Use of red nocardia cell wall skeleton in the treatment of recurrent aphthous ulcers

Abstract

Use of red nocardia cell wall skeleton in the treatment of recurrent aphthous ulcer. The recurrent aphthous ulcer is selected from any one or combination of the following: mild recurrent aphthous ulcer, herpetic recurrent aphthous ulcer, severe recurrent aphthous ulcer. After administration, the ulcer can be observed to disappear, the hyperemia can be observed to disappear, and the pain symptoms can be observed to disappear.

Description

[0001] This application claims priority to patent application filed on January 9, 2019 (application number 2019100177396), which is cited and herein. Technical Field

[0002] This application relates to the use of the Nocardia rubra cell wall skeleton in the preparation of a medicament for treating recurrent aphthous ulcers. Background Technology

[0003] Nocardia rubra is a type of Nocardia. The cell wall skeleton of Nocardia rubra (hereinafter referred to as Nr-CWS or N-CWS) can be obtained by fermentation, cell disruption, and protease degradation of the cells.

[0004] Nocardia bacteria are pleomorphic, appearing as spherical, rod-shaped, or filamentous forms. The bacteria are non-motile; some strains are weakly acid-resistant and obligate aerobes. On ordinary agar plates, colonies appear after 3 days of incubation; after 7 to 10 days, the colonies rise and form aerial hyphae with a velvety surface. Colonies of different strains can be yellow, orange, or red. The G+C content in their DNA is 60-72%. Most Nocardia bacteria are saprophytic and exist in soil.

[0005] In the prior art, the Nocardia rubrum cell wall scaffold can be commercially available, such as a product manufactured by Liaoning Grestech Biopharmaceutical Co., Ltd. (trade name "Nakejia"), or provided by Fujian Shanhe Pharmaceutical Co., Ltd., Fujian Guang Shengtang Pharmaceutical Co., Ltd., Fujian Institute of Microbiology, etc. The Nocardia rubrum cell wall scaffold has been used to treat cervical erosion, cervical precancerous lesions (CN101073583A), anti-human papillomavirus (CN1935262A), skin lesions (CN101209267A), skin diseases (eczema, neurodermatitis, nonspecific dermatitis, atopic dermatitis, psoriasis) (CN108938674A), acne (CN108295095A), fungal infections, herpes simplex, and herpes zoster (CN1879661A).

[0006] Recurrent aphthous ulcers are small, painful ulcers that occur on the oral mucosa and are characterized by periodicity, recurrence, and self-limitation. Recurrent aphthous ulcers mostly occur on the lips, cheeks, and tongue margins. Modern medicine believes that the ulcers will heal spontaneously within 7 to 10 days. The problem is that many patients experience recurring episodes. The etiology and pathogenesis of recurrent aphthous ulcers remain unclear. Precipitating factors may include local trauma, mental stress, food, medication, changes in hormone levels, and deficiencies in vitamins or trace elements. Systemic diseases, genetics, immunity, and microorganisms may play important roles in the occurrence and development of recurrent aphthous ulcers (Natah S et al., Recurrent aphthous ulcers today: a review of the growing knowledge. Int J Oral Max Surg, 2004, 33(3):221.; Li Yuanyuan, Chinese Journal of Experimental Traditional Medical Formulae, Vol.17, No.11, 2011).

[0007] The diagnosis of recurrent aphthous ulcers can be referenced in the "Guidelines for the Diagnosis and Treatment of Recurrent Aphthous Ulcers" formulated by the Oral Mucosal Diseases Committee of the Chinese Stomatological Association. Recurrent aphthous ulcers mainly include:

[0008] 1. Mild recurrent aphthous ulcers

[0009] The lesion is sensitive or presents as a pinhead-sized or slightly larger area of ​​congestion; within a short period, it forms a small, shallow ulcer, approximately 2 to 4 mm in diameter, round or oval, with well-defined borders. The center is slightly depressed, and the surface is covered with a pale yellow pseudomembrane; the surrounding mucosa is congested, giving it a reddish halo. There are usually 2 to 3 ulcers. After ulceration, there is a severe burning pain. The ulcer can gradually heal spontaneously within 7 to 10 days without leaving a scar. However, it may recur after a period of varying length.

