WO2020147530A1 - 分离的赤红球菌细胞壁骨架在制备治疗人乳头瘤病毒感染的药物中的用途 - Google Patents

分离的赤红球菌细胞壁骨架在制备治疗人乳头瘤病毒感染的药物中的用途 Download PDF

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WO2020147530A1
WO2020147530A1 PCT/CN2019/127895 CN2019127895W WO2020147530A1 WO 2020147530 A1 WO2020147530 A1 WO 2020147530A1 CN 2019127895 W CN2019127895 W CN 2019127895W WO 2020147530 A1 WO2020147530 A1 WO 2020147530A1
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rhodococcus
cell wall
wall skeleton
isolated
human papillomavirus
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PCT/CN2019/127895
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English (en)
French (fr)
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WO2020147530A8 (zh
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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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Priority to EP19909992.0A priority Critical patent/EP3909595B1/en
Priority to KR1020217025003A priority patent/KR20210114966A/ko
Priority to CN201980010480.0A priority patent/CN111787931B/zh
Priority to US17/421,718 priority patent/US12097224B2/en
Priority to JP2021539950A priority patent/JP7572062B2/ja
Publication of WO2020147530A1 publication Critical patent/WO2020147530A1/zh
Publication of WO2020147530A8 publication Critical patent/WO2020147530A8/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A61L15/44—Medicaments
    • 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
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • C12N1/06—Lysis of microorganisms
    • C12N1/066—Lysis of microorganisms by physical methods
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    • 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
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)

Definitions

  • the present disclosure relates to the medical field, the microbiological field, and the biopharmaceutical field.
  • the present invention relates to the use of the isolated Rhodococcus rhodococcus cell wall skeleton and the pharmaceutical composition containing the same in the preparation of a medicine for treating human papillomavirus infection.
  • HPV Human papillomavirus
  • HPV subtypes have been isolated. Different types of HPV cause different clinical manifestations, which are divided into skin type and mucosal type according to the location of the infected epithelium; different types of HPV and the risk of causing tumors are different, and are divided into high-risk type and low-risk type. Skin low-risk HPV is related to common warts, flat warts, toe warts, etc.; skin high-risk HPV may cause vulvar cancer, penile cancer, anal cancer, prostate cancer, bladder cancer, etc.
  • Mucosal low-risk HPV can infect the genitals, anus, oropharynx, and esophageal mucosa; mucosal high-risk HPV can cause cervical cancer, rectal cancer, oral cancer, tonsil cancer, etc. About 35 types are related to genital tract infections, and about 20 types are related to tumors.
  • low-risk HPV types include: HPV6, 11, 42, 43, 44, CP8304, etc., which can cause benign lesions such as genital warts, including cervical epithelium
  • Low-grade disease (CINI) and high-risk HPV include: HPV16, 18, 31, 33, 35, 45, 51, 52, 56, 58, 59, 68, etc., which can cause high-grade cervical epithelial disease (CINII, CINIII) and Cervical cancer, and the most common HPV subtypes in cervical cancer are type 16 and type 18.
  • HPV infection is one of the most common sexually transmitted diseases in the world and is related to sexual factors.
  • persistent high-risk HPV infection is the primary cause of cervical cancer, which has been recognized worldwide. Therefore, human papillomavirus infection seriously endangers human health and even life.
  • cervical cancer ranks second in the global mortality rate of women from cancer, and even ranks first in some developing countries. Every year, there are 500,000 new cases of cervical cancer worldwide, and about 200,000 people die from cervical cancer, 80% of which occur in developing countries. Cervical cancer accounts for 24% of malignant tumors in women in developing countries and 7% in developed countries. In the 21st century, cervical cancer is becoming one of the major diseases affecting women's health worldwide.
  • Rhodococcus ruber also called Rhodococcus ruber
  • Rhodococcus ruber is a Gram-positive bacteria. Generally speaking, 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 complete gene sequencing of Rhodococcus rubrum. For example, Fan Xin et al. sequenced the entire genome of Rhodococcus rubrum SD3 strain and performed bioinformatics analysis. The whole genome length of the SD3 strain is about 5.37Mb, the GC content is about 70.63%, and the 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 wide degradation spectrum. Therefore, Rhodococcus is widely used in pollution remediation, organic compound degradation, sewage treatment and other fields.
  • Rhodococcus rubrum lies in environmental management, see CN108862590A, CN107151635A, CN102250796A, CN1519312A, CN103627653A, CN101033454A, CN108130288A, CN104830738A, CN101619299A, CN103509833A, CN106434466A, CN101580808A, CN168A491, CN101580808A, CN102591A, CN102591106A, CN108862590A, CN106434466A, CN101580808A.
  • 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.
  • the present inventors unexpectedly discovered that the Rhodococcus rubrum cell wall skeleton and the composition containing the same have beneficial uses in the treatment of human papillomavirus (HPV virus) infections.
  • HPV virus human papillomavirus
  • an isolated Rhodococcus ruber is provided.
  • Rhodococcus rubrum which was deposited at the China General Microbiological Culture Collection Center of the China General Microbiological Culture Collection Center on March 22, 2019, Beichen, Chaoyang District, Beijing No. 3, No. 1 West Road, Institute of Microbiology, Chinese Academy of Sciences; Zip code: 100101), and the deposit number is CGMCC 17431.
  • the deposit meets the requirements of the Budapest Treaty on the International Recognition of Microorganisms 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 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 the deposit number of CGMCC 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 undergoing purification (fat removal, nucleic acid removal, protein removal).
