WO2020182180A1 - 赤红球菌产品在治疗外阴白色病变中的用途 - Google Patents

赤红球菌产品在治疗外阴白色病变中的用途 Download PDF

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WO2020182180A1
WO2020182180A1 PCT/CN2020/078932 CN2020078932W WO2020182180A1 WO 2020182180 A1 WO2020182180 A1 WO 2020182180A1 CN 2020078932 W CN2020078932 W CN 2020078932W WO 2020182180 A1 WO2020182180 A1 WO 2020182180A1
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rhodococcus
cell wall
weeks
vulva
product
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PCT/CN2020/078932
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English (en)
French (fr)
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盖波
窦春艳
张轶
张国英
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辽宁格瑞仕特生物制药有限公司
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Publication of WO2020182180A1 publication Critical patent/WO2020182180A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/365—Nocardia

Definitions

  • the present disclosure relates to the medical field, the microbiological field, and the biopharmaceutical field. Specifically, it relates to Rhodococcus erythropolis and its cell wall components, preparations, pharmaceutical compositions, preparation methods, and the use of Rhodococcus erythrophilus cell wall components in the preparation of drugs/medical devices for treating white lesions of the vulva.
  • Rhodococcus ruber also called Rhodococcus ruber
  • Rhodococcus ruber a gram-positive bacteria.
  • the colony is orange or orange, round; the size of the colony is about 1mm to 2mm; the cell morphology is spherical or short rod; it can form primary branched mycelium; it has no flagella.
  • Rhodococcus is aerobic and chemically heterotrophic.
  • Rhodococcus rubrum researchers have performed whole-gene foci of Rhodococcus rubrum.
  • Fan Xin et al. sequenced the entire genome of Rhodococcus rubrum SD3 strain and conducted bioinformatics analysis.
  • the whole genome length of SD3 strain is about 5.37Mb
  • GC content is about 70.63%
  • GenBank accession number is CP029146 (Fan Xin, Rhodococcus rubrum SD3 whole genome sequencing and its heat shock protein DnaK expression analysis, genomics and applied biology, January 2019).
  • Rhodococcus is a genus of Rhodococcus, which can adapt to a variety of living environments due to its strong tolerance to organic matter and a broad degradation spectrum. Therefore, Rhodococcus is widely used in pollution remediation, organic compound degradation, sewage treatment and other fields.
  • Rhodococcus rubra lies in environmental management, see CN108862590A, CN107151635A, CN102250796A, CN1519312A, CN103627653A, CN101033454A, CN108130288A, CN104830738A, CN101619299A, CN103509833A, CN106434466A, CN101580808A, CN102491, 875, CN102591106A105, CN102604466A, CN101580808A, CN102604, 875, CN101580808A, CN102491, 875, CN102591106A, CN103160.
  • CN109576180A discloses a bacterium RDC-01 selected from the red soil in the suburbs near Panyu District, Guangzhou City. After 16S rRNA gene sequence analysis and identification of culture characteristics, the strain was identified as Rhodococcus rubrum. After the bacterium was inactivated, it was added as an immune adjuvant to an inactivated vaccine for animals, and it was found to promote the production of antibodies in animals.
  • Rhodococcus rubrum in the field of human medicine has not yet been reported.
  • White lesions of the vulva include white lesions of the vulva, leukoplakia or vulvar dystrophy. Because the vulvar mucosa of patients with lichen sclerosus and squamous cell hyperplasia is white, it is called vulvar white lesions (non-tumor-like lesions of the vulvar epithelium).
  • Non-neoplastic epithelial disorders of skin and mucosa non-neoplastic epithelial disorders of skin and mucosa
  • Mild dysplasia Mild dysplasia
  • Moderate dysplasia (moderate dysplasia);
  • Treatments for white lesions of the vulva usually include:
  • Drug therapy Commonly used drugs for lichen sclerosus are pyruvate ointment, compound vitamin A ointment and progesterone ointment, glucocorticoid ointment or immunotherapy. Squamous epithelial hyperplasia of the vulva can be treated with topical corticosteroids to control itching. Treatment is effective for most patients, but long-term medication must be adhered to.
  • Physiotherapy It is suitable for those who are ineffective or severely ill. Microwave therapy, carbon dioxide laser, helium-neon laser, Pomer light, high-frequency electrosurgical knife, local electrocautery therapy, and liquid nitrogen local cryotherapy.
  • an isolated Rhodococcus ruber is provided.
  • Rhodococcus rubrum which was deposited at the General Microbiology Center of the China Microbial Culture Collection Management Committee on March 22, 2019 (No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing) , Institute of Microbiology, Chinese Academy of Sciences; Zip Code: 100101; China General Microbiological Culture Collection Center. Address: Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing China, and the deposit number is CGMCC No. 17431 .
  • the deposit satisfies the provisions of the Budapest Treaty on the International Recognition of Microorganism Preservation for Patent Procedures (Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure).
  • Rhodococcus rubrum and its derivative products are provided.
  • the derivative product is derived from Rhodococcus rubrum and contains the components of Rhodococcus rubrum (such as protein, nucleic acid, lipid, cell wall and its constituent components, carbohydrates, metabolites).
  • an isolated Rhodococcus rubrum cell wall is provided.
  • an isolated Rhodococcus rhodococcus cell wall is provided, and the Rhodococcus rhodococcus refers to a strain with the deposit number of CGMCC No. 17431.
  • an isolated Rhodococcus rubrum cell wall skeleton is provided.
  • an isolated Rhodococcus rhodococcus cell wall skeleton is provided, and the Rhodococcus rhodococcus refers to a strain with a deposit number of CGMCC No. 17431.
  • a pharmaceutical composition comprising the cell wall of Rhodococcus or the cell wall skeleton of Rhodococcus according to the present disclosure.
  • Rhodococcus Rhodococcus product which comprises a product obtained by pulverizing Rhodococcus Rhodococcus.
  • Rhodococcus Rhodococcus product which comprises a product obtained by crushing Rhodococcus Rhodococcus and purifying (fat removal, nucleic acid removal, protein removal).
  • Rhodococcus rubrum product which comprises the cell wall of Rhodococcus rubrum.
  • Rhodococcus rubra product which comprises the cell wall skeleton of Rhodococcus rubra.
  • a pharmaceutical composition or medical device which comprises a product obtained by crushing Rhodococcus rubrum.
  • a pharmaceutical composition or medical device which comprises a product obtained by crushing and purifying Rhodococcus rubrum (removing fat, and/or nucleic acid, and/or protein).
  • a pharmaceutical composition or medical device which comprises the cell wall of Rhodococcus rubrum.
  • a pharmaceutical composition or medical device which comprises the cell wall skeleton of Rhodococcus rubrum.
  • a pharmaceutical composition or medical device which comprises the above-mentioned Rhodococcus product.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.
  • the rhodococcus product in the pharmaceutical composition is 1 part by weight
  • the pharmaceutically acceptable excipient is 100 to 1000 parts by weight (100, 200, 300, 400, 500, 600, 700 , 800, 900, 1000), preferably 200 to 500 parts by weight, more preferably 200 to 300 parts by weight (for example, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 and Any point value within any two numerical ranges).
  • the Rhodococcus rhodochrous cell wall in the pharmaceutical composition is 1 part by weight
  • the pharmaceutically acceptable excipient is 100 to 1000 parts by weight (100, 200, 300, 400, 500, 600, 700, 800, 900, 1000), preferably 200 to 500 parts by weight, more preferably 200 to 300 parts by weight (e.g., 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 And any point value within any two numerical ranges).
  • the Rhodococcus rubrum cell wall skeleton in the pharmaceutical composition is 1 part by weight
  • the pharmaceutically acceptable excipient is 100 to 1000 parts by weight (100, 200, 300, 400, 500, 600 , 700, 800, 900, 1000), preferably 200 to 500 parts by weight, more preferably 200 to 300 parts by weight (e.g., 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 and any point value within any two numerical ranges).
  • the pharmaceutical composition may be prepared as a liquid (liquid formulation).
  • the pharmaceutical composition can be prepared as a solid (dry powder formulation or lyophilized powder formulation).
  • the liquid preparation and the dry powder preparation can be converted into each other, and the difference is only in the water content. Remove most or all of the water in the liquid formulation to obtain a dry powder formulation (or freeze-dried powder formulation). The dry powder preparation (or freeze-dried powder preparation) is dissolved (or reconstituted) to obtain a liquid preparation.
  • the drug or pharmaceutical composition is prepared into a dosage form selected from the group consisting of ointments, creams, emulsions, suspensions, pastes, gels, lotions, tinctures, oils, tablets, gas Fog, spray, liniment, powder, suppository; wherein the ointment is selected from: ointment, plaster, and cream.
  • the dosage form is a form suitable for application to the lesion.
  • sprays, ointments, lotions, dressings, patches are examples of suitable for application to the lesion.
  • ointments for example, sprays, ointments, lotions, dressings, patches.
  • the pharmaceutically acceptable excipient relates to, but is not limited to: fillers, stabilizers, flavoring agents, disintegrants, binders, and lubricants.
  • the pharmaceutically acceptable excipient such as but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythrose Alcohol, gelatin, magnesium stearate, propellant, humectant, solvent, solubilizer, emulsifier, antioxidant, pH regulator, preservative.
  • non-limiting examples of the pharmaceutically acceptable excipients also include: white petrolatum, carbomer, hypromellose, methyl cellulose, sodium hydroxymethyl cellulose, chitosan Sugar, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerin, propylene glycol, deionized water, water for injection, distilled water, ethanol, ten Hexanol, stearyl alcohol, p-aminobenzoic acid, acetamide, isopropanol, Tween, polyoxyethyl hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, Fatty acid sucrose esters, sucrose esters, sucrose acetate isobutyrate, sorbitan tristearate,
  • a method for preparing a Rhodococcus rubrum product which includes or consists of the following steps:
  • remove the water in the purified product preferably by freeze-drying to remove the water in the purified product;
  • steps 5.1), 5.2), and 5.3) can be interchanged in order or parallel; step 6) and step 7) can be interchanged in order.
