CN111971054A

CN111971054A - Application of Rhodococcus ruber product in treating white lesions of vulva

Info

Publication number: CN111971054A
Application number: CN202080002080.8A
Authority: CN
Inventors: 盖波; 窦春艳; 张轶; 张国英
Original assignee: Liaoning Gerui Shite Bio Pharmacy Co ltd
Current assignee: Liaoning Gerui Shite Bio Pharmacy Co ltd
Priority date: 2019-03-14
Filing date: 2020-03-12
Publication date: 2020-11-20
Also published as: WO2020182180A1; WO2020182181A1; CN112040962A; CN117482118A

Abstract

Use of Rhodococcus ruber product for treating leukoplakia vulvae is provided. A Rhodococcus ruber product comprising a cell wall or component thereof is provided. The red blood coccus product is applied to a subject, and has good treatment effect on exoleukosis (including lichen sclerosus and squamous cell hyperplasia); and has no side effect.

Description

Application of Rhodococcus ruber product in treating white lesions of vulva

The present application claims priority from a patent application filed 2019 on month 03 and 14 (application No. CN201910191961.8), which is incorporated herein by reference.

Technical Field

The present disclosure relates to the fields of medicine, microbiology and biopharmaceuticals. In particular to rhodococcus ruber, a cell wall component, a preparation, a pharmaceutical composition and a preparation method thereof, and application of the cell wall component of the rhodococcus ruber in preparing a medicine/medical device for treating white lesions of vulva.

Background

Rhodococcus ruber (also known as Rhodococcus ruber), a gram-positive bacterium. Generally, colonies are orange or orange, round; colony size is about 1mm to 2 mm; the cell shape is spherical or short rod-shaped; primary branched mycelium can be formed; without flagellum. Rhodococcus ruber is aerobic and heterotrophic.

Currently, researchers have performed whole gene profiling on Rhodococcus ruber. For example, fan Xin et al sequenced the entire genome of Rhodococcus ruber strain SD3 and performed bioinformatic analyses. The total genome length of the SD3 strain is about 5.37Mb, the GC content is about 70.63%, and the GenBank accession number is CP029146 (Sword wins, Rhodococcus ruber SD3 total genome sequencing and the expression analysis, genomics and application biology of heat shock protein DnaK thereof, 1 month in 2019).

Rhodococcus Rhodococcus is capable of adapting to various living environments due to its very strong organic tolerance and wide degradation spectrum. Therefore, Rhodococcus is widely used in the fields of pollution remediation, organic compound degradation, sewage treatment, and the like. Currently, the main application field of rhodococcus ruber is in environmental management, see CN108862590A, CN107151635A, CN102250796A, CN1519312A, CN103627653A, CN101033454A, CN108130288A, CN104830738A, CN101619299A, CN103509833A, CN106434466A, CN101580808A, CN102604875A, CN103160491A, CN106591168A, CN106591172A, and CN 105820982A.

CN109576180A discloses a bacterium RDC-01 screened from suburban red soil near the area of Muyu wine, Guangzhou city, which is identified as Rhodococcus ruber by 16S rRNA gene sequence analysis and culture characteristic identification. After the inactivation of the bacterium, the bacterium was added to an inactivated vaccine for animals as an immunoadjuvant, and it was found that the production of antibodies by animals can be promoted.

However, the application of Rhodococcus ruber in the field of human medicine has not been reported.

The vulvar white lesions include vulvar white lesions, vulvar leukoplakia or vulvar dystrophy. The vulvar mucosa of patients with lichen sclerosus and squamous cell hyperplasia is white, so it is called vulvar white lesion (non-neoplastic lesion belonging to vulvar epithelium).

The lichen sclerosus and squamous epithelial cell hyperplasia are known in different ages due to different clinical and pathological knowledge (leukoplakia vulvae, leukoplakia vulvitis, vulvar xerosis, hyperplastic or atrophic vulvitis, lichen sclerosus, etc.). Due to the confusion of disease nomenclature, it was collectively called chronic vulvar dystrophy by the international society for the study of vulvar diseases (ISSVD) in 1975. In 1987 ISSVD was discussed in conjunction with the International society of gynecology pathologists (ISGYP), and a new classification of vulvar dermatoses was formulated (ISSVD, 1987), including:

(1) non-neoplastic epithelial changes in the skin and mucosal epithelium (non-neoplastic epithelial disorders of skin and mucosas)

1) Lichen sclerosus (lichen sclerosus);

2) squamous cell hyperplasia (squamous cell hyperplasia);

(2) intraepithelial neoplasia (intra-epithelial neoplasma)

1) Squamous intraepithelial neoplasia (squamous intra-epithelial neoplasma);

a. mild dysplasia (mil dysplasia);

b. moderate atypical hyperplasia (modete dyssplasia);

c. severe dysplasia or carcinoma in situ (severe dysplasia or carcinoma in situ);

2) non-squamous intraepithelial neoplasia (non-squamous intra-epithelial neoplasma);

a. paget's disease;

b. non-invasive melanoma (non-invasive tumors of melanosomes);

(3) infiltrating cancers (invasive tumors).

Methods of treating vulvar white lesions generally include:

1. keep vulva clean and dry, forbid wearing airtight underpants, avoid eating spicy and allergic food. For patients with insomnia due to pruritus, sedative, hypnotic and antiallergic drugs can be added.

2. And (3) drug treatment: common drugs for lichen sclerosus include pyruvic acid ointment, compound vitamin A ointment, progesterone ointment, glucocorticoid ointment, or immunotherapy. Topical application of cortical hormone to the vulva squamous epithelium may be used to control pruritus. Most patients are therapeutically effective, but need to adhere to long-term medication.

3. Physical therapy: it is applicable to the cases with ineffective or serious medical treatment. Microwave therapy, carbon dioxide laser, helium neon laser, Bohm light, high-frequency electrotome, local fulguration therapy, liquid nitrogen local cryotherapy and the like.

4. And (3) surgical treatment: it is only suitable for patients with severe disease and ineffective repeated medication or physical therapy. When malignant change is suspected, surgical treatment is required.

In view of the above, there is still a need in the art to provide a drug effective in treating vulvar white lesions.

Disclosure of Invention

According to some embodiments of the present disclosure, there is provided an isolated Rhodococcus ruber.

