CN114209808A - Application of polypeptide RK12 in preparation of medicine for treating acne - Google Patents
Application of polypeptide RK12 in preparation of medicine for treating acne Download PDFInfo
- Publication number
- CN114209808A CN114209808A CN202111596950.1A CN202111596950A CN114209808A CN 114209808 A CN114209808 A CN 114209808A CN 202111596950 A CN202111596950 A CN 202111596950A CN 114209808 A CN114209808 A CN 114209808A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- propionibacterium acnes
- clindamycin
- resistant
- inflammatory
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/10—Anti-acne agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides application of a polypeptide RK12 in preparing a medicine for treating acne. The new application of the polypeptide RK12 is used for preparing the medicine for treating acne, the polypeptide RK12 has obvious inhibition effect on propionibacterium acnes and clindamycin-resistant propionibacterium acnes, and compared with antibiotics, the polypeptide RK12 has low drug resistance and is concentration and time-dependent, the research result of an in vitro antibacterial spectrum experiment also proves that the polypeptide RK12 can quickly inhibit the growth of propionibacterium acnes and clindamycin-resistant propionibacterium acnes, the research result of an in vitro anti-inflammatory experiment also proves that the polypeptide RK12 has obvious anti-inflammatory effect, and the antibacterial mechanism different from the antibiotics is less prone to generate drug-resistant bacteria. According to the novel strategy and thought for treating acne provided by the invention, the antibacterial action mechanism of the novel strategy and thought is helpful for clinically limiting the influence of antibiotic resistance.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, and in particular relates to application of a polypeptide RK12 in preparation of a medicine for treating acne.
Background
Acne vulgaris is a chronic bacterial sebaceous gland inflammatory disease that commonly forms on the face and upper torso (anterior chest and back) of adolescents and is characterized by the formation of comedones, papules, nodules, pustules and cysts. It is a common skin disease, accounting for over 35% to 90% of adolescent infected individuals. Acne lesions are usually classified as non-inflammatory (open and closed comedones) or inflammatory (papules and pustules). Inflammatory acne is difficult to treat, often increases in severity, and may lead to scarring. Current studies indicate that the pathophysiology of acne involves three factors: seborrheic excess of sebum, abnormal keratinization of hair follicles and the proliferation of propionibacterium acnes in pilosebaceous glands. Due to their interaction, the microenvironment of the skin changes and leads to an inflammatory response in the host, contributing to acne lesions. Among them, the overgrowth of Propionibacterium acnes is widely recognized as a key factor in the formation of acne vulgaris.
Propionibacterium acnes (p.acnes) is a gram-positive bacterium associated with dermatological acne, an anaerobic bacterium with pleomorphism belonging to the phylum actinomycetales, which is parasitic in the cell and is part of the normal microflora that form the skin, the oral cavity, and the gastrointestinal and genitourinary tracts.
Traditional methods of treating acne use antibiotics, which are essential conditions for the growth or survival of microorganisms, such as inactivation and denaturation of enzymes, to achieve bactericidal effects, and bacteria can be sufficiently resistant to attack by such antibiotics by changing only one gene. In recent years, due to the wide application of antibiotics, the drug resistance of some countries reaches 60%, and the antibiotic resistance phenomenon has become an important threat in public health.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides application of the polypeptide RK12 in preparing a medicine for treating acne.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of a polypeptide RK12 as an active ingredient in preparing a medicine for treating acne, wherein an amino acid sequence of the polypeptide RK12 is shown as SEQ ID NO. 1.
Further, the small molecular polypeptide has anti-propionibacterium acnes and anti-inflammatory effects, and the minimum inhibitory concentration of the polypeptide RK12 on propionibacterium acnes is 8 mug/mL.
Furthermore, the small molecular polypeptide also has the effect of resisting clindamycin-resistant propionibacterium acnes, and the minimum inhibitory concentration of the polypeptide RK12 to the clindamycin-resistant propionibacterium acnes is 8 mug/mL.
Further, the dose of the polypeptide RK12 as an effective component is 50-200 mug. The small molecular polypeptide RK12 provided by the invention is soluble in aqueous solution, and exists in the preparation of the medicine for treating acne as an effective active ingredient, the effective concentration for treating acne is in the range of about 0.0002% -90%, in other embodiments, the polypeptide RK12 is used as the only active ingredient for preparing the medicine for treating acne, and the effective dose for treating acne is 50-200 mug.
Further, the polypeptide RK12 is prepared into a pharmaceutical preparation. The medicinal preparation takes small molecular polypeptide as an effective active ingredient, and comprises a liquid preparation, a solid preparation, a semi-solid preparation or a gas preparation and the like.
Furthermore, the dosage form of the pharmaceutical preparation comprises any pharmaceutically acceptable dosage form.
Further, the pharmaceutical preparation also comprises any one or more pharmaceutically acceptable pharmaceutical excipients.
Further, the small molecule polypeptide is used for preparing a medicament for applying to skin or skin wounds.
