Use of Nocardia rubra cell wall skeleton in the treatment of radiation sickness

Abstract

Use of Nocardia rubra cell wall skeleton in the treatment of radiation sickness. A pharmaceutical composition, which contains components derived from the cell wall of Nocardia rubra, can effectively treat or relieve radiation sickness and its symptoms.

Description

Technical Field

[0001] The present disclosure relates to the fields of medicine, microbiology, and biopharmaceuticals. Specifically, the present application relates to the use of Nocardia rubra cell wall skeleton in the preparation of a medicament for treating radiation sickness. Background Art

[0002] Nocardia rubra is one of the Nocardia. The cell bodies of Nocardia rubra can be prepared into Nocardia rubra cell wall skeleton (hereinafter referred to as Nr-CWS) after fermentation, cell disruption, and protease degradation.

[0003] Nocardia is polymorphic, with spherical, rod-shaped, or filamentous forms. The cell bodies are non-motile, some strains are weakly acid-fast, and are obligate aerobes. On a common agar plate, colonies can be seen after 3 days of culture, and after 7 to 10 days, the colonies bulge, forming aerial hyphae, and the surface is villous. The colonies of different strains are yellow, orange, or red. The G + C molar content in DNA is 60 to 72%. Most Nocardia are saprophytes and exist in the soil.

[0004] In the prior art, Nocardia rubra cell wall skeleton can be obtained commercially. For example, it is a product produced by Liaoning Gerist Biological Pharmaceutical Co., Ltd. (trade name "Nacojia"), or provided by Fujian Shanhe Pharmaceutical Co., Ltd., Fujian Guangshengtang Pharmaceutical Co., Ltd., Fujian Institute of Microbiology, etc. Nocardia rubra cell wall skeleton has been used in the treatment of cervical erosion, cervical intraepithelial neoplasia (CN101073583A), anti-human papillomavirus (CN1935262A), skin injury or skin ulcer (CN101209267A), skin lesions (eczema, neurodermatitis, non-specific dermatitis, atopic dermatitis, psoriasis) (CN108938674A), acne (CN108295095A), fungal infection, herpes simplex, herpes zoster (CN1879661A).

[0005] Radiation sickness is a systemic or local disease (including its symptoms) caused by the body being exposed to a large dose of radiation. Radiation sickness can occur due to both internal radiation and external radiation. The rays that cause radiation sickness include γ-rays, neutrons, and X-rays, etc.

[0006] In the initial stage after radiation, there are symptoms such as nausea and loss of appetite, and the irradiation dose may be greater than 1 Gy; in the case of vomiting, it may be greater than 2 Gy. If multiple vomitings occur within several hours after radiation and severe diarrhea quickly occurs, but there are no neurological symptoms, it can be considered as intestinal radiation sickness. If frequent vomiting, disorientation, ataxia, limb tremors, and increased muscle tone occur within 1 hour after radiation, it can be diagnosed as cerebral radiation sickness.

[0007] In clinical treatment, radiotherapy is a local treatment method that uses rays to treat tumors or cancers. The rays include α, β, γ, and x-rays generated by radiotherapy machines or accelerators, electron beams, proton beams, and other particle beams. However, radiotherapy often has various side effects or complications:

[0008] - Systemic reactions: manifested as fatigue, dizziness, headache, nausea, vomiting, etc.

[0009] - Blood changes: mainly manifested as a decrease in white blood cells and platelets.

[0010] - Skin reactions: The irradiated area may show hyperemia, pigmentation, dry desquamation, wet desquamation, skin atrophy, epidermal capillary dilation, fibrosis of subcutaneous fat fibrous tissue, etc.

[0011] - Mucosal reactions: There may be flushing, swelling, dryness, superficial ulcers, and the formation of a pale pseudomembrane; patients with lung cancer often develop esophagitis.

[0012] - Reactions in other tissues: After a large amount of irradiation to the central nervous system, brain necrosis, radiation myelitis, etc. may occur; high-dose pelvic irradiation can cause bladder and rectal irritation symptoms such as urgency, dysuria, frequency of urination, hematuria, increased frequency of defecation, and tenesmus.

