WO2020216281A1 - 赤红球菌产品在治疗热损伤中的用途 - Google Patents

赤红球菌产品在治疗热损伤中的用途 Download PDF

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WO2020216281A1
WO2020216281A1 PCT/CN2020/086353 CN2020086353W WO2020216281A1 WO 2020216281 A1 WO2020216281 A1 WO 2020216281A1 CN 2020086353 W CN2020086353 W CN 2020086353W WO 2020216281 A1 WO2020216281 A1 WO 2020216281A1
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rhodococcus
cell wall
weeks
product
optionally
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PCT/CN2020/086353
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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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Priority to EP20794551.0A priority Critical patent/EP3960845A4/en
Priority to CN202080002633.XA priority patent/CN112218647B/zh
Priority to US17/604,364 priority patent/US20220175852A1/en
Priority to KR1020217037350A priority patent/KR20220004091A/ko
Priority to PCT/CN2020/086353 priority patent/WO2020216281A1/zh
Priority to JP2021562147A priority patent/JP2022531109A/ja
Publication of WO2020216281A1 publication Critical patent/WO2020216281A1/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
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present disclosure relates to the medical field, the microbiological field, and the biopharmaceutical field. Specifically, it relates to Rhodococcus erythrophilus 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 heat injury.
  • 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
  • the GC content is about 70.63%
  • the GenBank accession number is CP029146 (Fan Xin, Rhodococcus spp 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.
  • 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 which comprises 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 excipients 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 can be 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 use in the treatment of heat injury.
  • Rhodococcus rubrum product for treating heat injury.
  • a pharmaceutical composition or medical device which is used to treat heat injury.
  • Rhodococcus rubrum cell wall according to the present disclosure in the treatment of heat injury.
  • Rhodococcus rubrum cell wall of the present disclosure in the preparation of a medicine/medical device for treating heat injury.
  • the use of the Rhodococcus product according to the present disclosure in the treatment of heat injury is provided; and the use of the Rhodococcus product of the present disclosure in the preparation of a medicine/medical device for the treatment of heat injury is also provided.
  • the use of the pharmaceutical composition according to the present disclosure in the treatment of thermal injury is provided.
  • the thermal injury may be caused by factors selected from the group consisting of burns, scalds, and chemical burns.
  • thermal injury can affect the following tissues: epidermis, dermis, mucosa, subcutaneous tissue.
  • the degree of thermal damage is: I, II (deep, shallow), or III degree damage.
  • the severity of thermal injury is divided into degree I, degree II shallow, degree II deep, and degree III according to the three-degree quartile:
  • Degree III Injuries the entire layer of the skin, even deep to the subcutaneous, muscle, bone, etc.
  • Rhodococcus Rhodococcus product/Rhodococcus cell wall/pharmaceutical composition according to the present application is especially used for the treatment of deep II degree heat damage.
  • the medicament is used to treat heat injury.
  • the medical device (such as dressing, patch, bandage, film, patch, etc.) is used to treat heat injury.
  • a method for treating heat injury comprising contacting a subject (lesion) with a therapeutically effective amount of any one selected from the following:
  • different dosages are used to administer the drug (or medical device) to the lesion.
  • the drug or medical device
  • Liquid medicine, ointment, and dry powder can be used alternately on the lesion.
  • the dosage for each application varies depending on the size and severity of the patient's lesion, 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 , 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 rubra 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 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 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.
  • technicians can perform taxonomic identification on a strain of bacteria.
  • the available identification techniques include morphology, physiological and biochemical characteristics, 16S rRNA, etc.
  • 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.
  • Chemical sensitivity test SV, pH5.0, 8% sodium chloride, lincomycin, fusidic acid, D-serine, vancomycin, tetrazolium violet, tetrazolium blue; sensitive to sodium bromate, 1% Sodium lactate, pH 6.0, 1%-4% sodium chloride, nalidixic acid, lithium chloride, potassium tellurite, aztreonam, sodium butyrate are not sensitive.
  • 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.
  • 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).
  • Excipients such as dextran 40, mannitol or trehalose are added to the product obtained in Preparation Example 3. After dispensing, it is the pharmaceutical composition.
  • 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.).