The technical scheme provided by the invention has the beneficial effects that:
the invention provides a new application of a small molecular polypeptide RK12, which is used for preparing acne treatment medicines, has obvious inhibiting effect on propionibacterium acnes and clindamycin-resistant propionibacterium acnes, has low drug resistance compared with antibiotics, and also proves that the small molecular polypeptide RK12 can quickly inhibit the growth of propionibacterium acnes and clindamycin-resistant propionibacterium acnes through the research result of in vitro antibacterial spectrum experiments, the bacteriostasis mechanism of the small molecular polypeptide RK12 relates to the interaction with the cell wall membrane of bacteria and induces the leakage of the content in the cells, the integrity of the bacteria is damaged to cause the death of the bacteria cells, the permeability of the cell wall membrane of the bacteria can be changed, the synthesis of cell walls, proteins and DNA and the enzyme activity can be reduced, the small molecular polypeptide RK12 can also participate in the immunoregulation function and reduce the infection besides directly killing the microorganisms and indirectly killing the microorganisms, for example, the secretion of proinflammatory cytokines is controlled to prevent tissue damage caused by over-high secretion of the cytokines, the recruitment and activation of immune cells, the promotion of angiogenesis and the like, and research results of in vitro anti-inflammatory experiments also prove that the proinflammatory cytokines have obvious anti-inflammatory effects, and are different from antibiotic bacteriostatic mechanisms and are less prone to generate drug-resistant bacteria. According to the novel strategy and thought for treating acne provided by the invention, the antibacterial action mechanism of the novel strategy and thought is helpful for clinically limiting the influence of antibiotic resistance.
Drawings
FIG. 1 is a graph of RK12 time-antimicrobial effect;
FIG. 2a is a scanning electron micrograph of a blank set without drug treatment;
FIG. 2b is a scanning electron micrograph of experimental group 1 after RK12 treatment;
FIG. 2c is a SEM image of experimental group 2 after RK12 treatment;
FIG. 2d is a scanning electron micrograph of experimental group 3 after RK12 treatment;
FIG. 3a is a graph of PBS injection into the ear endothelium of mice in an inflammatory animal model treated with Propionibacterium acnes;
figure 3b is a blank control plot of an inflammatory animal model treated with propionibacterium acnes;
FIG. 3c is a graph of ear inflammation in mice administered 20 μ g/ml RK12 in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 3d is a graph of ear inflammation in mice administered 100 μ g/ml RK12 in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 3e is a graph of ear inflammation in mice administered 200 μ g/ml RK12 in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 3f is a graph of ear inflammation in mice administered with 0.05 μ g/ml clindamycin in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 4a is a graph showing HE staining results of ear tissues of mice injected with PBS in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 4b is a graph of HE staining of ear tissue of naive mice in an animal model of inflammation treated with Propionibacterium acnes;
FIG. 4c is a graph of HE staining of mouse ear tissue following treatment with 20 μ g/ml RK12 in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 4d is a graph of HE staining of mouse ear tissue following treatment with 100 μ g/ml RK12 in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 4e is a graph of HE staining of mouse ear tissue following treatment with 200 μ g/ml RK12 in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 4f is a graph of HE staining of mouse ear tissue following treatment with 0.05 μ g/ml clindamycin in an inflammatory animal model treated with Propionibacterium acnes;
FIG. 5a is a graph of PBS injection into the ear endothelium of mice in an inflammatory animal model treated with clindamycin-resistant Propionibacterium acnes;
FIG. 5b is a blank control plot of an inflammatory animal model treated with clindamycin-resistant Propionibacterium acnes;
FIG. 5c is a graph of ear inflammation in mice administered 200 μ g/ml RK12 in an inflammatory animal model treated with clindamycin-resistant Propionibacterium acnes;
FIG. 5d is a graph of ear inflammation in mice administered with 10 μ g/ml clindamycin in an inflammatory animal model treated with clindamycin-resistant Propionibacterium acnes;
FIG. 6a is a graph of HE staining of ear tissue in mice injected with PBS in an inflammatory animal model treated with clindamycin-resistant Propionibacterium acnes;
FIG. 6b is a graph of HE staining of ear tissue from naive mice in an animal model of inflammation treated with clindamycin-resistant Propionibacterium acnes;
FIG. 6c is a graph of HE staining of mouse ear tissue following treatment with 200 μ g/ml RK12 in an inflammatory animal model treated with clindamycin-resistant Propionibacterium acnes;
FIG. 6d is a graph of HE staining of mouse ear tissue after treatment with 100 μ g/ml RK12 in an inflammatory animal model treated with clindamycin-resistant Propionibacterium acnes.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in detail below with reference to the accompanying drawings and specific examples.
The amino acid sequence of the small molecule polypeptide RK12 is RWWRFFKFWWKK, as shown in SEQ ID NO.1, the antibacterial activity of the small molecule polypeptide RK12 has high correlation with specific amino acid at a specific position, and therefore the antibacterial activity of RK12 can be influenced by physicochemical parameters including hydrophile/hydrophobicity, isoelectric point, amino acid composition and the like. AMPs initially target microbial membranes through electrostatic interactions, and small-molecule cationic polypeptides RK12 have a hydrophobicity of-1.325, and are capable of recognizing anionic microbial membranes and binding and folding to form an amphibious structure, and the hydrophobic surface of the amphibious structure is fused into a phospholipid bilayer interface, so that the membranes are cracked and collapsed. In addition, the small molecular polypeptide RK12 is rich in tryptophan and phenylalanine with high hydrophobic residues, so that the antimicrobial activity of the peptide is improved, the capability of penetrating bacterial membranes is increased, the membrane structure is changed, and the antibacterial peptide is not easy to generate drug resistance.