According to some specific embodiments of the present disclosure, Rhodococcus ruber is provided, which was deposited at the General Microbiological Culture Collection center of China Committee for Culture Collection of microorganisms at 22.03.2019 (Institute of Microbiology, China Academy of Sciences, Institute of Microbiology, No. 3, West beer, Chaoyang District, Beijing, Chaoji China), and the deposit number is CGMCC No.17431 (Beijing Institute of Microbiology, national Institute of Microbiology, No. 3, North Cheng West beer, and China). This Deposit satisfies the provisions of the Budapest Treaty on the International Recognition of the destination of Microorganisms for the Purposes of Patent Procedure.

According to some embodiments of the present disclosure, there is provided rhodococcus ruber and derivatives thereof. The derivative product is derived from Rhodococcus ruber and contains constituent components (such as protein, nucleic acid, lipid, cell wall and its constituent components, carbohydrate, and metabolite) of Rhodococcus ruber.

In a specific embodiment, an isolated Rhodococcus ruber cell wall is provided.

In a specific embodiment, an isolated Rhodococcus ruber cell wall is provided, wherein the Rhodococcus ruber is a strain with a preservation number of CGMCC No. 17431.

In a specific embodiment, an isolated Rhodococcus ruber cell wall scaffold is provided.

In a specific embodiment, an isolated Rhodococcus ruber cell wall skeleton is provided, wherein the Rhodococcus ruber is a strain with a collection number of CGMCC No. 17431.

According to some embodiments of the present disclosure, there is provided a pharmaceutical composition comprising a cell wall of rhodococcus ruber or a cell wall scaffold of rhodococcus ruber according to the present disclosure.

According to some embodiments of the present disclosure, there is provided a rhodococcus ruber product comprising a product obtained after a rhodococcus ruber has been comminuted.

According to other embodiments of the present disclosure, there is provided a Rhodococcus ruber product comprising a product of Rhodococcus ruber after pulverization and purification (lipid removal, nucleic acid removal, protein removal).

According to other embodiments of the present disclosure, there is provided a rhodococcus ruber product comprising a cell wall of rhodococcus ruber.

According to other embodiments of the present disclosure, there is provided a rhodococcus ruber product comprising a cell wall skeleton of rhodococcus ruber.

According to some embodiments of the present disclosure, there is provided a pharmaceutical composition or medical device comprising a product obtained after pulverization of rhodococcus ruber.

According to other embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device comprising a product obtained by pulverizing and purifying (defatting, and/or removing nucleic acid, and/or removing protein) Rhodococcus ruber.

According to other embodiments of the present disclosure, there is provided a pharmaceutical composition or medical device comprising a cell wall of rhodococcus ruber.

According to other embodiments of the present disclosure, there is provided a pharmaceutical composition or medical device comprising a cell wall scaffold of rhodococcus ruber.

According to further embodiments of the present disclosure, there is provided a pharmaceutical composition or medical device comprising the above-described rhodococcus ruber product.

In a specific embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In some embodiments, the rhodococcus ruber product is present in the pharmaceutical composition in 1 part by weight and the pharmaceutically acceptable excipient is present in 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 within any two numerical ranges).

In other embodiments, the rhodococcus ruber cell wall is 1 part by weight and 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 within any two numerical ranges).

In still other embodiments, the rhodococcus ruber cell wall skeleton is 1 part by weight and 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 within any two numerical ranges).

In some embodiments, the pharmaceutical composition may be prepared in a liquid state (liquid formulation).

In other embodiments, the pharmaceutical composition may be prepared as a solid (dry powder formulation or lyophilized powder formulation).

The skilled artisan understands that for the pharmaceutical compositions of the present disclosure, both a liquid formulation and a dry powder formulation (or lyophilized powder formulation), can be interconverted, differing only in water content. Removing most or all of water in the liquid preparation to obtain dry powder preparation (or lyophilized powder preparation). Dissolving (or redissolving) the dry powder preparation (or the freeze-dried powder preparation) to obtain a liquid preparation.

In some embodiments, the medicament or pharmaceutical composition is prepared in a dosage form selected from the group consisting of: ointment, cream, lotion, suspension, paste, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, powder, suppository; wherein the paste is selected from: ointment, plaster, cream.

In some embodiments, the dosage form is in a form suitable for application to a lesion. For example, sprays, ointments, lotions, dressings, patches.

In some embodiments, the pharmaceutically acceptable excipient relates to, but is not limited to: filler, stabilizer, flavoring agent, disintegrating agent, binder, and lubricant.

In some embodiments, the pharmaceutically acceptable excipient, such as, but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethylcellulose, erythritol, gelatin, magnesium stearate, a propellant, a humectant, a solvent, a solubilizer, an emulsifier, an antioxidant, a pH regulator and a preservative.

In some embodiments, non-limiting examples of the pharmaceutically acceptable excipient further include: white petrolatum, carbomer, hypromellose, methylcellulose, sodium carboxymethylcellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerol, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, acetamide, isopropyl alcohol, tween, polyoxyethylene hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid ester, sucrose acetate isobutyrate, sucrose anhydride tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol ester, sulfite, cysteine, di-tert-butyl hydroxytoluene, potassium sorbate, phosphate buffer solution, Triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salt.

According to some embodiments of the present disclosure, there is provided a method of preparing a rhodococcus ruber product comprising or consisting of the steps of:

1) providing Rhodococcus ruber;

2) optionally, culturing said Rhodococcus ruber;

3) optionally, collecting the cultured Rhodococcus ruber;

4) pulverizing the cultured Rhodococcus ruber to obtain a pulverized product;

5.1) optionally, subjecting the comminuted product to an operation to remove lipids;

5.2) optionally, subjecting the pulverized product to a nucleic acid removal operation;

5.3) optionally, subjecting the comminuted product to an operation to remove protein;

5.4) obtaining a purified product;

6) optionally, removing water from the purified product, preferably by freeze-drying;

7) optionally, performing split charging;

8) harvesting said Rhodococcus ruber product;

wherein the steps 5.1), 5.2), 5.3) can be interchanged in sequence or can be parallel; step 6) and step 7) may be interchanged in sequence.

Optionally, a step of removing the cell membrane (e.g., with a nonionic surfactant) may be further included in step 5), as necessary.