  • step 5) may further include (for example, using a nonionic surfactant) a step of removing the cell membrane.
  • Rhodococcus rubrum is not limited to specific culture media and culture parameters, and the skilled person can culture it in a well-known and appropriate manner, and can use petri dishes, culture bottles, and fermenters according to the scale of preparation.
  • Technicians have the ability to adjust the specific parameters and equipment of cultivation, crushing, separation, collection, impurity removal, and packaging according to the subsequent application (external application, etc.) of the active ingredients (cell wall and its components), so as to avoid the introduction of influence in the preparation step Factors for subsequent applications.
  • an organic solvent is used, for example, to remove lipids in the fragmented product.
  • DNA and RNA in the fragmented product are removed using, for example, nuclease.
  • a hydrolase is used to degrade the protein in the fragmented product.
  • a surfactant is used to remove the cell membrane in the disrupted product.
  • the average particle size of pulverization is 10 nm to 1000 nm; mention may be made of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190nm ⁇ 10nm, and the range between any two of the above values.
  • particle size testing Hu Songqing et al., Modern Particle Size Measurement Technology, Modern Engineering, 2002 22:1).
  • the average particle size of the pulverization is 10 nm to 800 nm.
  • the average particle size of the pulverization is 10 nm to 500 nm.
  • the dispensing refers to dispensing into a container or placing on a solid support.
  • the container is selected from: bottles, tubes, bags, bags, plates, ampoules, injection devices, aluminum film packaging, dressings, and capsules.
  • the dispensing refers to dispensing into bottles/ampoules. Just before use, add solvent to the bottle/ampule.
  • Rhodococcus rubrum product which is prepared by the method according to the present disclosure.
  • a pharmaceutical composition or medical device which includes the Rhodococcus product prepared according to the method of the present disclosure.
  • an isolated Rhodococcus rubrum cell wall for the treatment of white lesions of the vulva.
  • Rhodococcus rubrum product for treating white lesions of the vulva.
  • a pharmaceutical composition or medical device for treating white lesions of the vulva for treating white lesions of the vulva.
  • the use of the Rhodococcus rubrum cell wall according to the present disclosure in the treatment of white lesions of the vulva is provided.
  • Rhodococcus rubrum cell wall of the present disclosure in preparing a medicine/medical device for treating white lesions of the vulva.
  • the use of the Rhodococcus product according to the present disclosure in the treatment of white lesions of the vulva is provided; the use of the Rhodococcus product of the present disclosure in the preparation of a medicine/medical device for the treatment of white lesions of the vulva is also provided .
  • the use of the pharmaceutical composition according to the present disclosure in the treatment of white lesions of the vulva is provided.
  • the white lesions of the vulva are intradermal non-neoplastic changes of the skin of the vulva and on the mucosa of the vulva, and are selected from: lichen sclerosus and squamous cell hyperplasia.
  • the drug is used to treat white lesions of the vulva.
  • the medical device (such as dressing, patch, bandage, membrane, patch, etc.) is used to treat white lesions of the vulva.
  • a method for treating white lesions of the vulva comprising contacting a subject (lesions) with a therapeutically effective amount of any one selected from the following:
  • different doses are used to administer the drug (or medical device) to the lesion according to the area and depth of the lesion.
  • the drug or medical device
  • different doses are used to administer the drug (or medical device) to the lesion according to the area and depth of the lesion. For example, but not limited to:
  • Liquid medicine, ointment, and dry powder can be used alternately on the lesion.
  • the dosage for each application is different depending on the size and depth of the patient's lesion, and is usually 5 ⁇ g to 800 ⁇ g/unit dose/each time, preferably 10 ⁇ g to 600 ⁇ g/unit dose/each time.
  • the period of contact is: 2 days to 4 months or longer. Specifically, for example 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 days; for another example, 1 week, 2 weeks, 3 weeks can be mentioned , 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks or longer.
  • the subject is administered the active ingredient for 3-5 weeks.
  • the administration is performed at the following frequency: 1 to 3 times a day, 1 to 6 times two days, 1 to 9 times three days, 1 to 14 times a week, 1 to 60 times a month. In some embodiments, it is administered twice a day, or once a day, or once every two days.
  • the dosage for each application varies depending on the specific conditions of the subject, usually from 1 ⁇ g to 1000 ⁇ g/unit dose/each application; specifically, such as 1, 5, 10, 15, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 ⁇ g/unit dose/ Each time, and the range between any two of the aforementioned values.
  • the Rhodococcus Rhodococcus product, pharmaceutical composition or medical device of the present disclosure is in contact with the lesion site: a few minutes to a few hours, for example, 30 minutes to 24 hours.
  • the period of contact between the Rhodococcus rhodococcus product, pharmaceutical composition or medical device of the present disclosure and the lesion site is: once a day, or twice a day, or once every two days.
  • the contact is achieved in the following ways, such as but not limited to: mucosal, transdermal, transdermal.
  • the aforementioned treatment methods are applicable to any animal with skin/mucosal structure, including but not limited to: humans, non-human primates, pigs, cows, horses, sheep, dogs, cats, rats, rabbits.
  • the subject is an animal other than humans, such as farm animals, pets, working animals, ornamental animals, and production animals.
  • the subject is a human.
  • the subject is suspected of having, confirmed to have, has had, or is susceptible to the target disease or its symptoms.
  • the only therapeutic (or preventive) active ingredient in the drug or medical device is a product derived from Rhodococcus, especially containing Rhodococcus components (such as protein, nucleic acid, lipid, cell wall and Its constituents, carbohydrates, metabolites), specifically products containing Rhodococcus cell wall (more preferably Rhodococcus cell wall skeleton or its composition).
  • Rhodococcus components such as protein, nucleic acid, lipid, cell wall and Its constituents, carbohydrates, metabolites
  • specifically products containing Rhodococcus cell wall more preferably Rhodococcus cell wall skeleton or its composition.
  • Figure 1 Colony morphology of Rhodococcus rubrum.
  • Isolation refers to the separation of the Rhodococcus rubrum of the present disclosure from its original growth environment.
  • the cell wall structures of Gram-positive bacteria and Gram-negative bacteria are different.
  • the cell wall of gram-positive bacteria is thick (usually 20nm to 80nm), containing about 90% peptidoglycan and about 10% teichoic acid (a polymer formed by alcohol and phosphoric acid molecules, usually sugar Exist in the form of ester or amino acid ester).
  • the peptidoglycan layer is dense, even up to 20 layers.
  • the cell wall of gram-negative bacteria is much thinner than that of gram-positive bacteria, and the structure is more complex. It is divided into outer membrane and peptidoglycan layer (usually 2nm to 3nm).
  • the peptidoglycan layer is a unique component of the bacterial cell wall and is a derivative of heteropolysaccharide.
  • the monomer of each peptidoglycan includes 3 parts: sugar unit (for example, at least two sugar molecules are connected by glycosidic bonds to form the framework structure of peptidoglycan), peptide tail (short peptide chain connected by several amino acids) , Which is connected to the N-acetylmuramic acid molecule), and peptide bridge (crosslinking adjacent "peptide tails" to form a high-strength network structure).
  • sugar unit for example, at least two sugar molecules are connected by glycosidic bonds to form the framework structure of peptidoglycan
  • peptide tail short peptide chain connected by several amino acids
  • peptide bridge crosslinking adjacent "peptide tails" to form a high-strength network structure.
  • Different bacteria have different peptide bridges, peptide tails, and cross-linking methods.
  • isolated Rhodococcus rhodococcus cell wall can be understood as both a complete cell wall and an incomplete cell wall (for example, broken or partially degraded).
  • the ingredient exhibiting the desired activity is derived from the cell wall of Rhodococcus (for example, the cell wall itself or its composition). Therefore, various forms such as complete cell walls, broken cell walls, incomplete degradation products of cell walls, components of cell walls, and cell wall extracts are allowed to be used in clinical applications, which are all included in the scope of the present disclosure.
  • Rhodococcus used in the embodiments of the present disclosure refers to Rhodococcus ruber of the genus Rhodococcus, and is not limited to a specific cell strain.
  • Non-limiting examples include TOY7 strain (Nanjing Agricultural University Agricultural Environmental Microbial Culture Collection), CGMCC No. 4795, DSM43338, CCTCC No. 2012035, CGMCC No. 16640, CGMCC No. 17431.
  • microbial identification techniques According to known or future microbial identification techniques, technicians can perform taxonomic identification on a strain of bacteria.
  • available identification techniques include morphology, physiological and biochemical characteristics, 16S rRNA, and so on.
  • Technicians understand that with the development of science and technology, identification techniques involve different methods. In the earlier period, morphological and biochemical identification methods were mainly used, but the reliability of this method is not high. After the emergence of sequencing technology, technicians can use more reliable methods to identify strains.
  • the medicine or pharmaceutical composition or active ingredient or product of the present disclosure can be embodied in but not limited to the following forms: ointment, cream, ointment, gel, tablet, lotion, tincture, liniment, oil, Pastes, powders, powders, sprays, aerosols, suppositories, patches, suspensions, mouthwashes, buccal tablets, accessories, patches, bandages, film, gauze.
  • the medicament or pharmaceutical composition or active ingredient or product of the present disclosure can be prepared in the form of a unit preparation (unit preparation).
  • the unit dose in the drug contains:
  • Rhodococcus rubrum cell wall skeleton -1 ⁇ g to 1000 ⁇ g of the Rhodococcus rubrum cell wall skeleton.