The small molecular polypeptide RK12 is a novel AMP designed and synthesized based on a natural AMP sequence, 10 novel short peptides are designed according to the amino acid sequence of a 20-residue peptide RanacyclinAJ in frog skin secretion, and the peptides are named as AKK1, AKK2 and the like until AKK 10. The small molecule polypeptide RK12 is a novel peptide which is derived from AKK8, the amino acid sequence of AKK8 is RWRFKWWKK, and the peptide is rich in tryptophan and lysine. On the basis of AKK8, the small molecule polypeptide RK12 improves the alpha-helix degree and the amphipathy of the polypeptide by increasing the residue tryptophan with high hydrophobicity, thereby improving the antibacterial activity and the selectivity of the polypeptide. Compared with AKK8, the small molecule polypeptide RK12 also increases hydrophobic phenylalanine, improves the ability of the peptide to penetrate bacterial membrane and changes the membrane structure, so that the antibacterial peptide is not easy to generate drug resistance.
The small molecular polypeptide RK12 can be prepared by artificial synthesis, namely a polypeptide solid phase synthesis method, can be used as a polypeptide of an antimicrobial agent and related compounds thereof, and solves the increasingly serious problem of drug resistance of bacteria and fungi and the pain of stubborn infection to patients. The small molecular polypeptide RK12 has stable antibacterial effect, no toxicity and easy acceptance by human bodies, can be applied to various stubborn infectious diseases and common infections, particularly shows strong inhibition effect on propionibacterium acnes and clinical drug-resistant propionibacterium acnes, has the minimum inhibitory concentration of 8 mug/mL, can kill the propionibacterium acnes or the drug-resistant propionibacterium acnes in a short time, can last for hours, and can be used as an excellent substitute drug or an auxiliary drug for treating acne.
The invention utilizes the polypeptide solid phase technology to design from the beginning, optimizes on the basis of AKK8 sequence, and synthesizes to obtain the small molecule polypeptide RK 12. It has simple structure, low hemolytic activity, low cytotoxicity and high physiological stability.
In clinical practice, microbial infections include infections caused by one or more pathogens of bacteria, viruses, fungi or protozoa. The small molecular polypeptide RK12 shows different antibacterial effects on propionibacterium acnes, drug-resistant propionibacterium acnes, staphylococcus aureus, streptococcus mutans, bacillus subtilis and escherichia coli, particularly has the strongest antibacterial effect on propionibacterium acnes and drug-resistant propionibacterium acnes, and simultaneously shows excellent anti-inflammatory effect.
In one exemplary embodiment of the present invention, a pharmaceutical formulation is provided. The pharmaceutical preparation comprises an effective dose of the small molecule polypeptide RK12, such as a pharmaceutically acceptable carrier, excipient or diluent.
The preferable dosage range of the raw material drug of the small molecular polypeptide RK12 is 50-200 mug parts by weight.
Common auxiliary materials for preparing the liquid preparation comprise: mannitol, ethanol, propylene glycol, glycerol, hydrochloric acid, acetic acid, sodium acetate, lactic acid, sodium hydroxide, citric acid, sodium citrate, tartaric acid, sodium tartrate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium carbonate, sodium sulfite, sodium metabisulfite, sodium thiosulfate, phenol, benzyl alcohol, thimerosal, sodium chloride, glucose, disodium ethylene diamine tetraacetate, gelatin, methyl cellulose, carboxymethyl cellulose, pectin, lactose, sucrose, polyoxyethylene castor oil, polysorbate, povidone, and the like.
The common auxiliary materials for preparing the solid preparation comprise: polyvinylpyrrolidone, talc, lactose, dextrin, sucrose, mannitol, glucose, sorbitol, fructose, erythrose, sodium chloride, starch, microcrystalline cellulose, monocalcium phosphate, magnesium carbonate, calcium sulfate, aluminum silicate, calcium silicate, methyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, ethyl cellulose, polyethylene glycol, polyvinyl alcohol, povidone, gelatin, gum arabic, tragacanth, sodium alginate, agar-agar, alginic acid, sodium alginate, hydroxypropyl starch, sodium carboxymethyl starch, calcium carboxymethyl cellulose, magnesium stearate, calcium stearate, stearic acid, colloidal silica, polyethylene glycol, sodium lauryl sulfate, magnesium lauryl sulfate, polyoxyethylene monostearate, polyoxyethylene lauryl ether, and the like.
