Rhodococcus ruber products and their pharmaceutical uses

Abstract

The present invention relates to an isolated Rhodococcus ruber, a product derived from Rhodococcus ruber (particularly a product derived from the cell wall of Rhodococcus ruber), a method for producing a Rhodococcus ruber product, and use of a Rhodococcus ruber product in the prevention and / or treatment of lichen planus. The Rhodococcus ruber product effectively reduces the area of ​​erosions caused by lichen planus and relieves the degree of pain.

Description

[Technical field]

[0001] This disclosure claims the benefit of priority from Chinese patent application "Cell wall skeleton of Rhodococcus ruber and use thereof" filed on January 15, 2019 (application number 201910036001.4) and Chinese patent application "Isolated Rhodococcus ruber" filed on October 25, 2019 (application number 201911022193.X), the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to the medical, microbiological and biopharmaceutical fields, and more particularly to Rhodococcus ruber and its cell wall components, formulations, pharmaceutical compositions, methods of manufacture and therapeutic uses of Rhodococcus ruber cell wall components. [Background technology]

[0003] Rhodococcus ruber is a gram-positive bacterium. The colonies are usually round, yellowish orange or reddish orange in color, about 1mm to 2mm in size, the cells are spherical or short rod-shaped, can form primary branched mycelium, and lack flagella. Rhodococcus ruber is aerobic and heterotrophic.

[0004] Currently, researchers are sequencing the whole genome of Rhodococcus ruber. For example, Fan Xin et al. sequenced the whole genome of Rhodococcus ruber SD3 strain and performed bioinformatics analysis. The whole genome of SD3 strain is about 5.37Mb long, with a GC content of about 70.63%, and the GenBank accession number is CP029146 (Fan Xin, Whole genome sequencing of Rhodococcus ruber SD3 and analysis of the expression of its heat shock protein DnaK, Genomics and Applied Biology, January 2019).

[0005] Rhodococcus itself has a very strong organic matter resistance and a wide decomposition spectrum, allowing it to adapt to a variety of living environments. Therefore, Rhodococcus is widely used in areas such as pollution remediation, decomposition of organic compounds, and wastewater treatment. At present, Rhodococcus louver is mainly applied in the field of environmental control, see CN108862590A, CN107151635A, CN102250796A, CN1519312A, CN103627653A, CN101033454A, CN108130288A, CN104830738A, CN101619299A, CN103509833A, CN106434466A, CN101580808A, CN102604875A, CN103160491A, CN106591168A, CN106591172A, CN105820982A.

[0006] CN109576180A discloses the bacterium RDC-01, which was screened from red soil in the suburbs near Panyu District, Guangzhou City, and was identified as Rhodococcus ruber through 16S rRNA gene sequence analysis and cultural characteristic identification. It has been shown that after inactivation, this bacterium can be added to animal inactivated vaccines as an immune adjuvant to promote the production of animal antibodies.

[0007] However, there have been no reports of the application of Rhodococcus ruber in human medicine.

[0008] Oral lichen planus (OLP) is a chronic inflammatory disease of the skin and mucosa caused by cell-mediated immunity (Oral Mucosal Pathology, Peking University Medical Press, 2014). It is a fairly common clinical disease, with a prevalence of 0.1%-4%. Oral lichen planus with long-term erosion is at potential risk of progression (Lodi G et al., Current controversies in oral lichen planus: Report of an international consensus meeting, part 2; clinical management and malignant transformation. Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics, 2005, 100(2): 164-78). In 2005, the WHO listed it as an oral potentially malignant disorder (OPMD), and the latest study in 2019 showed that the malignant transformation rate of OLP is 0.8%-1.5%.

[0009] For the diagnosis of lichen planus, one can refer to the "Guideline for the Treatment of Oral Lichen Planus" jointly formulated by the Chinese Association of Stomatology Special Committee on Oral Mucosal Diseases and the Chinese Association of Stomatology Special Committee on Integrated Chinese and Western Medicine, or the WHO's 2003 diagnostic criteria for oral lichen planus. In "Oral Mucosal Pathology," lichen planus is divided into reticular, annular, linear, papular, erosive, macular, vesicular, and atrophic types.

[0010] The cause of OLP is still unclear, and the disease tends to last a long time and recur. There is no cure yet, so symptomatic treatment and immunomodulatory treatment are the main treatments. In clinical practice, glucocorticoids are administered locally as the main therapeutic agent (Garcia-Pola MJ et al., Treatment of oral lichen planus. Systematic review and therapeutic guide. Medicina Clinica, 2017, 149(8):351-362), and severe patients are administered glucocorticoids or immunosuppressants systemically for a short period of time. However, long-term local administration of glucocorticoids has significant side effects, such as a series of complications such as mucosal atrophy and worsening infection, electrolyte imbalance, delayed wound healing, and growth suppression in children, causing adverse reactions throughout the body, making this medicine unsuitable for long-term administration in clinical practice (Oray M et al., Long-term side effects of glucocorticoids. Expert Opinion on Drug Safety, 2016, 15(4):457-65).

[0011] Therefore, finding a medicine that is safe, effective and has no significant side effects is a clinical problem that needs to be solved quickly. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] China Patent Application Publication No. 108862590 [Patent Document 2] China Patent Application Publication No. 107151635 [Patent Document 3] China Patent Application Publication No. 102250796 [Patent Document 4] China Patent Application Publication No. 1519312 [Patent Document 5] China Patent Application Publication No. 103627653 [Patent Document 6] China Patent Application Publication No. 101033454 [Patent Document 7] China Patent Application Publication No. 108130288 [Patent Document 8] China Patent Publication No. 104830738 [Patent Document 9] China Patent Publication No. 101619299 [Patent Document 10] China Patent Application Publication No. 103509833 [Patent Document 11] China Patent Application Publication No. 106434466 [Patent Document 12] China Patent Publication No. 101580808 [Patent Document 13] China Patent Application Publication No. 102604875 [Patent Document 14] China Patent Application Publication No. 103160491 [Patent Document 15] China Patent Publication No. 106591168 [Patent Document 16] China Patent Application Publication No. 106591172 [Patent Document 17] China Patent Application Publication No. 105820982 [Patent Document 18] China Patent Application Publication No. 109576180 [Patent Document 19] China Patent Application Publication No. 101250490 [Patent Document 20] China Patent Application Publication No. 101323865 [Non-patent literature]