  • unit doses are 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 65, 66, 67, 68, 69, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 ⁇ g ⁇ 10%, and the range between any two of the above values.
  • administering refers to drugs or medical devices and animals, humans, cells, tissues, organs or biological samples, refers to drugs or medical devices and animals, humans, cells, tissues, organs or Biological sample contact.
  • Treatment means to give a subject an internal or external drug (therapeutic agent, active ingredient or composition) (for example, the Rhodococcus Rhodococcus cell wall or a pharmaceutical composition thereof according to the present disclosure) or a medical device, in the treatment of the subject Alleviate (relieve, delay, ameliorate, cure) one or more symptoms of a disease in a person (or group) to a clinically measurable degree, wherein the subject has, is suspected of suffering, or is susceptible For one or more diseases or their symptoms.
  • an internal or external drug for example, the Rhodococcus Rhodococcus cell wall or a pharmaceutical composition thereof according to the present disclosure
  • a medical device in the treatment of the subject Alleviate (relieve, delay, ameliorate, cure) one or more symptoms of a disease in a person (or group) to a clinically measurable degree, wherein the subject has, is suspected of suffering, or is susceptible For one or more diseases or their symptoms.
  • the amount of the drug (therapeutic agent, active ingredient or composition) that is effective to relieve the symptoms of any disease is called the therapeutically effective amount. It can vary depending on a variety of factors, such as the disease state, age, and weight of the subject. It should be understood that the drug (therapeutic agent, active ingredient, or composition) may be ineffective in relieving the target disease or its symptoms of a single subject, but according to any statistical test method known in the art (such as Student's T test, card The prescription test, according to Mann and Whitney's U test), determines that the drug (therapeutic agent, active ingredient or composition) is statistically effective for the target disease or its symptoms.
  • any statistical test method known in the art such as Student's T test, card The prescription test, according to Mann and Whitney's U test
  • “Optional” means that what is described later can happen, but does not have to happen; it depends on the situation.
  • sub-package means that the product is allowed to be sub-packaged, but it is not required to be sub-packaged; whether the product is sub-packaged or not does not affect the realization of the technical effect.
  • the inventor deposited the master strains preserved in the laboratory on March 22, 2019 at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, the General Microbiology Center of the China Microbial Culture Collection Management Committee, and the deposit number is CGMCC No.17431 . The test showed that the deposited strains survived.
  • the size of the colony is about 1mm to 2mm (it will vary slightly depending on the culture conditions).
  • the bacteria When cultured for 4 to 5 days, the bacteria will become short rod-shaped or spherical (different depending on the culture conditions).
  • Gram stain is positive.
  • Negative Inositol, inulin, lactose, sucrose, starch, maltose, glycogen, xylitol, gluconate, trehalose, erythritol, melezitose, melibiose, raffinose, cellobiose , Amygdalin, gentiobiose, adonol, arbutin, D-arabinose, L-arabinose, ⁇ -methyl-D-glucoside, ⁇ -methyl-D-mannoside, D -Ribose, D-xylose, L-xylose, N-acetyl-glucosamine, D-turbiose, D-lyxose, ⁇ -methyl-D-xyloside, D-galactose, D -Tagatose, D-fucose, L-fucose, D-mannose, L-sorbose, L-arabinitol
  • alkaline phosphatase alkaline phosphatase, lipid esterase (C8), lipase (C14), leucine araminase, valine araminase, cystine araminase, trypsin, chymotrypsin Protease, acid phosphatase, naphthol-AS-B1-phosphohydrolase, ⁇ -glucosidase;
  • Negative N-acetyl-glucosaminidase, esterase (C4), ⁇ -galactosidase, ⁇ -uronidase, ⁇ -glucosidase, ⁇ -galactosidase, ⁇ -mannosidase , ⁇ -Fucosidase.
  • Nitrate reduction reaction is positive, contact enzyme is positive, tyrosinase is positive, amylase is negative, oxidase is negative, gelatin liquefaction is negative.
  • the 15 strains isolated from the working seed tube and the 10 different strains isolated from the original seed tube were subjected to genome extraction, 16Sr RNA amplification, and sequencing.
  • the 16Sr RNA gene identity of 25 strains in total is 100%. This means that 25 strains are of the same species ( Figure 2).
  • the neighbor-joining strain evolutionary tree constructed based on the Kimura2-parameter algorithm showed that the strain was classified as Rhodococcus ruber.
  • Rhodococcus rubrum can be cultivated by conventional microbial production methods.
  • the culture method can be solid culture or liquid culture (such as shake flask, fermentor).
  • the culture medium contains carbon sources, nitrogen sources and other nutrient sources commonly used for culturing Gram-positive bacteria.
  • the carbon source is any carbon source available to Rhodococcus rubrum. For example, fructose, glucose, etc.
  • -Nitrogen source meat extract, peptone, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds.
  • inorganic salts can be added appropriately.
  • composition of the culture medium includes:
  • Peptone beef extract, sodium chloride, phosphate, glycerin (and, optionally, agar, when in solid culture).
  • the working strain After the working strain is recovered, it is transferred to solid culture medium for 3-5 days, and then transferred to liquid culture (30-37°C, maintained for 3-5 days).
  • the fed-batch semi-continuous mode can also be used. Batch mode. Monitor pH, bacterial density, dissolved oxygen, and carbon source consumption during cultivation.
  • Collect the bacteria obtained in Preparation Example 1 and pulverize the cells (for example, but not limited to ultrasonic disruption). It is also allowed to use any appropriate well-known method in the art to disrupt the bacteria, such as CN101250490A or CN101323865A.
  • a common protease such as trypsin
  • Organic reagents such as but not limited to one or a combination of acetone, ether, and ethanol are added to the precipitate, and lipids are removed according to conventional operations in the art.
  • TritonX-100 was added to the precipitate, and the precipitate was collected by centrifugation according to conventional operations in the art, and rinsed with PBS.
  • the technician can adjust the sequence to make the steps compatible.
  • the precipitate was re-dissolved in water for injection and set aside. Optionally, it can be sterilized at 115°C for 20-30 minutes as the original solution of the cell wall skeleton (including the cell wall skeleton and its components).
  • the pharmaceutical composition can be formulated in various forms
  • the pharmaceutical composition of item 1 is lyophilized to prepare lyophilized powders (numbered as lyophilized powder compositions 1 to 7 respectively).
  • the pharmaceutical composition of item 1 (active ingredient 60 ⁇ g to 120 ⁇ g, such as 60 ⁇ g, 70 ⁇ g, 80 ⁇ g, 90 ⁇ g, 100 ⁇ g, 110 ⁇ g, 120 ⁇ g) is coated on a dressing (for example, sterile gauze) to prepare an external medical device.
  • a dressing for example, sterile gauze
  • the film is prepared using methods known in the art (for example, the methods disclosed in Chinese application numbers 201610605617.5, 201510614414.8, 200610200450.0, 201610511974.5, 201610471977.0, etc.).
  • film-forming materials such as polyvinyl alcohol, carbomer and hydroxypropyl cellulose to water, swelling to form a homogeneous viscous liquid
  • adding the pharmaceutical composition of item 1 (respectively, compositions 1 to 7), and mix well; let stand to defoam; cast the formed mixed viscous liquid without bubbles; cast on a mold coated with a small amount of paraffin, dry for 5-20min, take out the film, and cut into the required area.
  • composition 3 and the esterifying agent are stirred and dissolved in a solvent, and hydroxyalkyl cellulose is added to swell it, and continue to stir to form a gel; then add the crosslinking agent and continue to stir until Completely uniform.
  • the preparation method of ointment for external use on the skin can also be found in but not limited to the methods disclosed in Chinese Application Nos. 201610856428.5, 01133296.4, 1133297.2, "Drug Formulation and Preparation Design” (Chemical Industry Press 2009).
  • mice After intravenous injection of 1000 times the human clinical dose, the coordinated movement and learning and memory functions of mice did not have obvious effects.
  • composition 1 to composition 7 have no significant effect on animal spirit, nervous system, cardiovascular system, and respiratory system.
  • mice 1. Acute toxicity test in mice:
  • the experimental group was administered by subcutaneous injection and intraperitoneal injection, and the dosage was 5 times that of humans.
  • the control group was treated with sterile normal saline 0.5ml per tube for continuous observation for 7-8 days.
  • the mice are in good condition and have no abnormal body weight; all organs of the mice are normal.
  • composition 1 to composition 7 30 times the clinical dose was applied once a day for three months of continuous vaginal administration. No toxic effects were found in dogs; the electrocardiogram and blood biochemical indicators were within normal ranges. Two weeks after stopping the drug, no delayed toxicity was seen (composition 1 to composition 7).
  • composition 1 to composition 7 Placed at room temperature (18-25°C) for 0, 1, 2, 3, 8, 14, 21 months, the alanine content, muramic acid content, and phagocytosis of the pharmaceutical composition (composition 1 to composition 7) Compared with the beginning of the experiment, there was no statistically significant difference in the rate and phagocytosis index (three batches were tested).
  • the pharmaceutical composition of the freeze-dried powder formulation can be stored stably for 24 months.
  • phagocytes are activated after being stimulated by an antigen, which can significantly enhance the phagocytic function.
  • an antigen which can significantly enhance the phagocytic function.
  • the mice were injected with chicken red blood cells in the peritoneal cavity. After 30 minutes, the mice were killed and the peritoneal fluid was taken out, stained, and the percentage of phagocytic red blood cells was counted under a microscope to determine the killing ability of phagocytes. Measure the body's non-specific immunity level.
  • the phagocytosis rate was 75%, and the phagocytosis index was 1.05.