The culture of Rhodococcus ruber is not limited to specific culture media and culture parameters, and the skilled person can culture in a known appropriate manner, and culture dishes, culture bottles, and fermentation tanks can be used according to the scale of preparation.

For the pulverization of Rhodococcus ruber, the purpose is to remove intracellular substances, so that techniques such as ultrasonication, lysozyme and the like can be employed. The skilled artisan will appreciate that any known or future method suitable for disrupting gram-positive bacteria is suitable for use in the presently disclosed embodiments.

The skilled person has the ability to adapt the specific parameters and equipment of culturing, disruption, separation, collection, removal of impurities, split charging, depending on the subsequent application (e.g. topical application, etc.) of the active ingredient (cell wall and its constituent components) in order to avoid introducing factors in the preparation step that affect the subsequent application.

In some embodiments, lipids are removed from the disrupted product using, for example, an organic solvent. In some embodiments, DNA and RNA in the disruption products are removed using, for example, a nuclease. In some embodiments, the protein in the disruption product is degraded using, for example, a hydrolase. In some embodiments, cell membranes in the disruption product are removed using, for example, a surfactant.

In some embodiments, the average particle size of the pulverization is from 10nm 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 ranges between any two of the foregoing values. There are many methods for measuring particle size (kusnezoff et al, modern particle size measurement techniques, modern chemical, 22: 1 2002).

In some specific embodiments, the average particle size of the pulverization is from 10nm to 800 nm.

In other specific embodiments, the milled average particle size is from 10nm to 500 nm.

In some embodiments, the dispensing refers to dispensing into containers or onto solid supports. The container is selected from: bottle, tube, bag, plate, ampoule, injection device, aluminum film package, dressing, capsule.

For example, in particular embodiments, the dispensing means into vials/ampoules. Just before use, the solvent is added to the bottle/ampoule.

According to some embodiments of the present disclosure, there is provided a rhodococcus ruber product prepared by a method according to the present disclosure.

According to some embodiments of the present disclosure, there is provided a pharmaceutical composition or medical device comprising a rhodococcus ruber product prepared according to a method of the present disclosure.

According to some embodiments of the present disclosure, there is provided an isolated rhodococcus ruber cell wall for use in treating a vulvar white lesion.

According to some embodiments of the present disclosure, there is provided a rhodococcus ruber product for use in the treatment of vulvar white lesions.

According to some embodiments of the present disclosure, there is provided a pharmaceutical composition or a medical device for treating vulvar white lesions.

According to some embodiments of the present disclosure, there is provided a use of a rhodococcus ruber cell wall according to the present disclosure in the treatment of vulvar whiteness.

Also provided is the use of the Rhodococcus ruber cell wall of the present disclosure in the manufacture of a medicament/medical device for the treatment of leukoplakia vulvae.

According to some embodiments of the present disclosure, there is provided a use of a rhodococcus ruber product according to the present disclosure in the treatment of vulvar white lesions; also provided is the use of a Rhodococcus ruber product of the present disclosure in the manufacture of a medicament/medical device for the treatment of leukoplakia vulvae.

According to some embodiments of the present disclosure, there is provided a pharmaceutical composition according to the present disclosure for use in treating vulvar white lesions.

In some embodiments, the vulvar white lesion is an intradermal non-neoplastic lesion of the vulvar skin and on the vulvar mucosa selected from: lichen sclerosus and squamous epithelial cell hyperplasia.

According to some embodiments of the present disclosure, there is provided a use of any one of the following in the manufacture of a medicament (or medical device):

rhodococcus ruber according to the present disclosure,

An isolated Rhodococcus ruber cell wall according to the present disclosure,

A Rhodococcus ruber product according to the present disclosure,

-a pharmaceutical composition according to the present disclosure.

In some specific embodiments, the medicament is for treating vulvar white lesions.

In some specific embodiments, the medical device (e.g., dressing, patch, bandage, film, patch, etc.) is used to treat vulvar white lesions.

There is also provided, according to some embodiments of the present disclosure, a method of treating a vulvar white lesion, comprising contacting a subject (lesion) with a therapeutically effective amount of any one selected from the group consisting of:

an isolated Rhodococcus ruber cell wall according to the present disclosure,

A Rhodococcus ruber product according to the present disclosure,

-a pharmaceutical composition according to the present disclosure,

-a medical device according to the present disclosure.

In some specific embodiments, the drug (or medical device) is administered to the lesion at different doses for different areas and depths of the lesion. For example, but not limited to:

-painting with a medicament comprising the cell wall skeleton of Rhodococcus ruber; or

-covering the lesion with a patch (or membrane, gauze) impregnated with the cell wall skeleton of rhodococcus ruber;

-the lyophilized powder comprising the cell wall skeleton of Rhodococcus ruber is administered directly at the lesion; or

-applying a cream comprising the cell wall skeleton of rhodococcus ruber on the lesion.

The medicinal liquid, paste, and dry powder can be alternately applied on the affected part.

The dose for each administration, which varies depending on the size and depth of the lesion area of the patient, is usually 5. mu.g to 800. mu.g per unit dose per one application, and preferably 10. mu.g to 600. mu.g per unit dose per application.

In some specific embodiments, the period of contacting is: for 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; as 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 can be mentioned. In specific embodiments, the subject is administered the active ingredient for 3-5 weeks.

In some embodiments, administration is performed at the following frequency: 1 to 3 applications a day, 1 to 6 applications a day, 1 to 9 applications a three day, 1 to 14 applications a week, 1 to 60 applications a month. In some embodiments, the administration is twice a day, or once every two days.

The amount administered at each time will vary depending on the particular condition of the subject, and will generally be from 1 μ g to 1000 μ g per unit dose per administration; specifically, for example, 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 μ g per unit dose per time, and ranges between any two of the foregoing values.

In some specific embodiments, the rhodococcus ruber product, pharmaceutical composition or medical device of the present disclosure is contacted with a focal site: from minutes to hours, for example from 30 minutes to 24 hours.

In some specific embodiments, the red coccus ruber product, pharmaceutical composition or medical device of the present disclosure is contacted with the focal site for a period of: once a day, or twice a day, or once every two days.

In some specific embodiments, the contacting is accomplished by, for example but not limited to: mucosal, transdermal.