[0013] [Non-Patent Document 1] Lodi G et al., “Current controversies in oral lichen planus:Report of an international consensus meeting,part 2;clinical management and malignant transformation,” Oral Surgery Oral Medicine Oral Pathology Oral Radiology and Endodontics 2005, 100(2), p.164-78 [Non-Patent Document 2] Garcia-Pola MJ et al., “Treatment of oral lichen planus, Systematic review and therapeutic guide,” Medicina Clinica 2017, 149(8), p.351-362 [Non-Patent Document 3] Oray M et al., "Long-term side effects of glucocorticoids", Expert Opinion on Drug Safety 2016, 15(4), p.457-65 [Non-Patent Document 4] Meij EHVD, Waal IVD, "Lack of clinicopathologic correlation in the diagnosis of oral lichen planus based on the presently available diagnostic criteria and suggestions for modifications", Journal of Oral Pathology&Medicine, 2003, 32(9), p.507-512 [Non-Patent Document 5] Gorouhi F et al., “Randomized trial of pimecrolimus cream versus triamcinolone acetonide paste in the treatment of oral lichen planus,” Journal of the American Academy of Dermatology, 2007, 57(5), p.806-813 [Non-Patent Document 6] Park HK et al., "Oral lichen planus: REU scoring system correlates with pain," Oral Surgery Oral Medicine Oral Pathology Oral Radiology, 2012, 114(1), p.75-82 Summary of the Invention [Problem to be solved by the invention]

[0014] In one aspect of some embodiments of the present disclosure, an isolated Rhodococcus ruber is provided. [Means for solving the problem]

[0015] In some specific embodiments of the present disclosure, there is provided Rhodococcus ruber, deposited on March 22, 2019 at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, Postal Code: 100101, with deposit number CGMCC No. 17431. This deposit complies with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.

[0016] In some embodiments of the present disclosure, Rhodococcus ruber and its derivative products are provided, which are derived from Rhodococcus ruber and contain components of Rhodococcus ruber, such as proteins, nucleic acids, lipids, cell walls and their components, carbohydrates, and metabolites.

[0017] In a specific embodiment, an isolated Rhodococcus ruber cell wall is provided.

[0018] In a specific embodiment, an isolated cell wall of Rhodococcus ruber is provided, said Rhodococcus ruber referring to the strain having the deposit number CGMCC No. 17431.

[0019] In a specific embodiment, an isolated Rhodococcus ruber cell wall skeleton is provided.

[0020] In a specific embodiment, an isolated cell wall skeleton of Rhodococcus ruber is provided, said Rhodococcus ruber referring to the strain having the deposit number CGMCC No. 17431.

[0021] In some embodiments of the present disclosure, a pharmaceutical composition is provided comprising a cell wall of Rhodococcus ruber or a cell wall skeleton of Rhodococcus ruber of the present disclosure.

[0022] In one embodiment of the present disclosure, a Rhodococcus ruber product is provided, comprising a product obtained by grinding Rhodococcus ruber.

[0023] In some other embodiments of the present disclosure, a Rhodococcus ruber product is provided, which includes a product obtained by crushing Rhodococcus ruber and then purifying (removing lipids, nucleic acids, and proteins).

[0024] In some further embodiments of the present disclosure, a Rhodococcus ruber product is provided, comprising a cell wall of Rhodococcus ruber.

[0025] In some further embodiments of the present disclosure, a Rhodococcus ruber product is provided, comprising a cell wall skeleton of Rhodococcus ruber.

[0026] In one embodiment of the present disclosure, there is provided a pharmaceutical composition or medical device comprising a product obtained by grinding Rhodococcus ruber.

[0027] In some other embodiments of the present disclosure, a pharmaceutical composition or medical device is provided, comprising a product obtained by crushing Rhodococcus ruber and then purifying (removing lipids, and / or nucleic acids, and / or removing proteins).

[0028] In some further embodiments of the present disclosure, there is provided a pharmaceutical composition or medical device comprising a cell wall of Rhodococcus ruber.

[0029] In some further embodiments of the present disclosure, there is provided a pharmaceutical composition or medical device comprising a cell wall skeleton of Rhodococcus ruber.

[0030] In some further embodiments of the present disclosure, there is provided a pharmaceutical composition or medical device comprising the Rhodococcus ruber product.

[0031] In a specific embodiment, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient.

[0032] In some embodiments, the Rhodococcus ruber product in the pharmaceutical composition is 1 part by weight and the pharma- ceutically acceptable excipient is 200-300 parts by weight (e.g., 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, and any value between any two of these numbers).

[0033] In some other embodiments, the cell wall of Rhodococcus ruber in the pharmaceutical composition is 1 part by weight and the pharma- ceutically acceptable excipient is 200 to 300 parts by weight (e.g., 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, and any value between any two of these numbers).

[0034] Further, in some embodiments, the cell wall skeleton of Rhodococcus ruber in the pharmaceutical composition is 1 part by weight and the pharma- ceutically acceptable excipient is 200-300 parts by weight (e.g., 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, and any value between any two of these numbers).

[0035] In some embodiments, the pharmaceutical composition may be prepared as a liquid (liquid formulation).

[0036] In some alternative embodiments, the pharmaceutical composition may be prepared as a solid (dry powder formulation or lyophilized powder formulation).

[0037] Those skilled in the art can understand that the pharmaceutical composition of the present disclosure can be converted into either liquid formulation or dry powder formulation (or freeze-dried powder formulation), with the only difference being the water content. The dry powder formulation (or freeze-dried powder formulation) is obtained by removing most or all of the water in the liquid formulation. The liquid formulation is obtained by dissolving (or redissolving) the dry powder formulation (or freeze-dried powder formulation).