The auxiliary materials for preparing the oral liquid preparation comprise: ethanol, ethylparaben, methylparaben, sodium benzoate, sorbic acid, honey, sucrose, sodium bisulfite, sodium thiosulfate, ascorbic acid, thiourea, disodium edetate, phosphoric acid, citric acid, citrate, tartaric acid, tartrate, glycerol, lactose, polyoxyethylene fatty acid esters, potassium iodide, sodium acetate, propylene glycol, polyethylene glycol, benzyl alcohol, parabens, benzalkonium bromide, o-phenylphenol, eucalyptus oil, cassia oil, peppermint oil, mannitol, aspartame, sodium saccharin, aspartame, essence, gum arabic, sodium carboxymethylcellulose, agar, gelatin, methyl cellulose, sodium bicarbonate, pigments, gum arabic, tragacanth, peach gum, sodium alginate, agar, starch slurry, bentonite, methyl cellulose, sodium carboxymethylcellulose, hydroxymethyl cellulose, carbopol, Povidone, dextran, aluminum monostearate, polysorbates, polyoxyethylene castor oils, poloxamers, sodium stearate, potassium stearate, sodium oleate, calcium stearate, sodium lauryl sulfate, cetyl sulphated castor oil, monoglycerides, triglycerides, polyglyceryl stearates, sucrose monolaurate, sorbitan fatty acids, polysorbates, calcium hydroxide, zinc hydroxide, cetyl alcohol, beeswax, glyceryl monostearate, stearic acid, stearyl alcohol and the like.
The common auxiliary materials for preparing the external preparation comprise: mannitol, polysorbate-80, polyoxyl stearate, triethanolamine, polyvinylpyrrolidone, sodium bicarbonate, sodium chloride, glucose, boric acid, borax, vaseline, paraffin, liquid paraffin, lanolin, beeswax, spermaceti wax, dimethicone, stearic acid, paraffin, stearyl alcohol, glycerin, propylene glycol, sorbitol, methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, sodium alginate, alginic acid, bentonite, carbomer, pectin, alkyl gallate, butylated hydroxyanisole, butylated hydroxytoluene, ascorbic acid, isoascorbic acid, sulfite, citric acid, tartaric acid, disodium ethylenediaminetetraacetate, mercaptopropionic acid, ethanol, isopropanol, chlorobutanol, phenyl-p-chlorophenylpropanediol, phenoxyethanol, benzoic acid, dehydroacetic acid, sodium chloride, potassium chloride, sodium chloride, potassium chloride, sodium chloride, potassium chloride, sodium chloride, Propionic acid, sorbic acid, cinnamic acid, anisole, citronellal, eugenol, vanillyl ester, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, phenol, cresol, thymol, p-chloro-o-cresol, p-chloro-m-xylenol, policosanoic acid, salicylic acid, esters of p-hydroxybenzoic acid (acetic acid, propionic acid, butyric acid), benzalkonium chloride, alkyltrimethylammonium bromide, theobromate, semisynthetic cocoate, semisynthetic palm oil, propylene glycol stearate, polyethylene glycol, monostearate, poloxamer, stearic acid, hydrogenated castor oil, glyceryl monostearate, aluminum stearate, and the like.
The small molecular polypeptide RK12 raw material medicine can be matched with the common pharmaceutic adjuvants in a certain proportion, and can be prepared into any one of oral agents, injections, tablets, powder, pills, emulsions, suspensions, capsules, granules, oral liquids, ointments, gels, eye drops, film sticking agents, suppositories, sprays, aerosols, film coating agents and lotions according to the conventional method in the field.
In a specific embodiment, the method for treating acne provided by the invention comprises the step of applying an effective dose of the small molecule polypeptide RK12 to skin or skin wounds, wherein the effective dose is 100-200 mug parts by weight.
The present invention will be described in further detail with reference to specific examples.
Example 1:
research on resistance of small-molecule polypeptide RK12 to Propionibacterium acnes
Preparation of small molecule polypeptide RK12
The invention adopts an Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase chemical synthesis method for alpha-amino to synthesize the antibacterial peptide with the amino acid sequence of RWWRFFKFWWKK, firstly puffing the resin (the purpose of puffing the resin is to expose active groups on the resin), then adding 2.5mL of 20 percent piperidine into the puffed resin for deprotection, and simultaneously activating the amino acid. Adding activated amino acid into resin which is subjected to deprotection and cleaning for coupling, draining liquid in a tube when the coupling is completed, cleaning the tube with DMF for 8 times, transferring the resin into a cracking tube after all the synthesis is completed, and adding a cracking solution (TFA: phenol: Tis: EDT: 87.5: 5: 5: 2.5) for cracking. And finally, adding glacial ethyl ether into the lysate to 45ml, centrifuging for 5min at the rotating speed of 4000r/min, taking out the supernatant, and repeating twice to obtain the lower-layer crude peptide chain product required by people.
The specific method for HPLC separation and purification comprises the following steps:
separating and purifying the synthesized crude polypeptide product by reverse phase high performance liquid chromatography (RP-HPLC, column type: XB-C18 reverse phase column) of preparative Waters2615, wherein the mobile phase uses ultrapure water and acetonitrile; detection wavelengths of 215nm and 280 nm; the flow rate is 20 mL/min; wherein the mobile phase A is acetonitrile, and the phase B is ultrapure water containing 0.1% TFA. Collecting each elution peak, identifying target peak by mass spectrum, collecting the elution peak of purer target protein, freeze-drying by a freeze dryer, and storing at-20 ℃.