  • the phagocytic rate and phagocytic index of the negative control (excipient) and blank control (physiological saline) were lower. It shows that the cell wall skeleton or its components of the present application have strong immunity-promoting phagocytic ability.
  • composition 3 (ointment, once every other day, 60 ⁇ g of active ingredients each time). Check up every week and follow up if you are unwell.
  • Histopathological examination local anesthesia with Primacaine STA, routine disinfection, take about 0.8 ⁇ 0.5 ⁇ 0.5cm 3 tissue from the lesion on the right vulva and suture 3 stitches. The samples were fixed with 10% formalin, sent to pathology, and the stitches were removed one week after the doctor ordered them.
  • the biopsy site is healed well, and the sutures are stored in 3 stitches, and they are not off;
  • composition 3 dry powder, once every other day, 60 ⁇ g active ingredient each time. Check up every week and follow up if you are unwell.
  • composition 3 gel, once every other day, 60 ⁇ g active ingredient each time. Check up every week and follow up if you are unwell.

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Abstract

赤红球菌产品在治疗外阴白色病变中的用途。提供了一种包含赤红球菌细胞壁或其成分的赤红球菌产品。向受试者施用该赤红球菌产品，对外阴白色病变(包括硬化性苔藓和鳞状上皮细胞增生)的治疗效果好；且无副作用。

Description

赤红球菌产品在治疗外阴白色病变中的用途
本申请要求2019年03月14日提交的专利申请(申请号CN201910191961.8)的优先权，通过引用并入此处。
技术领域
本公开涉及医学领域、微生物领域、生物制药领域。具体而言，涉及赤红球菌及其细胞壁成分、制剂、药物组合物、制备方法、以及赤红球菌的细胞壁成分在制备治疗外阴白色病变的药物/医疗装置中的用途。
背景技术
赤红球菌(Rhodococcus ruber)(也称为红色红球菌)，革兰氏阳性菌。通常而言，菌落呈桔黄色或呈桔红色，圆形；菌落大小约1mm至2mm；细胞形态为球状或短杆状；可形成初级分枝菌丝体；无鞭毛。赤红球菌好氧，化能异养。
目前，已有研究人员对赤红球菌进行了全基因侧学。例如，樊欣等人对赤红球菌SD3株的全基因组进行测序，并进行生物信息学分析。SD3株的全基因组长度大约为5.37Mb，GC含量约为70.63％，GenBank登录号为CP029146(樊欣，赤红球菌SD3全基因组测序及其热休克蛋白DnaK的表达分析，基因组学与应用生物学，2019年1月)。
红球菌属Rhodococcus，因其自身具有非常强的有机物耐受性，以及较宽的降解谱，能够适应多种生存环境。因此，红球菌属被广泛应用于污染修复、有机化合物降解、污水处理等领域。目前，赤红球菌的主要应用领域在于环境治理，参见CN108862590A、CN107151635A、CN102250796A、CN1519312A、CN103627653A、CN101033454A、CN108130288A、CN104830738A、CN101619299A、CN103509833A、CN106434466A、CN101580808A、CN102604875A、CN103160491A、CN106591168A、CN106591172A、CN105820982A。
CN109576180A中公开了一种从广州市番禺区附近郊野红土中筛选到的菌RDC-01，经16S rRNA基因序列分析和培养特性鉴定，该菌 株鉴定为赤红球菌。将该菌灭活后，作为免疫佐剂添加入动物用的灭活疫苗中，发现可以促进动物生产抗体。
然而，赤红球菌在人类医学领域中的应用，尚未有报道。
外阴白色病变包括外阴白色病损、外阴白斑或外阴营养不良。由于硬化性苔藓及鳞状上皮细胞增生患者的外阴黏膜呈白色，故称为外阴白色病变(属于外阴上皮的非瘤样病变)。
硬化性苔藓及鳞状上皮细胞增生在不同年代，由于对其临床、病理认识不同而几易其名(外阴白斑、白斑性外阴炎、外阴干枯症、增生性或萎缩性外阴炎、硬化萎缩性苔藓等)。因病的命名混乱，1975年国际外阴疾病研究协会(ISSVD)将其统称为慢性外阴营养不良。1987年ISSVD与国际妇科病理学家协会(ISGYP)共同讨论、制订新的外阴皮肤病分类(ISSVD，1987)，包括：
(1)皮肤和黏膜上皮内非瘤样变(non-neoplastic epithelial disorders of skin and mucosa)
1)硬化性苔藓(lichen sclerosus)；
2)鳞状上皮细胞增生(squamous cell hyperplasia)；
(2)上皮内瘤样变(intra-epithelial neoplasia)
1)鳞状上皮内瘤样变(squamous intra-epithelial neoplasia)；
a.轻度不典型增生(mild dysplasia)；
b.中度不典型增生(moderate dysplasia)；
c.重度不典型增生或原位癌(severe dysplasia or carcinoma in situ)；
2)非鳞状上皮内瘤样变(non-squamous intra-epithelial neoplasia)；
a.派杰氏病(Paget`sdisease)；
b.非浸润性黑色素细胞瘤(noninvasive tumors of melanocytes)；
(3)浸润癌(invasive tumors)。
外阴白色病变的治疗方法通常包括：
1.保持外阴清洁干燥，禁用刺激性大的药物，忌穿不透气的内裤， 不食辛辣和易过敏食物。对瘙痒症状明显以致失眠者，可加用镇静、安眠和抗过敏药物。
2.药物治疗：硬化性苔藓的常用药物有丙酮酸油膏、复方维生素A膏及黄体酮油膏、糖皮质激素软膏或免疫治疗。外阴鳞状上皮增生可局部应用皮质激素控制瘙痒。多数患者治疗有效，但是需坚持长期用药。
3.物理治疗：适用于药物治疗无效或病情严重者。微波治疗、二氧化碳激光、及氦氖激光、波姆光、高频电刀、局部电灼治疗以及液氮局部冷冻治疗等。
4.手术治疗：仅适用于病情严重、反复药物或物理治疗无效者。怀疑恶变时，需行手术治疗。
鉴于此，本领域仍需要提供一种有效治疗外阴白色病变的药物。
发明内容
根据本公开的一些实施方案，提供了一种分离的赤红球菌(Rhodococcus ruber)。
根据本公开的一些具体的实施方案，提供了一种赤红球菌，其于2019年03月22日在中国微生物菌种保藏管理委员会普通微生物中心保藏(北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所；邮政编码：100101；China General Microbiological Culture Collection Center.Address：Institute of Microbiology,Chinese Academy of Sciences,No.1West Beichen Road,Chaoyang District,Beijing China)，保藏编号为CGMCC No.17431。该保藏满足《国际承认用于专利程序的微生物保存布达佩斯条约(Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure)》的规定。
根据本公开的一些实施方案，提供了赤红球菌及其衍生产品。所述衍生产品源自赤红球菌，包含赤红球菌的组成成分(如蛋白、核酸、脂质、细胞壁及其组成成分、碳水化合物、代谢物)。
在具体的实施方案中，提供了一种分离的赤红球菌细胞壁。
在具体的实施方案中，提供了一种分离的赤红球菌细胞壁，所述赤红球菌是指保藏号为CGMCC No.17431的株。