[0038] In some embodiments, the medicament or pharmaceutical composition is prepared in a dosage form selected from the group consisting of an ointment, cream, gel, wash, tincture, liniment, oil, paste, aerosol, lozenge, patch, lyophilized powder, and suspension.

[0039] In some embodiments, the dosage form is a lozenge, for example obtained by compressing a lyophilized powder.

[0040] In some embodiments, the pharma- ceutically acceptable excipient is, but is not limited to, a filler, a stabilizer, a flavoring agent, a disintegrant, an adhesive, or a lubricant.

[0041] In some embodiments of the present disclosure, a method for producing a Rhodococcus louvre product is provided, the method comprising or consisting of the steps of: 1) Prepare Rhodococcus louvre; 2) optionally culturing said Rhodococcus ruber; 3) Optionally, harvest the cultured Rhodococcus ruber; 4) crushing the cultured Rhodococcus ruber to obtain a crushed product; 5.1) Optionally, subjecting the milled product to a lipid removal operation; 5.2) Optionally, subjecting the disruption product to a nucleic acid removal procedure; 5.3) Optionally subjecting the ground product to a protein removal operation; 5.4) Obtaining a purified product; 6) optionally removing water in the purified product, preferably by freeze-drying; 7) In some cases, individual packaging; 8) obtaining the Rhodococcus louvre product; Steps 5.1), 5.2), and 5.3) may be performed in any order or simultaneously. Steps 6) and 7) may be performed in any order or simultaneously.

[0042] Optionally, step 5) may include removing cell membranes (eg, with a non-ionic detergent).

[0043] The culture medium and culture parameters for culturing Rhodococcus ruber are not limited to specific ones, and those skilled in the art can culture the bacteria in any suitable manner known in the art. Depending on the production scale, petri dishes, culture flasks, and fermentation tanks can be used.

[0044] The purpose of disrupting Rhodococcus ruber is to remove intracellular material, so techniques such as ultrasonic disruption, lysozyme, etc. Those skilled in the art can understand that any known or future method for disrupting Gram-positive bacteria is suitable for the technical solution of the present disclosure.

[0045] Those skilled in the art have the ability to adjust the specific parameters and equipment for culturing, disrupting, isolating, harvesting, removing impurities, and packaging depending on the subsequent administration (e.g., oral administration, injection, topical application, etc.) of the active ingredient (cell walls and their constituent components) so as to avoid introducing factors into the manufacturing steps that will affect the subsequent administration.

[0046] In some embodiments, lipids in the disrupted product are removed with an organic solvent. In some embodiments, DNA and RNA in the disrupted product are removed with a nuclease. In some embodiments, proteins in the disrupted product are degraded with a hydrolase. In some embodiments, cell membranes in the disrupted product are removed with a detergent.

[0047] In some embodiments, the average particle size after milling is between 10 nm and 1000 nm, including 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 nm ± 10 nm, and any two of the above values. There are many methods for measuring particle size (Hu Songqing et al., Modern Particle Size Measurement Technology, Modern Chemical Engineering, 2002, 22:1).

[0048] In some specific embodiments, the average particle size after milling is between 10 nm and 800 nm.

[0049] In some other specific embodiments, the average particle size after milling is from 10 nm to 500 nm.

[0050] In some embodiments, the individually packaged refers to being individually packaged in a container, the container being selected from the group consisting of a bottle, a tube, a pack, a bag, a plate, an ampoule, an injection device, a foil package, a dressing, and a capsule.

[0051] For example, in a specific embodiment, the individual packaging refers to individual packaging in a bottle / ampule, to which a solvent is added prior to administration.

[0052] In some specific embodiments of the present disclosure, a Rhodococcus louvre product is provided that is produced by the method of the present disclosure.

[0053] In some specific embodiments of the present disclosure, a pharmaceutical composition or medical device is provided that includes a Rhodococcus ruber product produced by a method of the present disclosure.

[0054] In some specific embodiments of the present disclosure, there is provided an isolated Rhodococcus ruber cell wall for the prevention and / or treatment of Lichen planus.

[0055] In some specific embodiments of the present disclosure, a Rhodococcus ruber product for the prevention and / or treatment of Lichen planus is provided.

[0056] In some specific embodiments of the present disclosure, a pharmaceutical composition or medical device for the prevention and / or treatment of lichen planus is provided.

[0057] In some specific embodiments of the present disclosure, there is provided a use of the cell wall of Rhodococcus ruber of the present disclosure in the prevention and / or treatment of Lichen planus, and further provided a use of the cell wall of Rhodococcus ruber of the present disclosure in the manufacture of a pharmaceutical / medical device for the prevention and / or treatment of Lichen planus.

[0058] In some specific embodiments of the present disclosure, there is provided a use of a Rhodococcus ruber product of the present disclosure in the prevention and / or treatment of lichen planus, and further provided is a use of a Rhodococcus ruber product of the present disclosure in the manufacture of a pharmaceutical / medical device for the prevention and / or treatment of lichen planus.

[0059] In some specific embodiments of the present disclosure, there is provided a use of a pharmaceutical composition of the present disclosure in the prevention and / or treatment of lichen planus, and further provided is a use of a pharmaceutical composition of the present disclosure in the manufacture of a medicament / medical device for the prevention and / or treatment of lichen planus.

[0060] In some embodiments of the present disclosure, there is provided a use of any one of the Rhodococcus ruber of the present disclosure, the isolated Rhodococcus ruber cell wall of the present disclosure, the Rhodococcus ruber product of the present disclosure, and the pharmaceutical composition of the present disclosure in the manufacture of a medicine (or medical device).

[0061] In some specific embodiments, said medicament is used for the prevention and / or treatment of lichen planus.

[0062] In some specific embodiments, said medical device (eg, a dressing, patch, bandage, film, etc.) is used to prevent and / or treat Lichen planus.

[0063] In some embodiments of the present disclosure, there is further provided a method of preventing and / or treating lichen planus, comprising contacting a subject with a therapeutically effective amount (or a prophylactically effective amount) of any one selected from the following: - Rhodococcus ruber in the present disclosure, - the cell wall of the isolated Rhodococcus ruber of the present disclosure, - Rhodococcus louvre product in this disclosure, - a pharmaceutical composition according to the present disclosure, - A medical device in the present disclosure.