[0010] 2. Herpetic recurrent aphthous ulcers

[0011] Also known as stomatitis-type aphthous ulcers. Except for the small size and large number of ulcers (up to 20 to 30), the phenotype is similar to that of mild recurrent aphthous ulcers. The ulcers are scattered and widely distributed, with significant mucosal congestion. Severe pain is present, accompanied by headache, fever, or local lymphadenopathy.

[0012] 3. Severe recurrent aphthous ulcers

[0013] Also known as recurrent necrotizing periglandular stomatitis or periglandular aphthous ulcers, this is the most severe type. Ulcers usually occur singly, rarely two or more. They commonly occur on the inner lip and corners of the mouth. Initially, the ulcers resemble mild recurrent aphthous ulcers, but their diameter gradually increases to 1-2 cm and extends deeper into the mucosal glands. The ulcers are purplish-red or dark red, with irregular, lobulated edges and a central depression. The base is uneven, slightly hard, and nodular, surrounded by a red halo. Severe local pain and possibly accompanied by local lymphadenopathy and fever are common. The course of the disease often lasts for several months or more. Scarring is left after healing, and in severe cases, tissue defects may occur.

[0014] Currently, the conventional treatment methods include:

[0015] -Local treatment:

[0016] Use mouthwashes or lozenges with antibacterial and anti-inflammatory effects, apply dressings or analgesics to the ulcer; other methods include local cauterization, local injection, or laser therapy, the main purpose of which is to reduce inflammation, relieve pain, and promote ulcer healing.

[0017] -Systemic treatment:

[0018] According to traditional Chinese medicine theory, systemic treatment has some efficacy, but it is slow to take effect and ineffective in treating relapses. Alternatively, immunosuppressants (such as thalidomide) (Muriel Hello et al. Use of Thalidomide for Severe Recurrent Aphthous Stomatitis: A Multicenter Cohort Analysis Medicine. Volume 89, 2010, 176-182), prednisone, dexamethasone, etc., can be used, but these have significant side effects and a slow onset of action. Immunomodulators can also be used. Because the etiology and pathogenesis are still unclear, systemic treatment is not ideal, and relapses are common after recovery.

[0019] Therefore, there is still a need in the art to provide an effective treatment for recurrent aphthous ulcers. Summary of the Invention

[0020] According to some embodiments of this application, the use of the Nocardia rubra cell wall skeleton in the preparation of a medicament for treating recurrent aphthous ulcers or their recurrence is provided.

[0021] According to some embodiments of this application, the use of the Nocardia rubrum cell wall skeleton in the preparation of a medical device for treating recurrent aphthous ulcers or their recurrence is provided. The medical device is an adjuvant, bandage, membrane, or patch.

[0022] In some implementations, recurrent aphthous ulcers are selected from any one or a combination of the following: mild recurrent aphthous ulcers, herpetic recurrent aphthous ulcers, and severe recurrent aphthous ulcers.

[0023] In some implementations, drugs or medical devices are administered via mucosal contact.

[0024] In some embodiments, the drug or medical device comprises a pharmaceutically acceptable carrier. Any suitable carrier known to those skilled in the art can be used to implement the technical solutions of this application.

[0025] In some embodiments, the drug is prepared in a dosage form selected from the following: ointment, cream, emulsion, suspension, paste, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, powder; wherein the ointment is selected from: ointment, plaster, cream.

[0026] According to some implementation schemes, a method for treating recurrent aphthous ulcers is provided, comprising the steps of: providing a subject with a therapeutically effective amount of Nocardia rubrum cell wall skeleton.

[0027] In some specific implementations, drugs (or medical devices) are applied to the lesion depending on the size and depth of the ulcer. For example, but not limited to, applying a drug containing the cell wall skeleton of Nocardia rubrum, covering the lesion with a patch impregnated with the cell wall skeleton of Nocardia rubrum, applying lyophilized powder containing the cell wall skeleton of Nocardia rubrum directly to the ulcer surface, or applying an ointment containing the cell wall skeleton of Nocardia rubrum to the ulcer surface, etc.

[0028] In some implementations, the drug is a lozenge. For example, it is prepared by directly compressing lyophilized powder at room temperature.

[0029] In some embodiments, the drug comprises:

[0030] - Nocardia redis cell wall skeleton, and

[0031] - A pharmaceutically acceptable carrier.