在具体的实施方案中，提供了一种分离的赤红球菌细胞壁骨架。
在具体的实施方案中，提供了一种分离的赤红球菌细胞壁骨架，所述赤红球菌是指保藏号为CGMCC No.17431的株。
根据本公开的一些实施方案，提供了药物组合物，其包含根据本公开的赤红球菌的细胞壁或赤红球菌的细胞壁骨架。
根据本公开的一些实施方案，提供了一种赤红球菌产品，其包含赤红球菌经粉碎后所得的产物。
根据本公开的另一些实施方案，提供了一种赤红球菌产品，其包含赤红球菌经粉碎并经过纯化(除脂、除核酸、除蛋白)所得的产物。
根据本公开的另一些实施方案，提供了一种赤红球菌产品，其包含赤红球菌的细胞壁。
根据本公开的另一些实施方案，提供了一种赤红球菌产品，其包含赤红球菌的细胞壁骨架。
根据本公开的一些实施方案，提供了一种药物组合物或医疗装置，其包含赤红球菌经粉碎后所得的产物。
根据本公开的另一些实施方案，提供了一种药物组合物或医疗装置，其包含赤红球菌经粉碎和经过纯化(除脂、和/或除核酸、和/或除蛋白质)后所得的产物。
根据本公开的另一些实施方案，提供了一种药物组合物或医疗装置，其包含赤红球菌的细胞壁。
根据本公开的另一些实施方案，提供了一种药物组合物或医疗装置，其包含赤红球菌的细胞壁骨架。
根据本公开的另一些实施方案，提供了一种药物组合物或医疗装置，其包含上述赤红球菌产品。
在具体的实施方案中，药物组合物还包含药学上可接受的赋形剂。
在一些实施方案中，药物组合物中所述赤红球菌产品为1个重量份，药学上可接受的赋形剂为100至1000个重量份(100、200、300、400、500、600、700、800、900、1000)，优选200个至500个重量份， 更优选200至300个重量份(例如，200、210、220、230、240、250、260、270、280、290、300以及任意两个数值范围内的任意点值)。
在另一些实施方案中，药物组合物中赤红球菌细胞壁为1个重量份，所述药学上可接受的赋形剂为100至1000个重量份(100、200、300、400、500、600、700、800、900、1000)，优选200个至500个重量份，更优选200至300个重量份(例如，200、210、220、230、240、250、260、270、280、290、300以及任意两个数值范围内的任意点值)。
在又一些实施方案中，药物组合物中赤红球菌细胞壁骨架为1个重量份，所述药学上可接受的赋形剂为100至1000个重量份(100、200、300、400、500、600、700、800、900、1000)，优选200个至500个重量份，更优选200至300个重量份(例如，200、210、220、230、240、250、260、270、280、290、300以及任意两个数值范围内的任意点值)。
在一些实施方案中，药物组合物可以制备成液态的(液体制剂)。
在另一些实施方案中，药物组合物可以制备成固体的(干粉制剂或冻干粉制剂)。
技术人员理解，对于本公开的药物组合物而言，液体制剂和干粉制剂(或冻干粉制剂)，二者可以相互转化，差别仅在于含水量。除去液体制剂中的绝大部分或全部水，得到干粉制剂(或冻干粉制剂)。干粉制剂(或冻干粉制剂)溶解(或复溶)后得到液体制剂。
在一些实施方案中，药物或药物组合物被制备成选自以下的剂型：膏剂、霜剂、乳液、混悬剂、糊剂、凝胶剂、洗剂、酊剂、油剂、片剂、气雾剂、喷雾剂、搽剂、粉剂、栓剂；其中，所述膏剂选自：软膏剂、硬膏剂、乳膏剂。
在一些实施方案中，剂型是适合应用至病灶上的形式。例如，喷雾、软膏、洗剂、敷料、贴片。
在一些实施方案中，所述药学上可接受的赋形剂涉及但不限于：填充剂、稳定剂、矫味剂、崩解剂、粘合剂、润滑剂。
在一些实施方案中，所述药学上可接受的赋形剂，例如但不限于： 右旋糖酐、乳糖、微晶纤维素、海藻糖、甘氨酸、木糖醇、羧甲基纤维素钠、赤藓糖醇、明胶、硬脂酸镁、抛射剂、保湿剂、溶剂、增溶剂、乳化剂、抗氧化剂、pH调节剂、防腐剂。
在一些实施方案中，所述药学上可接受的赋形剂的非限制示例还包括：白凡士林、卡波姆、羟丙甲纤维素、甲基纤维素、羟甲基纤维素钠、壳聚糖、硫糖铝壳聚糖、聚乙烯吡咯烷酮、聚乙烯醇、玻璃酸钠、二甲醚、四氟乙烷、氢氟烷烃、甘油、丙二醇、去离子水、注射用水、蒸馏水、乙醇、十六醇、十八醇、对氨基苯甲酸、乙酰胺、异丙醇、吐温、聚氧乙基氢化蓖麻油、硬脂酸、单硬脂酸甘油酯、三聚甘油单硬脂酸酯、脂肪酸蔗糖酯、蔗糖酯、乙酸异丁酸蔗糖糖酯、山梨醇酐三硬脂酸酯、肉豆蔻酸异丙酯、胆固醇、角鲨烯、角鲨烷、正丁醇、乙二醇、乙醇、丙二醇、聚甘油酯、亚硫酸盐、半胱氨酸、二叔丁基羟基甲苯、山梨酸钾、磷酸缓冲溶液、三乙醇胺、氢氧化钠、乙二胺、月桂胺、碳酸氢钠、盐酸、尼泊金类、硫柳汞、氯甲酚、三氯叔丁醇、苯甲酸及其钠盐。
根据本公开的一些实施方案，提供一种赤红球菌产品的制备方法，其包括以下步骤或由以下步骤组成：
1)提供赤红球菌；
2)任选，培养所述赤红球菌；
3)任选，收集经培养的赤红球菌；
4)粉碎所述经培养的赤红球菌，得到粉碎产物；
5.1)任选，对所述粉碎产物进行去除脂质的操作；
5.2)任选，对所述粉碎产物进行去除核酸的操作；
5.3)任选，对所述粉碎产物进行去除蛋白质的操作；
5.4)得到纯化产物；
6)任选地，除去所述纯化产物中的水，优选通过冷冻干燥除去所述纯化产物中的水；
7)任选地，进行分装；
8)收获所述的赤红球菌产品；
其中，步骤5.1)、5.2)、5.3)可互换顺序或可并行；步骤6)和步 骤7)可互换顺序。
任选地，根据需要，步骤5)中还可以包含(例如用非离子型表面活性剂)去除细胞膜的步骤。
赤红球菌的培养不限于具体的培养介质和培养参数，技术人员可以采用公知的适当方式进行培养，可以根据制备规模采用培养皿、培养瓶、发酵罐。
对于赤红球菌的粉碎，其目的在于去除细胞内的物质，因此可以采用超声破碎、溶菌酶等技术。技术人员理解，任何适用于破碎革兰氏阳性菌的已知或未来方法，均适用于本公开技术方案。
技术人员有能力根据活性成分(细胞壁及其组成成分)的后续应用(例如外敷等)，来调整培养、破碎、分离、收集、除杂质、分装的具体参数和设备，以免制备步骤中引入影响后续应用的因素。
在一些实施方案中，利用例如有机溶剂去除破碎产物中的脂质。在一些实施方案中，利用例如核酸酶去除破碎产物中的DNA和RNA。在一些实施方案中，利用例如水解酶降解破碎产物中的蛋白质。在一些实施方案中，利用例如表面活性剂去除破碎产物中的细胞膜。
在一些实施方案中，粉碎的平均粒度为10nm至1000nm；可以提及10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190nm±10nm，以及上述任意两个数值之间的范围。粒度的测试方法有很多(胡松青等人，现代颗粒粒度测量技术，现代化工，2002年22：1)。
在一些具体的实施方案中，粉碎的平均粒度为10nm至800nm。
在另一些具体的实施方案中，粉碎的平均粒度为10nm至500nm。
在一些实施方案中，所述分装是指分装至容器或置于固体支持物上。所述容器选自：瓶、管、包、袋、板、安瓿、注射装置、铝膜包装、敷料、胶囊。
例如，在具体的实施方案中，所述分装是指分装至瓶/安瓿中。临用前，向瓶/安瓿中添加溶剂。
根据本公开的一些实施方案，提供了一种赤红球菌产品，其是通过根据本公开的方法制备所得。
根据本公开的一些实施方案，提供了一种药物组合物或医疗装置，其包含根据本公开的方法制备所得的赤红球菌产品。
根据本公开的一些实施方案，提供了一种分离的赤红球菌细胞壁，其用于治疗外阴白色病变。
根据本公开的一些实施方案，提供了一种赤红球菌产品，其用于治疗外阴白色病变。
根据本公开的一些实施方案，提供了一种药物组合物或医疗装置，其用于治疗外阴白色病变。
根据本公开的一些实施方案，提供根据本公开的赤红球菌细胞壁在治疗外阴白色病变中的用途。
还提供了本公开的赤红球菌细胞壁在制备用于治疗外阴白色病变的药物/医疗装置中的用途。
根据本公开的一些实施方案，提供根据本公开的赤红球菌产品在治疗外阴白色病变中的用途；还提供了本公开的赤红球菌产品在制备用于治疗外阴白色病变的药物/医疗装置中的用途。
根据本公开的一些实施方案，提供根据本公开的药物组合物在治疗外阴白色病变中的用途。
在一些实施方案中，所述外阴白色病变是外阴皮肤的和外阴黏膜上的皮内非瘤样变，选自：硬化性苔藓和鳞状上皮细胞增生。
根据本公开的一些实施方案，提供选自以下的任一项在制备药物(或医疗装置)中的用途：
-根据本公开的赤红球菌、
-根据本公开的分离的赤红球菌细胞壁、
-根据本公开的赤红球菌产品、
-根据本公开的药物组合物。
在一些具体的实施方案中，所述药物用于治疗外阴白色病变。
在一些具体的实施方案中，所述医疗装置(如敷料、贴片、绷带、膜、贴片等)用于治疗外阴白色病变。
根据本公开的一些实施方案，还提供了一种治疗外阴白色病变的方法，包括使受试者(病灶)接触治疗有效量的选自以下的任一项：
-根据本公开的分离的赤红球菌细胞壁、
-根据本公开的的赤红球菌产品、
-根据本公开的药物组合物、
-根据本公开的医疗装置。
在一些具体的实施方案中，针对病灶的面积和深度的不同，采用不同的剂量，将药物(或医疗装置)施用至病灶。例如，但不限于：
-用包含赤红球菌细胞壁骨架的药物涂抹；或者