  • Rhodococcus rubra product which comprises the cell wall of Rhodococcus rubra.
  • 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 (removing fat, and/or nucleic acid, and/or protein) of Rhodococcus rubrum.
  • 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 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 in the pharmaceutical composition is 1 part by weight
  • the pharmaceutically acceptable excipient is 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 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 lies 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 ointment, cream, gel, lotion, tincture, liniment, oil, paste, aerosol, oral Tablets, patches, freeze-dried powders, suspensions.
  • the dosage form is a buccal tablet.
  • lyophilized powder is used for tableting.
  • the pharmaceutically acceptable excipient relates to, but is not limited to: fillers, stabilizers, flavors, disintegrants, binders, and lubricants.
  • 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), 5.3) can be interchanged in order or can be parallel; step 6) and step 7) can be interchanged in order or can be parallel.
  • step 5) may further include (for example, using a non-ionic surfactant) to remove the cell membrane.
  • Rhodococcus rubrum is not limited to specific 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 fermentation tanks according to the preparation scale.
  • 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 (such as oral, injection, topical application, etc.) of the active ingredients (cell wall and its components), so as to avoid preparation Introduce factors that affect subsequent applications in the steps.
  • an organic solvent is used to remove lipids in the fragmented product.
  • nucleases are used to remove DNA and RNA from the broken products.
  • hydrolytic enzymes are used to degrade proteins in the broken products.
  • a surfactant is used to remove cell membranes in the disrupted product.
  • the crushed average particle size 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 Chemical Industry, 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 containers.
  • 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. Add solvent to the bottle/ampule just before use.
  • the present invention provides a use of an isolated Rhodococcus ruber cell wall skeleton in the preparation of a medicine for treating human papillomavirus infection, wherein:
  • the isolated Rhodococcus rhodococcus cell wall skeleton is derived from the General Microbiology Center of the China Microbial Culture Collection Management Committee, No. 3, Beichen West Road, Chaoyang District, Beijing on March 22, 2019, and the preservation number is CGMCC Rhodococcus 17431;
  • Rhodococcus rubrum cell wall skeleton is obtained by the following steps:
  • the present invention provides a use of a pharmaceutical composition in the preparation of a medicament for the treatment of human papillomavirus infection, wherein the pharmaceutical composition comprises:
  • the isolated Rhodococcus cell wall skeleton preferably, the isolated Rhodococcus cell wall skeleton is derived from the China Microbial Species Preservation Management Committee at No. 3 Beichen West Road, Chaoyang District, Beijing on March 22, 2019. The Center for Microbiology, the collection number of Rhodococcus erythrococcus with the deposit number CGMCC 17431;
  • the isolated Rhodococcus rubrum cell wall skeleton is 1 part by weight, and the pharmaceutically acceptable excipient is 200 to 300 parts by weight;
  • the pharmaceutically acceptable excipient is selected From dextran, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythritol, gelatin, magnesium stearate and mannitol.
  • the isolated Rhodococcus ruber cell wall skeleton or the composition containing the same provided by the present invention can be used for topical application; optionally, the topical application may be local skin or submucosal injection, or local skin or Mucosal coating.
  • the isolated Rhodococcus ruber cell wall skeleton provided by the present invention or the composition containing the same can be formulated into ointments, creams, plasters, gels, lotions, tinctures, liniments, oils , Paste, lyophilized powder, aerosol, suppository, patch or suspension, preferably, wherein the drug is formulated into a lyophilized powder preparation, and the lyophilized powder preparation is diluted in physiological saline before use to make
  • the concentration of the cell wall skeleton of Rhodococcus rubrum is 15 ⁇ g/ml-500 ⁇ g/ml, preferably the concentration is 30-240 ⁇ g/ml, more preferably the concentration is 30-120 ⁇ g/ml, more preferably the concentration is 30-60 ⁇ g/ml.
  • the present invention provides a use of an isolated Rhodococcus cell wall skeleton in the preparation of a medical device for the treatment of human papillomavirus infection, wherein:
  • the isolated Rhodococcus rhodococcus cell wall skeleton is derived from the General Microbiology Center of the China Microbial Culture Collection Management Committee, No. 3, Beichen West Road, Chaoyang District, Beijing on March 22, 2019, and the preservation number is CGMCC Rhodococcus 17431;
  • the medical device comprises the isolated Rhodococcus rhodococcus cell wall skeleton, and
  • the medical device is an external medical device, preferably selected from the following forms: dressing, patch, bandage or film, preferably, the dressing, patch, bandage or film has an infiltration concentration of 15 ⁇ g/ml -500 ⁇ g/ml, preferably at a concentration of 30-240 ⁇ g/ml, more preferably at a concentration of 30-120 ⁇ g/ml, more preferably at a concentration of 30-60 ⁇ g/ml in a suspension of Rhodococcus cell wall skeleton.
  • the present invention provides the isolated Rhodococcus erythrococcus cell wall skeleton or composition containing the same according to the present invention for the treatment of human skin, cervix, vagina, labia, penis, perianal, oropharynx, tonsils and oral cavity.
  • the present invention provides an isolated Rhodococcus rubrum cell wall skeleton for the treatment of human papillomavirus infection, wherein:
  • the isolated Rhodococcus rhodococcus cell wall skeleton is derived from the General Microbiology Center of the China Microbial Culture Collection Management Committee, No. 3, Beichen West Road, Chaoyang District, Beijing on March 22, 2019, and the preservation number is CGMCC 17431 Rhodococcus.