The purity of the prepared small molecular polypeptide RK12 is not less than 95%.
Secondly, performing antibacterial spectrum determination on the prepared small molecular polypeptide RK12
And (3) culturing bacteria: propionibacterium acnes (ATCC6919) was grown under anaerobic conditions in BHI medium; staphylococcus aureus (Staphylococcus aureus), Candida albicans (Albus strain-coccus), Bacillus subtilis, and Escherichia coli (Escherichia coli) were grown in an agar medium under aerobic conditions, and all strains were cultured at 37 ℃.
Preparing a bacterial suspension: the concentration of bacteria is generally measured by using a Mach's turbidimeter, which has a turbidity of about 0.5 Mach's turbidity, in which case the number of bacterial colonies is about 1X 108cfu/ml, then diluted to 10 at a ratio of 1:10005-106cfu/ml bacterial suspension.
And (3) antibacterial experiment: the peptide concentration was 128-0.5. mu.g/mL. The bacterial suspension was taken at logarithmic growth phase and Propionibacterium acnes (P.acnes ATCC6919) and clindamycin-Resistant Propionibacterium acnes (Resistant P.acnes) were diluted to a final concentration of 1X 106CFU/mL, other strains were diluted to 1X 105CFU/mL. 100 μ L of bacterial suspension was added to each well. After 96-hour static culture of propionibacterium acnes and 17-20 hour static culture of other strains, the absorbance of the bacteria liquid at 600nm is measured by a microplate reader, the MIC value is the lowest observed drug concentration, no sign of bacterial growth exists, and the average value of the sample concentration of a hole in which the bacterial growth cannot be detected and the sample concentration of an adjacent hole is taken as the MIC value. Test results 4 to 6 independent experiments were repeated.
The results are shown in table 1, and the antimicrobial activity of small molecule polypeptides against propionibacterium acnes was tested by using MIC. The MIC of polypeptide RK12 for Propionibacterium acnes was 8 μ g/mL, which was the lowest among the other tested bacterial strains such as Staphylococcus aureus and Streptococcus mutans.
TABLE 1 measurement of antibacterial spectrum of small molecule polypeptide RK12
The result shows that the small molecular polypeptide RK12 has specificity on resisting propionibacterium acnes, in addition, the propionibacterium acnes resistant to clindamycin is cultured according to different sterilization mechanisms of antibacterial peptide and antibiotic, and the MIC determination is carried out on the resistant bacteria, the result is shown in Table 1, the MIC of the small molecular polypeptide RK12 on the propionibacterium acnes resistant to clindamycin is 8 mug/mL, and is consistent with the MIC result of propionibacterium acnes vulgaris, and the peptide also has a good bacteriostatic effect on the propionibacterium acnes resistant to clindamycin.
Next, the kinetics of Propionibacterium acnes killing at various time points with the small molecule polypeptide RK12 was measured, as shown in FIG. 1, by treating Propionibacterium acnes with a concentration of RK12 of 1 × MIC (1 × 10)6CFU/mL) for 45 minutes, the antibacterial effect is obvious compared with that of clindamycin group under the same condition, and the rapid antibacterial effect of the small molecular polypeptide RK12 is far higher than that of clindamycin which is a clinical common antibiotic. Compared with broad-spectrum antibiotics, the small-molecule polypeptide therapy focuses more on pathogens, has a rapid antibacterial effect on bacteria, and reduces the development of bacterial drug resistance.
Thirdly, researching mechanism of micromolecule polypeptide for resisting propionibacterium acnes
Propionibacterium acnes (5X 10)6CFU/mL) was treated with 10. mu.g/mL RK12 for 2 hours. The bacteria were harvested by centrifugation (1000 Xg, 5min), washed twice with PBS buffer, and then treated with 2.5% glutaraldehyde overnight. Cells were washed with 0.1M phosphate for 15 minutes at least three times and fixed with 1% osmium tetroxide for 2-3 hours. The dehydration conditions were as follows: 50% ethanol 15-20 minutes, 70% ethanol 15-20 minutes, 90% ethanol 15-20 minutes and 90% acetone (1: 1, v/v). The incubation conditions were as follows: acetone and embedding medium (2: 1, v/v) were incubated at room temperature for 3-4 hours, acetone and embedding medium (1: 2, v/v) were incubated at room temperature overnight, 100% embedding medium was incubated at 37 ℃ for 2-3 hours. The curing conditions were 37 ℃ overnight, 45 ℃ for 12 hours and 60 ℃ for 24 hours. After slicing, the sample was observed by scanning electron microscopy using 3% uranyl acetate and lead citrate as dyes.
The results are shown in FIGS. 2a-2d, revealing RK12 killer by using Transmission Electron Microscopy (TEM)The possible mechanisms of bacteria, fig. 2a is a blank group without drug treatment, and fig. 2b, 2c and 2d are experimental group 1, experimental group 2 and experimental group 3 after treatment with RK 12. Propionibacterium acnes (1X 10) was compared with blank group6CFU/mL) was treated with 10. mu.g/mL RK12 for 2 hours, the arrows indicated that Propionibacterium acnes had significant leakage of bacterial contents, indicating that the bacterial wall membrane integrity of Propionibacterium acnes treated with RK12 was compromised.