[0064] In some specific embodiments, the cycle of contact is 2 days to 2 months.Specifically, it can be, for example, 2, 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 days, or, for example, 1, 2, 3, 4, 5 weeks.In a specific embodiment, the subject is used with the active ingredient for 4 weeks.

[0065] In some embodiments, it is used 1 to 3 times a day, 1 to 6 times every 2 days, 1 to 9 times every 3 days, 1 to 14 times a week, or 1 to 60 times a month. In some embodiments, it is used twice a day, or once a day, or once every 2 days.

[0066] The dosage per administration may vary depending on the specific condition of the subject, and is usually 1 μg to 1000 μg / unit dose / 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 μg / unit dose / administration, and any two of the above values ​​in between.

[0067] In some specific embodiments, the contacting is performed by means of, for example, but not limited to, oral ingestion, intravenous injection, intramuscular injection, transdermal injection, intraperitoneal injection, puncture, nasal drops, eye drops, or suppository.

[0068] In some specific embodiments, the subject is a non-human animal, such as a farm animal, a pet animal, a working animal, an ornamental animal, or an industrial animal.

[0069] In a specific embodiment, the subject is a human.

[0070] In some specific embodiments, the subject is one suspected of having, diagnosed with, already having, or susceptible to the disease of interest or a symptom thereof.

[0071] In the context of this application, the only therapeutically (or prophylactically) active ingredient in the medicament or pharmaceutical composition is a product derived from Rhodococcus ruber, in particular a product containing components of Rhodococcus ruber (e.g. proteins, nucleic acids, lipids, cell walls and their components, carbohydrates, metabolites), specifically a product containing the cell wall of Rhodococcus ruber (more preferably the cell wall skeleton of Rhodococcus ruber or its components). [Brief description of the drawings]

[0072] [Figure 1] Colony morphology of Rhodococcus ruber. [Diagram 2] These are the results of 16S rRNA identification. [Diagram 3] It is effective in treating lichen planus. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] "Isolation" refers to removing the Rhodococcus ruber of the present disclosure from its original growth environment.

[0074] It is known to those skilled in the art that the cell walls of gram-positive bacteria and gram-negative bacteria have different structures. Specifically, gram-positive bacteria have thicker cell walls (usually 20 nm to 80 nm) and contain about 90% peptidoglycan and about 10% teichoic acid (a polymer formed of alcohol molecules and phosphate molecules, usually existing in the form of sugar esters or amino acid esters). The peptidoglycan layer is dense and can reach 20 layers in some cases. However, the cell walls of gram-negative bacteria are often thinner than those of gram-positive bacteria, have a complex structure, and are divided into an outer membrane and a peptidoglycan layer (usually 2 nm to 3 nm).

[0075] The peptidoglycan layer is a unique component of bacterial cell walls and is a derivative of heteropolysaccharides. Each peptidoglycan monomer contains three parts: sugar units (i.e., at least two sugar molecules connected by glycosidic bonds to form the framework of peptidoglycan), peptide ends (short peptide chains composed of multiple amino acids connected to N-acetylmuramic acid molecules), and peptide bridges (crosslinking adjacent "peptide ends" to form a high-strength network structure). Different bacteria have different peptide bridges, peptide ends, and crosslinking methods.

[0076] Isolated Rhodococcus ruber cell walls In the present disclosure, the term "isolated cell wall of Rhodococcus ruber" can be interpreted not only as a complete cell wall, but also as an incomplete cell wall (e.g., a crushed or partially decomposed cell wall). Based on the teachings of the present disclosure, a person skilled in the art can understand that the component exhibiting the desired activity is derived from the cell wall of Rhodococcus ruber (e.g., the cell wall itself or its constituent components). Therefore, various forms such as a complete cell wall, a crushed cell wall, an incomplete decomposition product of the cell wall, a constituent component of the cell wall, and an extract of the cell wall can be adopted in clinical applications, and all of these are included in the scope of the present disclosure.

[0077] Cell wall skeleton Although these are constituent components that constitute the main structure of the cell wall, they should not be construed as representing only the substantial cross-linked network within the cell wall, and those skilled in the art will understand that they do not exclude other cell wall components that are adsorbed, bound, or held by the substantial cross-linked network.

[0078] Rhodococcus ruber As used in the embodiments of the present disclosure, Rhodococcus ruber refers to the species Rhodococcus ruber of the genus Rhodococcus, and is not limited to a particular cell line.

[0079] Non-limiting examples include TOY7 strain (Nanjing Agricultural University Agricultural Environmental Microbial Seed Collection Center), CGMCC No. 4795, DSM43338, CCTCC No. 2012035, CGMCC No. 16640, and CGMCC No. 17431.

[0080] Identification of Rhodococcus ruber With known or future microbial identification techniques, a person skilled in the art can make a taxonomic identification of a bacterial strain, for example, the identification techniques that can be adopted include morphological characteristics, physiological and biochemical characteristics, 16S rRNA, etc. As understood by a person skilled in the art, with the development of science and technology, identification techniques involve various means, and in the early stages, morphological identification and biochemical identification methods were mainly used, but these methods are unreliable. Since the advent of sequencing technology, a person skilled in the art can make a more reliable method of identifying a strain. For example, if the DNA sequence of 16S rRNA has a homology of 97% or more, two bacteria are determined to belong to the same species (Hua Xu et al., Progress in the classification and application research of the genus Rhodococcus, Microbiology Communications, 2003:30(4)). For Rhodococcus ruber, a known strain deposited at the International (or National) Culture Collection Center is used as a model strain for comparison.

[0081] Dosage form The medicaments or pharmaceutical compositions or active ingredients or products of the present disclosure may be in the form of, but are not limited to, an ointment, cream, plaster, gel, cleanser, tincture, liniment, oil, paste, lyophilized powder, aerosol, suppository, patch, suspension, oral liquid, lozenge, skin care product (facial cleanser, lotion, serum, emulsion, face cream, face mask).