  • the present invention provides a pharmaceutical composition for treating human papillomavirus infection, wherein the pharmaceutical composition comprises:
  • the isolated Rhodococcus cell wall skeleton preferably, the isolated Rhodococcus cell wall is derived from the common microorganisms of the China Microbial Species Preservation Management Committee, which was deposited at No. 3, Beichen West Road, Chaoyang District, Beijing on March 22, 2019 Center, the deposit number is CGMCC 17431 Rhodococcus; and
  • the cell wall skeleton of Rhodococcus rubrum is 1 part by weight, and the pharmaceutically acceptable excipient is 200 to 300 parts by weight;
  • Rhodococcus rhodochrous cell wall skeleton is 1 part by weight
  • the pharmaceutically acceptable excipient is 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 and any Any point value within two numerical ranges.
  • Rhodococcus rhodococcus cell wall skeleton provided in the present invention for the treatment of human papillomavirus infection is obtained by the following steps:
  • the present invention provides a method of treating human papillomavirus infection, the method comprising administering to a subject in need thereof a therapeutically effective amount of isolated Rhodococcus cell wall skeleton, wherein:
  • the isolated Rhodococcus rhodococcus cell wall skeleton is derived from the General Microbiology Center of the China Microbial Culture Collection Management Committee, No. 3, Beichen West Road, Chaoyang District, Beijing on March 22, 2019, and the preservation number is CGMCC Rhodococcus Rhodococcus of 17431; or a pharmaceutical composition comprising the isolated Rhodococcus Rhodococcus cell wall skeleton; preferably, the application is topical; preferably, the topical application is skin or mucosal coating, or skin or submucosal injection ;
  • the unit dose of the therapeutically effective amount is 1 ⁇ g to 1000 ⁇ g; preferably, 15 ⁇ g-500 ⁇ g, preferably 30-240 ⁇ g, more preferably 30-120 ⁇ g, more preferably 30-60 ⁇ g;
  • the treatment is performed at a frequency selected from: 1 to 3 times a day, 1 to 6 times for two days, 1 to 9 times for three days, 1 to 14 times a week, 1 to 14 times a month, 60 times; more preferably, once a day;
  • the treatment lasts for 2 days to 2 months, more preferably, lasts for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks.
  • the only therapeutic (or preventive) active ingredient in a drug or pharmaceutical composition 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
  • 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. Specifically, the cell wall of gram-positive bacteria is thicker (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 complicated, 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.
  • Each peptidoglycan monomer consists of 3 parts: sugar unit (for example, at least two sugar molecules are connected by glycosidic bonds to form the framework of the peptidoglycan), peptide tail (a short peptide chain connected by several amino acids) , Which is connected to the N-acetylmuramic acid molecule), and the 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 of the peptidoglycan
  • peptide tail a short peptide chain connected by several amino acids
  • the 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 rhodochrous cell wall can be understood as both a complete cell wall and an incomplete cell wall (for example, broken or partially degraded).
  • the ingredients exhibiting the desired activity are derived from the cell wall of Rhodococcus (for example, the cell wall itself or its composition). Therefore, in clinical applications, various forms such as intact cell walls, broken cell walls, incomplete degradation products of cell walls, components of cell walls, extracts of cell walls, and the like are all included within 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 17431.
  • technicians can perform taxonomic identification on a strain of bacteria.
  • the 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 advent of sequencing technology, technicians can identify strains in a more reliable way.
  • 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, plaster, gel, lotion, tincture, liniment, oil, paste, Freeze-dried powders, aerosols, suppositories, patches, suspensions, oral liquids, lozenges, skin care products (cleansers, lotions, essences, lotions, creams, masks).
  • Excipients suitable for the present disclosure such as but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythritol, gelatin, magnesium stearate , Mannitol, propellant, humectant, solvent, solubilizer, emulsifier, antioxidant, pH regulator, preservative.
  • non-limiting examples also include: white petrolatum, carbomer, hypromellose, methyl cellulose, sodium hydroxymethyl cellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, Polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerin, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, ethyl Amide, isopropanol, Tween, polyoxyethyl hydrogenated castor oil, stearic acid, glyceryl monostearate, triglyceride monostearate, sucrose fatty acid ester, sucrose ester, sucrose acetate isobutyrate Esters, sorbitan tristearate, isopropyl myristate,
  • 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 of the drug (or formulation, or therapeutic agent, or medical device) contains:
  • Rhodococcus rubrum cell wall skeleton 500 mg of the Rhodococcus rubrum cell wall skeleton.
  • unit doses are 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg ⁇ 10%, and the range between any two of the above values.
  • administer refers to drugs (therapeutic agents, active ingredients or compositions) and animals, humans , Cells, tissues, organs or biological samples.
  • Treatment means to give a subject an internal or external drug (therapeutic agent, active ingredient, or composition) (eg, the Rhodococcus cell wall or pharmaceutical composition thereof according to the present disclosure) in the subject (or The population) alleviate (relieve, delay, improve, cure) one or more disease symptoms to a clinically measurable degree, wherein the subject has, is suspected of having, or is susceptible to one Or multiple diseases or their symptoms.