[0013] However, in the prior art, there has been no report on the role of Nocardia rubra cell wall skeleton in radiation sickness, nor has there been a report on its use in improving or alleviating skin and mucosal side effects or complications caused by radiotherapy. Summary of the Invention

[0014] According to some embodiments of the present application, there is provided the use of Nocardia rubra cell wall skeleton in the preparation of a medicament or medical device for treating radiation sickness.

[0015] In some embodiments, the radiation sickness is radiation-induced radiation sickness.

[0016] In some embodiments, the radiation is ionizing radiation or non-ionizing radiation.

[0017] In some embodiments, the radiation sickness is caused by any one or a combination selected from the following: α rays, β rays, x rays, γ rays, neutron radiation.

[0018] In some embodiments, the radiation sickness is side effects caused by radiotherapy and / or complications caused by radiotherapy.

[0019] In some embodiments, side effects caused by radiotherapy and / or complications caused by radiotherapy particularly refer to side effects and / or complications related to the epidermis, dermis, and / or mucosa.

[0020] In some embodiments, radiation sickness can affect the following tissues: the epidermal layer, the dermal layer, mucous membranes (such as those of the digestive tract), and subcutaneous tissue.

[0021] In some embodiments, a drug or medical device is administered by contact at the site of injury.

[0022] 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.

[0023] In some embodiments, the drug is formulated into any dosage form selected from the following: suppositories, ointments, creams, emulsions, suspensions, pastes, gels, lotions, tinctures, oils, tablets, aerosols, sprays, liniments, powders; wherein, the ointments are selected from: ointments, plasters, creams.

[0024] According to some embodiments, a method for treating radiation sickness is provided, comprising the step of providing a therapeutically effective amount of Nocardia rubra cell wall skeleton to a subject.

[0025] In some specific embodiments, depending on the area or severity of the lesion, a drug (or medical device) is administered to the lesion. For example, but not limited to, applying a drug containing Nocardia rubra cell wall skeleton, or covering the lesion with a patch impregnated with Nocardia rubra cell wall skeleton, or directly administering the freeze-dried powder containing Nocardia rubra cell wall skeleton at the lesion, or applying a paste, lotion, etc. containing Nocardia rubra cell wall skeleton on the lesion. When the lesion is in the digestive tract, capsules, tablets, solutions, etc. containing Nocardia rubra cell wall skeleton can be administered orally.

[0026] In some embodiments, the drug comprises:

[0027] - Nocardia rubra cell wall skeleton, and

[0028] - a pharmaceutically acceptable carrier.

[0029] In some embodiments, the pharmaceutically acceptable carrier is selected from, but not limited to: fillers, stabilizers (such as trehalose, glycine), flavoring agents (such as xylitol), disintegrants (such as sodium carboxymethylcellulose), binders (such as gelatin), lubricants (such as magnesium stearate).

[0030] In some embodiments, the stabilizer is selected from one or a combination of the following: glycine, lysine, arginine, hydroxyethyl starch, hydroxymethyl starch, trehalose, dextran.

[0031] In some embodiments, the flavoring agent is selected from one or a combination of the following: sucrose, monosaccharides, sodium saccharin, aspartame, sorbitol, xylitol, mannitol.

[0032] In some embodiments, the binder is selected from one or a combination of the following: sodium carboxymethyl cellulose, hypromellose, gelatin.

[0033] In some embodiments, the lubricant is selected from one or a combination of the following: talc, magnesium stearate, colloidal silica.

[0034] In some specific embodiments, carriers applicable to the present application may also be mentioned, such as but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, sodium carboxymethyl cellulose, erythritol, gelatin, magnesium stearate, propellants, humectants, solvents, solubilizers, emulsifiers, antioxidants, pH regulators, preservatives. Specifically, non-limiting examples also include: white petrolatum, carbomer, hypromellose, methylcellulose, sodium hydroxymethylcellulose, chitosan, chitosan sucralfate, 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, isopropanol, Tween, polyoxyethylated hydrogenated castor oil, stearic acid, glycerol monostearate, polyglycerol 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 esters, sulfites, cysteine, dibutylhydroxytoluene, potassium sorbate, phosphate buffer solution, triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, chlorobutanol, benzoic acid and its sodium salt.