In some embodiments, the foregoing treatment methods are applicable to any animal having a skin/mucosal structure, including but not limited to: human, non-human primate, pig, cow, horse, sheep, dog, cat, mouse, rabbit.

In some specific embodiments, the subject is an animal other than a human, e.g., for farm animals, pets, work animals, ornamental animals, production animals.

In a specific embodiment, the subject is a human.

In some specific embodiments, the subject is suspected of having, diagnosed with, has had, or is susceptible to the target disease or a symptom thereof.

In some specific embodiments, the only therapeutically (or prophylactically) active ingredient in the medicament or medical device is a product derived from rhodococcus ruber, in particular a product comprising constituent components of rhodococcus ruber, such as proteins, nucleic acids, lipids, cell walls and their constituent components, carbohydrates, metabolites, in particular a product comprising the cell wall of rhodococcus ruber, more preferably the cell wall skeleton of rhodococcus ruber or its constituents.

Drawings

FIG. 1: colony morphology of Rhodococcus ruber.

FIG. 2: 16S rRNA identification results.

Detailed Description

By "isolated" is meant that the Rhodococcus ruber of the present disclosure is removed from its original growth environment.

The skilled worker knows that gram-positive and gram-negative bacteria differ in their cell wall structure. In particular, gram-positive bacteria have a thick cell wall (typically 20nm to 80nm) and contain about 90% peptidoglycan and about 10% teichoic acid (a polymer formed from an alcohol and a phosphate molecule, typically in the form of a sugar ester or an amino acid ester). The peptidoglycan layer was dense, even up to 20 layers. However, the cell wall of gram-negative bacteria is much thinner and more complex than that of gram-positive bacteria, and is divided into an outer membrane (outer membrane) and a peptidoglycan layer (typically 2nm to 3 nm).

The peptidoglycan layer is a unique component in bacterial cell walls and is a derivative of heteropolysaccharides. Each monomer of peptidoglycan includes 3 moieties: sugar units (e.g., at least two sugar molecules linked together by glycosidic bonds, constituting the framework structure of peptidoglycan), peptide tails (short peptide chains of several amino acids linked together, which are linked to N-acetylmuramic acid molecules), and peptide bridges (which crosslink adjacent "peptide tails" to form a high-strength network). The peptide bridges, peptide tails, and cross-linking patterns are different for different bacteria.

Isolated Rhodococcus ruber cell wall

In the present disclosure, "isolated Rhodococcus ruber cell wall" is understood to mean both an intact cell wall and an incomplete cell wall (e.g., disrupted, or partially degraded). The skilled artisan, in light of the present disclosure, will appreciate that the component exhibiting the desired activity is derived from the cell wall of Rhodococcus ruber (e.g., is the cell wall itself or a component thereof). Therefore, various forms of intact cell walls, disrupted cell walls, incomplete degradation products of cell walls, constituents of cell walls, extracts of cell walls, etc., which are allowed to be used in clinical applications, are included in the scope of the present disclosure.

Cell wall skeleton

A constituent constituting a cell wall body structure; but are not to be understood as merely representing cross-linked network entities within the cell wall, and the skilled person will understand that other cell wall components adsorbed, bound, carried on the cross-linked network entities are not excluded.

Rhodococcus ruber

The Rhodococcus ruber used in the embodiments of the present disclosure refers to Rhodococcus ruber (Rhodococcus ruber) of the genus Rhodococcus (Rhodococcus), and is not limited to a specific cell strain.

Non-limiting examples include strain TOY7 (Nanjing agriculture university agricultural environmental microorganism culture Collection), CGMCC No.4795, DSM43338, CCTCC No.2012035, CGMCC No.16640, and CGMCC No. 17431.

Identification of Rhodococcus ruber

The skilled person can taxonomically identify a strain of bacteria according to known or future microbial identification techniques, e.g. available identification techniques include morphological, physiological and biochemical characteristics, 16S rRNA, etc. The skilled person understands that as technology advances, identification techniques involve different means, and in earlier times morphological and biochemical identification methods were mainly used, but the reliability of such methods is not high. After the advent of sequencing technology, the skilled artisan could identify strains in a more reliable manner. For example, when the DNA sequence of 16S rRNA was identified as having a similarity of 97% or more, it was judged that two genera belong to the same species (Huagougen et al, progress in classification and application research of Rhodococcus, microbiological report, 2003: 30 (4)). In the case of Rhodococcus ruber, known strains deposited in the International (or national) Collection of species are used as model strains and compared therewith.

Dosage forms

The medicament or pharmaceutical composition or active ingredient or product of the present disclosure may be embodied in, but not limited to, the following forms: ointment, cream, plaster, gel, tablet, lotion, tincture, liniment, oil, paste, powder, spray, aerosol, suppository, patch, suspension, collutory, buccal tablet, adjuvant, patch, bandage, patch, and gauze.

Formulation unit

The medicament or pharmaceutical composition or active ingredient or product of the present disclosure may be prepared in the form of a unit preparation (unit preparation).

In some embodiments, the unit dose in the medicament (or formulation, or therapeutic agent, or medical device) contains:

-1 μ g to 1000 μ g of said Rhodococcus ruber product; or

-1 μ g to 1000 μ g of said Rhodococcus ruber cell wall; or

-1 μ g to 1000 μ g of said Rhodococcus ruber cell wall skeleton.

Specific examples of 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 μ g ± 10%, and ranges between any two of the foregoing values.

"administering," "providing," "treating," when applied to an animal, human, cell, tissue, organ, or biological sample, means that the drug or medical device is in contact with the animal, human, cell, tissue, organ, or biological sample.

By "treating" is meant administering an internal or external drug (therapeutic agent, active ingredient or composition) (e.g., a rhodococcus ruber cell wall or pharmaceutical composition thereof according to the present disclosure) or medical device to a subject who has, is suspected of having, or is susceptible to one or more diseases or symptoms thereof, to alleviate (reduce, delay, ameliorate, cure) one or more symptoms of the disease in the subject (or population) being treated so as to reach a clinically measurable degree.