[0032] In some embodiments, the pharmaceutically acceptable carrier is selected from, but not limited to, fillers, stabilizers (e.g., trehalose, glycine), flavoring agents (e.g., xylitol), disintegrants (e.g., sodium carboxymethyl cellulose), binders (e.g., gelatin), and lubricants (e.g., magnesium stearate).

[0033] In some embodiments, the stabilizer is selected from one or a combination of the following: glycine, lysine, arginine, hydroxyethyl starch, hydroxymethyl starch, trehalose, and dextran.

[0034] In some embodiments, the flavoring agent is selected from one or a combination of the following: sucrose, monosaccharides, sodium saccharin, aspartame, sorbitol, xylitol, and mannitol.

[0035] In some embodiments, the adhesive is selected from one or a combination of the following: sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, and gelatin.

[0036] In some implementations, the lubricant is selected from one or a combination of the following: talc, magnesium stearate, and micronized silica gel.

[0037] In some embodiments, the carriers suitable for this application include, but are not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythritol, gelatin, magnesium stearate, propellants, humectants, solvents, solubilizers, emulsifiers, antioxidants, pH adjusters, and preservatives. Specifically, non-limiting examples also include: white petrolatum, carbomer, hydroxypropyl methylcellulose, methylcellulose, sodium carboxymethyl cellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluorocarbons, glycerin, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, acetamide, isopropanol, Tween, polyoxyethyl hydrogenated castor oil, stearic acid, glyceryl monostearate, and triglycerides. Oil monostearate, fatty acid sucrose esters, sucrose esters, sucrose isobutyrate esters, sorbitan tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol esters, sulfites, cysteine, di-tert-butylhydroxytoluene, potassium sorbate, phosphate buffer, triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salt.

[0038] In some implementation schemes, the pharmaceutically acceptable carrier is dextran.

[0039] In some implementation schemes, the medication is administered twice a day, once a day, or once every two days. The dosage for each administration varies depending on the size and depth of the patient's ulcer, typically ranging from 1 μg to 1000 μg per unit dose per administration. Specifically, this includes, 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 per unit dose per administration, and any range between any two of the aforementioned values.

[0040] In some implementations, the application lasts from 2 days to 2 months, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks or longer, and any range between any two of the aforementioned values.

[0041] In some embodiments, the Nocardia rubrum cell wall skeleton is a commercially available Nocardia rubrum cell wall skeleton.

[0042] In other embodiments, the Nocardia rubrum cell wall skeleton is obtained by means of or consisting of the following steps:

[0043] 1) Provides Nocardia rubes red;

[0044] 2) The red Nocardia bacteria were pulverized to obtain a pulverized product;

[0045] 3.1) Optionally, the pulverized product is subjected to a lipid removal process;

[0046] 3.2) Optionally, the pulverized product may be subjected to a nucleic acid removal operation;

[0047] 3.3) Optionally, the pulverized product is subjected to a protein removal process;

[0048] 3.4) Obtain products derived from the cell walls of Nocardia rubrum;

[0049] 4) Optionally, the product derived from the cell wall of Nocardia rubrum is freeze-dried;

[0050] 5) Optional repackaging;

[0051] in,

[0052] Steps 3.1), 3.2), and 3.3) can be interchanged or performed in parallel.

[0053] Steps 4) and 5) can be interchanged;

[0054] The average particle size of the pulverized material is 10 nm to 1000 nm, preferably 10 nm to 800 nm, and more preferably 10 nm to 500 nm.

[0055] Preferably, the dispensing refers to dispensing into containers;

[0056] The container is selected from: bottles, tubes, bags, bladders, ampoules, injection devices, aluminum foil packaging, dressings, capsules, and membranes.

[0057] The purpose of pulverizing Nocardia rubrum is to remove intracellular substances; therefore, techniques such as ultrasonic disruption and lysozyme extraction can be employed. Those skilled in the art understand that any known or future methods suitable for disrupting Gram-positive bacteria are applicable to the technical solutions disclosed herein.

[0058] Technicians are able to adjust the specific parameters and equipment for culturing, breaking down, separating, collecting, removing impurities, and dispensing based on the subsequent application of the active ingredient (cell wall and its components) (e.g., topical application), so as to avoid introducing factors that may affect subsequent applications during the preparation process.