[0035] In some embodiments, the pharmaceutically acceptable carrier is dextran.

[0036] In some embodiments, the drug or medical device of the present application is administered 1 - 3 times a day, or once a day, or once every two days. Different doses per administration are adopted depending on the area and degree of the patient's lesion, usually 1 μg / unit dose / per administration to 1000 μg / unit dose / per administration. Specifically, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 μg / unit dose / per administration, and ranges between any two of the foregoing values.

[0037] In some embodiments, the administration period lasts from 2 days to 6 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 ranges between any two of the foregoing values.

[0038] In some embodiments, the Nocardia rubra cell wall skeleton is a commercially available Nocardia rubra cell wall skeleton.

[0039] In other embodiments, the Nocardia rubra cell wall skeleton is obtained by a method comprising the following steps or consisting of the following steps:

[0040] 1) Provide Nocardia rubra;

[0041] 2) Crush the Nocardia rubra to obtain a crushed product;

[0042] 3.1) Optionally, perform an operation to remove lipids from the crushed product;

[0043] 3.2) Optionally, perform an operation to remove nucleic acids from the crushed product;

[0044] 3.3) Optionally, perform an operation to remove proteins from the crushed product;

[0045] 3.4) Obtain a product derived from the cell wall of Nocardia rubra;

[0046] 4) Optionally, lyophilize the product derived from the cell wall of Nocardia rubra;

[0047] 5) Optionally, perform sub-packaging;

[0048] Wherein,

[0049] Steps 3.1), 3.2), and 3.3) can be interchanged in order or performed in parallel,

[0050] Steps 4) and 5) can be interchanged in order;

[0051] The average particle size of the crushing is from 10 nm to 1000 nm, preferably from 10 nm to 800 nm, more preferably from 10 nm to 500 nm;

[0052] Preferably, the sub-packaging refers to sub-packaging into a container;

[0053] The container is selected from: bottles, tubes, packages, bags, plates, ampoules, injection devices, aluminum foil packages, dressings, capsules, membranes.

[0054] For the comminution of Nocardia rubra, the purpose is to remove the substances inside the cells, so techniques such as ultrasonic disruption and lysozyme can be adopted. Those skilled in the art understand that any known or future method applicable to the disruption of Gram-positive bacteria is applicable to the technical solution of the present disclosure.

[0055] Those skilled in the art are capable of adjusting the specific parameters and equipment for cultivation, disruption, separation, collection, impurity removal, and sub-packaging according to the subsequent application of the active ingredient (cell wall and its components), such as external application, so as to avoid introducing factors that affect the subsequent application in the preparation steps.

[0056] In some embodiments, organic solvents are used to remove lipids in the disrupted product. In some embodiments, nucleases are used to remove DNA and RNA in the disrupted product. In some embodiments, hydrolases are used to degrade proteins in the disrupted product. In some embodiments, surfactants are used to remove cell membranes in the disrupted product.

[0057] In some embodiments, the average particle size of the comminution is from 10 nm to 1000 nm; 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 nm ± 10 nm can be mentioned, as well as the ranges between any two of the above values. The test method for particle size is well-known in the prior art.

[0058] In some specific embodiments, the average particle size of the comminution is from 10 nm to 800 nm.

[0059] In some other specific embodiments, the average particle size of the comminution is from 10 nm to 500 nm.

[0060] In a specific embodiment, the sub-packaging refers to sub-packaging into bottles / ampoules. Before use, a solvent (such as sterile water) is added to the bottles / ampoules.

[0061] In some specific embodiments, the subject is an animal other than a human, such as for farm animals, pets, working animals, ornamental animals, production animals, and experimental animals.

[0062] In a specific embodiment, the subject is a human; especially a radiated subject. As an example, the subject is a patient treated with radiotherapy; especially, a patient who has developed skin, dermal, and / or mucosal symptoms after radiotherapy.

[0063] In the context of the present application, the only therapeutic active ingredient in the medicament or medical device is a product derived from Nocardia rubra, in particular a product comprising the components of Nocardia rubra (such as proteins, nucleic acids, lipids, cell walls and their components, carbohydrates, metabolites), specifically a product comprising the cell wall of Nocardia rubra (more preferably the Nocardia rubra skeleton or its components). Description of the Drawings

[0064] Figures 1A to 1C : The pharmaceutical composition of the present application improves wound healing caused by chemotherapy.