[0082] Excipients The excipients of the present disclosure include, but are not limited to, dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethylcellulose, erythritol, gelatin, magnesium stearate, propellants, moisturizers, solvents, solubilizers, emulsifiers, antioxidants, pH adjusters, and preservatives.Specific, non-limiting examples include white petrolatum, carbomer, hypromellose, methylcellulose, sodium hydroxymethylcellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerol, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetanol, stearyl alcohol, p-aminobenzoic acid, amidoethyl, isopropanol, Tween, polyoxyethylene hydrogenated castor oil, stearic acid, glyceryl monostearate, monostearin Further included are triglycerol acid, sucrose fatty acid esters, sucrose esters, sucrose acetate isobutyrate, sorbitan tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol esters, sulfites, cysteic acid, dibutylhydroxytoluene, potassium sorbate, phosphate buffer, triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salts.

[0083] Formulation Unit The medicament or pharmaceutical composition or active ingredient or product of the disclosure may be manufactured in the form of a unit dosage form.

[0084] In some embodiments, the unit dose of the medicament (or formulation, or therapeutic agent, or medical device) comprises: -0.001 mg to 500 mg of the Rhodococcus ruber product; or -0.001 mg to 500 mg of the cell wall of said Rhodococcus ruber; or -0.001 mg to 500 mg of the cell wall skeleton of Rhodococcus ruber.

[0085] Specific examples of unit doses are 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg ± 10%, and ranges between any two of the above numbers.

[0086] The terms "using," "giving," "supplying," and "treating," when applied to an animal, human, cell, organ, or biological sample, refer to contacting a medicine or medical device with the animal, human, cell, organ, or biological sample.

[0087] "Treating" refers to administering to a subject a medicine (therapeutic agent, active ingredient or composition) (e.g., a Rhodococcus ruber cell wall or a pharmaceutical composition thereof in the present disclosure) or medical device to be administered orally or topically, thereby alleviating (relieving, delaying, ameliorating, curing) to a clinically measurable extent one or more disease symptoms in the subject (or population) being treated, said subject being one who already has, is suspected of having, or is susceptible to one or more diseases or symptoms thereof.

[0088] The amount of a medicine (therapeutic agent, active ingredient or composition) that effectively relieves the symptoms of any disease is called a therapeutically effective amount. It depends on various factors, such as the state of the disease, age and weight changes of the subject. It should be understood that a medicine (therapeutic agent, active ingredient or composition) may not be effective in alleviating a target disease or its symptoms in a single subject, but that a medicine (therapeutic agent, active ingredient or composition) can be determined to be statistically effective against a target disease or its symptoms by any statistical test method known in the art (Student's t-test, chi-square test, Mann-Whitney U test).

[0089] "In some cases" means that the matters described in the latter sentence may occur, but do not necessarily occur, and need to be set according to the circumstances. For example, "individually packaged" means that it is permissible to package the product individually, but it is not necessary to package it individually, and the individual packaging does not affect the achievement of the technical effect.

[0090] "A", "a single" and "this" include plural forms unless expressly stated otherwise.

[0091] The following examples, preparations and tests are combined to further illustrate the present disclosure, but these examples, preparations and tests are not limited to the scope of the present disclosure. Unless specific conditions are noted, the operations are carried out according to general conditions and conditions suggested by raw material suppliers. Reagents whose specific sources are not noted are common reagents purchased on the market.

[0092] Those skilled in the art will particularly appreciate that although a specific cell line may be used in the following specific examples, the achievement of the technical effect is not limited to this specific cell line, and any species of Rhodococcus ruber may be applicable.

[0093] Working Example Example 1. Preservation of strains There is no way to document where the strain was originally isolated. The inventor deposited the contemporary strain, which had been stored in the laboratory, at the Center for Ordinary Microorganisms, China Microbial Species Conservation and Management Committee, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, on March 22, 2019, under the deposit number CGMCC No. 17431. Test results showed that the deposited strain was viable.

[0094] Example 2. Identification of strains 1. Visual observation of colony morphological characteristics When cultured on glycerol agar medium at 30-37°C (specifically 32-35°C) for 12-72 (specifically 36-60, e.g. 40-50) hours, the following was found (Figure 1): -Colony rises; - Reddish orange color (there may be slight differences depending on the light, the color of the medium, etc.); - The surface is dry and cracked, slightly shiny (there may be slight differences due to different culture conditions); -Crumbles easily when touched; -Colony size is approximately 1mm to 2mm (there may be slight differences depending on the culture conditions).

[0095] 2. Microscopic Observation - The fungus is branched, has diaphragms, and forms a mycelium (there may be slight differences depending on the culture conditions); -The mycelium divides to form short, thick, regular cells (there may be slight differences depending on the culture conditions); -After 4 to 5 days of cultivation, the cells will become short rod-shaped or spherical (there may be slight differences depending on the cultivation conditions).

[0096] 3.Stainability Gram staining is positive.

[0097] 4. Biochemical reactions A glycerol agar slant medium is prepared and cultured at 30 to 37° C. (specifically, 32 to 35° C.) for 12 to 72 hours (specifically, 36 to 60 hours, for example, 40 to 50 hours). Next, the culture is subjected to each of the following tests.

[0098] 4.1 Acidogenesis of carbohydrates: [Table 1] 4.2 Measurement of enzyme activity (API ZYM): [Table 2] 4.3 It shows positive results in nitrate reduction reaction, positive results in catalase, positive results in tyrosinase, negative results in amylase, negative results in oxidase, and negative results in gelatin liquefaction reaction.

[0099] 4.4 Sole Carbon Source: [Table 3] 4.5. 16S rRNA Identification Genome extraction was performed on the 15 strains isolated in the working seed tube and the 10 strains isolated in the original seed tube, and 16Sr RNA was amplified and sequenced. The 16Sr RNA gene homology of the total 25 strains was 100%, which means that the 25 strains belong to the same species (Figure 2).

[0100] In addition, the neighbor-joining phylogenetic tree of the strains constructed by the Kimura two-parameter algorithm indicated that the strains belonged to Rhodococcus ruber.