-用浸有赤红球菌细胞壁骨架的贴片(或膜、纱布)覆盖在病灶处；
-包含赤红球菌细胞壁骨架的冻干粉在病灶直接施用；或
-在病灶上施用包含赤红球菌细胞壁骨架的膏体/霜。
可以在病灶上交替使用药液、膏剂、干粉剂。
每次的施用量，视患者病灶的面积大小及深度不同采用不同的剂量，通常为5μg至800μg/单位剂量/每次，优选为10μg至600μg/单位剂量/每次。
在一些具体的实施方案中，接触的周期为：持续2天至4个月或更长。具体而言，例如2、4、6、8、10、15、20、25、30、35、40、45、50、55、60天；再比如，可以提及1周、2周、3周、4周、5周、6周、7周、8周、9周、10周、11周、12周、13周、14周、15周、16周、17周、18周或更长。在具体的实施方案中，向受试者施用3-5周活性成分。
在一些实施方案中，按照以下频率进行施用：一天施用1至3次、两天施用1至6次、三天施用1至9次、一周施用1至14次、一月施用1至60次。在一些实施方案中，一天施用两次，或一天施用一次，或二天施用一次。
每次施用量视受试者具体情况的不同采用不同的剂量，通常为1μg至1000μg/单位剂量/每次施用；具体而言，例如1、5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、200μg/单位剂量/每次、以及前述任意两个数值之间的范围。
在一些具体的实施方案中，本公开的赤红球菌产品、药物组合物 或医疗装置与病灶部位接触：数分钟至数小时，例如30分钟至24小时。
在一些具体的实施方案中，本公开的赤红球菌产品、药物组合物或医疗装置与病灶部位接触的周期为：一天一次，或者一天两次，或者两天一次。
在一些具体的实施方案中，接触是通过以下方式实现的，例如但不限于：粘膜、经皮、透皮。
在一些实施方案中，前述治疗方法适用于任何具有皮肤/粘膜结构的动物，包括但不限于：人、非人灵长动物、猪、牛、马、羊、犬、猫、鼠、兔。
在一些具体的实施方案中，受试者是人以外的动物，例如用于农场动物、宠物、工作动物、观赏动物、生产动物。
在具体的实施方案中，受试者是人。
在一些具体的实施方案中，受试者疑似患有、确诊患有、已经患有、或易感于目标疾病或其症状。
在一些具体的实施方案中，药物或医疗装置中的唯一治疗性(或预防性)活性成分是源自赤红球菌的产品，尤其是包含赤红球菌组成成分(如蛋白、核酸、脂质、细胞壁及其组成成分、碳水化合物、代谢物)的产品，具体而言包含赤红球菌细胞壁(更优选赤红球菌细胞壁骨架或其组成)的产品。
附图说明
图1：赤红球菌的菌落形态。
图2：16S rRNA鉴定结果。
具体实施方式
“分离”是指本公开的赤红球菌脱离其原始生长环境。
技术人员知晓，革兰氏阳性菌和革兰氏阴性菌的细胞壁结构不同。具体而言，革兰氏阳性菌的细胞壁较厚(通常20nm至80nm)，含有约90％的肽聚糖和约10％的磷壁酸(一种由醇和磷酸分子形成的聚合物，通常以糖酯或氨基酸酯的形式存在)。肽聚糖层致密，甚至多达 20层。然而，革兰氏阴性菌的细胞壁比革兰氏阳性菌的细胞壁要薄很多，结构较复杂，分为外膜(outer membrane)和肽聚糖层(通常2nm至3nm)。
肽聚糖层是细菌细胞壁中特有成分，是一种杂多糖的衍生物。每一个肽聚糖的单体包括3部分：糖单元(例如，至少两种糖分子通过糖苷键连接起来，构成肽聚糖的框架性结构)、肽尾(由若干氨基酸连接成的短肽链，其连接在N-乙酰胞壁酸分子上)、和肽桥(将相邻“肽尾”交联形成高强度的网状结构)。不同细菌的肽桥、肽尾、交联方式是不同的。
分离的赤红球菌细胞壁
在本公开中，“分离的赤红球菌细胞壁”既可以理解为完整的细胞壁，也可以理解为不完整的细胞壁(例如，破碎的、或部分降解的)。在本公开的教导下，技术人员将理解，显示出所需活性的成分来自赤红球菌的细胞壁(例如，是细胞壁本身或其组成)。因此，在临床应用中允许采用完整的细胞壁、经破碎的细胞壁、细胞壁的不完全降解产物、细胞壁的组成成分、细胞壁的提取物等各种形式，这些都包含在本公开范畴之内。
细胞壁骨架
构成细胞壁主体结构的组成成分；但不能理解为仅仅表示细胞壁当中的交联网状实体，技术人员理解不排除交联网状实体上所吸附、结合、携带的其他细胞壁成分。
赤红球菌
本公开实施方案中所用的赤红球菌是指红球菌属(Rhodococcus)的赤红球菌种(Rhodococcus ruber)，不限于特定的细胞株。
非限制性示例包括TOY7株(南京农业大学农业环境微生物菌种保藏中心)、CGMCCNo.4795、DSM43338、CCTCC No.2012035、CGMCC No.16640、CGMCC No.17431。
赤红球菌的鉴定
根据已知的或未来的微生物鉴定技术，技术人员可以对一株细菌进行分类学鉴定，例如可用的鉴定技术包括形态学、生理生化特征、 16S rRNA等。技术人员理解，随着科技的发展，鉴定技术涉及不同的手段，在较早的时期主要采用形态学和生化鉴定方式，但是这种方法的可靠程度不高。测序技术出现后，技术人员可以利用更为可信的方式鉴定菌株。例如，当16S rRNA的DNA序列被鉴定为具有97％(含)以上相似性时，判定两个菌属于相同的种(华苟根等人，红球菌属的分类及应用研究进展，微生物学通报，2003：30(4))。就赤红球菌而言，将保藏在国际(或国家级)菌种保藏单位中的已知菌株作为模式菌株，并与其进行比对。
剂型
本公开的药物或药物组合物或活性成分或产品，可以体现为但不限于以下形式：软膏剂、乳膏剂、硬膏剂、凝胶剂、片剂、洗剂、酊剂、搽剂、油剂、糊剂、散剂、粉剂、喷雾剂、气雾剂、栓剂、贴片、悬液、漱口液、口含片、辅料、贴片、绷带、贴膜、纱布。
制剂单元
本公开的药物或药物组合物或活性成分或产品，可以制备成单位制剂(单元制剂)的形式。
在一些实施方案中，所述药物(或制剂、或治疗剂、或医疗装置)中的单位剂量含有：
-1μg至1000μg所述的赤红球菌产品；或
-1μg至1000μg所述的赤红球菌细胞壁；或
-1μg至1000μg所述的赤红球菌细胞壁骨架。
单位剂量的具体示例是1、2、5、10、15、20、25、30、40、50、55、56、57、58、59、60、61、62、63、65、66、67、68、69、70、80、90、100、150、200、250、300、350、400、450、500μg±10％、以及上述任意两个数值之间的范围。
“施用”、“给予”、“提供给”、“处理”当应用于动物、人、细胞、组织、器官或生物样品时，是指药物或医疗装置与动物、人、细胞、组织、器官或生物样品接触。
“治疗”意指给予受试者内用或外用药物(治疗剂、活性成分或组合物)(如，根据本公开的赤红球菌细胞壁或其药物组合物)或医疗 装置，在被治疗的受试者(或群体)中缓解(减轻、延迟、改善、治愈)一种或多种疾病症状，以至于达到临床可测量的程度，其中所述的受试者已经患有、疑似患有或易感于一种或多种疾病或其症状。
有效缓解任何疾病症状的药物(治疗剂、活性成分或组合物)的量称为治疗有效量。可根据多种因素变化，例如受试者的疾病状态、年龄和体重。应当理解，在缓解单个受试者的目标疾病或其症状时，药物(治疗剂、活性成分或组合物)可能无效，但是根据本领域已知的任何统计学检验方法(如Student T检验、卡方检验、依据Mann和Whitney的U检验)确定，药物(治疗剂、活性成分或组合物)在统计学意义上对目标疾病或其症状是有效的。
“任选”意味着其随后所描述的事项可以发生，但不必须发生；需要视情况而定。例如，“任选地，进行分装”意味着允许对产品进行分装，但是不是必须进行分装；分装与否不影响技术效果的实现。
“一个”、“一”、“单个”、“该”，如果没有明确说明，也包括复数形式。
以下结合实施例、制备例和测试例，进一步描述本公开。但这些实施例、制备例和测试例并非限制着本公开的范围。当未注明具体条件时，按照常规条件、按照原料供应商所建议的条件操作。未注明具体来源的试剂，为市场购买的常规试剂。
技术人员尤其理解，虽然以下具体示例采用了特定的细胞株，但是技术效果的实现不限于该特定的细胞株，任何属于红球菌属赤红球菌种的(Rhodococcus ruber)物种均适用。
实施例
实施例1.菌株保藏
发明人将实验室保存的主代菌株于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号，中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.17431。经检测表明，所寄存的菌株存活。
实施例2.菌株鉴定
1.菌落形态特征的裸眼观测
在甘油琼脂培养介质上，30-37℃(具体为32-35℃)培养12至72(具体为36至60，例如40-50)小时，可见(图1)：
-菌落隆起；
-呈桔红色(受光线、培养介质颜色等影响，则略有差别)；
-表面干燥皱起、稍显光泽(随培养条件差别，则略有差别)；
-轻触易碎；
-菌落大小约1mm至2mm(随培养条件差别，则略有差别)。
2.显微镜观察
-菌体呈分枝状，有横隔膜，形成菌丝体(随培养条件差别，则略有差别)；
-菌丝分裂形成规则的短粗细胞(随培养条件差别，则略有差别)；
-培养4至5天时，菌体成短杆状或球形(随培养条件差别，则略有差别)。
3.染色性
革兰氏染色阳性。
4.生化反应
