CN115089617A - Application of Nocardia rubrum cell wall skeleton in the treatment of chronic cervicitis - Google Patents

Abstract

The present disclosure relates to the application of Nocardia rubrum cell wall skeleton in the treatment of chronic cervicitis. In particular, the present disclosure provides Nocardia rubrum cell wall skeleton for use in the prevention or treatment of chronic cervicitis associated with infection by a high-risk HPV type selected from any one of the following in a subject or a combination thereof: Types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68.

Description

Application of Nocardia rubrum cell wall skeleton in the treatment of chronic cervicitis

technical field

The present disclosure relates to the fields of medicine and biopharmaceuticals. In particular, the present disclosure relates to the use of Nocardia rubrum cell wall skeleton in the treatment of chronic cervicitis.

Background technique

Chronic cervicitis is the most common disease in women of childbearing age and is often caused by Streptococcus, Enterococcus, Escherichia coli and Staphylococcus. Special pathogenic microorganisms include Chlamydia trachomatis and Neisseria gonorrhoeae. In addition, childbirth and mechanical injury are also predisposing factors for chronic cervicitis.

Nocardia rubra is a species of Nocardia. The cell wall skeleton of Nocardia rubrum can be obtained after fermentation, cell fragmentation and protease degradation of the nocardia rubra cells.

In the prior art, the cell wall skeleton of Nocardia rubrum can be obtained commercially, specifically, a commodity produced by Liaoning Grace Bio-Pharmaceutical Co., Ltd. Nocardia rubrum cell wall skeleton has been used to treat cervical precancerous lesions (CN101073583A), anti-human papillomavirus (CN1935262A), skin lesions or skin ulcers (CN101209267A), fungal infections, herpes simplex, herpes zoster (CN1879661A).

SUMMARY OF THE INVENTION

In a first aspect, the present disclosure provides a Nocardia rubrum cell wall skeleton.

Nocardia rubra refers to Nocardia rubra of the genus Nocardia.

Identification of Nocardia rubrum: According to known or future microbial identification techniques, technicians can carry out taxonomic identification of a bacterium, for example, available identification techniques include morphology, physiological and biochemical characteristics, 16S rRNA, etc. The skilled person understands that, with the development of science and technology, identification techniques involve different means. In the earlier period, morphological and biochemical identification methods were mainly used, but the reliability of this method was not high. With the advent of sequencing technologies, technicians can identify strains in a more confident manner. For example, when the DNA sequences of 16S rRNA are identified as having a similarity of 97% (inclusive) or more, it is determined that the two bacteria belong to the same species. For Nocardia rubrum, known strains deposited in international (or national) collections of strains are used as type strains and compared with them.

In the present disclosure, "Nocardia rubrum cell wall" can be understood as either an intact cell wall or an incomplete cell wall (eg, broken, or partially degraded). Given the teachings of the present disclosure, the skilled artisan will appreciate that the component exhibiting the desired activity is derived from the cell wall of Nocardia rubrum (eg, the cell wall itself or its constituents). Therefore, various forms of intact cell walls, broken cell walls, incomplete degradation products of cell walls, constituents of cell walls, extracts of cell walls, etc. are allowed to be used in clinical applications, which are all included in the scope of the present disclosure.

The cell wall skeleton of the present disclosure cannot be understood as only representing the cross-linked network entities in the cell wall, and the skilled person should understand that the term does not exclude other cell wall components adsorbed, bound and carried on the cross-linked network entities.

In a specific example, the cell wall skeleton of the present disclosure is a product obtained by bacteria after breaking and removing impurities (protein, nucleic acid, cell membrane, lipid).

In a specific embodiment, the cell wall skeleton is the Nocardia erythraea cell wall skeleton corresponding to the standard Chinese medicine S20030009 or its equivalent.

Technical personnel should understand that S20030009 is the initial number of the administrative license issued by the drug regulatory department, and this number changes with the update of the certificate, the adjustment of laws and numbering rules. However, no matter how the number changes, the product standard it represents (product parameters and/or quality requirements) remains the same. Therefore, in this disclosure, S20030009 should be understood to also include: S20030009 itself, its equivalent number, the cell wall skeleton corresponding to the update number, and also the cell wall skeleton with the same product parameters and/or quality requirements as S20030009.

"Cell wall skeleton with the same product parameters and/or quality requirements" or "cell wall skeleton corresponding to equivalent standards" means that under the same test method, the cell wall skeleton to be tested and the cell wall skeleton shown in S20030009 have no statistically significant difference in activity Components (muramic acids, sugars, lipids).

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

1) Provide Nocardia rubrum;

2) crushing described Nocardia red to obtain a crushed product;

3.1) removing lipid from the crushed product;

3.2) removing nucleic acid from the fragmented product;

3.3) removing protein from the crushed product;

3.4) removing the cell membrane from the fragmented product;

3.5) obtain the Nocardia rubrum cell wall skeleton;

4) optionally, subpackage;

5) optionally, freeze-drying the Nocardia rubrum cell wall skeleton;

Steps 3.1), 3.2), 3.3), 3.4) can be interchanged or parallel, and steps 4) and 5) can be interchanged.

For the disruption of Nocardia rubrum, the purpose is to remove intracellular substances. Therefore, technologies such as ultrasonic crushing, high-pressure homogenizer crushing, and lysozyme can be used. The skilled person understands that any known or future method suitable for disrupting Gram-positive bacteria is applicable to the technical solution of the present disclosure.

Technicians have the ability to adjust the specific parameters and equipment of culture, crushing, separation, collection, impurity removal, and sub-packaging according to the subsequent application (such as external application) of the active ingredient (cell wall and its constituent components), so as to avoid the introduction in the preparation step that affects the follow-up. applied factors.

In some embodiments, an organic solvent is used to remove lipids from the disrupted product. In some embodiments, nucleases are used to remove DNA and RNA from the fragmented product. In some embodiments, a hydrolase is used to degrade the protein in the crushed product. In some embodiments, a surfactant is used to remove cell membranes in the disrupted product.

In some embodiments, the average particle size of the fragmentation is 10 nm 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, 190 nm ± 10 nm, and the range between any two of the above values. Test methods for particle size are well known in the art.

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

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

In specific embodiments, said dispensing refers to dispensing into bottles or ampoules. Just before use, add a solvent (eg, sterile water) to the bottle or ampoule. As an example, the vial refers to a vial (vial, made of borosilicate glass or soda lime glass).

In a second aspect, the present disclosure provides the use of the aforementioned Nocardia rubrum cell wall skeleton in the preparation of a medicament for preventing or treating chronic cervicitis associated with high-risk HPV infection in a subject.

The high-risk HPV type is selected from any one or a combination of the following: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 types.

Described chronic cervicitis is selected from the following any one or its combination: cervical columnar ectopic, cervical polyps, cervical mucositis, cervical gland cyst, cervical hypertrophy (categorization can be found in, for example, "Obstetrics and Gynecology" published by People's Health Publishing House. , seventh edition).

In some embodiments, cervical columnar heterotopia is the most common local feature during inflammatory lesions of chronic cervicitis. The red lesions on the surface of the cervix are the result of the exfoliation of the squamous epithelium and the replacement of the columnar epithelium and the exposure of the subepithelial blood vessels. Clinically, it is often divided into mild I° (the lesion area is less than 1/3 of the total area of the cervix), moderate II° (1/3-1/2), and severe III° (more than 1/2) according to the lesion area.

In some embodiments, the cervical polyp is one of the manifestations of chronic cervicitis. Due to long-term stimulation of chronic inflammation, the local mucosa of the cervical canal proliferates, and because the uterus tends to exclude foreign bodies, the hyperplastic mucosa can gradually protrude from the base to the external os of the cervix to form polyps.

In some embodiments, cervical mucositis is one of the common manifestations of chronic cervicitis. The lesions are located in the mucous membrane and submucosal tissue of the cervical canal.

In some embodiments, cervical gland cysts are one of the common manifestations of chronic cervicitis. During the recovery of cervical columnar ectopic epithelium, the new squamous epithelium covers the orifice of the cervical gland or extends into the gland, blocking the opening of the gland; the hyperplasia or scarring of the connective tissue around the gland compresses the gland and narrows the gland. Even blocked, gland secretion drainage is blocked, and the cyst formed by retention is called cervical Nessler cyst. Cervical gland cysts are inflammations, not tumors.

In some embodiments, the cervical hypertrophy is a type of chronic cervicitis. Inflammatory changes caused by pathogenic infection of the cervical mucosa. Squamous epithelium exfoliation and columnar epithelial hyperplasia may also occur on the hypertrophic cervical surface due to injury or inflammatory stimulation.

In some embodiments, the medicament is prepared in a dosage form selected from any of the following: injections, ointments, creams, emulsions, suspensions, pastes, gels, lotions, tablets, aerosols, Sprays, liniments, powders, dressings, bandages, films, patches, suppositories.

In a third aspect, the present disclosure provides a pharmaceutical composition or medicament for preventing or treating chronic cervicitis associated with high-risk HPV infection in a subject, comprising: a pharmaceutically acceptable carrier and the disclosed Nocardia rubrum cell wall skeleton.

The pharmaceutical compositions or medicaments of the present disclosure may be prepared in unit dosage (or unit formulation) form.

In some embodiments, the pharmaceutical composition or drug can be prepared in liquid form (liquid formulation).

In other embodiments, the pharmaceutical composition or drug can be prepared as a solid (dry powder formulation or lyophilized powder formulation).

The skilled person understands that liquid preparations and dry powder preparations (or lyophilized powder preparations) can be converted into each other, and the difference is only in the water content. Most or all of the water in the liquid formulation is removed to obtain a dry powder formulation (or a lyophilized powder formulation). The dry powder preparation (or freeze-dried powder preparation) is dissolved (or reconstituted) to obtain a liquid preparation.

In some embodiments, the pharmaceutically acceptable carrier is selected from, but is not limited to, fillers, stabilizers (eg trehalose, glycine), flavoring agents (eg xylitol), disintegrants (eg carboxylate) sodium methylcellulose), binders (eg, gelatin), lubricants (eg, magnesium stearate).

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

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

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

In some embodiments, the lubricant is selected from one or a combination of the following: talc, magnesium stearate, micronized silica gel.

In some specific embodiments, pharmaceutically acceptable carriers suitable for use in the present disclosure may also be mentioned, such as, but not limited to: dextran, lactose, microcrystalline cellulose, trehalose, glycine, xylitol, carboxymethyl Sodium cellulose, erythritol, gelatin, magnesium stearate, propellant, humectant, solvent, solubilizer, emulsifier, antioxidant, pH adjuster, preservative. Specifically, non-limiting examples also include: white petrolatum, carbomer, hypromellose, 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, cetyl alcohol, stearyl alcohol, para-aminobenzoic acid, ethyl alcohol Amide, isopropyl alcohol, tween, polyoxyethyl hydrogenated castor oil, stearic acid, glycerol monostearate, triglycerol monostearate, fatty acid sucrose ester, sucrose ester, sucrose acetate isobutyrate Esters, Sorbitan Tristearate, Isopropyl Myristate, Cholesterol, Squalene, Squalane, n-Butanol, Ethylene Glycol, Ethanol, Propylene Glycol, Polyglyceride, Sulfite, Cysteine Amino acid, di-tert-butylhydroxytoluene, potassium sorbate, phosphate buffer solution, triethanolamine, sodium hydroxide, ethylenediamine, laurylamine, sodium bicarbonate, hydrochloric acid, parabens, thimerosal, chlorocresol, tris Chlorobutanol, benzoic acid and its sodium salt.

In a fourth aspect, the present disclosure provides a method for preventing or treating chronic cervicitis associated with high-risk HPV infection in a subject, comprising the step of administering to the subject a therapeutically effective amount of Nocardia rubrum of the present disclosure Cell wall skeleton or pharmaceutical composition.

"Administering", "providing to", "treating" when applied to an animal, human, cell, tissue, organ or biological sample refers to contact of a drug or medical device with the animal, human, cell, tissue, organ or biological sample.

"Treatment" means administering to a subject an internal or external drug (therapeutic agent, active ingredient or composition) (eg, exosomes or pharmaceutical compositions of the present disclosure) or medical device, in the subject being treated ( alleviate (reduce, delay, ameliorate, cure) one or more symptoms of a disease to a clinically measurable extent in a population) in which the subject has, is suspected of having, or is susceptible to a one or more diseases or their symptoms.

The amount of a drug (therapeutic agent, active ingredient or composition) effective to relieve the symptoms of any disease is referred to as a therapeutically effective amount. It can vary depending on a variety of factors, such as the subject's disease state, age, and weight. It should be understood that a drug (therapeutic agent, active ingredient or composition) may be ineffective in alleviating the target disease or symptoms thereof in a single subject, but may be ineffective according to any statistical test known in the art (e.g. Student's T-test, Cardiac The drug (therapeutic agent, active ingredient or composition) is statistically effective against the target disease or its symptoms, as determined by the square test, according to Mann and Whitney's U test.

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, laboratory animals (eg, rats, mice, guinea pigs, rabbits, dogs, primates).

In some specific embodiments, 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 symptoms thereof.

In some embodiments, the administration is 1-3 times a day, or once a day, or once every two days. Depending on the size and extent of the patient's lesions, different doses are used.

In some embodiments, the administration cycle lasts from 2 days to 6 months, eg, 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.

The same pharmaceutically active ingredient or different pharmaceutically active ingredients may be administered at one time, or may be divided into a number of smaller unit doses administered at timed intervals. It is understood that the exact dose, duration, interval of treatment is a function of the disease being treated and can be determined by extrapolation using animal or clinical trial data. The administration may comprise a single administration, or two or more administrations spaced at appropriate time intervals. Wherein, two adjacent administrations can be separated by 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, one and a half days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months.

"Optional" means that the event it describes subsequently can, but need not, occur; it depends on the circumstances. For example, "optionally, subpackaged" means that subpackaged product is permitted, but not required to be subpackaged.

"A", "an", "single", "the", if not expressly stated, also include plural forms.

When referring to a numerical range (eg, 60 μg to 120 μg), this is a shorthand way of treating each value within the range as being explicitly referred to, including decimals and whole numbers.

Detailed ways

The present disclosure is further described below in conjunction with the examples. However, these examples do not limit the scope of the present disclosure. When no specific conditions are specified, operate in accordance with the conventional conditions and the conditions suggested by the raw material supplier. Reagents with no specific source indicated are conventional reagents purchased in the market.

Example

Example 1. Commercially available Nocardia rubrum cell wall skeleton

The cell wall skeleton of Nocardia rubrum was purchased from Liaoning Gressite Bio-Pharmaceutical Co., Ltd., with the national medicine approval number S20030009 (the solid content of the cell wall skeleton in each tube should not be less than 60 μg, of which the content of muramic acid should not be less than 1.0 μg, and the sugar content should not be less than 1.0 μg. The content is not less than 4.0μg; the reconstituted volume is 2.0ml).

Example 2. Preparation of Nocardia rubrum cell wall skeleton

The preparation method of S20030009 cell wall skeleton is essentially no significant difference from the following steps, but it can be adjusted due to different production scales.

Thus, as an alternative, the following steps can be used to prepare the cell wall skeleton:

1. The cells are cultured according to a known method and collected. The cells are disrupted (eg, sonication or high pressure homogenizer disruption). It is also allowed to disrupt the cells by any suitable method known in the art. Check the broken situation under the microscope, no more than 5 visible bacteria in each field of view, and if several (10 to 30) fields of view meet this standard, it is qualified.

2. Nucleic acid removal: centrifuge the crushed supernatant, add DNase and RNase to the obtained precipitate, and remove nucleic acid according to the operation recommended by the enzyme supplier.

3. Removal of protein: common protease (eg trypsin) is added to the precipitate, and the protein is removed according to the operation suggested by the supplier of the enzyme.

4. Removing lipids: adding organic reagents (such as one or a combination of acetone, ether, and ethanol) to the precipitate, and removing lipids according to routine operations in the art.

5. Removal of cell membrane: TritonX-100 was added to the precipitate, the precipitate was collected by centrifugation, and rinsed with PBS.

It should be understood that, between the above steps of removing impurities, a skilled person can adjust the sequence to make the steps compatible. After removing the non-cell wall components, the precipitate was reconstituted in water for injection and used. Optionally, it can be sterilized at 115°C for 20-30 minutes as a stock solution of cell wall skeleton.

Example 3. Exemplary Preparation of Pharmaceutical Compositions

1. Coat the product obtained in Example 1 or 2 on a dressing (eg, sterile gauze) to prepare a drug for external use.

2. The product obtained in Example 2 was made into lyophilized powder.

3. The preparation method of lotion known in the art can also be used, for example: the lotion is mostly made of water and alcohol as the dispersion medium; it is made from active ingredients, electrolytes, isotonicity regulators, etc. in the dispersion medium.

4. The product obtained in Example 1 or 2 was prepared into capsules.

5. The product obtained in Example 1 or 2 was dissolved in water for injection to prepare an injection.

6. The products obtained in Example 1 or 2 were prepared into vaginal suppositories.

Example of effect

The cell wall skeleton of Example 1 was administered to a subject clinically diagnosed with chronic cervicitis. The treatment results are shown in Table 1.

Table 1

*HPV(-) means all high-risk HPV tests were negative.

Claims (4)

1. The use of Nocardia rubrum cell wall skeleton in the preparation of medicine, wherein: The medicament is used to prevent or treat chronic cervicitis associated with high-risk HPV infection in a subject; The high-risk HPV type is selected from any one or a combination of the following: 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68; The chronic cervicitis is selected from any one or a combination of the following: cervical columnar heterotopia, cervical polyps, cervical mucositis, cervical gland cysts, cervical hypertrophy. 2. The use according to claim 1, wherein the medicament is prepared in a dosage form selected from the group consisting of ointments, creams, suppositories, emulsions, suspensions, pastes, gels, lotions, tablets , Aerosols, Sprays, Liniments, Powders, Dressings, Bandages, Films, Patches, Injections. 3. The use according to claim 1 or 2, wherein the Nocardia rubra cell wall skeleton is the Nocardia rubra cell wall skeleton corresponding to the national medicine standard S20030009 or its equivalent standard. 4. The use according to claim 1 or 2, wherein the Nocardia rubrum cell wall skeleton is obtained by the following method comprising or consisting of the following steps: 1) Provide Nocardia rubrum; 2) crushing described Nocardia red to obtain a crushed product; 3.1) removing lipid from the crushed product; 3.2) removing nucleic acid from the fragmented product; 3.3) removing protein from the crushed product; 3.4) removing the cell membrane from the fragmented product; 3.5) obtain the Nocardia rubrum cell wall skeleton; 4) optionally, subpackage; 5) optionally, freeze-drying the Nocardia rubrum cell wall skeleton; in, Steps 3.1), 3.2), 3.3), 3.4) can be interchanged or paralleled, Step 4) and step 5) can be interchanged; The average particle size of the crushing is 10 nm to 1000 nm, preferably 10 nm to 800 nm, more preferably 10 nm to 500 nm.