The amount of drug (therapeutic agent, active ingredient or composition) that is effective to alleviate any symptoms of the disease is referred to as a therapeutically effective amount. May vary depending on a number of factors, such as the disease state, age and weight of the subject. It is understood that a drug (therapeutic agent, active ingredient or composition) may not be effective in alleviating a target disease or symptom thereof in an individual subject, but the drug (therapeutic agent, active ingredient or composition) is statistically effective against the target disease or symptom thereof as determined by any statistical test method known in the art, such as Student's T test, chi-square test, U-test by Mann and Whitney.

"optional" means that the subsequently described events thereof can occur, but need not occur; as the case may be. For example, "optionally, portioned" means that a product is allowed to be portioned, but not necessarily; whether the split charging or not does not influence the realization of the technical effect.

The terms "a", "an", "the", and "the" include plural references unless expressly stated otherwise.

The present disclosure is further described below with reference to examples, preparation examples and test examples. These examples, preparations and test examples do not limit the scope of the present disclosure. When the specific conditions are not specified, the operation is carried out under the conventional conditions, as recommended by the raw material supplier. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.

The skilled person understands in particular that, although the following specific examples employ a specific cell strain, the achievement of the technical effect is not limited to this specific cell strain, any species belonging to the Rhodococcus erythropolis species (Rhodococcus ruber) of the genus Rhodococcus being suitable.

Examples

Example 1 Strain preservation

The inventor reserves the main generation strain stored in the laboratory in No. 3 of Xilu No.1 of Beijing, Chaoyang district, China general microbiological culture Collection center with the collection number of CGMCC No.17431, 22.03 and 22 days in 2019. The detection shows that the deposited strain survives.

Example 2 Strain identification

1. Naked eye observation of morphological characteristics of bacterial colonies

Incubation on glycerol agar medium at 30-37 deg.C (specifically 32-35 deg.C) for 12 to 72 (specifically 36 to 60, e.g., 40-50) hours, as seen (FIG. 1):

-colony uplift;

orange (slightly different, influenced by light, culture medium colour, etc.);

dry, wrinkled, slightly shiny (slightly different depending on the culture conditions);

-frangible to touch;

colony sizes from about 1mm to 2mm (slightly different depending on culture conditions).

2. Observation by microscope

The mycelium is branched and has a diaphragm, and mycelium is formed (slightly different according to different culture conditions);

hyphal division into regular, stubby cells (slightly different depending on culture conditions);

when cultured for 4 to 5 days, the cells were in the form of short rods or spheres (slightly different depending on the culture conditions).

3. Dyeability

Gram staining positive.

4. Biochemical reaction

Culturing on glycerol agar slant culture medium at 30-37 deg.C (specifically 32-35 deg.C) for 12-72 (specifically 36-60, such as 40-50) hr. The cultures were then tested as follows.

4.1 carbohydrate acid production:

positive: glycerol, mannitol, sorbitol, D-arabitol, D-fructose, D-glucose;

negative results are shown: inositol, inulin, lactose, sucrose, starch, maltose, glycogen, xylitol, gluconate, trehalose, erythritol, melezitose, melibiose, raffinose, cellobiose, amygdalin, gentiobiose, adonitol, arbutin, D-arabinose, L-arabinose, alpha-methyl-D-glucoside, alpha-methyl-D-mannoside, D-ribose, D-xylose, L-xylose, N-acetyl-glucosamine, D-turanose, D-lyxose, beta-methyl-D-xyloside, D-galactose, D-tagatose, D-fucose, L-fucose, D-mannose, L-sorbose, L-arabitol, L-rhamnose, D-fucose, D-xyloside, D-mannose, D-arabinosyl, L-rhamnose, glucose, 2-keto-gluconate.

4.2 enzyme Activity assay (API ZYM):

positive: alkaline phosphatase, lipoid esterase (C8), lipoid esterase (C14), leucine arylamine, valine arylamine, cystine arylamine, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-B1-phosphohydrolase, alpha-glucosidase;

negative results are shown: n-acetyl-glucosaminidase, esterase (C4), beta-galactosidase, beta-glucuronidase, beta-glucosidase, alpha-galactosidase, alpha-mannosidase, beta-fucosidase.

4.3 nitrate reduction reaction positive, catalase positive, tyrosinase positive, amylase negative, oxidase negative, gelatin liquefaction negative.

4.4 sole carbon source:

4.5.16S rRNA identification

Genome extraction, 16Sr RNA amplification, and sequencing were performed on 15 strains isolated in the working seed tube and 10 different strains isolated in the original seed tube. The 16Sr RNA gene identity for a total of 25 strains was 100%. This indicates that 25 strains are of the same species (FIG. 2).

Meanwhile, the neighbor-join strain evolutionary tree constructed based on the Kimura 2-parameter algorithm showed that the strain was assigned to Rhodococcus ruber.

Preparation example

Preparation example 1 culture method

1. Rhodococcus ruber can be cultured by a conventional microbial production method.

2. The culture mode can be solid culture or liquid culture (such as shake flask and fermentation tank).

3. The nutrient source in the culture medium is not particularly limited, and the culture medium contains a carbon source, a nitrogen source and other nutrient sources which are commonly used for culturing gram-positive bacteria.

The carbon source is any carbon source that can be utilized by Rhodococcus ruber. For example, fructose, glucose, and the like.

-nitrogen source: meat extract, peptone, ammonium salts, nitrates and other organic or inorganic nitrogen-containing compounds.

-other nutrient sources: inorganic salts may be added as appropriate. Such as NaCl, phosphate, etc.

4. The culture conditions (temperature, time, etc.) are not critical, and the skilled person can select the conditions that will maximize the yield based on preliminary small-scale pilot plant data.

5. As an example, rhodococcus ruber is fermented using the following culture conditions:

(1) the culture medium composition comprises:

peptone, beef extract, sodium chloride, phosphate, glycerol (and, optionally, agar when cultured as a solid).

(2) The method parameters of the culture are as follows:

after the recovery of the working strain, transferring to a solid culture medium for 3-5 days, and then transferring to liquid culture (30-37 ℃ for 3-5 days), wherein a fed-batch semi-continuous mode or a batch mode can be adopted. During the cultivation, pH, bacterial density, dissolved oxygen, carbon source consumption were monitored.