取甘油琼脂斜面培养介质上，30-37℃(具体为32-35℃)培养12至72(具体为36至60，例如40-50)小时培养。然后，对培养物进行以下各项测试。
4.1碳水化合物产酸：
呈阳性：甘油、甘露醇、山梨醇、D-阿拉伯糖醇、D-果糖、D-葡萄糖；
呈阴性：肌醇、菊糖、乳糖、蔗糖、淀粉、麦芽糖、糖原、木糖醇、葡萄糖酸盐、海藻糖、赤藓醇、松三糖、蜜二糖、棉子糖、纤维二糖、苦杏仁甙、龙胆二糖、阿东醇、熊果甙、D-阿拉伯糖、L-阿拉伯糖、α-甲基-D-葡萄糖甙、α-甲基-D-甘露糖甙、D-核糖、D-木糖、L-木糖、N-乙酰-葡糖胺、D-松二糖、D-来苏糖、β-甲基-D-木糖甙、 D-半乳糖、D-塔格糖、D-岩藻糖、L-岩藻糖、D-甘露糖、L-山梨糖、L-阿拉伯糖醇、L-鼠李糖、2-酮基-葡萄糖酸盐。
4.2酶活性测定(API ZYM)：
呈阳性：碱性磷酸酶、类脂酯酶(C8)、类脂酶(C14)、白氨酸芳胺酶、缬氨酸芳胺酶、胱氨酸芳胺酶、胰蛋白酶、胰凝乳蛋白酶、酸性磷酸酶、萘酚-AS-B1-磷酸水解酶、α-葡萄糖苷酶；
呈阴性：N-乙酰-葡萄糖胺酶、酯酶(C4)、β-半乳糖苷酶、β-糖醛酸苷酶、β-葡萄糖苷酶、α-半乳糖苷酶、α-甘露糖苷酶、β-岩藻糖苷酶。
4.3硝酸盐还原反应阳性、接触酶阳性、酪氨基酸酶阳性、淀粉酶阴性、氧化酶阴性、明胶液化阴性。
4.4唯一碳源：
Figure PCTCN2020078932-appb-000001
4.5. 16S rRNA鉴定
对工作种子管中分离的15株菌和原始种子管中分离的10株不同菌株，进行基因组提取、16Sr RNA扩增、并测序。总计25个菌株的16Sr RNA基因同一性为100％。这表示25个菌株为相同种属(图2)。
同时，基于Kimura2-参数算法构建的neighbor-joining菌株进化树， 结果显示菌株归属为Rhodococcus ruber。
制备例
制备例1.培养方法
1.可以通过常规的微生物生产方法，培养赤红球菌。
2.培养方式既可为固体培养，也可为液体培养(比如摇瓶、发酵罐)。
3.对培养介质中的营养源并无特殊的限制，培养介质中含有培养革兰氏阳性菌常用的碳源、氮源及其它营养源。
-碳源是赤红球菌可以利用的任何碳源。例如，果糖、葡萄糖等。
-氮源：肉膏、蛋白胨、铵盐、硝酸盐以及其它有机或无机含氮化合物。
-其它营养源：可适当添加无机盐。例如NaCl、磷酸盐等。
4.对培养条件(温度、时间等)并无严格的限制，技术人员可以根据初步的小规模的中试数据，自行选择使其产量最高的条件。
5.作为一个示例，采用以下培养条件发酵赤红球菌：
(1)培养介质组成包含：
蛋白胨、牛肉膏、氯化钠、磷酸盐、甘油(以及，任选琼脂，当固体培养时)。
(2)培养的方法参数：
工作菌种复苏后，转移至固体培养介质上维持3-5天，再转移至液体培养(30-37℃，维持3-5天)，可以采用补料分批的半连续模式，也可以采用分批模式。培养期间监控pH、细菌密度、溶解氧、碳源消耗。
制备例2.菌体破碎
收集制备例1所得到的菌，对细胞进行粉碎(例如但不限于通过超声波破碎)。也允许采用本领域任何适当的公知方法对菌体进行破碎，例如CN101250490A或CN101323865A。
显微镜下检查粉碎的情况，每个视野有形菌不得超过5个，检查若干(10至30个)视野均达到此标准为合格。
制备例3.去除非细胞壁成分
1.除核酸：
将破碎产物进行离心，获得的沉淀物中加入DNA酶和RNA酶，按照酶的供应商建议的操作去除核酸。
2.除蛋白质：
沉淀物加入常见的蛋白酶(例如胰蛋白酶)，按照酶的供应商建议的操作去除蛋白质。
3.除脂质：
沉淀物中加入有机试剂(如但不限于丙酮、乙醚、乙醇中的一种或组合)，按照本领域常规操作去除脂质。
4.除细胞膜：
沉淀物中加入TritonX-100，按照本领域常规操作，离心收集沉淀物，用PBS漂洗。
应当理解，上述除去杂质的步骤之间，技术人员可以调整先后顺序，使得步骤之间兼容。去除非细胞壁成分后，将沉淀物复溶于注射用水，待用。任选地，可以在115℃下灭菌20-30分钟，作为细胞壁骨架的原液(包含细胞壁骨架及其组成成分)。
除了上述方法，技术人员还可以使用本领域已知的或未来的方法以去除非细胞壁成分，例如CN105779326A中公开的萃取细胞壁成分的方法。
5.产量
从159个克氏瓶中共收集菌液653ml(破碎后)；湿重产量为138g；细胞壁骨架产量为约0.87g/克氏瓶。
制备例4.药物组合物(医疗装置)的制备方法
1.液体组合物
向制备例3所得产物中加入赋形剂(如右旋糖酐40、甘露醇或海藻糖)。分装后，即为药物组合物。
表1.药物组合物可以配制成多种形式
Figure PCTCN2020078932-appb-000002
Figure PCTCN2020078932-appb-000003
2.冻干粉组合物
将第1项的药物组合物冻干，制得冻干粉(分别编号为冻干粉组合物1至7)。
3.制剂的制备方法
(1)敷料
将第1项的药物组合物(活性成分60μg至120μg，例如60μg、70μg、80μg、90μg、100μg、110μg、120μg)涂覆在敷料(例如无菌纱布)上，制备成外用医疗装置。
(2)贴片或贴膜
采用本领域公知的方法，制备贴膜(例如中国申请号201610605617.5、201510614414.8、200610200450.0、201610511974.5、201610471977.0等公开的方法)。
例如：将成膜材料聚乙烯醇、卡波姆、羟丙基纤维素加如水中，溶胀，形成均质粘稠液；向其中加入第1项的药物组合物(分别地，组合物1至7)，并混匀；静置消泡；将形成的无气泡的混合黏稠液；浇铸在涂抹少量石蜡的模具上，干燥5-20min，取出起膜，切割成所需面积。
(3)凝胶剂
也可以制成凝胶的形式。例如参照中国申请号200510028076.6公开的方法，将组合物3和酯化剂搅拌溶解在溶媒中，加入羟烷基纤维 素，使之溶胀，继续搅拌形成凝胶状；然后加入交联剂继续搅拌至完全均匀。
(4)膏剂
用于皮肤外用软膏的制备方法，还见于但不限于中国申请号201610856428.5、01133296.4、1133297.2、《药物剂型与制剂设计》(化学工业出版社2009)公开的方法。
4.质量检验(以冻干粉组合物3为例)
表2.质量检验项目
Figure PCTCN2020078932-appb-000004
测试例
测试例1.药理实验
1.用相当于人类临床剂量的20、40、80倍静脉注射后，对麻醉猫的血压、呼吸、心率和心电进行监测，未产生明显影响；
2.用相当于人类临床剂量的1000倍静脉注射后，小鼠的协调运动和学习记忆功能未产生明显的影响。
可见本公开的药物组合物(组合物1至组合物7)对动物精神、神经系统、心血管系统、呼吸系统均无明显影响。
测试例2.安全实验
1.无菌实验：
结果阴性，证明无菌。
2.小鼠急性毒性实验：
实验组分别以皮下注射及腹腔注射方式给药，其剂量是人用量的5倍，对照组以无菌生理盐水0.5ml/支，连续观察7-8天。小鼠状态良好，体重无异常；小鼠各脏器无异常。
3.长期毒性实验：
应用临床剂量的30倍，每日一次，连续阴道给药三个月，未发现狗有毒性作用；心电图、血液生化指标在正常范围内。停药两周后，未见到延迟毒性(组合物1至组合物7)。
测试例3.稳定性实验
在常温(18-25℃)放置0、1、2、3、8、14、21个月，该药物组合物(组合物1至组合物7)的丙氨酸含量、胞壁酸含量、吞噬率、吞噬指数与实验起始时相比，无统计学显著差异(测试三个批次)。
综上，冻干粉制剂的药物组合物可以稳定保存24个月。
测试例4.吞噬实验
现有技术中，吞噬细胞受抗原刺激后活化，可使吞噬功能明显增强。在小鼠体内诱导腹腔巨噬细胞产生后，给小鼠腹腔注射鸡血红细胞，30min后处死小鼠取出腹腔液，染色，显微镜下计数吞噬红细胞的百分数，以判断吞噬细胞的杀伤能力，间接地测定机体的非特异性免疫水平。
利用本申请的组合物3进行测试，其吞噬率为75％，吞噬指数1.05。而阴性对照(赋形剂)和空白对照(生理盐水)的吞噬率和吞噬指数都较低。说明本申请的细胞壁骨架或其组成成分的促免疫吞噬能力强。
效果例
效果例1
1.初诊(2019-06-07)：
主诉：外阴皮肤变白增生4个月；
现病史：4个月前患者无明显诱因出现外阴瘙痒，之后出现外阴皮肤变白，出现增生，局部皮肤粗糙，无创伤史，无高血压糖尿病等全身性疾病；
既往史：无；
家族史：无；
体格检查：外阴发育正常，左侧外阴处可见1.5×1.5cm 2大小的白色增生病损，略高于皮肤表面，病损未见明显破溃面，未见明显脓性分泌物。
初步诊断：外阴白色病变。
2.治疗计划：
告知患者病情，取得知情同意。
保持会阴部清洁；避免挠抓等物理刺激；局部施用组合物3(软膏，隔日一次，每次60μg活性成分)。每周复查，不适随诊。
3.第一次复诊(2019-06-14)：
复诊：患者上次就诊后遵医嘱用药，自觉左侧外阴处病损面积减小。
检查：外阴发育正常，左侧外阴处可见0.8×1.5cm 2大小的白色病损，较治疗前减小，局部萎缩，未见明显脓性分泌物。
4.第二次复诊(2019-06-21)：
复诊：患者上次就诊后遵医嘱用药，自觉左侧外阴处病损面积减小。
