Product derived from <i>Rhodococcus ruber</i>, and pharmaceutical use thereof

Abstract

Isolated Rhodococcus ruber, a product derived from the Rhodococcus ruber (in particular, a product derived from the Rhodococcus ruber cell wall), a method for preparing the product derived from the Rhodococcus ruber, and use of the product derived from the Rhodococcus ruber in prevention and/or treatment of lichen planus. The product derived from the Rhodococcus ruber effectively reduce the erosion area of lichen planus and relieve pain.

Claims

1. A method for preventing and/or treating erosive oral lichen planus in a subject in need thereof, comprising topically administering to the subject a therapeutically effective amount of Rhodococcus ruber cell wall skeleton.

2. The method of claim 1, wherein the Rhodococcus ruber cell wall skeleton is derived from the isolated Rhodococcus ruber deposited with CGMCC with Accession No. 17431.

3. The method of claim 1, wherein the therapeutically effective amount is in a unit dose of 1 ?g to 1000 ?g.

4. The method of claim 1, wherein a therapeutically effective amount of Rhodococcus ruber cell wall skeleton is administered at a frequency selected from the group consisting of: 1 to 3 times per day, 1 to 6 times in two days, 1 to 9 times in three days, 1 to 14 times per week, and 1 to 60 times per month.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: The colony morphology of Rhodococcus ruber.

(2) FIG. 2: The identification results of 16S rRNA.

(3) FIG. 3: The therapeutic effect of lichen planus.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

(4) Isolation refers to the separation of the Rhodococcus ruber of the present disclosure from its original growth environment.

(5) The skilled person knows that the cell wall structures of gram-positive bacteria and gram-negative bacteria are different. Specifically, the cell wall of gram-positive bacteria is thicker (usually 20 nm to 80 nm), comprising about 90% peptidoglycan and about 10% teichoic acid (a polymer formed by alcohol and phosphoric acid molecules, usually existing in the form of sugar ester or amino acid ester). The peptidoglycan layer is dense, even as many as 20 layers. However, the cell wall of gram-negative bacteria is much thinner than that of gram-positive bacteria, and the structure is more complex, divided into outer membrane and peptidoglycan layer (usually 2 nm to 3 nm).

(6) The peptidoglycan layer is a unique component of the bacterial cell wall and is a derivative of heteropolysaccharide. Each peptidoglycan monomer comprises 3 parts: the sugar unit (for example, at least two sugar molecules are connected by glycosidic bonds to form the framework of peptidoglycan), the peptide tail (a short peptide chain formed by linking several amino acids, which is connected to a N-acetylmuramic acid molecule), and the peptide bridge (crosslinking adjacent peptide tails to form a high-strength network structure). Different bacteria have different peptide bridges, peptide tails and cross-linking manners.

(7) Isolated Rhodococcus ruber Cell Wall Skeleton

(8) In the present disclosure, isolated Rhodococcus ruber cell wall can be interpreted as either a complete cell wall or an incomplete cell wall (for example, disrupted or partially degraded). Under the teaching of the present disclosure, the skilled person will understand that the components exhibiting the desired activity are derived from Rhodococcus ruber cell wall (for example, the cell wall itself or components thereof). Therefore, various forms are allowed to be used in clinical applications, including complete cell walls, disrupted cell walls, incomplete degradation products of cell walls, cell wall components, cell wall extracts, etc., which are all included in the scope of the present disclosure.

(9) Cell Wall Skeleton

(10) A component that constitute the main structure of the cell wall; however, it cannot be interpreted as merely representing the cross-linked network-like entity in the cell wall, and the skilled person understands that other cell wall components adsorbed on, bound to and carried by the cross-linked network-like entity are not excluded.

(11) Rhodococcus ruber

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

(13) Non-limiting examples include the TOY7 strain (Agricultural Environment Microbiological Culture Collection, Nanjing Agricultural University), CGMCC No. 4795, DSM43338, CCTCC No. 2012035, CGMCC No. 16640 and CGMCC No. 17431.