[0059] In some embodiments, organic solvents are used to remove lipids from the fragmented products. In some embodiments, nucleases are used to remove DNA and RNA from the fragmented products. In some embodiments, hydrolases are used to degrade proteins in the fragmented products. In some embodiments, surfactants are used to remove cell membranes from the fragmented products.

[0060] In some implementations, the average particle size of the pulverized material is from 10 nm to 1000 nm; values ​​of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 nm ± 10 nm, and any range between any two of these values, may be mentioned. There are many methods for particle size measurement (Hu Songqing et al., Modern Particle Size Measurement Technology, Modern Chemical Industry, 2002, 22:1).

[0061] In some specific implementations, the average particle size of the pulverized material is between 10 nm and 800 nm.

[0062] In some other specific implementations, the average particle size of the pulverized material is between 10 nm and 500 nm.

[0063] In the specific implementation plan, the term "dispensing" refers to dispensing into bottles / ampoules. Before use, a solvent (such as sterile water) is added to the bottle / ampoule.

[0064] In some specific implementations, the subjects are animals other than humans, such as farm animals, pets, working animals, ornamental animals, and production animals.

[0065] In the specific implementation plan, the subjects are human beings.

[0066] In some specific implementation plans, subjects are suspected of having, diagnosed with, already have, or are susceptible to the target disease or its symptoms.

[0067] In the context of this application, the sole therapeutic (or preventative) active ingredient in a medicament or medical device is a product derived from Nocardia rubrum, particularly a product containing components of Nocardia rubrum (such as proteins, nucleic acids, lipids, cell walls and their components, carbohydrates, metabolites), specifically a product containing the cell wall of Nocardia rubrum (more preferably the Nocardia rubrum skeleton or its components). Detailed Implementation

[0068] Nocardia rubrum cell wall

[0069] In this disclosure, "Nocardia rubrum cell wall" can be understood as both an intact cell wall and an incomplete cell wall (e.g., a broken or partially degraded cell wall). Under the teachings of this disclosure, those skilled in the art will understand that the component exhibiting the desired activity derives from the cell wall of Nocardia rubrum (e.g., the cell wall itself or a component thereof). Therefore, in clinical applications, various forms are permitted, including intact cell walls, broken cell walls, incompletely degraded cell wall products, components of the cell wall, and extracts of the cell wall, all of which are included within the scope of this disclosure.

[0070] Cell wall skeleton

[0071] The components that make up the main structure of the cell wall; however, it should not be understood as merely referring to the cross-linked network entities within the cell wall. Those skilled in the art understand that it does not exclude other cell wall components that are adsorbed, bound, or carried on the cross-linked network entities.

[0072] unit dose

[0073] The drug or medical device disclosed herein can be prepared in the form of a unit dose (or unit formulation).

[0074] "Optional" means that the event described thereafter may occur, but is not required; it depends on the circumstances. For example, "optionally, repackaging" means that repackaging of the product is permitted, but not mandatory; whether or not repackaging is performed does not affect the achievement of the technical effect.

[0075] "One," "one," "single," and "that," unless otherwise specified, also include the plural form.

[0076] The present disclosure is further described below with reference to embodiments. However, these embodiments are not intended to limit the scope of the present disclosure. Unless otherwise specified, operations are performed under conventional conditions and as recommended by the raw material supplier. Reagents not specifically named are commercially available, conventional reagents.

[0077] Technicians especially understand that although the specific examples below use specific commercially available cell wall products, the technical effects are not limited to those specific commercially available products and are applicable to any species classified as Nocardia rubrum.

[0078] Example 1. Commercially available Nocardia rubescens cell wall skeleton

[0079] Nocardia rubrum cell wall skeleton (trade name: Nacojia) was purchased from Liaoning Grestech Biopharmaceutical Co., Ltd., with approval number S20030009 (2ml / ampoule; lyophilized powder), which contains 60μg of active ingredient and 15mg of dextran 40.