Preparation example 2 disruption of cells

The bacteria obtained in preparation example 1 were collected, and the cells were pulverized (for example, but not limited to, by ultrasonic disruption). It is also permissible to disrupt the bacterial cells by any suitable method known in the art, for example, CN101250490A or CN 101323865A.

The crushed condition is checked under a microscope, the number of the visible bacteria in each visual field is not more than 5, and the standard is met in a plurality of visual fields (10 to 30) to be qualified.

Preparation example 3 removal of non-cell wall Components

1. Removing nucleic acid:

the disrupted product was centrifuged, and DNase and RNase were added to the obtained precipitate to remove nucleic acids according to the procedures recommended by the enzyme supplier.

2. Removing protein:

the precipitate is added with a common protease (e.g. trypsin) and the protein is removed according to the procedures recommended by the supplier of the enzyme.

3. Removing lipid:

adding organic reagent (such as but not limited to one or combination of acetone, diethyl ether and ethanol) into the precipitate, and removing lipid according to conventional operation in the field.

4. Removing cell membranes:

TritonX-100 was added to the pellet, and the pellet was collected by centrifugation and rinsed with PBS according to a routine procedure in the art.

It should be understood that, between the above steps of removing impurities, the skilled person can adjust the order so as to make the steps compatible. After removing the non-cell wall components, the precipitate was redissolved in water for injection for use. Optionally, sterilization can be performed at 115 ℃ for 20-30 minutes as a stock solution of the cell wall skeleton (comprising the cell wall skeleton and its constituents).

In addition to the above methods, the skilled person may also use methods known in the art or future to remove non-cell wall components, such as the method of extracting cell wall components disclosed in CN 105779326A.

5. Yield of the product

653ml of the bacterial liquid was collected from 159 Kirschner flasks (after disruption); the wet weight yield was 138 g; the cell wall skeleton yield was about 0.87 g/kjeldahl flask.

Preparation example 4 preparation of pharmaceutical composition (medical device)

1. Liquid composition

To the product obtained in preparation example 3, an excipient (e.g., dextran 40, mannitol, or trehalose) was added. Subpackaging to obtain the pharmaceutical composition.

TABLE 1 pharmaceutical compositions may be formulated in a variety of forms

2. Freeze-dried powder composition

Lyophilizing the pharmaceutical composition of item 1 to obtain lyophilized powder (numbered lyophilized powder compositions 1 to 7, respectively).

3. Preparation method of preparation

(1) Dressing material

The pharmaceutical composition of item 1 (active ingredient 60 μ g to 120 μ g, e.g., 60 μ g, 70 μ g, 80 μ g, 90 μ g, 100 μ g, 110 μ g, 120 μ g) is coated on a dressing (e.g., sterile gauze) to prepare an external medical device.

(2) Patches or films

The patches are prepared using methods known in the art (e.g., as disclosed in chinese application nos. 201610605617.5, 201510614414.8, 200610200450.0, 201610511974.5, 201610471977.0, etc.).

For example: adding film-forming materials such as polyvinyl alcohol, carbomer and hydroxypropyl cellulose into water, and swelling to form homogeneous viscous liquid; adding the pharmaceutical composition of item 1 (compositions 1 to 7, respectively) thereto and mixing; standing for defoaming; mixing the formed bubble-free viscous liquid; casting on a mold coated with a small amount of paraffin, drying for 5-20min, taking out, stripping, and cutting into required area.

(3) Gel agent

It can also be made into gel. For example, referring to the method disclosed in chinese application No. 200510028076.6, composition 3 and an esterifying agent are dissolved in a solvent under stirring, hydroxyalkyl cellulose is added to swell the hydroxyalkyl cellulose, and the stirring is continued to form a gel; then adding the cross-linking agent and continuing stirring until the mixture is completely uniform.

(4) Ointment preparation

Methods for preparing ointments for external application to the skin are also disclosed in, but not limited to, chinese application nos. 201610856428.5, 01133296.4, and 1133297.2, and methods disclosed in pharmaceutical dosage forms and formulation design (chemical industry press 2009).

4. Quality control (taking lyophilized powder composition 3 as an example)

TABLE 2 quality test items

Test example

Test example 1 pharmacological test

1. After intravenous injection with 20, 40 and 80 times of clinical dose of human, blood pressure, respiration, heart rate and electrocardio of the anaesthetized cat are monitored without obvious influence;

2. after intravenous injection, which is 1000 times the clinical dose of human, the coordination of movement and learning and memory functions of mice did not have significant effect.

It can be seen that the pharmaceutical compositions of the present disclosure (compositions 1 to 7) had no significant effect on the mental, nervous, cardiovascular, and respiratory systems of the animals.

Test example 2 safety experiment

1. And (3) sterile experiment:

the results were negative, demonstrating sterility.

2. Acute toxicity test in mice:

the experimental group was administered by subcutaneous injection and intraperitoneal injection, the dose was 5 times of the human dose, and the control group was continuously observed with sterile physiological saline 0.5 ml/branch for 7-8 days. The state of the mouse is good, and the body weight is not abnormal; the mouse has no abnormality in each organ.

3. Long-term toxicity test:

30 times of clinical dosage is applied once a day, and the vaginal administration is continued for three months, so that no toxic effect is found in dogs; the electrocardiogram and blood biochemical indexes are in normal range. After two weeks of withdrawal, no delayed toxicity was seen (compositions 1 to 7).

Test example 3 stability test

The alanine content, muramic acid content, phagocytosis rate, phagocytosis index of the pharmaceutical compositions (composition 1 to composition 7) were not statistically significantly different from those at the start of the experiment (three batches tested) when left at room temperature (18-25 ℃) for 0, 1, 2, 3, 8, 14, 21 months.

In conclusion, the pharmaceutical composition of the lyophilized powder preparation can be stably stored for 24 months.

Test example 4 phagocytosis assay

In the prior art, phagocyte is activated after being stimulated by antigen, so that the phagocytic function is obviously enhanced. After the abdominal cavity macrophage is induced to generate in a mouse body, chicken blood red cells are injected into the abdominal cavity of the mouse, the mouse is killed after 30min, abdominal cavity liquid is taken out, staining is carried out, the percentage of phagocytic red cells is counted under a microscope to judge the killing capacity of phagocytic cells, and the nonspecific immunity level of the body is indirectly measured.