检查：外阴发育正常，左侧外阴处可见0.8×0.6cm 2大小的白色病损，较治疗前减小，局部萎缩，未见明显脓性分泌物。
5.第三次复诊(2019-07-04)：
复诊：患者上次就诊后遵医嘱用药，自觉左侧外阴处病损面积减小。
检查：外阴发育正常，左侧外阴处皮肤恢复正常，局部稍伴萎缩，未见明显脓性分泌物。
效果例2
1.初诊(2019-07-02)
主诉：外阴皮肤变白伴瘙痒2个月；
现病史：2个月前患者无明显诱因出现外阴瘙痒，奇痒难忍，之后出现外阴皮肤变白，局部皮肤粗糙，无阴道出血，无创伤史，无高血压糖尿病等全身性疾病；
既往史：无；
家族史：无；
体格检查：外阴双侧可见约1.2×1.0cm 2大小色素减退斑(hypopigmentation spot)，双侧大阴唇萎缩，未见明显破溃及出血，无明显脓性分泌物。
初步诊断：外阴白色病变。
2.治疗计划：
告知患者病情，取得知情同意。
组织病理学检查：必兰(Primacaine)STA局麻，常规消毒，于右侧外阴病损处取约0.8×0.5×0.5cm 3组织，缝合3针。样本用10％福尔马林固定标本，送病理，医嘱术后一周拆线。
3.第一次复诊(2019-07-09)：
复诊：患者上次取活检后无不适。病理结果显示符合外阴白色病变。
检查：活检部位愈合良好，缝线3针存，未脱落；
处置：保持会阴部清洁；避免挠抓等物理刺激；局部施用组合物3(干粉，隔日一次，每次60μg活性成分)。每周复查，不适随诊。
4.第二次复诊(2019-07-16)：
复诊：患者上次就诊后遵医嘱用药，自觉双侧外阴病损面积减小，粗糙程度减轻。
检查：双侧外阴可见约1.0×0.7cm 2大小色素减退斑，双侧大阴唇萎缩，未见明显破溃及出血，无脓性分泌物。
5.第三次复诊(2019-07-23)：
复诊：患者上次就诊后遵医嘱用药，自觉双侧外阴病损减小。
检查：双侧外阴可见约0.8×0.5cm 2大小色素减退斑，双侧大阴唇 萎缩，未见明显破溃及出血，无脓性分泌物。
6.第四次复诊(2019-07-31)：
复诊：患者上次就诊后遵医嘱用药，自觉双侧外阴病损基本愈合。
检查：双侧外阴色素减退斑基本恢复消失，双侧大阴唇稍萎缩，未见明显破溃及出血，无脓性分泌物。
效果例3
1.初诊(2019-05-10)：
主诉：外阴发白1个月；
现病史：患者1个月前发现外阴发白，伴有外阴瘙痒，不断挠抓止痒，自用药后未见明显好转，局部皮肤较为粗糙，无脓性分泌物及阴道出血，无其它皮肤病损；
既往史：无；
家族史：无；
体格检查：外阴双侧可见约1.0×2.0cm 2大小的白色病损，双侧大阴唇萎缩，病损中央可见约0.4×0.6cm 2大小破溃面，周缘脱屑，未见明显脓性分泌物。
初步诊断：外阴白色病变。
2.治疗计划：
告知患者病情，取得知情同意。
保持会阴部清洁；避免挠抓等物理刺激；局部用组合物5的洗剂冲洗，并随后施用组合物3(凝胶剂，隔日一次，每次60μg活性成分)。每周复查，不适随诊。
3.第一次复诊(2019-05-17)：
复诊：患者上次就诊后遵医嘱用药，自觉外阴双侧病损面积减小，破溃面愈合。
检查：外阴发育正常，外阴双侧可见0.6×2cm 2大小的白色病损，较治疗前减小，局部萎缩，病损中央破溃面愈合，未见明显脓性分泌物。
4.第二次复诊(2019-05-24)：
复诊：患者上次就诊后遵医嘱用药，自觉外阴双侧病损面积减小。
检查：外阴发育正常，外阴双侧处可见0.8×1.2cm 2大小的白色病损，较治疗前减小，局部萎缩，未见明显脓性分泌物。
5.第三次复诊(2019-06-02)：
复诊：患者上次就诊后遵医嘱用药，自觉外阴双侧病损面积减小。
检查：外阴发育正常，双侧外阴处可见0.2×0.3cm 2大小的白色病损，较治疗前减小，局部萎缩，未见明显脓性分泌物。

Claims (9)

  1. 赤红球菌细胞壁或赤红球菌细胞壁骨架在制备药物中的用途，其中所述药物用于治疗外阴白色病变或预防其复发。
  2. 根据权利要求1所述的用途，其中所述外阴白色病变是外阴硬化性苔藓和/或外阴鳞状上皮细胞增生。
  3. 根据权利要求1所述的用途，其中所述药物制备成选自以下的剂型：膏剂、霜剂、乳液、混悬剂、糊剂、凝胶剂、洗剂、酊剂、油剂、片剂、气雾剂、喷雾剂、搽剂、粉剂、辅料、绷带、膜、贴片、栓剂。
  4. 根据权利要求1所述的用途，其中：
    所述药物包含1μg至1000μg单位剂量的赤红球菌细胞壁或赤红球菌细胞壁骨架；
    优选地，包含5μg至800μg单位剂量的赤红球菌细胞壁或赤红球菌细胞壁骨架；
    更优选地，包含10μg、20μg、30μg、40μg、50μg、60μg、70μg、80μg、90μg、100μg、150μg、200μg、250μg、300μg、350μg、400μg、500μg、600μg、700μg、800μg每单位剂量。
  5. 根据权利要求1所述的用途，其中所述赤红球菌是2019年03月22日保藏在中国微生物菌种保藏管理委员会普通微生物中心的赤红球菌Rhodococcus ruber，保藏编号为CGMCC No.17431。
  6. 根据权利要求1所述的用途，其中：
    所述药物还包含药学上可接受的赋形剂；
    所述赤红球菌细胞壁或赤红球菌细胞壁骨架为1个重量份，所述药学上可接受的赋形剂为100个至1000个重量份，优选200个至500个重量份，更优选200个至300个重量份；
    优选，所述赤红球菌细胞壁或赤红球菌细胞壁骨架为1个重量份，所述药学上可接受的赋形剂为250个重量份；
    所述药物是液态、干粉制剂、或冻干粉制剂。
  7. 根据权利要求1至6任一项所述的用途，所述赤红球菌细胞壁或赤红球菌细胞壁骨架能够通过以下方法获得，所述方法包括以下步骤或由以下步骤组成：
    1)提供赤红球菌；
    2)粉碎所述赤红球菌，得到粉碎产物；
    3.1)任选地，对所述粉碎产物进行去除脂质的操作；
    3.2)任选地，对所述粉碎产物进行去除核酸的操作；
    3.3)任选地，对所述粉碎产物进行去除蛋白质的操作；
    3.4)得到源自赤红球菌细胞壁的产物；
    4)任选地，除去所述源自赤红球菌细胞壁的产物中的水，优选对所述源自赤红球菌细胞壁的产物进行冷冻干燥；
    5)任选地，分装；
    其中，步骤3.1)、3.2)、3.3)能够互换顺序或并行；步骤4)和步骤5)能够互换顺序；
    优选，所述粉碎的平均粒度为10nm至1000nm，更优选10nm至800nm，更优选10nm、20nm、30nm、40nm、50nm、60nm、70nm、80nm、90nm、100nm、110nm、120nm、130nm、140nm、150nm、160nm、170nm、180nm、190nm、200nm、250nm、270nm、300nm、320nm、350nm、370nm、400nm、420nm、450nm、470nm、500nm；
    优选地，所述分装是指分装至容器中或固体支撑物上；
    所述容器选自：瓶、管、包、袋、板、安瓿、注射装置、铝膜包装、敷料、胶囊。
  8. 一种治疗外阴白色病变或预防其复发的方法，包括：
    使受试者接触治疗有效量的赤红球菌细胞壁或赤红球菌细胞壁骨架；
    所述外阴白色病变是外阴硬化性苔藓和/或外阴鳞状上皮细胞增 生；
    所述赤红球菌细胞壁或赤红球菌细胞壁骨架制备成选自以下的形式：膏剂、霜剂、乳液、混悬剂、糊剂、凝胶剂、洗剂、酊剂、油剂、片剂、气雾剂、喷雾剂、搽剂、粉剂、辅料、绷带、膜、贴片、栓剂；
    所述接触为一天两次、或一天一次、或二天一次、或二天三次、或三天一次、或一周一次；
    所述接触为每次30分钟至每次24小时；
    所述接触持续2天、3天、4天、5天、6天、1周、2周、3周、4周、5周、6周、7周、8周、9周、10周、11周、12周、13周、14周、15周、16周、17周、18周或更长。
  9. 根据权利要求8所述的方法，所述赤红球菌细胞壁或赤红球菌细胞壁骨架能够通过以下方法获得，所述方法包括以下步骤或由以下步骤组成：
    1)提供赤红球菌；
    2)粉碎所述赤红球菌，得到粉碎产物；
    3.1)任选地，对所述粉碎产物进行去除脂质的操作；
    3.2)任选地，对所述粉碎产物进行去除核酸的操作；
    3.3)任选地，对所述粉碎产物进行去除蛋白质的操作；
    3.4)得到源自赤红球菌细胞壁的产物；
    4)任选地，除去所述源自赤红球菌细胞壁的产物中的水，优选对所述源自赤红球菌细胞壁的产物进行冷冻干燥；
    5)任选地，分装；
    其中，步骤3.1)、3.2)、3.3)能够互换顺序或并行；步骤4)和步骤5)能够互换顺序；
    所述粉碎的平均粒度为10nm至1000nm，优选10nm至800nm，更优选10nm至500nm；
    优选地，所述分装是指分装至容器中或固体支撑物上；
    所述容器选自：瓶、管、包、袋、板、安瓿、注射装置、铝膜包装、敷料、胶囊。
PCT/CN2020/078932 2019-03-14 2020-03-12 赤红球菌产品在治疗外阴白色病变中的用途 WO2020182180A1 (zh)

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