Example 2:
anti-inflammatory study of Small molecule polypeptide RK12
(1) To study the anti-inflammatory effect of RK12 on propionibacterium acnes-induced skin disease, the present invention validated the anti-inflammatory effect of the peptide by monitoring changes in the animal ear using an inflammatory animal model of propionibacterium acnes treatment.
Female Kunming mice (22-25g) (n ═ 5) at 6 weeks of age were randomly divided into 5 groups of 5 mice each. The group injected with PBS in the ear endothelium of mice is shown in FIG. 3a, the blank control is shown in FIG. 3b, and the injection concentration in the ear endothelium of the remaining three groups of mice is 1X 107CFU/20. mu.L of Propionibacterium acnes. Thereafter, RK12(20,100,200 μ g) (as shown in FIGS. 3c, 3d, 3 e) and clindamycin (0.05 μ g) (as shown in FIG. 3 f) were applied separately to the surface of the right ear. After 72 hours, the animals were sacrificed by cervical dislocation and the ears were quickly excised. The results showed that erythema and swelling of the skin of the mouse ear was observed 24h after injection of propionibacterium acnes, and no swelling was observed in the ears of the control mice injected with PBS only. After 72 hours of treatment, the change of the degree of the ear inflammation of the mice by different drug groups can be observed, wherein the right ear inflammatory response of the mice in the RK12 group is obviously reduced, and the right ears of the mice in the clindamycin group and the control group have necrosis conditions of different degrees. Next, histological testing was used to confirm the anti-inflammatory effect of RK12 in the mouse model.
The results are shown in FIGS. 4a-4f, FIG. 4a shows HE staining of ear tissue of mice injected with PBS, FIG. 4b shows HE staining of ear tissue of blank mice, FIGS. 4c, 4d, and 4e show HE staining of ear tissue of mice treated with 20,100, 200. mu.g/ml RK12, and FIG. 4f shows HE staining of ear tissue of mice treated with 0.05. mu.g/ml clindamycin. HE staining histological analysis showed a significant increase in the number of inflammatory cells infiltrated by the control group, especially at the site of bacterial injection. When RK12 is applied to the upper epidermis of the mouse ear, infiltration inflammatory cells and inflammation induced by Propionibacterium acnes are reduced, and the treatment effect is changed according to the concentration of RK12, and the treatment effect is changed in dependence on the concentration of RK 12. Thus, RK12 is proved to have certain treatment effect on Propionibacterium acnes.
(2) To study the anti-inflammatory effect of RK12 on clindamycin-resistant propionibacterium acnes-induced skin disease, the present invention verifies the anti-inflammatory effect of the peptide by monitoring changes in the animal ear using an inflammatory animal model of clindamycin-resistant propionibacterium acnes treatment.
Female Kunming mice (22-25g) (n ═ 5) at 6 weeks of age were randomly divided into 3 groups of 5 mice each. As shown in fig. 5a, the PBS group was injected into the endothelium of the mouse ear; as shown in FIG. 5b, the blank control was injected at a concentration of 1X 10 in the ear endothelium of the remaining three groups of mice7CFU/20. mu.L of Propionibacterium acnes resistant to clindamycin. Thereafter, 200. mu.g/ml RK12 (as shown in FIG. 5 c) and 10. mu.g/ml clindamycin (as shown in FIG. 5 d) will be administered to the surface of the right ear, respectively. After 72 hours, the animals were sacrificed by cervical dislocation and the ears were quickly excised. The results showed that erythema and swelling of the ear skin of the mice was observed 24 hours after injection of propionibacterium acnes, and no swelling was observed in the ears of the control mice injected with PBS only. After 72 hours of treatment, the change of the degree of the ear inflammation of the mice by different drug groups can be observed, wherein the inflammatory response of the right ear of the mice in the RK12 group with 200 mug/ml is obviously reduced, and the necrosis of the right ear of the mice in the clindamycin group and the blank group with different degrees appears. Next, histological testing was used to confirm the anti-inflammatory effect of RK12 in the mouse model.
Results are shown in FIGS. 6a-6d, in which FIG. 6a shows HE staining of ear tissue of mice injected with PBS, FIG. 6b shows HE staining of ear tissue of blank mice, FIG. 6c shows HE staining of ear tissue of mice treated with 200. mu.g/ml RK12, and FIG. 6d shows HE staining of ear tissue of mice treated with 10. mu.g/ml clindamycin. HE staining histological analysis showed a significant increase in the number of inflammatory cells infiltrated by the control group, especially at the site of bacterial injection. Whereas the infiltration of inflammatory cells and inflammation induced by clindamycin-resistant propionibacterium acnes was reduced after administration of RK12 on the epithelium of the mouse ear, thereby demonstrating that RK12 has some therapeutic effect on clindamycin-resistant propionibacterium acnes.