[0101] Manufacturing Example Production example 1.Culture method 1. Cultivate Rhodococcus ruber using a common microbial production method.

[0102] 2. The culture method may be solid culture or liquid culture.

[0103] 3. There are no particular restrictions on the nutrient sources in the medium, as long as the medium contains carbon sources, nitrogen sources, and other nutrient sources that are commonly used in microbial culture.

[0104] - The carbon source can be any carbon source that can be utilized by Rhodococcus ruber, such as fructose, glucose, etc.

[0105] - Nitrogen sources are bouillon, peptone, ammonium salts, nitrates and other organic or inorganic nitrogen compounds.

[0106] -Several inorganic salts can be appropriately added as other nutrient sources, e.g. NaCl, phosphates.

[0107] 4. There are no strict limitations on the culture conditions (temperature, time, etc.), and those skilled in the art can select the conditions that maximize the production yield based on the data from initial small-scale pilot tests.

[0108] 5. As an example, ferment Rhodococcus ruber under the following culture conditions: (1) The medium composition includes the following: Peptone, beef bouillon, sodium chloride, phosphate, glycerol (and sometimes agar for solid cultures).

[0109] (2) The culture method parameters are as follows: The working strain is regenerated, then transferred to solid culture medium for 3-5 days, and then transferred to liquid for cultivation (30-37℃, maintained for 3-5 days). Semi-continuous batch feeding mode of auxiliary materials can be adopted, and batch feeding mode can also be adopted. During the cultivation period, pH, bacterial density, dissolved oxygen, and carbon source consumption are monitored.

[0110] Manufacturing 2. Disruption of bacteria The bacteria obtained in Preparation Example 1 are collected and the cells are disrupted (for example, by ultrasonic disruption, but not limited thereto). The bacteria can be disrupted by any suitable method known in the art, for example, CN101250490A or CN101323865A.

[0111] Check the crushing condition under a microscope. The number of bacteria that retain their shape in each field of view should not exceed five. It is sufficient to check multiple fields (10-30) and ensure that all of them meet this criterion.

[0112] Production Example 3. Removal of nucleic acids, lipids, contaminating proteins, and cell membranes 1. Removal of nucleic acids: The disruption supernatant is centrifuged, and DNA and RNA enzymes are added to the resulting precipitate to remove nucleic acids according to the procedure suggested by the enzyme supplier.

[0113] 2. Protein Removal: A common protease (eg, trypsinase) is added to the precipitate and deproteinized according to the procedure suggested by the enzyme supplier.

[0114] 3. Lipid Removal: An organic reagent (such as, but not limited to, one or a combination of acetone, ether, and ethanol) is added to the precipitate to remove lipids according to common procedures in the art.

[0115] 4. Removal of cell membranes: TritonX-100 is added to the precipitate, and the precipitate is collected by centrifugation and washed with PBS according to common procedures in the field.

[0116] It should be understood that those skilled in the art can adjust the order of the above steps for removing impurities and make the steps compatible with each other. After removing components other than the cell wall, the precipitate is redissolved in water for injection. In some cases, it can be sterilized at 115°C for 20 to 30 minutes to obtain a stock solution of the cell wall skeleton (mainly containing the cell wall skeleton and its constituent components).

[0117] 5. Yield From 159 coleflasks, 653 ml of bacterial solution (after disruption) was collected, with a wet weight yield of 138 g and a cell wall skeleton yield of approximately 0.87 g / coleflask.

[0118] Production Example 4: Method for producing pharmaceutical composition 1. An excipient (e.g., dextran 40, mannitol, or trehalose) is added to the product obtained in Production Example 3. The mixture is filled into a container to obtain a pharmaceutical composition.

[0119] [Table 4] 2. The product obtained in Manufacturing Example 3 (active ingredient 60 μg to 120 μg) is applied to a dressing material to manufacture it as an external medical device.

[0120] 3. The pharmaceutical composition of item 1 is freeze-dried to obtain a freeze-dried powder (numbered compositions 1 to 7, respectively).

[0121] 4. Quality Inspection (using freeze-dried powder composition 1 as an example) [Table 5] Test Example Test Example 1. Pharmacological test 1. After intravenous injection of 20, 40, and 80 times the human clinical dose into cats, the cats were anesthetized and blood pressure, respiration, heart rate, and electrocardiogram were measured, but no obvious effects were found.

[0122] 2. When mice were intravenously injected with 1,000 times the clinical dose in humans, there was no obvious effect on motor coordination or learning and memory abilities.

[0123] It was found that the pharmaceutical compositions of the present invention (Compositions 1 to 7) had no obvious effect on the mental, nervous, cardiovascular, or respiratory systems of animals.

[0124] Test example 2. Safety test 1. Sterility test: The results were negative and the specimen was proven sterile.

[0125] 2. Acute toxicity test in mice: The test group was administered subcutaneous and intraperitoneal injections at a dose five times that of humans, while the control group was injected with 0.5ml of sterile saline per bottle and observed for 7-8 days. The mice were in good condition, with no abnormalities in their weight or any of their organs.

[0126] 3. Chronic Toxicity Tests: Thirty times the clinical dose was administered into the vagina of dogs once a day for three months, but no toxic effects were observed, and electrocardiograms and blood biochemical indices were within normal ranges. No delayed toxicity was observed even two weeks after administration was terminated (Compositions 1 to 7).

[0127] Test Example 3. Stability test The pharmaceutical composition was left at room temperature (18-25°C) for 0, 1, 2, 3, 8, 14, and 21 months, but there was no statistically significant difference in the alanine content, muramic acid content, phagocytosis rate, and phagocytosis index compared to the start of the test (the test was performed three times).

[0128] From the above, the pharmaceutical compositions in the form of freeze-dried powder formulations can be stably stored for 24 months (Compositions 1 to 7).

[0129] Test Example 4. Phagocytosis test Macrophages are the main cells of the mononuclear phagocyte system, and phagocytes can be activated and significantly enhance phagocytic function when stimulated by antigen. After inducing the production of peritoneal macrophages in mice, the mice are intraperitoneally injected with chicken blood red blood cells, and the mice are killed 30 minutes later to extract the peritoneal fluid, which is stained and counted under a microscope to determine the killing ability of macrophages and indirectly measure the level of non-specific immunity in the body.

[0130] When the test was performed using the composition 1 of the present application, the phagocytosis rate was 75% and the phagocytosis index was 1.05. The phagocytosis rate and phagocytosis index of the negative control (excipient) and the blank control (saline) were both low. This indicates that the cell wall skeleton of the present application has a high immune phagocytosis-promoting ability.

[0131] Effectiveness example (treatment of oral lichen planus) 1. Case diagnosis and inclusion and exclusion criteria 1.1 Evidence for diagnosis Based on the 2003 WHO diagnostic criteria for oral lichen planus (Meij EHVD, Waal IVD. Lack of clinicopathologic correlation in the diagnosis of oral lichen planus based on the presently available diagnostic criteria and suggestions for modifications. Journal of Oral Pathology & Medicine, 2003, 32 (9): 507-512).

[0132] 1.2 Acceptance Criteria 1) Patients diagnosed with erosive oral lichen planus based on medical history, clinical presentation, and pathology; 2) Patients aged 18-75 years at onset and who had not taken any related medications within one month prior to onset; 3) Patients without visual field defects, fundus lesions, or systemic diseases (Liu Qinglan et al., Observation of the therapeutic effect and safety of triamcinolone acetonide oral ointment in treating hyperemic erosive oral lichen planus, Journal of Practical Oral Medicine, 2017, 33(04):536-540).

[0133] 1.3 Exclusion criteria 1) Patients with other definite oral mucosal diseases; 2) Patients with uncontrolled diabetes, tumors, etc. 3) Patients who have used antibiotics within the past month or immunosuppressants within the past 3 months; 4) Patients whose lichenoid reaction may have been caused by any medicine or amalgam inlay; 5) Patients with allergies and those who have had drug or food allergies; 6) Patients who are unable to follow doctor's medication instructions or who have incomplete records of the trial process, which may affect the judgment of the treatment effect (Gorouhi F et al., Randomized trial of pimecrolimus cream versus triamcinolone acetonide paste in the treatment of oral lichen planus, Journal of the American Academy of Dermatology, 2007, 57(5):806-813).

[0134] 1.4 Research Subjects Select patients with erosive oral lichen planus who meet the above diagnostic criteria. A randomized, double-blind, placebo-controlled clinical trial will be used to randomly divide the patients into a test group and a control group. The research plan will be submitted to the ethical committee for approval. All subjects will sign a consent form.

[0135] 1.5 Sample size The effective number of small samples to be collected in this study is at least 60 cases, of which 30 are in the test group and 30 in the control group (considering that the dropout rate is about 20%, it is recommended to collect 75 cases).

[0136] 2. Treatment methods and grouping 2.1 Treatment groups The lyophilized powder of Composition 1 is administered topically to the erosion site of the patient once every other day, one bottle (60μg) at a time, before going to bed, in combination with rinsing with saline for one course of 28 days. Administration method: Before administration, rinsing with saline is performed every time, then the erosion site is scrubbed and cleaned with a cotton swab, and the lyophilized powder is placed on the erosion site of the patient with erosive oral lichen planus for 2 hours.

[0137] 2.2 Control group Placebo (vehicle only) was administered topically to the erosion site, one bottle at a time, once every other day before going to bed, in combination with saline rinsing, for one course of 28 days.

[0138] 3. Evaluation index of treatment effect 3.1 Physical Signs Scoring The REU scoring system (Table 3) is used to score physical signs (Park HK et al., Oral lichen planus: REU scoring system correlates with pain, Oral Surgery Oral Medicine Oral Pathology Oral Radiology, 2012, 114(1):75-82).

[0139] [Table 6] 3.2 Pain symptom scoring For subjective assessment, a visual analogue scale (VAS) was used, divided into 10 levels, and the degree of pain was recorded on a scale of 1 to 10. After taking the medication, patients were asked to record their evaluation on a subject diary card every morning (Table 4).

[0140] [Table 7] 3.3 OHIP-14 scale The OHIP-14 scale is made up of 14 items in 7 parts: oral functional limitations, physiological pain, psychological discomfort, physiological disorders, psychological disorders, social anxiety disorders, and physical disorders. Each item on the scale is divided into 5 levels and the corresponding score is recorded (0: none, 1: a little, 2: sometimes, 3: often, 4: always), and the total is divided into 0 to 56 points, with the lower the score, the better the oral health.

[0141] The study involves patients completing the questionnaire at their first visit and at follow-up visits 1, 2, and 4 weeks after starting the medication (see Sampogna F et al., Comparison of patients' and providers' severity evaluation of oral mucosal conditions. Journal of the American Academy of Dermatology, 2011, 65(1):69-76).

[0142] 3.4 Evaluation criteria for treatment efficacy - Complete response: After treatment, congestion and erosion disappear completely, and the white lines disappear or become faint (physical signs score of 0 or 1).

[0143] Pain disappears completely (symptom score 0).

[0144] - Effective: Less congestion and erosion after treatment (fewer physical signs scored).

[0145] Pain is reduced (symptom scores are reduced).

[0146] - No effect - No change or increase in congestion and erosion after treatment (no change or increase in physical sign score).

[0147] Pain does not decrease or gets worse (symptom scores remain unchanged or increase).

[0148] Total effectiveness rate = (marked effectiveness + effective) × number of cases / total number of cases × 100%.

[0149] See Zhou Gang et al., Evaluation criteria for the treatment effect of oral lichen planus (atrophic type, erosive type), Chinese Journal of Stomatology, 2005, 40(2):92-93.

[0150] 3.5 Safety Assessment Laboratory indicators include blood cell analysis, kidney function tests, and blood glucose measurement.

[0151] 3.6 Timing of evaluation of treatment effect The symptoms and physical signs scores and OHIP-14 scale scores will be observed 1, 2, and 4 weeks after taking the medication, the therapeutic effect will be evaluated 4 weeks after the start of treatment, and the occurrence of adverse events during that period will be observed.