[0065] Figures 2A to 2D : The pharmaceutical composition of the present application improves wound healing caused by chemotherapy.

[0066] Figures 3A to 3H : The promoting effect of the pharmaceutical composition of the present application on the healing of skin injury wounds in a rat radiation model. 1 week after 45Gy radiation ( Figure 3A and Figure 3E ), 2 weeks ( Figure 3B and Figure 3F ), 3 weeks ( Figure 3C and Figure 3G ), 4 weeks ( Figure 3D and Figure 3H ); Figures 3A to 3D is the control group (only the control composition is administered after irradiation, that is, without the cell wall skeleton compared with the composition of the present application); Figures 3E to 3H is the treatment group, and the composition of the present application is administered. Detailed Description of the Invention

[0067] Nocardia rubra cell wall

[0068] In the present disclosure, the "Nocardia rubra cell wall" can be understood as either a complete cell wall or an incomplete cell wall (e.g., broken or partially degraded). Under the teachings of the present disclosure, those skilled in the art will understand that the ingredient showing the desired activity is from the cell wall of Nocardia rubra (e.g., is the cell wall itself or its components). Therefore, in clinical applications, various forms such as a complete cell wall, a broken cell wall, an incomplete degradation product of the cell wall, components of the cell wall, extracts of the cell wall, etc. are allowed, and these are all within the scope of the present disclosure.

[0069] Cell wall skeleton

[0070] The component constituting the main structure of the cell wall; however, it should not be understood as merely indicating the cross-linked network entity in the cell wall, and those skilled in the art understand that other cell wall components adsorbed, bound, or carried on the cross-linked network entity are not excluded.

[0071] Unit dose

[0072] The drug or medical device of the present disclosure can be prepared in the form of unit doses (or unit preparations).

[0073] "Optionally" means that the subsequent described matter can occur, but does not have to occur; it depends on the circumstances. For example, "Optionally, sub-packaging is carried out" means that sub-packaging of the product is allowed, but it is not necessary to carry out sub-packaging; whether or not sub-packaging is carried out does not affect the achievement of the technical effect.

[0074] "A", "an", "single", "the", if not clearly stated, also include the plural forms.

[0075] The present disclosure is further described below in conjunction with embodiments. However, these embodiments do not limit the scope of the present disclosure. When specific conditions are not indicated, operations are carried out according to conventional conditions and according to the conditions recommended by the raw material suppliers. Reagents without specific sources indicated are conventional reagents purchased from the market.

[0076] Those skilled in the art particularly understand that although the following specific examples use specific commercially available cell wall products, the achievement of the technical effect is not limited to this specific commercially available product, and any species belonging to Nocardia rubra are applicable.

[0077] Example 1. Commercially available cell wall skeleton of Nocardia rubra

[0078] The cell wall skeleton of Nocardia rubra (trade name: Nacojia) was purchased from Liaoning Gerist Biological Pharmaceutical Co., Ltd., with the approval number of national drug approval letter S20030009 (2 ml / vial; freeze-dried powder), which contains 60 μg of active ingredient and 15 mg of dextran 40.

[0079] Example 2. Preparation of cell wall skeleton of Nocardia rubra

[0080] 1. Cultivate the bacterial cells according to a well-known method and collect them. Crush the cells (for example, but not limited to, by ultrasonic disruption). Any other appropriate well-known method in the art for disrupting the bacterial cells is also allowed, such as CN101250490A or CN101323865A. Check the crushing situation under a microscope. The number of visible bacteria in each field of view should not exceed 5. If the standard is met after checking several (10 to 30) fields of view, it is considered qualified.

[0081] 2. Remove nucleic acids: Centrifuge the disrupted supernatant, add DNAse and RNAse to the obtained precipitate, and remove nucleic acids according to the operation recommended by the enzyme supplier.

[0082] 3. Remove proteins: Add common proteases (such as trypsin) to the precipitate and remove proteins according to the operation recommended by the enzyme supplier.

[0083] 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 lipids according to conventional operations in the art.