  • compositions 1 to 7 For example: adding 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; set aside to defoam; cast the formed mixed viscous liquid without bubbles; cast on a mold coated with a small amount of paraffin, dry for 5-20 minutes, take out the film, and cut into the required area.
  • film-forming materials such as polyvinyl alcohol, carbomer and hydroxypropyl cellulose
  • composition 3 and the esterification 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).
  • Jingwanhong ointment (Tianjin Darentang Jingwanhong Pharmaceutical Co., Ltd., National Medicine Standard Z20023137, specification 30g/bottle);
  • mice were adapted to the environment and reared for one week. After cutting the back skin of the mouse with a shaver, use a depilatory cream (about 3.5cm ⁇ 3.5cm) to remove the hair, leave it for 180s, wash the depilated area with 30-40°C warm water, and dry it with absorbent cotton.
  • a depilatory cream about 3.5cm ⁇ 3.5cm
  • mice After depilation, the mice were fed normally for 24 hours and observed, and it was confirmed that the depilation site had no abnormalities such as redness, inflammation, and damage. After 24 hours, anesthetized with ether, placed on the operating table, and disinfected the skin of the experimental area with 75% ethanol to construct a scald model.
  • the injured mice were reared in separate cages, and the wounds were treated with physiological saline, and fed with feed and distilled water to ensure that the litter was dry, clean and well ventilated.
  • the burn/scald is classified as deep II degree.
  • -Wound undamaged treatment group first treat the wound with normal saline, apply 3 (1 bottle of composition for each topical each day; inject 0.25ml of normal saline into the bottle to completely dissolve the freeze-dried powder, with 3 layers of 1.2cm ⁇ 1.2cm The gauze is completely infiltrated, and after applying it on the wound surface, fix it with medical desensitization tape);
  • -Wound damage treatment group first treat the wound with normal saline, and treat the damaged wound, apply 3 (1 bottle of external composition every day; inject 0.25ml of normal saline into the bottle to completely dissolve the freeze-dried powder, and use 3 layers of 1.2 cm ⁇ 1.2cm gauze is completely infiltrated, and after applying it on the wound, fix it with medical desensitization tape);
  • -Positive control group first treat the wound with normal saline, and then apply Jingwanhong ointment to each animal once a day.
  • the wound healing was observed on 1, 3, 7, 11, and 15 days of administration.
  • Judgment criteria include:
  • scabs are intermittently removed, the surface of the new tissue is not flat enough and there is a small amount of exudate in a small area, but no obvious infection foci;
  • mice The wound healing of mice was observed and recorded on days 3, 7, 11, and 15 of the administration. Complete epithelialization and no exudation of the wound were regarded as complete wound healing. At the same time, the wound was observed for redness, swelling, infection, etc. Calculate the wound healing rate at each time point: From the ruler in the photo, the wound diameter is obtained through the graphics processing software, and the wound area is calculated.
  • Wound healing rate% (original wound area-unhealed wound area)/original wound area ⁇ 100%.
  • SPSS 17.0 statistical software was used for analysis, and the groups were compared by one-way analysis of variance. The data obtained is based on Said. P ⁇ 0.05 indicates that the difference is statistically significant.
  • mice lived until the wounds healed, and there were no obvious differences in eating, drinking, mental state, activity, etc., and no obvious infection.
  • mice The wound surface of mice is completely healed and the tissue surface is flat as the healing standard.
  • the wound healing time of the wound damage treatment group and the positive group was shortened, and the difference was statistically significant P ⁇ 0.05. Comparing the wound damage treatment group and the positive group, the healing time was slightly earlier than the positive group, the difference was statistically significant P ⁇ 0.05.
  • the administration was started 24 hours after the model was established.
  • the wound area before the administration was the baseline area.
  • the wound area was recorded on 3, 7, 11, and 15 days, and the healing rate was calculated.
  • the wound healing rate of the wound damage treatment group was significantly higher than that of the model group after 7 days of administration, and the healing rate of the positive group and the undamaged treatment group also began to be higher than that of the model group at the 11th day of administration, which was statistically significant. ⁇ 0.05. Comparing the wound damage treatment group with the positive group, the wound healing rate began to be higher than that of the positive group at 7 days after administration, and it was statistically significant P ⁇ 0.05.
  • the cell wall skeleton of Rhodococcus rubrum can effectively improve the healing rate, shorten the healing time, reduce the formation of scars after wound healing, and have a significant repair effect on skin hair follicles and other accessories during the healing process of deep second-degree scalds in mice.

