CN111683670A - Use of nocardia rubra cell wall skeleton in treatment of recurrent aphthous ulcers - Google Patents

Abstract

Use of a nocardia rubra cell wall scaffold in the treatment of recurrent aphthous ulcers. The recurrent aphthous ulcer is selected from any one or a combination of: minor recurrent aphthous ulcer, herpes-type recurrent aphthous ulcer, and major recurrent aphthous ulcer. After the medicine is taken, the ulcer can be healed and disappeared, the congestion can be disappeared, and the pain symptom can be disappeared.

Description

Use of nocardia rubra cell wall skeleton in treatment of recurrent aphthous ulcers

This application claims priority from a patent application filed on 09.01.2019 (application No. 2019100177396), incorporated herein by reference.

Technical Field

The application relates to the use of a nocardia rubra cell wall skeleton for the preparation of a medicament for the treatment of recurrent aphthous ulcers.

Background

Nocardia rubra (Nocardia rubra) is one of Nocardia. The red nocardia thallus can be fermented, broken and degraded by protease to obtain the red nocardia cell wall skeleton (hereinafter referred to as Nr-CWS or N-CWS).

The Nocardia is in a polymorphic form, and has a spherical shape, a rod shape or a filiform shape. The thallus has no motility, and some strains have weak acid resistance and are obligately aerobic. On a common agar plate, colonies can be seen after 3 days of culture, and the colonies are raised after 7 to 10 days to form aerial hyphae with villous surface. Colonies of different strains have yellow, orange, or red color. The molar content of G + C in the DNA is between 60 and 72%. Most nocardia are saprophytic bacteria, which are present in soil.

In the prior art, the nocardia rubra cell wall skeleton is commercially available, for example, from the Liaoning Grace Shite biopharmaceutical Co., Ltd ("Nakejia" trade name), or Fujian province, Shanhe pharmaceutical industry Co., Ltd, Fujian Guangsheng pharmaceutical industry Co., Ltd, Fujian province microorganism research institute, etc. Nocardia rubra cell wall skeleton has been used for the treatment of cervical erosion, precancerous lesions of cervical cancer (CN101073583A), anti-human papilloma virus (CN1935262A), skin lesions (CN101209267A), skin lesions (eczema, neurodermatitis, nonspecific dermatitis, atopic dermatitis, psoriasis) (CN108938674A), acne (CN108295095A), fungal infections, herpes simplex, herpes zoster (CN 1879661A).

Recurrent aphthous ulcers (Recurrent aphthous ulcers) are ulcers that cause small and painful oral mucosa, and have the characteristics of periodicity, recurrence, self-limitation and the like. Recurrent aphthous ulcers occur mostly on the lips, cheeks, and tongue margin. Modern medicine believes that ulcers heal spontaneously within 7 to 10 days. The problem is that the condition of many patients is often recurrent. The etiology and pathogenesis of recurrent aphthous ulcers remain unclear. The causes may be local trauma, mental stress, changes in food, medication, hormone levels, and vitamin or trace element deficiencies. Systemic diseases, genetics, immunity and microorganisms may play an important role in the development and progression of Recurrent aphthous ulcers (Natah S et al: secure of the growing knowledgebase. int J Oral Max Surg, 2004, 33(3): 221.; Yuyuan, J.Experimental Proc., Vol.17, No.11, 2011).

The diagnosis of recurrent aphthous ulcer can refer to the diagnosis and treatment guideline for recurrent aphthous ulcer prepared by the professional Committee of oral mucosa disease of the Chinese oral medical society, and the recurrent aphthous ulcer mainly comprises:

1. mild recurrent aphthous ulcer

Sensitive lesion or hyperemia area with needle-point-like size or slightly larger; in a short period, a circular or elliptical ulcer with a diameter of about 2 to 4mm is formed, and a shallow small ulcer with a clear boundary is formed. The center is slightly sunken, and a layer of faint yellow false film is covered on the surface of the center; the mucous membrane around the ulcer is red-halo-shaped. The number of ulcers is typically around 2 to 3. After the ulcer forms, there is a more severe burning pain. The ulcer can be self-healed gradually in about 7 to 10 days without scar. But after intermittent periods of different lengths, the disease can recur.

2. Recurrent aphthous ulcer of herpes type

Also known as aphtha due to stomatitis. The remaining phenotypes were similar to those of mild recurrent aphthous ulcers, except for small and numerous ulcers (up to 20 to 30). The ulcer is scattered and distributed widely, and the mucous membrane is obvious in congestion. There is severe pain, and headache, fever, or swelling of local lymph nodes.