[0080] Example 2. Preparation of the cell wall skeleton of Nocardia rubrum

[0081] 1. Culture and collect the bacterial cells according to known methods. Disrupt the cells (e.g., but not limited to sonication). Disruption of the bacterial cells may also be performed using any suitable known method in the art, such as CN101250490A or CN101323865A. Examine the disruption under a microscope; no more than 5 formed bacteria should be present in each field of view. It is considered acceptable if this standard is met in several fields (10 to 30).

[0082] 2. Nucleic acid removal: Centrifuge the supernatant and add DNase and RNase to the precipitate. Remove nucleic acid according to the enzyme supplier's instructions.

[0083] 3. Protein removal: Add a common protease (such as trypsin) to the precipitate and remove the protein according to the enzyme supplier's instructions.

[0084] 4. Lipid removal: Add an organic reagent (such as, but not limited to, one or a combination of acetone, ether, and ethanol) to the precipitate and remove the lipids according to conventional procedures in the art.

[0085] 5. Cell membrane removal: Add Triton X-100 to the precipitate, centrifuge to collect the precipitate according to standard procedures in the art, and wash with PBS.

[0086] It should be understood that the order of the above-mentioned impurity removal steps can be adjusted by the technician to ensure compatibility between the steps. After removing non-cell wall components, the precipitate is reconstituted in water for injection and set aside for later use. Optionally, it can be sterilized at 115°C for 20-30 minutes as the stock solution for the cell wall skeleton (mainly containing the cell wall skeleton and its components).

[0087] Example 3. Preparation of the formulation

[0088] 1. The product obtained in Example 2 (60 μg to 120 μg of active ingredient, such as 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg) or the commercially available product of Example 1 is coated onto a dressing (e.g., sterile gauze) to prepare a topical medical device.

[0089] 2. Alternatively, the product obtained in Example 2 (60 μg of active ingredient) can be prepared into a lyophilized powder and applied directly to the ulcer surface.

[0090] 3. Alternatively, methods for preparing oral patches known in the art (such as those disclosed in CN201610605617.5, CN201510614414.8, CN200610200450.0, CN201610511974.5, CN201610471977.0, etc.) may be used, for example:

[0091] The film-forming materials polyvinyl alcohol, carbomer, and hydroxypropyl cellulose are added to water and swelled to form a homogeneous viscous liquid. The active ingredient of this application is added to the liquid and mixed well. The mixture is allowed to stand to defoam. The resulting bubble-free viscous liquid is poured onto a mold coated with a small amount of paraffin wax, dried for 5-20 minutes, removed, and cut into the required area.

[0092] Test case: Treatment efficacy of recurrent aphthous ulcers

[0093] Table 1. Patient Demographic Information

[0094]

[0095]

[0096] Table 2. First follow-up visit

[0097]

[0098] Table 3. Second follow-up visit

[0099]

[0100] Table 4. Third follow-up visit

[0101]

[0102] Experimental group: Continued administration at the same frequency for two weeks, then discontinued. Follow-up for three weeks after discontinuation showed no recurrence.

[0103] In the control group (three patients, with no significant difference in demographics from the experimental group), the same administration regimen was used for the drug-grade carrier, and no therapeutic benefit was observed in the ulcers during follow-up (data not shown here).

Claims

1. Use of red Nocardia cell wall skeleton of State Food and Drug Administration (SFDA) No. S20030009 in the preparation of a medicament, wherein the medicament is used for treating or preventing recurrence of severe recurrent aphthous ulcer.

2. Use of red Nocardia cell wall skeleton of State Food and Drug Administration (SFDA) No. S20030009 in the preparation of a medical device, wherein the medical device is used for treating or preventing recurrence of severe recurrent aphthous ulcer, and the medical device is a dressing, a bandage, a film, or a patch.

3. The use according to claim 1, wherein the medicament is prepared into a dosage form selected from the group consisting of an ointment, a cream, an emulsion, a suspension, a paste, a gel, a lotion, a tincture, an oil, a tablet, an aerosol, a spray, a liniment, and a powder; and the ointment is selected from the group consisting of an ointment, a plaster, and a cream.

4. The use according to claim 1 or 2, wherein a unit dose of the medicament or the medical device comprises 1 μg to 1000 μg of the red Nocardia cell wall skeleton. wherein 5. The use according to claim 4, wherein a unit dose of the medicament or the medical device comprises 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, or 200 μg of the red Nocardia cell wall skeleton. ​ ​

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