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Abstract

赤红球菌细胞壁或赤红球菌细胞壁骨架在制备治疗热损伤药物中的用途。所述热损伤包括烧伤、烫伤、化学灼伤等。向受试者施用该赤红球菌产品，对热损伤的治疗效果好，且无副作用。

Description

赤红球菌产品在治疗热损伤中的用途
本申请要求2019年04月25日提交的专利申请(申请号CN201910333119.3)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热损伤的药物/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。
在创伤外科领域里，对于热损伤，特别是烧伤或烫伤的创面大多采取抗感染治疗原则。皮肤热损伤的愈合是一个复杂的生物学过程，受多种细胞因子、血管内皮细胞、成纤维细胞、角质细胞等各种因素影响。
然而，赤红球菌在人类医学领域中的应用，尚未有报道。
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.1 West 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，其用于治疗热损伤。
根据本公开的一些实施方案，提供了一种赤红球菌产品，其用于治疗热损伤。
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，其用于治疗热损伤。
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热损伤中的用途。
还提供了本公开的赤红球菌细胞壁在制备用于治疗热损伤的药物/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热损伤中的用途；还提供了本公开的赤红球菌产品在制备用于治疗热损伤的药物/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热损伤中的用途。
在一些实施方案中，热损伤可以是选自以下因素所致：烧伤、烫伤、化学灼伤。
在一些实施方案中，热损伤可以累及至以下组织：表皮层、真皮层、粘膜、皮下组织。
在一些实施方案中，热损伤程度为：I、II(深、浅)、或III度损伤。
在本申请中，热损伤的严重程度按照三度四分法分为I度、浅II度、深II度和III度：
I度：仅伤及表皮，局部呈现红肿；
II度：深达真皮，局部出现水疱；
浅II度：仅伤及表皮生发层及真皮乳头层；
深II度：伤及真皮层，尚残留皮肤附件；
III度：伤及皮肤全层，甚至可深达皮下、肌肉、骨骼等。
在具体的实施方案中，根据本申请赤红球菌产品/赤红球菌细胞壁/药物组合物尤其用于治疗深II度的热损伤。
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，所述药物用于治疗热损伤。
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)用于治疗热损伤。
根据本公开的一些实施方案，还提供了一种治疗热损伤的方法，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，针对病灶的面积和严重程度的不同，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严重程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，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：
Biolog Gen II 葡糖醛酰胺、β-羟基-DL丁酸、D-果糖-6-磷
生长实验：酸、α-D-葡萄糖、D-果糖、D-甘露醇、D-阿糖醇、D-山梨醇、奎宁酸、γ-氨基丁酸、柠檬酸、L-苹果酸、溴代丁二酸、吐温40、丙酸、乙酸呈阳性；
Biolog Gen III 对二甲胺四环素、十四烷硫酸钠、利福霉素
化学敏感实验：SV、pH5.0、8％氯化钠、林可霉素、夫西地酸、D-丝氨酸、万古霉素、四唑紫、四唑蓝敏感；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 PCTCN2020086353-appb-000001
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 PCTCN2020086353-appb-000002
测试例
1.材料
1.1药物及主要试剂
-测试药物：前述制备例中的组合物3冻干粉(60μg活性成分/瓶)；
-对照药物：京万红软膏(天津达仁堂京万红药业有限公司，国药 准字Z20023137，规格30g/支)；
薇婷脱毛膏(利杰时家化(中国)有限公司)；
乙醚(国药集团化学试剂有限公司)。
1.2实验动物
清洁级昆明小鼠16只，体质量28至36g，由辽宁长生生物技术股份有限公司提供，合格证号211002300051326。
2.实验方法
2.1实验设计
取昆明小鼠16只，随机分为4组，每组4只：
1)模型组；
2)创面未破损治疗组(赤红球菌组)；
3)创面破损治疗组(赤红球菌组)；
4)阳性对照组(对照药物)。
2.2小鼠II度烫伤模型制备
小鼠适应环境饲养一周后。小鼠背部皮肤用剃毛器剪毛后，使用脱毛膏脱毛(范围约3.5cm×3.5cm)，放置180s，用30至40℃温水对脱毛区进行清洗，脱脂棉擦干。
脱毛后的小鼠，正常饲养24h观察，确定脱毛部位无红肿、炎症和破损等异常情况。24h后采用乙醚麻醉，置于操作台上，75％乙醇消毒实验区皮肤，进行烫伤模型的构建。