3. Severe recurrent aphthous ulcer

Also known as recurrent necrotizing periglandular inflammation of the mucosa or periglandular aphtha, is the most severe type. Ulcers often occur singly, rarely in 2 or more than 2. It is mostly found on the mucous membrane of the inner side of the lip and the corner area of the mouth. At the beginning, the ulcer resembles a mild recurrent aphthous ulcer, but it gradually enlarges to 1 to 2cm in diameter and progresses deeper into the mucosal glands. The ulcer is purple red or dark red, and has irregular edge, lobe-like bulge, and depressed center. Uneven, slightly hard, nodular, reddish-brown ulcer, severe local pain, swelling of local lymph nodes, fever, etc. The course of disease is often more than several months. Scar remains after healing, and severe cases can form tissue defects.

The current conventional treatment methods are:

-local treatment:

applying gargle or buccal tablet with antibacterial and antiinflammatory effects, or applying adjuvant or analgesic on ulcer wound surface; in addition, there are local cauterization, local sealing or laser treatment, which mainly aims at diminishing inflammation, relieving pain and promoting ulcer healing.

-systemic treatment:

according to the traditional Chinese medicine theory, the whole body treatment has certain curative effect, but the effect is slow, and the relapse treatment is ineffective; or immunosuppressant (such as Thalidomide) (mud Hello et al Use of novel for Severe recovery Aphthous Stomatosis: A Multicenter Cohort Analysis medicine volume 89, 2010, 176-182), prednisone, dexamethasone and the like are adopted, but the side effect is large and the effect is slow; or treatment with an immunomodulator. Because the etiology and pathogenic mechanism are not clear, the treatment effect of the whole body is not ideal enough, and the disease is easy to recur after healing.

In view of the above, there remains a need in the art to provide a drug effective in treating recurrent aphthous ulcers.

Disclosure of Invention

According to some embodiments of the present application, there is provided a use of a nocardia rubra cell wall scaffold in the manufacture of a medicament for treating a recurrent aphthous ulcer or a recurrence thereof.

According to some embodiments of the present application, there is provided a use of a nocardia rubra cell wall scaffold in the preparation of a medical device for treating or recurrence of a recurrent aphthous ulcer. The medical device is an accessory, bandage, film, or patch.

In some embodiments, the recurrent aphthous ulcer is selected from any one or combination of: minor recurrent aphthous ulcer, herpes-type recurrent aphthous ulcer, and major recurrent aphthous ulcer.

In some embodiments, the drug or medical device is administered by mucosal contact.

In some embodiments, the pharmaceutical or medical device comprises a pharmaceutically acceptable carrier. Any suitable vector known to those skilled in the art may be used in the practice of the present embodiments.

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

According to some embodiments, there is provided a method of treating recurrent aphthous ulcers, comprising the steps of: providing a therapeutically effective amount of nocardia rubra cell wall skeleton to a subject.

In some specific embodiments, the drug (or medical device) is administered to the lesion for differences in the area and depth of the ulcer. For example, but not limited to, smearing with a drug containing nocardia rubra cell wall skeleton, covering a lesion with a patch impregnated with nocardia rubra cell wall skeleton, directly applying lyophilized powder containing nocardia rubra cell wall skeleton to an ulcer surface, applying a paste containing nocardia rubra cell wall skeleton to an ulcer surface, and the like.

In some embodiments, the medicament is a buccal tablet. For example, it is prepared by direct compression of lyophilized powder at room temperature.

In some embodiments, the medicament comprises:

-nocardia rubra cell wall skeleton, and

-a pharmaceutically acceptable carrier.

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), disintegrating agents (e.g., sodium carboxymethylcellulose), binders (e.g., gelatin), lubricants (e.g., magnesium stearate).

In some embodiments, the stabilizing agent is selected from one or a combination of: glycine, lysine, arginine, hydroxyethyl starch, hydroxymethyl starch, trehalose, and dextran.

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

In some embodiments, the binder is selected from one or a combination of: sodium carboxymethylcellulose, hypromellose, and gelatin.

In some embodiments, the lubricant is selected from one or a combination of: comprises talcum powder, magnesium stearate and superfine silica powder.

In some embodiments, vectors suitable for use in the present application, such as, but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethylcellulose, erythritol, gelatin, magnesium stearate, a propellant, a humectant, a solvent, a solubilizer, an emulsifier, an antioxidant, a pH regulator and a preservative. Specifically, non-limiting examples also include: white petrolatum, carbomer, hypromellose, methylcellulose, sodium carboxymethylcellulose, chitosan, sucralfate chitosan, polyvinylpyrrolidone, polyvinyl alcohol, sodium hyaluronate, dimethyl ether, tetrafluoroethane, hydrofluoroalkane, glycerol, propylene glycol, deionized water, water for injection, distilled water, ethanol, cetyl alcohol, stearyl alcohol, p-aminobenzoic acid, acetamide, isopropyl alcohol, tween, polyoxyethylene hydrogenated castor oil, stearic acid, glyceryl monostearate, triglycerol monostearate, sucrose fatty acid ester, sucrose acetate isobutyrate, sucrose anhydride tristearate, isopropyl myristate, cholesterol, squalene, squalane, n-butanol, ethylene glycol, ethanol, propylene glycol, polyglycerol ester, sulfite, cysteine, di-tert-butyl hydroxytoluene, potassium sorbate, phosphate buffer solution, Triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salt.

