in that glycerol is replaced with polyethylene glycol.

[0109] The stability test was conducted in the same manner as in Example 3, and the results are shown in Table 8.

[0110] Table 8

[0111]

[0112] According to Table 8, based on the results of samples 1-9 in Example 3 and Examples 5-11, the pH range of the antimicrobial peptide is 4.5-6.5. Carbomer 980, Carbomer 934, and Carbomer 941 are suitable gelling agents, with Carbomer 980 being the best. Glycerin, propylene glycol, ethanol, sorbitol, and polyethylene glycol are suitable moisturizers, with glycerin being the best.

Claims

1. An antimicrobial peptide having the amino acid sequence shown in SEQ ID No.

1.

2. A composition comprising the antimicrobial peptide of claim 1.

3. The composition according to claim 2, characterized in that, The composition also includes a gelling agent, a moisturizing agent, a pH adjuster, and water.

4. The composition according to claim 3, characterized in that, 0.5 to 2.0 parts by weight of antimicrobial peptide, 1 to 2 parts by weight of gelling agent, 10 to 20 parts by weight of moisturizer, and 500 to 700 parts by weight of sterile water.

5. The composition according to claim 3, characterized in that, The gelling agent is selected from carbomer.

6. The composition according to claim 3, characterized in that, The gelling agent is selected from at least one of carbomer 980, carbomer 934 and carbomer 941.

7. The composition according to claim 3, characterized in that, The moisturizer is selected from at least one of glycerin, propylene glycol, ethanol, sorbitol and polyethylene glycol.

8. The composition according to claim 3, characterized in that, The pH adjuster is selected from triethanolamine and / or sodium hydroxide.

9. The composition according to claim 3, characterized in that, The pH value of the composition is 4.7 to 7.

3.

10. The use of the antimicrobial peptide according to claim 1 or the composition according to any one of claims 2 to 9 in the preparation of a medicament against at least one of Staphylococcus spp., Streptococcus spp., Enterococcus spp., Micrococcus spp., Moraxella spp., Corynebacterium spp., Klebsiella spp., Enterobacter spp., Serratia spp., Proteus spp., Pseudomonas spp., Morganella morganii, Haemophilus spp., Xanthomonas spp., Acinetobacter spp., and Propionibacterium spp.; Preferably, the application is in the preparation of a medicament for use against at least one of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa; Preferably, the application is in the preparation of a medicament for treating at least one of bacterial vaginosis.

Images