The phagocytic rate, tested with composition 3 of the present application, was 75% with a phagocytic index of 1.05. While negative controls (vehicle) and placebo (saline) had lower phagocytic rates and lower phagocytic indices. Indicating that the cell wall skeleton or its constituent components of the present application have a strong immunophagic ability.

Examples of effects

Effect example 1

1. Preliminary diagnosis (2019-06-07):

the main complaints are: whitening and hyperplasia of vulvar skin for 4 months;

the current medical history: before 4 months, patients have no obvious cause and have pruritus vulvae, and then have the phenomena of whitening vulvae skin, hyperplasia, local rough skin, no wound history, no systemic diseases such as hypertension and diabetes mellitus and the like;

history of the past: none;

family history: none;

physical examination: the vulva develops normally, and the left vulva is 1.5 × 1.5cm²The white hyperplastic lesion is slightly higher than the skin surface, and no obvious lacerated surface and no obvious purulent secretion are observed.

And (3) preliminary diagnosis: white lesions of vulva.

2. Treatment planning:

the patient is informed of the disease condition and informed consent is obtained.

Keeping the perineum clean; physical stimulation such as scratching is avoided; composition 3 (ointment, 60 μ g active ingredient each time, once every other day) was applied topically. The patients were reviewed every week and were not suitable for follow-up.

3. First double-diagnosis (2019-06-14):

and (3) return diagnosis: the patient follows the medical advice to take the medicine after the last visit, and consciously feels that the lesion area at the left vulva is reduced.

And (4) checking: the vulva develops normally, 0.8 × 1.5cm is visible in the left vulva²The white lesions of the size are reduced compared with those before treatment, and local atrophy is realized, and obvious purulent secretion is not shown.

4. Second double-diagnosis (2019-06-21):

And (4) checking: the vulva develops normally, 0.8X 0.6cm is visible in the left vulva²The white lesions of the size are reduced compared with those before treatment, and local atrophy is realized, and obvious purulent secretion is not shown.

5. The third double-diagnosis (2019-07-04):

And (4) checking: the vulva develops normally, the skin of the left vulva recovers normally, the local part is slightly atrophic, and no obvious purulent secretion is found.

Effect example 2

1. First diagnosis (2019-07-02)

The main complaints are: the vulvar skin turns white with pruritus for 2 months;

the current medical history: before 2 months, patients have no obvious inducement to pruritus vulvae and intolerable pruritus vulvae, and then have the phenomena of whitening vulvae skin, rough local skin, no vaginal bleeding, no wound history, no systemic diseases such as hypertension and diabetes mellitus, and the like;

history of the past: none;

family history: none;

physical examination: the vulva is seen bilaterally at about 1.2X 1.0cm²Large and small pigment reducing spot (hypo-pigmentation spot), bilateral labia majora atrophy, no obvious ulceration and bleeding, and no obvious purulent secretion.

And (3) preliminary diagnosis: white lesions of vulva.

2. Treatment planning:

Histopathological examination: bilan (Primacaine) STA local anesthesia, and sterilizing conventionally, taking about 0.8 × 0.5 × 0.5cm at right vulva lesion³Tissue, 3 needles were sutured. Specimens were fixed with 10% formalin, submitted for pathology, and the lines were removed one week after the order.

3. First double-visit (2019-07-09):

and (3) return diagnosis: the patient had no discomfort after the last biopsy. The pathological result shows that the external genitalia white lesion is conformed.

And (4) checking: the biopsy part is well healed, and 3 stitches are stored without falling off;

treatment: keeping the perineum clean; physical stimulation such as scratching is avoided; composition 3 (dry powder, once every other day, 60 μ g active ingredient) was applied topically. The patients were reviewed every week and were not suitable for follow-up.

4. Second double diagnosis (2019-07-16):

and (3) return diagnosis: the patients follow the medical advice to take the medicine after the last visit, the lesion area of the vulva at both sides is consciously reduced, and the roughness is reduced.

And (4) checking: bilateral vulva is visible at about 1.0 × 0.7cm²The large and small pigments reduce the macula, the labia majora atrophy on both sides, no obvious ulceration and bleeding are seen, and no purulent secretion exists.

5. Third consultation (2019-07-23):

and (3) return diagnosis: the patients follow the medical advice to take the medicine after the last visit, and the bilateral vulva lesions are consciously reduced.

And (4) checking: bilateral vulva is visible at about 0.8 × 0.5cm²The large and small pigments reduce the macula, the labia majora atrophy on both sides, no obvious ulceration and bleeding are seen, and no purulent secretion exists.

6. Fourth double diagnosis (2019-07-31):

and (3) return diagnosis: after the patient has a doctor at the last time, the bilateral vulva lesions are basically healed by taking the medicine according to the advice of the doctor.

And (4) checking: the bilateral vulvar hypopigmentation spots basically recover and disappear, and the bilateral labia majora slightly atrophy without obvious ulceration and bleeding and purulent secretion.

Effect example 3

1. Preliminary diagnosis (2019-05-10):

the main complaints are: the vulva is whitish for 1 month;

the current medical history: the patients find that the vulva is whitish before 1 month, accompanied by pruritus vulvae, and the patients continuously scratch and relieve itching, have no obvious improvement after self-administration, have rough local skin, no purulent secretion and vaginal bleeding, and no other skin lesions;

history of the past: none;

family history: none;

physical examination: the vulva is seen bilaterally at about 1.0X 2.0cm²White lesions of size, atrophy of labia majora on both sides, with a lesion size of about 0.4X 0.6cm visible in the center²The large and small ulcerated surfaces have no obvious purulent secretion due to desquamation at the periphery.

And (3) preliminary diagnosis: white lesions of vulva.

2. Treatment planning:

Keeping the perineum clean; physical stimulation such as scratching is avoided; the topical composition 5 was rinsed with the lotion and then composition 3 (gel, once every other day, 60 μ g active ingredient each) was applied. The patients were reviewed every week and were not suitable for follow-up.

3. First double-visit (2019-05-17):

and (3) return diagnosis: the patients follow the medical advice after the last visit, the lesion area on the two sides of the vulva is consciously reduced, and the ulcerated surface is healed.

And (4) checking: the vulva develops normally, and the bilateral vulva can be seen at 0.6X 2cm²The white lesion with the size is reduced compared with that before treatment, the local atrophy and the central ulcerated surface of the lesion are healed, and no obvious purulent secretion is found.

4. Second double-visit (2019-05-24):

and (3) return diagnosis: the patients follow the medical advice to take the medicine after the last visit, and the affected areas on the two sides of the vulva are consciously reduced.

And (4) checking: the vulva develops normally, and the bilateral parts of the vulva can be seen at 0.8X 1.2cm²The white lesions of the size are reduced compared with those before treatment, and local atrophy is realized, and obvious purulent secretion is not shown.

5. The third re-diagnosis (2019-06-02):

And (4) checking: the vulva develops normally, 0.2X 0.3cm is seen at bilateral vulva²The white lesions of the size are reduced compared with those before treatment, and local atrophy is realized, and obvious purulent secretion is not shown.

Claims

Use of a Rhodococcus ruber cell wall or Rhodococcus ruber cell wall skeleton in the manufacture of a medicament for the treatment of vulvar whiteness disorder or prevention of recurrence thereof.
The use according to claim 1, wherein the vulvar whites lesion is vulvar lichen sclerosus and/or vulvar squamous epithelial cell hyperplasia.
The use according to claim 1, wherein the medicament is prepared in a dosage form selected from the group consisting of: ointment, cream, lotion, suspension, cataplasm, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, powder, adjuvant, bandage, membrane, patch, and suppository.
Use according to claim 1, wherein:

the medicament comprises a unit dose of 1 to 1000 μ g of a Rhodococcus ruber cell wall or Rhodococcus ruber cell wall skeleton;

preferably, a unit dose of 5 μ g to 800 μ g of the Rhodococcus ruber cell wall or Rhodococcus ruber cell wall skeleton is included;

more preferably, 10. mu.g, 20. mu.g, 30. mu.g, 40. mu.g, 50. mu.g, 60. mu.g, 70. mu.g, 80. mu.g, 90. mu.g, 100. mu.g, 150. mu.g, 200. mu.g, 250. mu.g, 300. mu.g, 350. mu.g, 400. mu.g, 500. mu.g, 600. mu.g, 700. mu.g, 800. mu.g per unit dose are contained.
The use according to claim 1, wherein the Rhodococcus ruber is Rhodococcus ruber deposited in the general microbiological center of the China Committee for culture Collection of microorganisms at 03 and 22 months in 2019 with the deposit number of CGMCC No. 17431.
Use according to claim 1, wherein:

the medicament further comprises a pharmaceutically acceptable excipient;

the cell wall or the cell wall skeleton of the rhodococcus ruber is 1 part by weight, and the pharmaceutically acceptable excipient is 100 to 1000 parts by weight, preferably 200 to 500 parts by weight, and more preferably 200 to 300 parts by weight;

preferably, the content of the rhodococcus ruber cell wall or the rhodococcus ruber cell wall skeleton is 1 part by weight, and the content of the pharmaceutically acceptable excipient is 250 parts by weight;

the medicament is a liquid, dry powder preparation or freeze-dried powder preparation.
Use according to any one of claims 1 to 6, the Rhodococcus ruber cell wall or Rhodococcus ruber cell wall skeleton being obtainable by a method comprising or consisting of the steps of:

1) providing Rhodococcus ruber;

2) crushing the Rhodococcus ruber to obtain a crushed product;

3.1) optionally, subjecting the comminuted product to an operation to remove lipids;

3.2) optionally, subjecting the comminuted product to an operation to remove nucleic acids;

3.3) optionally, subjecting the comminuted product to an operation to remove proteins;

3.4) obtaining a product derived from the cell wall of Rhodococcus ruber;

4) optionally, removing water from the product derived from the cell wall of Rhodococcus ruber, preferably freeze-drying the product derived from the cell wall of Rhodococcus ruber;

5) optionally, subpackaging;

wherein, the steps 3.1), 3.2), 3.3) can be interchanged in sequence or in parallel; step 4) and step 5) can be interchanged in sequence;

preferably, the milled average particle size is from 10nm to 1000nm, more preferably from 10nm to 800nm, more preferably 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, 500 nm;

preferably, the dispensing means into containers or onto solid supports;

the container is selected from: bottle, tube, bag, plate, ampoule, injection device, aluminum film package, dressing, capsule.
A method of treating or preventing recurrence of vulvar white lesions, comprising:

contacting the subject with a therapeutically effective amount of a Rhodococcus ruber cell wall or Rhodococcus ruber cell wall skeleton;

the vulvar white lesion is vulvar lichen sclerosus and/or vulvar squamous epithelial cell proliferation;

the Rhodococcus ruber cell wall or Rhodococcus ruber cell wall skeleton is prepared in a form selected from the group consisting of: ointment, cream, lotion, suspension, paste, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, powder, adjuvant, bandage, membrane, patch, and suppository;

the contacting is twice a day, or once every two days, or three times every two days, or once every three days, or once a week;

the contacting is from 30 minutes each time to 24 hours each time;

the contacting is for 2 days, 3 days, 4 days, 5 days, 6 days, 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 more.
The method according to claim 8, wherein the Rhodococcus ruber cell wall or Rhodococcus ruber cell wall skeleton is obtainable by a method comprising or consisting of the steps of:

1) providing Rhodococcus ruber;

2) crushing the Rhodococcus ruber to obtain a crushed product;

3.1) optionally, subjecting the comminuted product to an operation to remove lipids;

3.2) optionally, subjecting the comminuted product to an operation to remove nucleic acids;

3.3) optionally, subjecting the comminuted product to an operation to remove proteins;

3.4) obtaining a product derived from the cell wall of Rhodococcus ruber;

4) optionally, removing water from the product derived from the cell wall of Rhodococcus ruber, preferably freeze-drying the product derived from the cell wall of Rhodococcus ruber;

5) optionally, subpackaging;

wherein, the steps 3.1), 3.2), 3.3) can be interchanged in sequence or in parallel; step 4) and step 5) can be interchanged in sequence;

the pulverized average particle size is 10nm to 1000nm, preferably 10nm to 800nm, more preferably 10nm to 500 nm;

preferably, the dispensing means into containers or onto solid supports;

the container is selected from: bottle, tube, bag, plate, ampoule, injection device, aluminum film package, dressing, capsule.