In some embodiments, the pharmaceutically acceptable carrier is dextran.

In some embodiments, twice a day, or once in two days; the dosage for each administration will vary depending on the area and depth of the ulcer in the patient and will generally be from 1 μ g to 1000 μ g per unit dose per application. Specifically, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μ g per unit dose per time, and ranges between any two of the foregoing values.

In some embodiments, administration is for 2 days to 2 months, e.g., 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 ranges between any two of the foregoing.

In some embodiments, the nocardia rubra cell wall skeleton is a commercially available nocardia rubra cell wall skeleton.

In other embodiments, the nocardia rubra cell wall skeleton is obtained by a method comprising or consisting of the steps of:

1) providing nocardia rubra;

2) crushing the nocardia rubra to obtain a crushed product;

3.1) optionally, subjecting said comminuted product to an operation of lipid removal;

3.2) optionally, subjecting the comminuted product to a nucleic acid removal operation;

3.3) optionally, subjecting said comminuted product to a protein removal operation;

3.4) obtaining a product derived from the cell wall of the red nocardia;

4) optionally, freeze-drying the product derived from the nocardia rubra cell wall;

5) optionally, subpackaging;

wherein the content of the first and second substances,

steps 3.1), 3.2), 3.3) can be interchanged in sequence or in parallel,

step 4) and step 5) can be interchanged in sequence;

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

preferably, the dispensing means into containers;

the container is selected from: bottles, tubes, bags, pouches, plates, ampoules, injection devices, aluminum film packaging, dressings, pouches, films.

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

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

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

In some embodiments, the average particle size of the pulverization is from 10nm to 1000 nm; mention may be made of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190nm ± 10nm, and ranges between any two of the foregoing values. There are many methods for measuring particle size (kusnezoff et al, modern particle size measurement techniques, modern chemical, 22: 1 2002).

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

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

In particular embodiments, the dispensing means into vials/ampoules. Just prior to use, a solvent (e.g., sterile water) is added to the vial/ampoule.

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

In a specific embodiment, the subject is a human.

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

In the context of the present application, the only therapeutically (or prophylactically) active ingredient in a medicament or medical device is a product derived from nocardia rubra, in particular a product comprising nocardia rubra constituents, such as proteins, nucleic acids, lipids, cell walls and constituents thereof, carbohydrates, metabolites, in particular a product comprising nocardia rubra cell walls, more preferably nocardia rubra skeleton or constituents thereof.

Detailed Description

Nocardia rubra cell wall

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

Cell wall skeleton

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

Unit dose

The pharmaceutical or medical device of the present disclosure may be prepared in the form of a unit dose (or unit formulation).

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

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

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

The skilled person understands in particular that, although the following specific examples employ a specific commercially available cell wall product, the achievement of the technical effect is not limited to this specific commercially available product, any species classified as belonging to nocardia rubra being suitable.

Example 1 commercially available Nocardia rubra cell wall skeleton

Nocardia rubra cell wall skeleton (trade name: Nakejia) was purchased from Liaoning Gersted biopharmaceutical GmbH, approved Wen national drug Standard S20030009(2 ml/ampoule; lyophilized powder) containing 60. mu.g of active ingredient and 15mg of dextran 40.

Example 2 preparation of cell wall skeleton of Nocardia rubra

1. The cells were cultured according to a known method and collected. The cells are crushed (e.g., without limitation, by sonication). It is also permissible to disrupt the bacterial cells by any suitable method known in the art, for example, CN101250490A or CN 101323865A. The crushed condition is checked under a microscope, the number of the visible bacteria in each visual field is not more than 5, and the standard is met in a plurality of visual fields (10 to 30) to be qualified.

2. Removing nucleic acid: the disrupted supernatant was centrifuged, and DNase and RNase were added to the obtained precipitate to remove nucleic acids according to the procedures recommended by the enzyme supplier.

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

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

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

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

EXAMPLE 3 preparation of the formulations

1. The product obtained in example 2 (active ingredient 60. mu.g to 120. mu.g, for example, 60. mu.g, 70. mu.g, 80. mu.g, 90. mu.g, 100. mu.g, 110. mu.g, 120. mu.g) or the commercial product of example 1 was coated on a dressing (for example, sterile gauze) to prepare an external medical device.