  • an internal or external drug eg, active ingredient, or composition
  • the amount of a drug (therapeutic agent, active ingredient or composition) that effectively relieves the symptoms of any disease is called a therapeutically effective amount. It can vary according to various factors, such as the disease state, age and weight of the subject. It should be understood that drugs (therapeutic agents, active ingredients, or compositions) may be ineffective in relieving the target disease or symptoms of a single subject, but according to any statistical test method known in the art (such as Student T test, card Fang test, U test according to Mann and Whitney) 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 T test, card Fang test, U test according to Mann and Whitney
  • the inventor deposited the master strain 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, with the preservation number CGMCC 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, ⁇ -glucuronidase, ⁇ -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.
  • Biolog Gen III is sensitive to serine, vancomycin, tetrazolium violet, and tetrazolium blue;
  • Chemical sensitivity test is not sensitive to sodium bromate, 1% sodium lactate, pH 6.0, 1%-4% sodium chloride, nalidixic acid, lithium chloride, potassium tellurite, aztreonam, and sodium butyrate.
  • 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 evolution tree constructed based on the Kimura2-parameter algorithm shows that the strain belongs to Rhodococcus ruber.
  • Rhodococcus erythrococcus with the deposit number CGMCC17431 can be cultivated by conventional microbial production methods.
  • the culture method can be solid culture or liquid culture.
  • the medium can contain carbon sources, nitrogen sources and other nutrient sources that are commonly used for microbial culture.
  • the carbon source can be any carbon source available to Rhodococcus rubrum. For example, fructose, glucose, etc.
  • the nitrogen source can be: meat extract, peptone, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds.
  • some inorganic salts can be added appropriately.
  • NaCl NaCl
  • phosphates NaCl
  • the medium composition 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.
  • the bacteria obtained in Preparation Example 1 were collected, and the cells were crushed (for example, but not limited to ultrasonic crushing). It is also allowed to use any suitable well-known method in the art to break up the bacterial cells, 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 order to make the steps compatible.
  • the precipitate is reconstituted in water for injection and is ready for use.
  • it can be sterilized at 115°C for 20-30 minutes as a stock solution of the cell wall skeleton (mainly including the cell wall skeleton and its constituent components).
  • the pharmaceutical composition can be formulated in various forms
  • mice After intravenous injection of 1000 times the equivalent of human clinical dose, the coordinated movement and learning and memory function of mice did not have obvious effects.
  • composition 1 to composition 7 has 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 dose 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 used, once a day, for three months of continuous vaginal administration, no toxic effects in dogs were found; the electrocardiogram and blood biochemical indicators were within the normal range. Two weeks after stopping the drug, no delayed toxicity was seen (composition 1 to composition 7).
  • composition 1 to composition 7 the pharmaceutical composition of the lyophilized powder formulation can be stored stably for 24 months (composition 1 to composition 7).
  • Macrophages are the main cells of the mononuclear phagocyte system. Phagocytes are activated after being stimulated by antigens, which can significantly enhance their phagocytic function. After inducing the production of peritoneal macrophages in mice, the mice were injected with chicken red blood cells. 30 minutes later, 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, indirectly 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 (normal saline) were both lower. It shows that the cell wall skeleton of the present application has a strong ability to promote immune phagocytosis.
  • the inventors used the following methods to identify the effect of the Rhodococcus Rhodococcus cell wall products obtained by the method of the present invention on the treatment of human papillomavirus (HPV) infection.
  • HPV human papillomavirus
  • the Pap stain detection method was usually used, also known as the smear detection method. That is, the collected cervical secretions are coated on a glass slide and observed with a microscope, mainly to check whether there are vacuolar cells or keratinocytes, the detection rate is 70-76%.
  • TCT Membrane liquid-based ultra-thin cytological detection method
  • the polyclonal antibody produced is used to check the tissue HPV antigen, and the PAP method is used to display the virus protein to prove the presence of the virus antigen.
  • HPV protein is positive, a light red weak positive reaction appears in epithelial cells, and brown granular sedimentation is seen in the vacuole cell nucleus as positive. But the detection rate is low 40-60%, the sensitivity is not high, and it cannot be typed.
  • HPV DNA molecular hybridization technology CP-14 immune hybridization method. With a fluorescence microscope 400 times, it can be seen that more than 3 fluorescent spots are positive. It can be typed, nucleic acid hybridization can detect HPV-DNA column, and PCR detection can show specific HPV-DNA amplification zone, but it is cumbersome and expensive. Certain equipment and conditions are required.
  • the present inventors used the freeze-dried powder formulation of composition 1 prepared in Table 1 of the present invention, which was diluted with normal saline to 30 ⁇ g/ml or 60 ⁇ g/ml before use.
  • the lyophilized powder was injected into the cervix.
  • the use of the composition containing the cell wall components of Rhodococcus rubrum of the present invention in the treatment of cervical HPV infection is discussed.
  • the above-mentioned cytological smear detection method and TCT method are used to confirm the diagnosis of HPV virus infection patients. After signing the "informed consent" with the patient, the patient is treated by external application of the combination of the present invention or local injection of the composition of the present invention at the infected site.
  • the comprehensive treatment method is to administer the composition of the present invention once a day, 20 days as a treatment cycle, and women should stop taking the medicine during menstruation.
  • the diluted preparation containing the Rhodococcus rubrum cell wall skeleton of the present invention can effectively treat human cervical HPV virus infection, and the virus conversion rate reaches 100%.

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Abstract

分离的赤红球菌细胞壁骨架或包含其的组合物在制备治疗人乳头瘤病毒感染的药物中的用途。该赤红球菌骨架从2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌中分离获得。

Description

分离的赤红球菌细胞壁骨架在制备治疗人乳头瘤病毒感染的药物中的用途
本公开要求2019年01月15日提交的中国专利申请《赤红球菌细胞壁骨架及其用途》(申请号201910036001.4)以及2019年10月25日提交的中国专利申请《一种分离的赤红球菌》(申请号201911022193.X)的优先权，它们的全部内容通过引用并入此处。
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，本发明涉及分离的赤红球菌细胞壁骨架以及包含其的药物组合物在制备用于治疗人乳头瘤病毒感染的药物中的用途。
背景技术
人乳头瘤病毒(HPV)是一类具有高度的特异性的嗜上皮性球形DNA病毒，仅感染人的皮肤和黏膜，能引起人体皮肤和黏膜的鳞状上皮增生，形成乳头状瘤。
目前，已分离出130多种HPV亚型。不同型别的HPV引起不同的临床表现，依其感染的上皮所在部位分为皮肤型和黏膜型；不同型别HPV与引发肿瘤的危险程度不同，又分为高危型和低危型。皮肤低危型HPV与寻常疣、扁平疣、趾疣等相关；皮肤高危型HPV可能引起外阴癌、阴茎癌、肛门癌、前列腺癌、膀胱癌等。黏膜低危型HPV可感染生殖器、肛门、口咽部、食道黏膜；黏膜高危型HPV可引起宫颈癌、直肠癌、口腔癌、扁桃体癌等。约有35种和生殖道感染有关，约20种与肿瘤相关，如低危型HPV有：HPV6、11、42、43、44、CP8304等型，可引发外生殖器湿疣等良性病变，包括宫颈上皮低度病变(CINI)，高危型HPV有：HPV16、18、31、33、35、45、51、52、56、58、59、68等型，可引发宫颈上皮高度病变(CINII、CINIII)及宫颈癌，而宫颈癌中最常见的HPV亚型是16型和18型。
HPV感染是一种世界上最常见的性传播疾病，与性行为因素相关。特别是高危型HPV持续感染是宫颈癌的首要病因，这一点已得到世界 行内公认。所以人乳头瘤病毒感染严重危及人类的健康，甚至危及生命。
全世界妇女中，每年约有10％-15％的新感染病例，在大多数国家，HPV病毒感染非常常见。年轻的性活跃妇女HPV病毒感染率最高，感染的高峰年龄在18-28岁。大部分妇女HPV感染期比较短，一般在8-10个月左右便可消失。但仍有10％-15％的35岁以上的妇女有持续感染的情况。这些持续感染HPV的妇女则成为宫颈癌的高危人群。
世界卫生组织的统计资料表明，宫颈癌在全球妇女癌症死亡率中位居第二，在一些发展中国家甚至居于首位。每年全球有50万例新发宫颈癌病例，约20万人死于宫颈癌，其中80％的死亡发生在发展中国家。宫颈癌占发展中国家妇女恶性肿瘤的24％，在发达国家则占7％。进入21世纪，宫颈癌正在成为影响全球妇女健康的重大疾病之一。
据不完全统计，我国现有宫颈癌病人约13.8万，每年约有5万人死于宫颈癌，占世界宫颈癌新发病例总数的四分之一。但令专家们苦恼的是，由于早期筛查工作尚不完美，大量的宫颈癌患者往往都是在晚期被发现的，失去了宝贵的早期治疗机会，而且现在已出现宫颈癌年轻化的趋势。
HPV感染的预防和治疗。
HPV感染的预防：防止过早性行为、避免不洁性生活、减少交叉感染、戒烟戒酒等，采取健康的生活方式，增强机体免疫力，适龄接种预防性HPV疫苗，能够减少疫苗中所包含的HPV类型的感染风险。
目前，尚无有效抗HPV感染的药物。FDA已经批准三种疫苗预防HPV感染，分别是二价疫苗、四价疫苗、和九价疫苗。这些疫苗能有效地防止部分HPV型别的感染，但是，对于已经明确的HPV感染或HPV引起的病变，预防性疫苗没有作用。因而，本领域中迫切希望有更有效、更安全的清除HPV感染的新药问世。
赤红球菌(Rhodococcus ruber)(也称为红色红球菌)是一种革兰氏阳性菌。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测序。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。本发明人意想不到地发现，赤红球菌细胞壁骨架以及包含其的组合物在治疗人乳头瘤病毒(HPV病毒)感染中的有益用途。
发明内容
根据本公开的一些实施方案，一方面，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(China General Microbiological Culture Collection Center，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)，保藏编号为CGMCC 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 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 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器械，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器械，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器械，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器械，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器械，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：软膏剂、乳膏剂、凝胶剂、洗剂、酊剂、搽剂、油剂、糊剂、气雾剂、口含片、贴片、冻干粉、悬液。
在一些实施方案中，剂型是口含片。例如，采用冻干粉经过压片而制得。
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。
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)培养所述赤红球菌；
3)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)对所述的粉碎产物进行去除脂质的操作；
5.2)对所述的粉碎产物进行去除核酸的操作；
5.3)对所述的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)可互换顺序或可并行。