Sequence listing
The features of the embodiments and embodiments described herein above may be combined with each other without conflict.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Paeder biomedical Co., Suzhou Ltd
<120> application of polypeptide RK12 in preparation of medicine for treating acne
<141> 2021-12-23
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> Artificial Sequence
<400> 1
Arg Trp Trp Arg Phe Phe Lys Phe Trp Trp Lys Lys
1 5 10
Claims (8)
1. Use of a polypeptide RK12 as active ingredient for the manufacture of a medicament for the treatment of acne.
2. The use of claim 1, wherein: the polypeptide RK12 has anti-propionibacterium acnes and anti-inflammatory effects, and the minimum inhibitory concentration of the polypeptide RK12 to propionibacterium acnes is 8 mug/mL.
3. The use of claim 1, wherein: the polypeptide RK12 also has the effect of resisting clindamycin-resistant propionibacterium acnes, and the minimum inhibitory concentration of the polypeptide RK12 to the clindamycin-resistant propionibacterium acnes is 8 mug/mL.
4. The use of claim 1, wherein: the dosage of the polypeptide RK12 as an effective component is 50-200 mug.
5. The use according to any one of claims 1 to 4, wherein: the polypeptide RK12 is prepared into a pharmaceutical preparation, and the pharmaceutical preparation comprises any one of a liquid preparation, a solid preparation, a semisolid preparation and a gas preparation.
6. Use according to any one of claims 5, characterized in that: the dosage form of the pharmaceutical preparation comprises any one pharmaceutically acceptable dosage form.
7. The use of claim 5, wherein: the pharmaceutical preparation also comprises any one or more pharmaceutically acceptable pharmaceutical excipients.
8. The use according to any one of claims 1 to 4, wherein: use of the polypeptide RK12 in the manufacture of a pharmaceutical formulation for administration to skin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111596950.1A CN114209808B (en) | 2021-12-24 | 2021-12-24 | Application of polypeptide RK12 in preparation of medicines for treating acne |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111596950.1A CN114209808B (en) | 2021-12-24 | 2021-12-24 | Application of polypeptide RK12 in preparation of medicines for treating acne |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114209808A true CN114209808A (en) | 2022-03-22 |
| CN114209808B CN114209808B (en) | 2023-08-18 |
Family
ID=80705533
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111596950.1A Active CN114209808B (en) | 2021-12-24 | 2021-12-24 | Application of polypeptide RK12 in preparation of medicines for treating acne |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114209808B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114209809A (en) * | 2021-12-24 | 2022-03-22 | 苏州佩德生物医药有限公司 | Combined pharmaceutical composition for resisting propionibacterium acnes and application thereof |
| CN116270966A (en) * | 2023-05-16 | 2023-06-23 | 成都佩德生物医药有限公司 | Application of polypeptide RK12 as active ingredient |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106589088A (en) * | 2016-11-21 | 2017-04-26 | 浙江大学 | Polypeptide having anti-bacterial and anti-inflammatory activity and application thereof |
| CN108570103A (en) * | 2018-04-03 | 2018-09-25 | 东北农业大学 | One kind is rich in tryptophan antibacterial peptide WK12 and its preparation method and application |
| CN109320585A (en) * | 2018-09-06 | 2019-02-12 | 南方医科大学 | A kind of short lipopeptid with specificity anti-acne Propionibacterium effect and anti-inflammatory effect |
| KR20190063777A (en) * | 2017-11-30 | 2019-06-10 | 주식회사 에버코스 | A cosmetic composition using antimicrobial peptide for anti-acne |
| CN110606871A (en) * | 2019-09-20 | 2019-12-24 | 湖南生达生物科技有限公司 | Small molecule polypeptide RK12 and application thereof |
| CN110627870A (en) * | 2019-09-20 | 2019-12-31 | 湖南生达生物科技有限公司 | Small molecule polypeptide RK12 and application thereof |
| WO2021165543A1 (en) * | 2020-02-21 | 2021-08-26 | Origimm Biotechnology Gmbh | Propionibacterium acnes prophylactic and therapeutic immune treatment |
-
2021
- 2021-12-24 CN CN202111596950.1A patent/CN114209808B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106589088A (en) * | 2016-11-21 | 2017-04-26 | 浙江大学 | Polypeptide having anti-bacterial and anti-inflammatory activity and application thereof |
| KR20190063777A (en) * | 2017-11-30 | 2019-06-10 | 주식회사 에버코스 | A cosmetic composition using antimicrobial peptide for anti-acne |
| CN108570103A (en) * | 2018-04-03 | 2018-09-25 | 东北农业大学 | One kind is rich in tryptophan antibacterial peptide WK12 and its preparation method and application |
| CN109320585A (en) * | 2018-09-06 | 2019-02-12 | 南方医科大学 | A kind of short lipopeptid with specificity anti-acne Propionibacterium effect and anti-inflammatory effect |