2. Alternatively, the product obtained in example 2 (60 μ g of active ingredient) is made into lyophilized powder, which is directly applied to the ulcer surface.

3. Alternatively, a method for producing an oral patch known in the art (for example, a method disclosed in CN201610605617.5, CN201510614414.8, CN200610200450.0, CN201610511974.5, CN201610471977.0, and the like) may be used, for example:

adding film-forming materials such as polyvinyl alcohol, carbomer and hydroxypropyl cellulose into water, and swelling to form homogeneous viscous liquid; adding the active ingredient of the present application thereto and mixing well; standing for defoaming; mixing the formed bubble-free viscous liquid; casting on a mold coated with a small amount of paraffin, drying for 5-20min, taking out, stripping, and cutting into required area.

Test example treatment of recurrent aphthous ulcer

TABLE 1 patient demographic information

TABLE 2 first double-visit

TABLE 3 second double diagnosis

TABLE 4. third consultation

Experimental groups: administration was continued at the same frequency for two weeks and then discontinued. After stopping taking the medicine, the patient is followed for three weeks without relapse.

In the control patient group (three cases, with no significant difference in demographic information from the experimental group), the treatment benefit of the ulcer surface was not shown by follow-up with the same administration regimen with the pharmaceutically acceptable carrier (data not shown here).

Claims (10)

1. Use of a nocardia rubra cell wall scaffold in the manufacture of a medicament for treating or preventing the recurrence of a recurrent aphthous ulcer.
2. Use of a nocardia rubra cell wall scaffold in the manufacture of a medical device for treating or preventing the recurrence of a recurrent aphthous ulcer, wherein the medical device is an adjuvant, bandage, film, or patch.
3. The use of claim 1 or 2, wherein the recurrent aphthous ulcer is selected from any one or a combination of: minor recurrent aphthous ulcer, herpes-type recurrent aphthous ulcer, and major recurrent aphthous ulcer.
4. The use according to claim 1, wherein the medicament is prepared in a dosage form selected from the group consisting of: ointment, cream, lotion, suspension, paste, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, powder;

   wherein the paste is selected from: ointment, plaster, cream.
5. The use of claim 1 or 2, wherein the unit dose of the medicament or medical device comprises from 1 μ g to 1000 μ g nocardia rubra cell wall skeleton; preferably 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μ g.
6. The use according to claim 1 or 2, wherein the nocardia rubra cell wall skeleton is a commercially available nocardia rubra cell wall skeleton.
7. Use according to claim 1 or 2, wherein the nocardia rubra cell wall skeleton is further obtainable by a method comprising or consisting of the steps of:

   1) providing nocardia rubra;

   2) crushing the nocardia rubra to obtain a crushed product;

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

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

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

   3.4) obtaining a product derived from the cell wall of the red nocardia;

   4) optionally, freeze-drying the product derived from the nocardia rubra cell wall;

   5) optionally, subpackaging;

   wherein the content of the first and second substances,

   steps 3.1), 3.2), 3.3) can be interchanged in sequence or in parallel,

   step 4) and step 5) can be interchanged in sequence;

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

   preferably, the dispensing means into containers;

   the container is selected from: bottles, tubes, bags, pouches, plates, ampoules, injection devices, aluminum film packaging, dressings, pouches, films.
8. A method of treating or preventing the recurrence of a recurrent aphthous ulcer, comprising the steps of:

   contacting the subject with a therapeutically effective amount of nocardia rubra cell wall skeleton;

   the recurrent aphthous ulcer is selected from any one or combination of: minor recurrent aphthous ulcers, herpetic recurrent aphthous ulcers, major recurrent aphthous ulcers;

   the nocardia rubra cell wall skeleton is prepared into a form selected from the group consisting of: an ointment, cream, emulsion, suspension, paste, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, powder, adjuvant, bandage, film, or patch; the ointment is selected from: ointments, plasters, creams;

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

   the contacting is for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, or more.
9. The method of claim 8, wherein the nocardia rubra cell wall skeleton is a commercially available nocardia rubra cell wall skeleton.
10. The method according to claim 8, wherein the nocardia rubra cell wall skeleton is obtained by a process comprising or consisting of the steps of:

    1) providing nocardia rubra;

    2) crushing the nocardia rubra to obtain a crushed product;

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

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

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

    3.4) obtaining a product derived from the cell wall of the red nocardia;

    4) optionally, freeze-drying the product derived from the nocardia rubra cell wall;

    5) optionally, subpackaging;

    wherein the content of the first and second substances,

    steps 3.1), 3.2), 3.3) can be interchanged in sequence or in parallel,

    step 4) and step 5) can be interchanged in sequence;

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

    preferably, the dispensing means into containers;

    the container is selected from: bottles, tubes, bags, pouches, plates, ampoules, injection devices, aluminum film packaging, dressings, pouches, films.