任选地，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基和培养参数，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(例如口服、注射、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，利用有机溶剂去除破碎产物中的脂质。在一些实施方案中，利用核酸酶去除破碎产物中的DNA和RNA。在一些实施方案中，利用水解酶降解破碎产物中的蛋白质。在一些实施方案中，利用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中。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。
具体地，本发明在一方面提供了一种分离的赤红球菌(Rhodococcus ruber)细胞壁骨架在制备用于治疗人乳头瘤病毒感染的药物中的用途，其中：
优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；
在优选的实施方案中，其中所述分离的赤红球菌细胞壁骨架通过以下步骤获得：
1)提供赤红球菌；
2)培养所述赤红球菌；
3)收集经培养的赤红球菌；
4)粉碎经培养的赤红球菌，得到粉碎产物；和
5)去除所得粉碎产物中的脂质、核酸和蛋白质。
另一方面，本发明提供了一种药物组合物在制备用于治疗人乳头瘤病毒感染的药物中的用途，其中所述药物组合物包含：
分离的赤红球菌细胞壁骨架，优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；和
药学上可接受的赋形剂；
优选地，其中所述分离的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200至300个重量份；
优选地，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；优选地，所述药学上可接受 的赋形剂选自右旋糖酐、海藻糖、甘氨酸、木糖醇、羧甲基纤维素钠、赤藓糖醇、明胶、硬脂酸镁和甘露醇。
另一方面，本发明提供的分离的赤红球菌(Rhodococcus ruber)细胞壁骨架或包含其的组合物可用于局部施用；任选地，所述局部施用可以是局部皮肤或粘膜下注射、或者局部皮肤或粘膜涂覆。
任选地，本发明提供的分离的赤红球菌(Rhodococcus ruber)细胞壁骨架或包含其的组合物可配制成软膏剂、乳膏剂、硬膏剂、凝胶剂、洗剂、酊剂、搽剂、油剂、糊剂、冻干粉、气雾剂、栓剂、贴片或悬液，优选地，其中所述药物配制成冻干粉制剂，并且在使用前将冻干粉制剂在生理盐水中稀释，使赤红球菌细胞壁骨架的浓度为15μg/ml-500μg/ml，优选浓度为30-240μg/ml，更优选浓度为30-120μg/ml，更优选浓度为30-60μg/ml。
另一方面，本发明提供了一种分离的赤红球菌细胞壁骨架在制备用于治疗人乳头瘤病毒感染的医疗器械中的用途，其中：
优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；
所述医疗器械包含所述分离的赤红球菌细胞壁骨架，和
任选地，药学上可接受的赋形剂；
任选地，所述医疗器械为外用医疗器械，优选地选自以下形式：敷料、贴片、绷带或膜，优选地，所述敷料、贴片、绷带或膜中浸润含浓度为15μg/ml-500μg/ml，优选浓度为30-240μg/ml，更优选浓度为30-120μg/ml，更优选浓度为30-60μg/ml的赤红球菌细胞壁骨架的混悬液。
具体地，本发明提供了根据本发明的分离的赤红球菌细胞壁骨架或包含其的组合物在制备用于治疗皮肤、宫颈、阴道、阴唇、阴茎、肛门肛周、口咽、扁桃体和口腔的人乳头瘤病毒感染的药物中的用途。
另一方面，本发明提供了一种用于治疗人乳头瘤病毒感染的分离的赤红球菌细胞壁骨架，其中：
优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌。
另一方面，本发明提供了一种用于治疗人乳头瘤病毒感染的药物组合物，其中所述药物组合物包含：
分离的赤红球菌细胞壁骨架，优选地，所述分离的赤红球菌细胞壁源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；和
药学上可接受的赋形剂；
优选地，其中所述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200至300个重量份；
优选地，其中所述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。
本发明中提供的用于治疗人乳头瘤病毒感染的分离的赤红球菌细胞壁骨架通过以下步骤获得：
1)提供赤红球菌；
2)培养所述赤红球菌；
3)收集经培养的赤红球菌；
4)粉碎经培养的赤红球菌，得到粉碎产物；和
5)去除所得粉碎产物中的脂质、核酸和蛋白质。
另一方面，本发明提供了一种治疗人乳头瘤病毒感染的方法，所述方法包括向有此需要的受试者施用治疗有效量的分离的赤红球菌细胞壁骨架，其中：
优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；或包含所述分离的赤红球菌细胞壁骨架的药物组合物；优选地，所述施用为局部施用；优选地，所述局部施用为皮肤或粘膜涂覆、或皮肤或粘膜下注射；
优选地，其中所述治疗有效量的单位剂量是1μg至1000μg；优选地，15μg-500μg，优选为30-240μg，更优选为30-120μg，更优选为30-60 μg；
优选地，所述治疗是按照选自以下频率进行的：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；更优选地，每天施用1次；
优选地，所述的治疗持续2天至2个月，更优选地，持续1周、2周、3周、4周、5周、6周、7周或8周。
在本申请的上下文中，药物或药物组合物中的唯一治疗性(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)、CGMCC No.4795、DSM43338、CCTCC No.2012035、CGMCC No.16640、CGMCC 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、洗剂、酊剂、搽剂、油剂、糊剂、冻干粉、气雾剂、栓剂、贴片、悬液、口服液、口含片、护肤品(洁面乳、化妆水、精华、乳液、面霜、面膜)。
赋形剂
适用于本公开的赋形剂，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、甘露醇、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。具体而言，非限制实例还包括：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。
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、或医疗器械)中的单位剂量含有：
-0.001mg至500mg所述的赤红球菌产品；或
-0.001mg至500mg所述的赤红球菌细胞壁；或
-0.001mg至500mg所述的赤红球菌细胞壁骨架。
单位剂量的具体示例是0.001、0.005、0.01、0.05、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、5、10、15、20、25、30、40、50、60、70、80、90、100、150、200、250、300、350、400、450、500mg±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(治疗剂、活性成分或组合物)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)，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 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
在甘油琼脂培养基上，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、培养基颜色等影响，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基上，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：葡糖醛酰胺、β-羟基-DL丁酸、D-果糖-6-磷酸、
Figure PCTCN2019127895-appb-000001
阳性；
对二甲胺四环素、十四烷硫酸钠、利福霉素SV、pH5.0、8％氯化钠、林可霉素、夫西地酸、D-
Biolog Gen III 丝氨酸、万古霉素、四唑紫、四唑蓝敏感；
化学敏感实验 对溴酸钠、1％乳酸钠、pH6.0、1％-4％氯化钠、萘啶酸、氯化锂、亚碲酸钾、氨曲南、丁酸钠不敏感。
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，培养保藏编号为CGMCC17431的赤红球菌。
2.培养方式既可为固体培养，也可为液体培养。
3.对培养基中的营养源并无特殊的规定，可使培养基中含有通常用于微生物培养的碳源、氮源及其它营养源。
-碳源可以是赤红球菌可以利用的任何碳源。例如，果糖、葡萄糖等。
-氮源可为：肉膏、蛋白胨、铵盐、硝酸盐以及其它有机或无机含氮化合物。
-其它营养源则可适当添加一些无机盐类。例如NaCl、磷酸盐类。
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)培养基组成包含：
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.除去核酸、除去脂质、除去杂蛋白、除去细胞膜
1.除核酸：
将破碎上清液进行离心，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(主要包含细胞壁骨架及其组成成分)。
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.药物组合物的制备方法
1.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)。灌装后，即为药物组合物。
表1.药物组合物可以配制成多种形式
Figure PCTCN2019127895-appb-000002
2.将制备例3所得产物(活性成分30μg至120μg)涂覆在敷料上，制备成外用医疗器械。
3.将第1项的药物组合物冻干，制得冻干粉(分别编号为组合物1至7)。
4.质量检验(以组合物1为例)
表2.质量检验项目
Figure PCTCN2019127895-appb-000003
测试例
测试例1.药理试验
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。
可见本发明的药物组合物(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.安全试验
1.无菌试验：
结果阴性，证明无菌。
2.小鼠急性毒性试验：
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.长期毒性试验：
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.稳定性试验
在常温(18-25℃)放置0、1、2、3、8、14、21个月，该药物组合物的丙氨酸含量、胞壁酸含量、吞噬率、吞噬指数与试验起始时相比，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(组合物1至组合物7)。
测试例4.吞噬试验
巨噬细胞是单核吞噬细胞系统的主要细胞，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1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的促免疫吞噬能力强。
效果例：本发明的赤红球菌细胞壁产物在治疗HPV感染者的用途
鉴于本发明人发现的本申请赤红球菌细胞壁骨架的促免疫吞噬能力，发明人采用以下方法鉴定了本发明方法获得的赤红球菌细胞壁产物对治疗人乳头瘤病毒(HPV病毒)感染的效果。
已知，本领域中HPV病毒的检测方法，通常有如下几种：
1、细胞学涂片检测方法：
也就是过去通常采用巴氏染色检测方法，也称之为涂片检测方法。就是将采集的宫颈分泌物涂在玻璃片上，用显微镜观察，主要是检查是否有空泡细胞或角化细胞，检出率70-76％。
2、膜式液基超薄细胞学检测方法(TCT)：
采取专用刷子在宫颈处取样，然后利用细胞保存液分离出被采集样本中杂质，形成超薄的清晰的细胞涂片，能检测出有没有HPV病毒，可确定何别何型。亦称为TCT。检出率70-95％。
3、免疫组织化学检测方法：
用HPV免疫动物后，产生的多克隆抗体查组织HPV抗原，采用PAP法显示病毒蛋白，以证明有病毒抗原。HPV蛋白阳性时，上皮细胞内出现淡红色的弱阳性反应，在空泡细胞核内见到棕褐色颗粒状沉着为阳性。但检测率低40-60％，敏感性不高，不能分型。
4、HPV病毒DNA检测方法：
(1)聚合酶链反应PCR：
用刮板或生理盐水浸润的棉棒从阴道和宫颈外口取分泌物，经过离心、洗涤等方法提取HPV的DNA，在经PCR扩增和凝胶电泳，观察电泳与样本比对即可做出诊断。虽然简便，但是不能定位，不知病毒死活。
(2)DNA杂交法PCK：
HPV DNA分子杂交技术、CP-14免疫杂交法。用荧光显微镜400倍可看到3个以上荧光点为阳性。可以分型，核酸杂交可检出HPV-DNA列，PCR检测可见特异性HPV-DNA扩增区带，但烦琐、昂贵。需要一定的设备和条件。
本发明人采用本发明表1中制备的组合物1的冻干粉制剂，在使用前用生理盐水稀释为30μg/ml或60μg/ml，在宫颈感染HPV的受试者中，通过宫颈局部注射或宫颈外敷，探讨了含有本发明赤红球菌细胞壁成分的组合物在治疗宫颈HPV感染中的用途。
采用上述细胞学涂片检测方法和TCT方法确诊HPV病毒感染患者，在与患者签署“知情同意书”之后，通过感染部位外敷本发明的组合或局部注射本发明的组合物，对患者进行治疗。
综合治疗方法，每天一次，施用本发明的组合物，20天为一个治疗周期，妇女月经期间应停止用药。
结果
Figure PCTCN2019127895-appb-000004
Figure PCTCN2019127895-appb-000005
在实验期间，同时对对照组的5个病例进行宫颈外敷或注射治疗不含有活性成分的右旋糖酐40溶液，在治疗前后HPV阳性均没有变化。
由上述结果可见，含本发明的赤红球菌细胞壁骨架稀释制剂可有效治疗人宫颈HPV病毒感染，病毒转阴率达100％。

Claims (14)

  1. 一种分离的赤红球菌(Rhodococcus ruber)细胞壁骨架在制备用于治疗人乳头瘤病毒感染的药物中的用途，其中：
    优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌。
  2. 根据权利要求1所述的用途，其中所述分离的赤红球菌细胞壁骨架通过以下步骤获得：
    1)提供赤红球菌；
    2)培养所述赤红球菌；
    3)收集经培养的赤红球菌；
    4)粉碎经培养的赤红球菌，得到粉碎产物；和
    5)去除所得粉碎产物中的脂质、核酸和蛋白质。
  3. 一种药物组合物在制备用于治疗人乳头瘤病毒感染的药物中的用途，其中所述药物组合物包含：
    分离的赤红球菌细胞壁骨架，优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；和
    药学上可接受的赋形剂；
    优选地，其中所述分离的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200至300个重量份；
    优选地，所述药学上可接受的赋形剂为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。
  4. 根据权利要求3所述的用途，其中所述药学上可接受的赋形剂选自右旋糖酐、海藻糖、甘氨酸、木糖醇、羧甲基纤维素钠、赤藓糖醇、明胶、硬脂酸镁和甘露醇。
  5. 根据权利要求1-4中任一项所述的用途，其中所述药物用于局部施用，优选地，所述局部施用为皮肤或粘膜涂覆、或皮肤或粘膜下注射。
  6. 根据权利要求5所述的用途，其中所述药物配制成软膏剂、乳膏剂、硬膏剂、凝胶剂、洗剂、酊剂、搽剂、油剂、糊剂、冻干粉、气雾剂、栓剂、贴片或悬液，优选地，其中所述药物配制成冻干粉制剂，并且在使用前将冻干粉制剂在生理盐水中稀释，使赤红球菌细胞壁骨架的浓度为15μg/ml-500μg/ml，优选浓度为30-240μg/ml，更优选浓度为30-120μg/ml，更优选浓度为30-60μg/ml。
  7. 一种分离的赤红球菌细胞壁骨架在制备用于治疗人乳头瘤病毒感染的医疗器械中的用途，其中：
    优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；
    所述医疗器械包含所述分离的赤红球菌细胞壁骨架，和
    任选地，药学上可接受的赋形剂。
  8. 根据权利要求7所述的用途，其中所述医疗器械为外用医疗器械，优选地选自以下形式：敷料、贴片、绷带或膜，优选地，所述敷料、贴片、绷带或膜中浸润含浓度为15μg/ml-500μg/ml，优选浓度为30-240μg/ml，更优选浓度为30-120μg/ml，更优选浓度为30-60μg/ml的赤红球菌细胞壁骨架的混悬液。
  9. 根据权利要求1-8中任一项所述的用途，其中所述人乳头瘤病毒感染选自皮肤、宫颈、阴道、阴唇、阴茎、肛门肛周、口咽、扁桃体和口腔的人乳头瘤病毒感染。
  10. 一种用于治疗人乳头瘤病毒感染的分离的赤红球菌细胞壁骨架，其中：
    优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌。
  11. 一种用于治疗人乳头瘤病毒感染的药物组合物，其中所述药物组合物包含：
    分离的赤红球菌细胞壁骨架，优选地，所述分离的赤红球菌细胞壁源自于2019年03月22日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；和
    药学上可接受的赋形剂；
    优选地，其中所述分离的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200至300个重量份；
    优选地，其中所述分离的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。
  12. 根据权利要求10所述的用于治疗人乳头瘤病毒感染的分离的赤红球菌细胞壁骨架、或根据权利要求11所述的用于治疗人乳头瘤病毒感染的药物组合物，其中所述分离的赤红球菌细胞壁骨架通过以下步骤获得：
    1)提供赤红球菌；
    2)培养所述赤红球菌；
    3)收集经培养的赤红球菌；
    4)粉碎经培养的赤红球菌，得到粉碎产物；和
    5)去除所得粉碎产物中的脂质、核酸和蛋白质。
  13. 一种治疗人乳头瘤病毒感染的方法，所述方法包括向有此需要的受试者施用治疗有效量的分离的赤红球菌细胞壁骨架或包含所述分离的赤红球菌细胞壁骨架的药物组合物，其中：
    优选地，所述分离的赤红球菌细胞壁骨架源自于2019年03月22 日保藏在北京市朝阳区北辰西路1号院3号的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 17431的赤红球菌；优选地，所述施用为局部施用；优选地，所述局部施用为皮肤或粘膜涂覆、或皮肤或粘膜下注射。
  14. 根据权利要求13所述的方法，其中所述治疗有效量的单位剂量是1μg至1000μg；优选地，15μg-500μg，优选为30-240μg，更优选为30-120μg，更优选为30-60μg；
    优选地，所述治疗按照选自以下的频率进行：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；更优选地，每天施用1次；
    优选地，所述治疗持续2天至2个月，更优选地，持续1周、2周、3周、4周、5周、6周、7周或8周。
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