(14) Identification of Rhodococcus ruber

(15) According to known or future microbial identification techniques, the skilled person can perform taxonomic identification on a strain of bacteria. For example, the available identification techniques include morphology, physiological and biochemical characteristics, 16S rRNA, and the like. The skilled person understands that with the development of science and technology, identification techniques involve different methods. In the earlier period, morphological and biochemical identification methods were mainly used, but the reliability of these methods is not high. After the advent of sequencing technology, the skilled person can identify bacteria strains in a more reliable way. For example, when the DNA sequences of 16S rRNA are identified as having more than 97% (inclusive) of identity, two bacteria would be deemed as belonging to the same species (Hua Gougen et al., The taxonomy and application of Rhodococcus, Microbiology China, 2003: 30 (4)). In terms of Rhodococcus ruber, the known strains deposited in international (or national) collection authorities of strains are used as model strains, and the strains to be identified are compared with the model strains.

(16) Dosage Form

(17) The medicament or pharmaceutical composition or active ingredient or product of the present disclosure can be formulated into, but not limited to, the following forms: ointment, cream, plaster, gel, lotion, tincture, liniment, oil, paste, lyophilized powder, aerosol, suppository, patch, suspension, oral solution, buccal tablet and skin care product (cleanser, toning lotion, serum, lotion, cream and mask).

(18) Excipient

(19) An excipient suitable for the present disclosure is for example but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythritol, gelatin, magnesium stearate, propellant, humectant, solvent, solubilizer, emulsifier, antioxidant, pH regulator and preservative. Specifically, non-limiting examples also include: albolene, carbomer, hydroxypropyl methylcellulose, methyl cellulose, sodium hydroxymethyl cellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerin, propylene glycol, deionized water, water for injection, distilled water, ethanol, hexadecanol, octadecanol, p-aminobenzoic acid, acetamide, isopropanol, Tween, polyoxyethyl hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid ester, sucrose ester, sucrose acetate isobutyrate, sorbitan 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, nipagins, thimerosal, chlorocresol, trichlorobutanol, benzoic acid and sodium salt thereof.

(20) Formulation Unit

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

(22) 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 Rhodococcus ruber cell wall; or 0.001 mg to 500 mg of the Rhodococcus ruber cell wall skeleton.

(23) Particular examples of the unit dose are 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 mg?10%, and the ranges between any two of the above values.

(24) Administrating, administrated, providing . . . to and treating, when applied to animals, humans, cells, tissues, organs or biological samples, refer to the contact of the medicament or medical device with the animals, humans, cells, tissues, organs or biological samples.

(25) Treatment means administrating an internal or external medicament (therapeutic agent, active ingredient or composition) (such as the Rhodococcus ruber cell wall or pharmaceutical composition thereof according to the present disclosure) or a medical device to a subject, in order to alleviate (relieve, delay, improve, cure) one or more disease symptoms to a clinically measurable degree in the subject to be treated (or population thereof), wherein the subject has, is suspected of having, or is susceptible to one or more diseases or symptoms thereof.

(26) The amount of the medicament (therapeutic agent, active ingredient or composition) that can effectively alleviate any disease symptoms is called the therapeutically effective amount. It can vary depending on a variety of factors, such as the disease state, age and body weight of the subject. It should be understood that the medicament (therapeutic agent, active ingredient or composition) may be ineffective in alleviating the target disease or symptoms thereof of a single subject, but is statistically effective for the target disease or symptoms thereof according to any statistical test method known in the art (such as Student's t test, chi-square test and U test according to Mann and Whitney).

(27) The term optionally means, the event that follows this term can happen, but not necessarily happen; it depends on the situation. For example, optionally, performing aliquoting means that the product is allowed to be aliquoted, but it is not necessarily to be aliquoted; whether the product is aliquoted or not does not affect the realization of the technical effects.

(28) A/an, one, single and the, if not explicitly stated, also involve the plural forms.

(29) The present disclosure is further described below with reference to the examples, preparation examples and test examples. However, these examples, preparation examples and test examples do not limit the scope of the present disclosure. When the particular conditions are not specified, operation should be performed in accordance with the normal conditions and the conditions recommended by the raw material supplier. The reagents without giving particular sources are conventional reagents purchased on the market.

(30) The skilled person especially understands that although a specific cell line is used in the following particular examples, the realization of the technical effects does not depend on the specific cell line; any species belonging to the Rhodococcus genus, Rhodococcus ruber species is applicable.

EXAMPLES

Example 1. Deposition of the Strain

(31) The original place for the isolation of the strain cannot be verified. The inventors deposited the laboratory-preserved seed strain at China General Microbiological Culture Collection Center (CGMCC, Yard No. 1(3) West Beichen Road, Chaoyang District, Beijing) on Mar. 22, 2019, under deposit number CGMCC No. 17431. Test showed that the deposited strain was viable.

Example 2. Identification of the Strain

(32) 1. Visual Observation of the Morphological Characteristics of the Colony

(33) The strain was cultured on a glycerol agar medium at 30? C. to 37? C. (specifically 32? C. to 35? C.) for 12 to 72 (specifically 36 to 60, such as 40 to 50) hours, and the following was observed (FIG. 1): the colonies plumped up; were orange-red in color (slightly different depending on influences of light, the color of the culture medium, etc.); the surface was dry and wrinkled, slightly shiny (slightly different depending on differences in culture conditions); were fragile to touch; the colony size was about 1 mm to 2 mm (slightly different depending on differences in culture conditions).
2. Microscope Observation The hyphae grew in a branching structure with septate, and formed mycelium (slightly different depending on differences in culture conditions); Division of the hyphae formed regular short and thick cells (slightly different depending on differences in culture conditions); After culturing for 4 to 5 days, the hyphae became short rod-shaped or spherical (slightly different depending on differences in culture conditions).
3. Staining Property

(34) The strain was gram stain positive.

(35) 4. Biochemical Reactions

(36) The strain was cultured on a glycerol agar slant medium at 30? C. to 37? C. (specifically 32? C. to 35? C.) for 12 to 72 (specifically 36 to 60, such as 40 to 50) hours. Then, the following tests were performed on the culture.

(37) 4.1 Acid Production from Carbohydrates:

(38) Positive: glycerin, mannitol, sorbitol, D-arabitol, D-fructose and D-glucose; Negative: inositol, inulin, lactose, sucrose, starch, maltose, glycogen, xylitol, gluconate, trehalose, erythritol, melezitose, melibiose, raffinose, cellobiose, amygdalin, gentiobiose, adonol, arbutin, D-arabinose, L-arabinose, ?-methyl-D-glucoside, ?-methyl-D-mannoside, D-ribose, D-xylose, L-xylose, N-acetyl-glucosamine, D-turbiose, D-lyxose, ?-methyl-D-xyloside, D-galactose, D-tagatose, D-fucose, L-fucose, D-mannose, L-sorbose, L-arabinitol, L-rhamnose and 2-keto-gluconate.
4.2 Enzyme Activity Determination (API ZYM): Positive: alkaline phosphatase, lipid esterase (C8), lipase (C14), leucine araminase, valine araminase, cystine araminase, trypsin, chymotrypsin, acid phosphatase, naphthol-AS-B1-phosphohydrolase and ?-glucosidase; Negative: N-acetyl-glucosaminidase, esterase (C4), ?-galactosidase, ?-uronidase, ?-glucosidase, ?-galactosidase, ?-mannosidase and ?-fucosidase.

(39) 4.3 Nitrate Reduction Reaction: Positive, Catalase: Positive, Tyrosinase: Positive, Amylase: Negative, Oxidase: Negative, Gelatin Liquefaction: Negative.

(40) 4.4 Sole Carbon Source:

(41) Biolog Gen II Positive for: glucuronamide, ?-hydroxy-DL butyric acid, growth experiment: D-fructose-6-phosphate, ?-D-glucose, D-fructose, D-mannitol, D-arabitol, D-sorbitol, quinic acid, ?-aminobutyric acid, citric acid, L-malic acid, bromosuccinic acid, Tween 40, propionic acid and acetic acid; Biolog Gen III Sensitive to: dimethylamine tetracycline, sodium tetradecyl chemical sensitivity sulfate, rifamycin SV, pH 5.0, 8% sodium chloride, lincomycin, experiment fusidic acid, D-serine, vancomycin, tetrazolium violet and tetrazolium blue; Tolerate to: sodium bromate, 1% sodium lactate, pH 6.0, 1%-4% sodium chloride, nalidixic acid, lithium chloride, potassium tellurite, aztreonam and sodium butyrate.
4.5. 16S rRNA Identification

(42) The 15 strains isolated from the working seed tube and the 10 different strains isolated from the original seed tube were subjected to genome extraction, 16S rRNA amplification and sequencing. The 16S rRNA gene identity of the 25 strains in total was 100%. This means that the 25 strains are of the same species (FIG. 2).

(43) Further, the neighbor-joining strain phylogenetic tree constructed based on the Kimura2-parameter algorithm showed that the strain was classified as Rhodococcus Tuber.

PREPARATION EXAMPLES

Preparation Example 1. Culture Methods

(44) 1. Rhodococcus ruber can be cultured by conventional microbial production methods.

(45) 2. The culture method can be solid culture or liquid culture.

(46) 3. There are no special requirements on the nutrient sources in the culture medium. The culture medium can contain carbon sources, nitrogen sources and other nutrient sources that are commonly used for microbial culture. The carbon source can be any carbon source that can be consumed by Rhodococcus ruber, for example, fructose, glucose, and the like. The nitrogen source can be broth, peptone, ammonium salt, nitrate and other organic or inorganic nitrogen-containing compounds. For other nutrient sources, some inorganic salts can be added appropriately, for example, NaCl and phosphates.

(47) 4. There are no strict limitations on the culture conditions (temperature, time, etc.). The skilled person can choose the conditions that maximize the yield based on the preliminary small-scale pilot test data.

(48) 5. As an example, the following culture conditions was used to ferment Rhodococcus ruber:

(49) (1) The Culture Medium Composition Comprising:

(50) peptone, broth, sodium chloride, phosphate, glycerin (and, optionally agar, when in solid culture).
(2) Parameters of the Culture Method:

(51) After the working strain was recovered, it was transferred to a solid culture medium for 3 to 5 days, and then transferred to liquid culture (30 to 37? C., maintained for 3 to 5 days). The fed-batch semi-continuous mode or the batch mode can be used. The pH, bacterial density, dissolved oxygen and carbon source consumption were monitored during culture.

Preparation Example 2. Bacteria Disruption

(52) The bacteria obtained in Preparation Example 1 were collected and the cells were disrupted (for example, but not limited to sonication). Any appropriate well-known method in the art were also allowed to disrupt the bacteria, such as CN101250490A or CN101323865A.

(53) The disruption state was checked under a microscope. There should not be more than 5 visible bacteria in each visual field. The disruption was considered as qualified when several (10 to 30) visual fields checked met this standard.

Preparation Example 3. Removal of Nucleic Acids, Lipids, Proteins and Cell Membranes

(54) 1. Removal of Nucleic Acids:

(55) The supernatant after disruption was centrifuged. DNase and RNase were added to the obtained precipitate, and nucleic acids were removed according to the operation recommended by the supplier of the enzymes.

(56) 2. Removal of Proteins:

(57) Commonly used protease (such as trypsin) was added to the precipitate, and proteins were removed according to the operation recommended by the supplier of the enzyme.

(58) 3. Removal of Lipids:

(59) Organic reagents (for example, but not limited to, any one of acetone, ether and ethanol or combination thereof) were added to the precipitate, and lipids were removed according to conventional operations in the art.

(60) 4. Removal of Cell Membranes:

(61) Triton X-100 was added to the precipitate, and the precipitate was collected by centrifugation according to conventional operations in the art, and rinsed with PBS.

(62) It should be understood that among the above steps for removing impurities; the skilled person can adjust their orders of steps to make them compatible with each other. After removing the non-cell wall components, the precipitate was reconstituted in water for injection, and then set aside till use. Optionally, it could be sterilized at 115? C. for 20 to 30 minutes as the stock solution of the cell wall skeleton (mainly containing the cell wall skeleton and components thereof).

(63) 5. Yield

(64) A total of 653 mL of bacterial liquid (after disruption) was collected from 159 Kolle flasks. The wet weight yield was 138 g; the cell wall skeleton yield was about 0.87 g/Kolle flask.

Preparation Example 4. Preparation Methods of the Pharmaceutical Compositions

(65) 1. Excipients (such as dextran 40, mannitol or trehalose) were added to the product obtained in Preparation Example 3. The resulting product was referred to the pharmaceutical composition, after filling into aliquots.

(66) TABLE-US-00001 TABLE 1 The pharmaceutical composition can be formulated in various forms Composition Capacity of each vial Components Composition 1 2 mL Active ingredient 60 ?g Dextran 40 15 mg Composition 2 2 mL Active ingredient 60 ?g Dextran 40 12 mg Composition 3 2 mL Active ingredient 120 ?g Dextran 40 36 mg Composition 4 2 mL Active ingredient 60 ?g Trehalose 12 mg Composition 5 2 mL Active ingredient 120 ?g Trehalose 36 mg Composition 6 2 mL Active ingredient 120 ?g Mannitol 36 mg Composition 7 2 mL Active ingredient 60 ?g Mannitol 12 mg

(67) 2. The product obtained in Preparation Example 3 (active ingredient 60 ?g to 120 ?g) was coated on the dressing to prepare a medical device for external use.

(68) 3. The pharmaceutical compositions in step 1 were lyophilized to prepare lyophilized powders (numbered as compositions 1 to 7, respectively).

(69) 4. Quality inspection (lyophilized powder composition 1 was taken as an example).

(70) TABLE-US-00002 TABLE 2 Quality inspection items Appearance White unconsolidated solid or powder Water content ?6% Solubility the product was deemed as being qualified, if it dissolved within 1 minute when 2.0 mL of NaCl injection was added Identification of saccharide The solution was blue-green in color Content of muramic acid 2.0 ?g/vial (criteria: ?1.0 ?g/vial) Residual amount of proteins 0.4 ?g/vial (criteria: ?9.0 ?g/vial) Residual amount of RNA 0.8% (criteria: not more than 5%) Residual amount of DNA 0.9% (criteria: not more than 5%) Residual amount of Triton X-100 Undetectable (criteria: not more than 5%) Residual amount of lipids 3.8% (criteria: not more than 5%) Phagocytosis rate 75% (criteria: ?40%) Phagocytic index 1.05 (criteria: ?0.50) Abnormal toxicity in mice All the mice should survive and have no abnormal reactions during the observation period. The composition was considered as qualified if the body weight of each mouse increased at the due date. Abnormal toxicity in guinea pigs All the guinea pigs should survive and have no abnormal reactions during the observation period. The composition was considered as qualified if the body weight of each guinea pig increased at the due date.

TEST EXAMPLES

Test Example 1. Pharmacological Tests

(71) 1. After intravenous injection (equivalent to 20, 40 and 80 times of human clinical dose), the blood pressure, respiration, heart rate and electrocardiogram of the anesthetized cats were monitored, and the compositions had no significant effect;

(72) 2. After intravenous injection (equivalent to 1000 times of the human clinical dose), the compositions had no significant effect on the coordinated movement and learning and memory function of mice.

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

Test Example 2. Safety Tests

(74) 1. Sterility Test:

(75) The results were negative, proving the sterility of the compositions.

(76) 2. Acute Toxicity Test in Mice:

(77) The experimental group was administrated by subcutaneous injection and intraperitoneal injection, and the dose was 5 times human dose. The control group was treated with sterile saline 0.5 mL/vial. The animals were continuously observed for 7 to 8 days. The mice were in good condition and had no abnormality in their body weight or their organs.

(78) 3. Long-Term Toxicity Test:

(79) The compositions were vaginally administrated for once per day for three months, at a dose which is equivalent to 30 times of the clinical dose. No toxic effects were observed in dogs; the electrocardiograms and blood biochemical indicators were within the normal range. Two weeks after cessation of administration, no delayed toxicity was observed (composition 1 to composition 7).

Test Example 3. Stability Tests

(80) The pharmaceutical compositions were placed at room temperature (18 to 25? C.) for 0, 1, 2, 3, 8, 14 and 21 months, and the alanine content, muramic acid content, phagocytosis rate and phagocytic index had no statistically significant difference compared to those at the start of the test (three batches were tested).

(81) In summary, the pharmaceutical composition of the lyophilized powder formulation can be stored stably for 24 months (composition 1 to composition 7).

Test Example 4. Phagocytosis Tests

(82) Macrophages are the main cells of the mononuclear phagocyte system. The activation of phagocytes after antigen stimulation can significantly enhance the phagocytic function. After inducing the production of peritoneal macrophages in the mice, the mice were intraperitoneally injected with chicken red blood cells. After 30 minutes, the mice were sacrificed and the peritoneal fluid was collected for staining. The percentage of phagocytes that phagocytized red blood cells was counted under a microscope to determine their killing ability, indirectly indicating the non-specific immunity level of the body.

(83) Composition 1 of the present application was used for testing. Its phagocytosis rate was 75% and phagocytic index was 1.05. While, the phagocytosis rate and phagocytic index of the negative control (excipient) and blank control (saline) were all relatively low. These results show that the cell wall skeleton of the present application has a strong ability to promote immunophagocytosis.

(84) Effect Example (Treatment of Oral Lichen Planus)

(85) 1. Criteria for Diagnosis, Inclusion and Exclusion of Cases

(86) 1.1 Criteria for Diagnosis

(87) Diagnosis was performed according to WHO 2003 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).

(88) 1.2 Criteria for Inclusion

(89) {circle around (1)} Patients diagnosed as erosive oral lichen planus based on medical history, clinical manifestations and pathology; {circle around (2)} the age of onset is 18-75 years old, and relevant treatment drugs have not been taken within 1 month before the onset; {circle around (3)} patients without visual field defects, fundus lesions and systemic diseases (Liu Qinglan et al., The effects and safety of triamcinolone acetonide oral ointment in the treatment of erosive oral lichen planus. Journal of Practical Stomatology, 2017, 33(04): 536-540).
1.3 Criteria for Exclusion {circle around (1)} patients with other confirmed oral mucosal diseases; {circle around (2)} patients with uncontrolled diabetes, tumors, etc.; {circle around (3)} patients who have been administrated with antibiotics within 1 month or immune formulations within 3 months; {circle around (4)} patients who may have lichen-like reactions induced by some drugs or amalgam fillings; {circle around (5)} patients with allergic constitution and a history of allergies to drugs and food; {circle around (6)} patients who are unable to follow the doctor's prescription or have incomplete records of the test process (which affect determination of the therapeutic 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).
1.4 Subjects to be Studied

(90) Patients with erosive oral lichen planus that met the above diagnostic criteria were selected. A randomized, double-blind, placebo-controlled clinical trial method was adopted, and the trial group and the control group were divided according to the random numbering. The research protocol was submitted to and approved by the ethics committee. All test subjects signed informed consent forms.

(91) 1.5 Sample Size

(92) The effective cases for small sample size collected in this test should not be less than 60, including 30 cases in the experimental group and 30 cases in the control group (considering the dropout rate was about 20%, it was recommended to collect 75 cases).

(93) 2. Treatment Methods and Groups

(94) 2.1 Treatment Group

(95) The lyophilized powder of composition 1 was topically applied to the erosion surface of the patient once every other day, 1 vial (60 ?g) each time, before going to bed, and combined with saline mouthwash for 28 days. Method of application: before each administration, the patient's mouth was rinsed by saline, then the erosion surface was wiped clean with a cotton swab, and the lyophilized powder was applied onto the erosion surface of the patient with erosive oral lichen planus for 2 hours.

(96) 2.2 Control Group

(97) The placebo (excipient only) was topically applied to the erosion surface of the patient once every other day, 1 vial each time, before going to bed, and combined with saline mouthwash for 28 days. Method of application: the same as above.

(98) 3. Efficacy Evaluation Indicators

(99) 3.1 Scoring of Signs

(100) REU scoring system was applied for scoring of signs (Table 3) (Park H K et al., Oral lichen planus: REU scoring system correlates with pain, Oral Surgery Oral Medicine Oral Pathology Oral Radiology, 2012, 114(1): 75-82).

(101) TABLE-US-00003 TABLE 3 REU scoring system of oral lichen planus Clinical classification Score Reticular/hyperkeratotic (R) 0 for no white stripes; 1 for with white stripes or keratotic plaques; Erosive/erythematous (E) 0 for no lesion; 1 for lesion < 100 mm.sup.2; 2 for lesion of 100 to 300 mm.sup.2; 3 for lesion > 300 mm.sup.2 Ulcerative (U) 0 for no lesion; 1 for lesion < 100 mm.sup.2; 2 for lesion of 100 to 300 mm.sup.2; 3 for lesion >300 mm.sup.2 Total score (?) ? = ?R + ?(E ? 1.5) + ?(U ? 2.0)
3.2 Scoring of Pain Symptoms

(102) The subjective indicators were based on visual analogue scale (VAS), dividing into 10 grades. The degree of pain was recorded as 1 to 10 points in a subject's diary card, and was evaluated by patient in the morning after administration every day (Table 4).

(103) TABLE-US-00004 TABLE 4 VAS scale for patients with oral lichen planus Degree of pain VAS score Score of symptoms No pain 0 0 Mild pain 1 to 3 1 Moderate pain 4 to 6 2 Severe pain 7 to 10 3
3.3 OHIP-14 Scale

(104) The OHIP-14 scale consists of 14 items in 7 parts (namely, restriction of oral function, physical pain, psychological discomfort, physical disorder, psychological disorder, social disorder and disability). Each item in the scale is divided into 5 levels and has a corresponding score (0 for never, 1 for rarely, 2 for sometimes, 3 for often, and 4 for very often). The total score is 0 to 56 points. The lower the value is, the better the oral health condition.

(105) In this trial, the scale was filled by the patient during the first visit and the follow-ups in the 1.sup.st, 2.sup.nd and 4.sup.th weeks after administration (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).

(106) 3.4 Criteria for Efficacy Evaluation

(107) TABLE-US-00005 Significantly The congestion and erosion disappeared completely after effective treatment, and the white stripes were absent or slight (score of signs was 0 or 1 point); the pain disappeared completely (score of symptoms was 0 point); Effective The congestion and erosion were reduced after treatment (score of signs decreased); the pain was alleviated (score of symptoms decreased); Invalid The congestion and erosion were unchanged or increased after treatment (score of signs unchanged or increased); the pain was not alleviated or increased after treatment (score of symptoms unchanged or increased);
Total efficacy rate=(significantly effective+effective)?number of cases/total number of cases?100%.

(108) See Zhou Gang et al., Oral lichen planus (atrophic, erosive) efficacy evaluation criteria, Chinese Journal of Stomatology, 2005, 40(2): 92-93.

(109) 3.5 Safety Evaluation

(110) Laboratory indicators: Blood cell analysis, liver and kidney function and blood sugar detection.

(111) 3.6 Time Period of Efficacy Evaluation

(112) The score of symptoms and score of signs after 1, 2, and 4 weeks of administration and the OHIP-14 scale score were observed to evaluate the efficacy after 4 weeks of treatment, and whether any side effect occurred during the observation period.

(113) 3.7 Statistical Methods

(114) SPSS 24.0 statistical software was used for data processing and analysis. The chi-square test was used for the comparison of enumeration data. The rank sum test or the group t test was used for the comparison of measurement data between groups. The rank sum test or paired t test was used for self-comparison before and after treatment within the group. The difference was deemed as statistically significant when P<0.05 (Huang Yueqin, Clinical Epidemiology, People's Medical Publishing House, 2014).

(115) 4. Results

(116) 4.1 General Information of the Subjects to be Studied

(117) A total of 66 patients were enrolled in this trial, of which 6 were lost to follow-up (patients were unable to apply the medicament due to business trips), and 60 completed the observation. Experimental group: 30 subjects, 9 males and 21 females; Control group (placebo): 30 subjects, 10 males and 20 females.

(118) There was no significant difference in the general condition and oral hygiene status between the two groups of patients, P>0.05.

(119) TABLE-US-00006 TABLE 5 Baseline characteristics of the population studied in the experimental group and the control group Item Treatment group Control group P Age 48.9 ? 9.60 51.23 ? 8.42 0.834 Female % 21 (70.00%) 20 (66.67%) 0.903 Course of disease 6.26 ? 4.96 5.93 ? 5.22 0.462 VAS score 2.11 ? 0.67 1.73 ? 0.56 0.238 OHIP score 18.99 ? 9.43 17.82 ? 10.24 0.434 REU score 3.08 ? 0.74 2.41 ? 0.23 0.154 The data is represented as x ? s (n = 2).
4.2 Effectiveness Analysis
(1) Comparison of REU Scores of Signs Between the Two Groups

(120) After 1, 2 and 4 weeks of treatment for patients in the experimental group and the control group, the differences in the scores of signs between the experimental group before and after treatment were statistically significant (P<0.05); the differences in the scores of signs between the two groups after 4 weeks of treatment were statistically significant (P<0.05) (Table 6).

(121) TABLE-US-00007 TABLE 6 Comparison of REU scores of signs between the two groups Item Week 0 Week 1 Week 2 Week 4 P value Experimental group 3.08 ? 0.74 2.37 ? 1.23 1.92 ? 1.13 1.48 ? 0.96 0.000* Control group 2.41 ? 0.23 2.36 ? 0.65 2.31 ? 0.59 2.26 ? 1.19 0.063* P 0.154.sup.+ 0.821.sup.+ 0.182.sup.+ 0.001.sup.+ *Comparison within the group, before and after treatment; .sup.+Comparison between groups after treatment; the data is represented as x ? s (n = 2).
(2) Comparison of Scores of Symptoms Between the Two Groups

(122) After 1, 2 and 4 weeks of treatment for patients in the experimental group and the control group, the differences in the scores of symptoms within the experimental group were statistically significant (P<0.05), and the differences in the scores of symptoms between the two groups after treatment were statistically significant (P<0.05) (Table 7).

(123) TABLE-US-00008 TABLE 7 Comparison of VAS scores between the two groups Item Week 0 Week 1 Week 2 Week 4 P value Experimental group 2.11 ? 0.67 1.21 ? 0.76 0.70 ? 0.54 0.32 ? 0.63 0.000* Control group 1.73 ? 0.56 1.67 ? 0.76 1.60 ? 0.93 1.56 ? 0.73 0.041* P 0.238.sup.+ 0.004.sup.+ 0.000.sup.+ 0.000.sup.+ *Comparison within the group, before and after treatment; .sup.+Comparison between groups after treatment; the data is represented as x ? s (n = 2).
(3) Comparison of OHIP-14 Scale Scores Between the Two Groups

(124) After 1, 2 and 4 weeks of treatment for patients in the experimental group and the control group, the OHIP-14 scale scores of the experimental group significantly decreased after treatment and were statistically significant (P<0.05), and the differences in the OHIP-14 scale scores between the two groups after treatment were statistically significant (P<0.05) (Table 8).

(125) TABLE-US-00009 TABLE 8 Comparison of OHIP-14 scale scores between the two groups Item Week 0 Week 1 Week 2 Week 4 P value Experimental group 18.99 ? 9.43 10.61 ? 4.97 8.32 ? 6.89 5.21 ? 3.99 0.000* Control group 17.82 ? 10.24 16.33 ? 10.22 15.55 ? 12.71 14.56 ? 11.28 0.001* P 0.434.sup.+ 0.013.sup.+ 0.000.sup.+ 0.000.sup.+ *Comparison within the group, before and after treatment; .sup.+Comparison between groups after treatment; the data is represented as x ? s (n = 2).
(4) Comparison of the Efficacy Rates Between the Two Groups after Treatment

(126) After 2 weeks of treatment and 4 weeks of treatment, the total efficacy rates of the experimental group were 70.00% and 86.67%, respectively, and the total efficacy rates of the control group were 23.33% and 26.67%, respectively.

(127) 4.3 Safety Analysis

(128) After 4 weeks of administration, there were no statistically significant differences between the experimental group and the control group in blood cell analysis, liver and kidney function, and blood glucose examination (P>0.05). No irritation or allergic reactions occurred in patients during administration, and no side effects occurred.

(129) 4.4 Follow-Up after Cessation of Administration

(130) 12 patients in the experimental group were followed up for 4 weeks after cessation of administration, and it was found that only 2 patients had recurrence of erosions. No irritation or allergic reactions occurred in patients after administration, and no side effects occurred.

(131) The study found that the total efficacy rates of the experimental group after administration were significantly higher than those of the control group. The erosion area, degree of pain and degree of oral health impact of the experimental group were all reduced after 1, 2 and 4 weeks of treatment. In the results of the study, the degree of pain and degree of oral health impact of the control group were slightly reduced than before treatment, which may be due to the psychological effect of placebo.

(132) Rhodococcus ruber cell wall skeleton used in the treatment of erosive lichen planus can effectively reduce the erosion area of OLP, reduce the degree of pain and show relatively favorable clinical effects after administration. There was no statistical difference between its safety indicators and those of the control group.