[0084] 5. Removal of cell membrane: Add TritonX-100 to the precipitate, collect the precipitate by centrifugation according to routine procedures in the art, and rinse with PBS.

[0085] It should be understood that between the above steps of removing impurities, the technician can adjust the order so that the steps are compatible. After removing the non-cell wall components, the precipitate is redissolved in water for injection and set aside. Optionally, it can be sterilized at 115°C for 20-30 minutes as a stock solution of the cell wall skeleton (mainly comprising the cell wall skeleton and its components).

[0086] Example 3. Preparation of pharmaceutical compositions or medical devices

[0087] 1. The product obtained in Example 2 (active ingredient 60 μg to 120 μg, for example 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg) or the commercially available product in Example 1 is coated on a dressing (for example, sterile gauze) to prepare an external medical device.

[0088] 2. The product obtained in Example 2 (active ingredient 60 μg) was made into freeze-dried powder and applied directly on the surface of the lesion.

[0089] 3. The preparation method of lotion known in the art may also be adopted. For example, lotion usually uses water and ethanol as dispersion medium; active ingredients, electrolytes, isotonicity regulators, etc. are prepared in the dispersion medium.

[0090] 4. The product obtained in Example 2 (active ingredient 60 μg to 120 μg, for example 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 110 μg, 120 μg) or the commercially available product in Example 1 is prepared into enteric-coated capsules.

[0091] Test case: The therapeutic effect of radiation sickness

[0092] Drugs and main reagents

[0093] Test drug: topical Nocardia rubrum cell wall skeleton (Liaoning Greishit Biotechnology Co., Ltd., national medicine standard S20030009, specification 60μg / vial, batch number 201809004);

[0094] Test Example 1

[0095] The patient, female, 56 years old. After surgery for right breast cancer, she started chemotherapy in February 2019 for a total of 8 times, and started radiotherapy in August for a total of 28 times. During the 20th time, she began to have red fluid and then ulcerated (Figure 1A )。

[0096] On September 30, 2019, the patient began to use the pharmaceutical composition of the present application, Figure 1B and Figure 1C were the wounds treated in the first and second weeks of administration. The wound healed 2 weeks after medication.

[0097] Test Example 2

[0098] Patient, female, 55 years old. Left breast cancer, bone metastasis, lung metastasis. Surgery was performed in June 2018, and chemotherapy began in July of the same year, for a total of 8 times. Radiation therapy began in March 2019, for a total of 25 times. Skin ulceration occurred at the 15th time ( Figure 2A )。

[0099] After seeing a doctor on October 17, 2019, the patient began to use the pharmaceutical composition of the present application. After medication, the wound significantly shrank and became smaller ( Figures 2B to 2D )。Due to the patient's poor mental and physical condition recently, a suspected recurrence of cancer occurred. Therefore, the healing speed was slower.

[0100] Test Example 3. Effect of Nocardia rubra cell wall skeleton on a rat radiation model

[0101] The back of the rat was irradiated with 45 Gy of X-rays once. The changes in the wounds at the irradiated sites of the rats were observed at 1, 2, 3, and 4 weeks after irradiation. The composition of the present application was applied to the wounds, and it was visually observed that the healing of the radioactive skin injury wounds was promoted ( Figures 3A to 3G )。

Claims

1. Use of the cell wall skeleton of Nocardia rubrum in the preparation of medicines, wherein: The drug is used to promote wound healing of skin damage caused by radiotherapy; The cell wall skeleton of Nocardia rubrum is the cell wall skeleton of Nocardia rubrum with national medicine standard number S20030009.

2. The method according to claim 1, wherein the skin lesion involves any one of the following tissues or a combination thereof: epidermis, dermis, mucosa, subcutaneous tissue.

3. The use according to claim 1, wherein the drug is prepared into a dosage form selected from any one of the following: ointment, cream, emulsion, suspension, paste, gel, lotion, tincture, oil, tablet, aerosol, spray, liniment, powder, excipient, bandage, film, patch, suppository.

4. The use according to claim 1, wherein a unit dose in the medicament comprises 1 μg to 1000 μg of Nocardia rubrum cell wall skeleton.

5. The method of claim 4, wherein a unit dose of the drug 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, 200 μg of Nocardia rubrum cell wall skeleton.

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