[0152] 3.7 Statistical methods Data processing and analysis were performed using SPSS 24.0 statistical software. Chi-square test was used to compare count data, and rank sum test or one-group t test was used to compare quantitative data groups. The rank sum test or paired t-test is used for self-comparison before and after treatment in the group. If P<0.05, the difference is statistically significant (Huang Yueqin, Clinical Epidemiology, People's Health Press, 2014).

[0153] 4.Results 4.1 General data of the study subject A total of 66 patients were admitted to the study, of which 6 were lost to follow-up (patients were unable to administer medication due to travel work), and 60 completed observation.

[0154] Study group: 30 participants, 9 men and 21 women.

[0155] Control group (placebo): 30 participants, 10 men and 20 women.

[0156] There was no significant difference in the general appearance and oral hygiene status of the patients in the two groups, P>0.05.

[0157] [Table 8]

[0158] 4.2 Efficacy Analysis (1) Comparison of REU physical sign scores between the two groups One, two and four weeks after treatment of the patients in the test and control groups, the differences in the scores of physical signs before and after treatment of the test group were all statistically significant, P<0.05, and the differences in the scores of physical signs 4 weeks after treatment of the two groups were statistically significant, P<0.05 (Table 6).

[0159] [Table 9]

[0160] (2) Comparison of symptom scores between the two groups One, two and four weeks after treatment of the patients in the test and control groups, the differences in symptom scores in the test group were all statistically significant (P<0.05), and the differences in symptom scores between the two groups after treatment were all statistically significant (P<0.05) (Table 7).

[0161] [Table 10]

[0162] (3) Comparison of OHIP-14 scale scores between the two groups One, two, and four weeks after treatment of the patients in the test and control groups, the OHIP-14 scale scores after treatment of the test group were significantly lower and statistically significant (P<0.05), and the differences in the OHIP-14 scale scores after treatment between the two groups were all statistically significant (P<0.05) (Table 8).

[0163] [Table 11]

[0164] (4) Comparison of efficacy rates after treatment between the two groups After 2 and 4 weeks of treatment, the total effective rates of the test group were 70.00% and 86.67%, respectively, and the total effective rates of the control group were 23.33% and 26.67%, respectively.

[0165] 4.3 Safety Analysis Four weeks after taking the medication, there were no statistically significant differences between the test and control groups in blood cell analysis, renal function tests, or blood glucose measurements (P>0.05). No patients experienced pain or allergic reactions when taking the medication, and there was not a single case of adverse reactions.

[0166] 4.4 Follow-up after treatment Furthermore, 12 patients in the study group were followed up 4 weeks after the end of treatment, and only two patients had recurrent erosions. None of the patients experienced pain or allergic reactions after taking the drug, and there were no adverse reactions.

[0167] The study found that the total effective rate of the test group after taking the medication was significantly higher than that of the control group, and the area of ​​erosion, the degree of pain, and the degree of impact on oral health of the test group 1, 2, and 4 weeks after taking the medication were all reduced compared to before treatment. The study results showed that the degree of pain and the impact on oral health of the control group were slightly reduced compared to before treatment, but this may be due to the psychological effect of the placebo.

[0168] Treating erosive lichen planus with cell wall skeleton of Rhodococcus ruber can effectively reduce the area of ​​erosion of OLP and reduce the degree of pain, and shows high clinical therapeutic efficacy after administration. There is no statistical difference in safety index compared with the control group.

Claims

1. A pharmaceutical composition for treating oral lichen planus, comprising: An isolated Rhodococcus ruber cell wall skeleton and a pharma- ceutically acceptable excipient, The isolated Rhodococcus ruber was deposited at the Center for Ordinary Microorganisms of the China Microbial Species Conservation and Management Committee at No. 3, Beichen West Road, Chaoyang District, Beijing on March 22, 2019, with the deposit number CGMCC No. 17431; A pharmaceutical composition comprising the isolated cell wall skeleton of Rhodococcus ruber in an amount of 1 part by weight and the pharma- ceutical acceptable excipient in an amount of 200-300 parts by weight.

2. the isolated Rhodococcus ruber cell wall skeleton is 1 part by weight, and the pharma- ceutically acceptable excipient is 250 parts by weight; The pharmaceutical composition of claim 1 which is a liquid.

3. A pharmaceutical composition for treating oral lichen planus, which is a dry powder formulation or a lyophilized powder formulation of the pharmaceutical composition according to claim 1 or 2.

4. A medical device for treating oral lichen planus, comprising: An isolated Rhodococcus ruber cell wall skeleton and a pharma- ceutically acceptable excipient, The isolated Rhodococcus ruber was deposited at the Center for Ordinary Microorganisms of the China Microbial Species Conservation and Management Committee at No. 3, Beichen West Road, Chaoyang District, Beijing on March 22, 2019, with the deposit number CGMCC No. 17431; A medical device selected from the group consisting of a dressing, a patch, a bandage, and a film.

5. 1. Use of an isolated cell wall skeleton of Rhodococcus ruber in the manufacture of a medicament or medical device for treating oral lichen planus, comprising: The isolated Rhodococcus ruber was deposited at the Center for Ordinary Microorganisms of the China Microbial Species Conservation and Management Committee at No. 3, Hall 1, Beichen West Road, Chaoyang District, Beijing on March 22, 2019, with the deposit number CGMCC No. 17431.

6. The use according to claim 5, wherein the oral lichen planus is one or a combination selected from the group consisting of reticular, annular, linear, papular, erosive, macular, bullous, and atrophic lichen planus.

7. 7. The use according to claim 5 or 6, wherein the medicament is prepared in a dosage form selected from the group consisting of salves, creams, emulsions, suspensions, pastes, gels, washes, tinctures, oils, tablets, aerosols, sprays, liniments, and powders.

8. The use according to claim 5 or 6, wherein the medical device is manufactured in a form selected from the group consisting of a dressing, a patch, a bandage, and a film.

9. The use according to claim 5 or 6, wherein the unit dose of the medicament or medical device contains from 1 μg to 1000 μg of active ingredient.

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