将小鼠置中间有1.5cm孔洞的烫伤装置上，使背部脱毛后皮肤处于孔洞部位，将裸露出背部的1.5cm孔洞置于75℃恒温水中持续12s,制成烫伤直径约为1.5cm的圆形创面。
伤后小鼠分笼饲养，创面经生理盐水处理，并给予饲料及蒸馏水，保证垫料干燥清洁，通风良好。根据三度四分鉴别烧/烫伤为深II度。
2.3分组给药
烫伤后24小时开始给药：
-模型组：每日每只烫伤皮肤创面涂抹生理盐水；
-创面未破损治疗组：先用生理盐水处理创面，每日每只外敷组合物3(1瓶；将0.25ml生理盐水注入瓶内，使冻干粉末完全溶解，以3 层1.2cm×1.2cm纱布完全浸润，敷于创面上后，用医用脱敏胶带固定)；
-创面破损治疗组：先用生理盐水处理创面，将创面处理破损，每日每只外敷组合物3(1瓶；将0.25ml生理盐水注入瓶内，使冻干粉末完全溶解，以3层1.2cm×1.2cm纱布完全浸润，敷于创面上后，用医用脱敏胶带固定)；
-阳性对照组：先用生理盐水处理创面，然后每日每只外敷京万红软膏1次。
分别于给药1、3、7、11、15天观察创面愈合情况。
3.实验观察及指标检测
3.1创面愈合时间的测定
针对各组，进行愈合时间的测定。判定标准包括：
-愈合：烫伤处结痂完整脱落，修复后组织表面新鲜且较平坦；
-基本愈合：烫伤处间断脱痂，新生组织表面不够平坦且有小范围的少许渗出物，但无明显感染灶；
-感染：痂周出现明显红肿，痂下有脓液或溃疡。
3.2创面愈合率
于给药3、7、11、15天观察并记录小鼠创面愈合情况，以创面完全上皮化、无渗出视为创面完全愈合，同时观察创面有无红肿、感染等情况。计算各时间点创面愈合率：由照片中的标尺，通过图形处理软件的得出创面直径，计算出创面面积。
创面愈合率％＝(原始创面面积－未愈合创面面积)/原始创面面积×100％。
3.3统计方法
采用SPSS 17.0统计软件分析，各组间用单因素方差分析比较。所得数据以
Figure PCTCN2020086353-appb-000003
表示。P<0.05为差异有统计学意义。
4.实验结果
4.1大致观察
所有小鼠均活至创面愈合，进食、饮水、精神状态、活动等均无明显差异，无明显感染现象。
4.2创面愈合情况
4.2.1创面愈合时间
以小鼠创面完全愈合，且组织表面较平坦为愈合标准。与模型组相比，创面破损治疗组和阳性组的创面愈合时间缩短，差异具有统计学意义P<0.05。创面破损治疗组和阳性组比较，愈合时间略提前于阳性组，差异具有统计学意义P<0.05。
表3.小鼠深II度烫伤创面愈合时间
Figure PCTCN2020086353-appb-000004
Figure PCTCN2020086353-appb-000005
注：与模型组比较，*P<0.05
4.2.2创面愈合率
模型建立24h后开始给药，给药前创面面积为基准面积，分别于3、7、11、15天记录创面面积，并计算愈合率。
创面破损治疗组与模型组比较，给药7d时创面愈合率开始明显高于模型组，给药11d时阳性组和创面未破损治疗组的愈合率也开始高于模型组，具有统计学意义P<0.05。创面破损治疗组与阳性组比较，给药7天时创面愈合率开始高于阳性组，并且具有统计学意义P<0.05。
表4.小鼠深II度烫伤创面愈合率
Figure PCTCN2020086353-appb-000006
Figure PCTCN2020086353-appb-000007
注：与模型组比较，*P<0.05
4.2.3创面愈合状态
创面破损治疗组在愈合时，创面无疤痕增生且平滑，而其它组均出现不同程度的疤痕增生；同时破损治疗组创面部位已开始生长毛发。
综上所述，赤红球菌细胞壁骨架在小鼠深II度烫伤愈合过程中， 可有效提高愈合率，缩短愈合时间，减少创面愈合后瘢痕的形成，同时对皮肤毛囊及其它附件的修复效果显著。

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热损伤。
  2. 根据权利要求1所述的用途，其中：
    所述热损伤选自：烧伤、烫伤、化学灼伤；
    优选，所述热损伤累及选自以下的组织：表皮层、真皮层、粘膜、皮下组织；
    优选，所述热损伤选自：I度、II度、III度，更优选深II度。
  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，所述药物包含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，所述药物包含10μg、20μg、30μg、40μg、50μg、60μg、70μg、80μg、90μg、100μg、150μg、200μg、250μg、300μg、350μg、400μg、500μg、600μg、700μg、800μg/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. 一种治疗热损伤的方法，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；
    所述热损伤选自：烧伤、烫伤、化学灼伤；
    优选，所述热损伤累及选自以下的组织：表皮层、真皮层、粘膜、皮下组织；
    优选，所述热损伤选自：I度、II度、III度，更优选深II度。
  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。
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US17/604,364 US20220175852A1 (en) 2019-04-24 2020-04-23 Use of rhodococcus ruber product in treating thermal injury
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