| CN110606871A (en) * | 2019-09-20 | 2019-12-24 | 湖南生达生物科技有限公司 | Small molecule polypeptide RK12 and application thereof |
| CN110627870A (en) * | 2019-09-20 | 2019-12-31 | 湖南生达生物科技有限公司 | Small molecule polypeptide RK12 and application thereof |
| WO2021165543A1 (en) * | 2020-02-21 | 2021-08-26 | Origimm Biotechnology Gmbh | Propionibacterium acnes prophylactic and therapeutic immune treatment |
Non-Patent Citations (5)
| Title |
|---|
| JURGEN HARDER, DAISUKE TSURUTA等: "What is the role of antimicrobial peptides (AMP) in acne vulgaris?", EXPERIMENTAL DERMATOLOGY, pages 386 * |
| ZHIYE ZHANG等: "A Small Peptide with Therapeutic Potential for Inflammatory Acne Vulgaris", PLOS ONE, vol. 8, no. 8, pages 72923 * |
| 吴赟，张广献等: "抗菌肽Chionodracine 对痤疮丙酸杆菌抑菌及抗炎作用的研究", 广东化工, vol. 48, no. 438, pages 26 - 28 * |
| 曾丹丹: "APDD-A2 治疗耐药痤疮的效果和机制研究", 万方 * |
| 熊唯琛，柴金为，吴洁娜，田茂林，卢万成，徐学清: "蛙皮肤来源抗菌肽Brevinin-2GHk的抗菌作用机制", J SOUTH MED UNIV,, vol. 41, no. 11, pages 1657 - 1663 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114209809A (en) * | 2021-12-24 | 2022-03-22 | 苏州佩德生物医药有限公司 | Combined pharmaceutical composition for resisting propionibacterium acnes and application thereof |
| CN114209809B (en) * | 2021-12-24 | 2023-09-05 | 成都佩德生物医药有限公司 | Combined pharmaceutical composition for resisting propionibacterium acnes and application thereof |
| CN116270966A (en) * | 2023-05-16 | 2023-06-23 | 成都佩德生物医药有限公司 | Application of polypeptide RK12 as active ingredient |
| CN116270966B (en) * | 2023-05-16 | 2023-08-18 | 成都佩德生物医药有限公司 | Application of polypeptide RK12 as active ingredient |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114209808B (en) | 2023-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9872885B2 (en) | Antimicrobial and anti-inflammatory peptides | |
| KR101306643B1 (en) | Antimicrobial hexapeptides | |
| US8466102B2 (en) | Antimicrobial cationic peptides and formulations thereof | |
| CN114209808B (en) | Application of polypeptide RK12 in preparation of medicines for treating acne | |
| KR101734064B1 (en) | Novel antimicrobial peptide derived from myxinidin peptide and uses thereof | |
| KR20180056226A (en) | Antimicrobial Peptide Analogues Derived From The Abalone, Haliotis Discus, And Antimicrobial Pharmaceutical Composition Containing The Same | |
| CN105017384A (en) | Novel antibacterial peptide and application thereof | |
| KR101595440B1 (en) | The effect of antimicrobial activity of CMA3 analogue peptide derived from CA-MA | |
| CN109320585B (en) | Short lipopeptide with specific anti-acne propionibacterium effect and anti-inflammatory effect | |
| JP2008533126A (en) | New hybrid oligomers, methods for their production and pharmaceutical compositions containing them | |
| US7135548B2 (en) | Modified α-MSH peptides and derivatives thereof | |
| CN101443352A (en) | Novel analogues of antimicrobial and anticancer peptide synthesized and produced from Gaegurin 5 | |
| KR102506674B1 (en) | Antimicrobial peptide H103B having antimicrobial actvity against antibiotic-resistant pathogen and uses thereof | |
| KR101977800B1 (en) | Pseudomonas specific antimicrobial peptide and antimicrobial composition comprising the same | |
| KR101627455B1 (en) | Chimeric peptide having antimicrobial activity and antimicrobial composition comprising the same | |
| US10100086B2 (en) | Peptide and uses thereof | |
| KR101980897B1 (en) | Novel antimicrobial peptide derived from LL37 peptide and uses thereof | |
| KR101329223B1 (en) | Antimicrobial peptide with improved bioavailability and reduced cytoxicity and antimicrobial composition comprising the same | |
| KR101998106B1 (en) | Novel antimicrobial peptide derived from Hp1404 peptide and uses thereof | |
| CN112110993B (en) | Chemically synthesized dimer polypeptide with antibacterial and antifungal effects, and preparation method and application thereof | |
| CN111909243B (en) | Antibacterial peptide, antibacterial and itching-relieving pharmaceutical composition and application | |
| KR102540598B1 (en) | Antibacterial peptide derived from Crassostrea gigas and its use | |
| KR102603281B1 (en) | Novel peptide derived from Hylin a1 peptide and uses thereof | |
| CN114209809A (en) | Combined pharmaceutical composition for resisting propionibacterium acnes and application thereof | |
| CN117024607A (en) | Polypeptide medicine for resisting bacteria and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 610200 Chengdu Tianfu international biological city, Chengdu, Sichuan (No. 18, Section 2, biological city middle road, Shuangliu District) Applicant after: Chengdu Peide biomedical Co.,Ltd. Address before: Room 503, 5 / F, building a, 108 Yuxin Road, Suzhou Industrial Park, Suzhou area, China (Jiangsu) pilot Free Trade Zone, Jiangsu 215000 Applicant before: Suzhou Peide biomedical Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |