RU2794363C1 - Pharmaceutical compositions for treatment of infectious-inflammatory diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and chemical-pharmaceutical industry, namely to improved gramicidin C substances containing certain amounts of new cyclic decapeptides from the gramicidin C family, as well as to pharmaceutical compositions containing them for the treatment of infectious and inflammatory diseases.

EFFECT: obtaining pharmaceutical compositions for treatment of infectious-inflammatory diseases.

27 cl, 4 tbl, 6 ex

Description

SUBSTANCE: invention relates to medicine and chemical-pharmaceutical industry, namely to improved gramicidin C substances containing certain amounts of new cyclic decapeptides from the gramicidin C family, as well as to pharmaceutical compositions containing them for the treatment of infectious and inflammatory diseases.

State of the art

Infectious and inflammatory diseases of the oral cavity and pharynx (pharyngitis, glossitis, stomatitis, gingivitis, and others) are among the most common reasons for patients to apply to local therapists, otorhinolaryngologists, pediatricians, which is associated with a high incidence rate among young people, working age and children, therapeutic tactics for infectious and inflammatory diseases of the mucous membrane of the oral cavity and pharynx includes the appointment of drugs with anti-inflammatory, analgesic, immunocorrective effects, local antiseptics, decongestants and hyposensitizing drugs [Morozova S.V. Pain in the throat: causes and possibilities of drug therapy. BC, Vol. 13, No. 21, 2005, p. 1447-1452].

Topical antibiotics remain one of the groups of drugs used to treat this group of diseases. New generations of antibiotics are being introduced into medical practice. At the same time, there is a tendency, along with the search for new pharmacological agents, to maximize the potential of already known agents.

These agents include gramicidin C, the first original domestic antibiotic polypeptide of the tyrothricin group, isolated from a culture of Bacillus brevis by G.F. Gause and M.G. Brazhnikova in 1942, intended exclusively for local use [Gause, G. G.; Brazhnikova, M. G. Gramicidin S and its use in the treatment of infected wounds. Nature 1944, 154, 703]. Gramicidin C preparations have a pronounced bactericidal effect against pathogenic gram-positive and gram-negative bacteria [Kondejewski, L. H., et al. Gramicidin S Is Active against Both GramPositive and Gram-Negative Bacteria. Int. J. Pept. Protein Res. 1996, 47(6), 460-466], which in most cases either directly cause diseases of the throat and oral cavity, or join secondarily with an initially viral infection - Streptococcus spp., Staphylococcus spp., Streptococcus pneumoniae, Neisseria, anaerobes and other Gramicidin C preparations also have some fungistatic and antiviral activity. According to modern ideas about rational antibiotic therapy, in order to prevent the emergence and spread of resistant strains of microorganisms, in all cases where it is possible to predict the causative agent of infection with a high degree of probability, it is recommended to give preference to just such drugs. Resistance to polypeptides rarely develops. There are very few reports in the literature on the isolation of gramicidin C-resistant strains of microorganisms from diseased or natural sources. It is very difficult to obtain strains resistant to this antibiotic in laboratory conditions [Polin A.N., Egorov N.S. Structural and functional features of gramicidin C in connection with its antibiotic activity // Antibiotics and Chemotherapy. - 2003. - No. 48 (12). - S. 29-32]. Due to the originality of the chemical structure, mechanism of action and method of application, the use of gramicidin C should not be accompanied by an increase in the level of resistance to antibiotics for systemic use.

Known preparations of gramicidin C in the form of tablets [RF patent No. 2213558, publ. 10.10.2003] and in the form of liquid compositions [RF patent No. 2633635, publ. 10/16/2017; RF patent No. 2604576, publ. 12/10/2016; RF patent No. 2627423, publ. 08.08.2017]. Combinations of gramicidin C with ambroxol are also known [RF patent No. 2749902, publ. 06/18/2021], gramicidin C with cetylpyridinium chloride [RF patent No. 2633635, publ. October 16, 2017] and with oxybuprocaine hydrochloride [RF patent No. 2627423, publ. 08.08.2017].

Three cyclic decapeptides from the gramicidin C family are currently known to have similar antibacterial activity: gramicidin C (also referred to as gramicidin C-1), gramicidin C-2, and gramicidin C-3 [Nozaki, S., & Muramatsu, I. ( 1985). Natural Homologs of Gramicidin S. II. Synthesis of Gramicidin S-2 and S-3. Bulletin of the Chemical Society of Japan, 58(1), 331-335.]. These Gramicidins C have the following structures:

gramicidin C: cyclo(-Val-Orn-Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-);

gramicidin C-2: cyclo(-Val-Orn-Leu-D-Phe-Pro-Abu-Orn-Leu-D-Phe-Pro-);

gramicidin C-3: cyclo(-Abu-Orn-Leu-D-Phe-Pro-Abu-Orn-Leu-D-Phe-Pro-);

where Abu is a 2-aminobutyric acid residue.

Gramicidin C-2 has the following structural formula:

Gramicidin C-3 has the following structural formula:

Given the prevalence of infectious and inflammatory diseases of the oral cavity and pharynx, there is a need to create new effective drugs with antibacterial activity.

Thus, the objective of the present invention is to expand the arsenal of gramicidin C drugs, including the development of new effective compositions based on gramicidin C, which have increased antibacterial activity.

The problem is solved by obtaining improved substances and compositions of gramicidin C containing certain amounts of cyclic decapeptides from the gramicidin C family, and creating effective antibacterial pharmaceutical compositions containing them.

Disclosure of the essence of the invention

The inventors obtained new, previously unknown cyclic decapeptides from the gramicidin C family, which were given the following names: gramicidin C-4-1, gramicidin C-4-2, gramicidin C-5.

Thus, the subject of the present invention is gramicidin C-4-1, which is a cyclic decapeptide from the gramicidin C family, characterized in that it has the following structure: cyclo(-Val-Orn-Leu-D-Phe-Pro-Val-Orn -2-amino-(5-methylhexanoic acid)-D-Phe-Pro-).

Gramicidin C-4-1 has the following structural formula:

The subject of the present invention is also gramicidin C-4-2, which is a cyclic decapeptide from the gramicidin C family, characterized in that it has the following structure: cyclo(-Val-Orn-Leu-D-Phe-Pro-Val-Orn-β -homoleucine-D-Phe-Pro-).

Gramicidin C-4-2 has the following structural formula:

The subject of the present invention is also gramicidin C-5, which is a cyclic decapeptide from the gramicidin C family, characterized in that it has the following structure: cyclo(-Val-α-methyl-L-ornithine-Leu-D-Phe-Pro-Val -Orn-Leu-D-Phe-Pro-).

Gramicidin C-5 has the following structural formula:

The subject of the present invention is also an active pharmaceutical substance of gramicidin C intended for the treatment of infectious and inflammatory diseases, containing gramicidin C or a pharmaceutically acceptable salt thereof, gramicidin C-4-1 and/or gramicidin C-4-2 and/or gramicidin C-5 in the following ratio (% wt.):

Gramicidin C - 91.5-99.9 wt%;

Gramicidin C-4-1 and/or C-4-2 - 0-3.5% wt.;

Gramicidin C-5 - 0-5.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-1 in an amount of not more than 3.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-1 in an amount of not more than 2.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-1 in an amount of not more than 2.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-1 in an amount of not more than 1.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-1 in an amount of not more than 1.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-1 in an amount of not more than 0.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-5 in an amount of not more than 4.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-5 in an amount of not more than 4.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-5 in an amount of not more than 3.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-5 in an amount of not more than 3.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-5 in an amount of not more than 2.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-5 in an amount of not more than 2.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-5 in an amount of not more than 1.5% of the mass.

In one of the variants of the present invention, the active pharmaceutical substance contains gramicidin C-5 in an amount of not more than 1.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-5 in an amount of not more than 0.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-2 in an amount of not more than 3.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-2 in an amount of not more than 2.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-2 in an amount of not more than 2.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-2 in an amount of not more than 1.5% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-2 in an amount of not more than 1.0% of the mass.

In one embodiment of the present invention, the active pharmaceutical ingredient contains gramicidin C-4-2 in an amount of not more than 0.5% of the mass.

In one of the embodiments of the present invention, gramicidin C-4-2 in the composition of the active pharmaceutical substance is in the form of its pharmaceutically acceptable salt.

In one of the embodiments of the present invention, gramicidin C-4-2 in the composition of the active pharmaceutical substance is in the form of dihydrochloride.

In one of the embodiments of the present invention, the active pharmaceutical substance contains additional unidentifiable impurities in an amount of not more than 2.0% of the mass.

In one embodiment of the present invention, gramicidin C in the composition of the active pharmaceutical substance is in the form of dihydrochloride.

In one of the embodiments of the present invention, gramicidin C-4-1 in the composition of the active pharmaceutical substance is in the form of its pharmaceutically acceptable salt.

In one embodiment of the present invention, gramicidin C-4-1 in the composition of the active pharmaceutical substance is in the form of dihydrochloride.

In one of the embodiments of the present invention, gramicidin C-5 in the composition of the active pharmaceutical substance is in the form of its pharmaceutically acceptable salt.

In one embodiment of the present invention, gramicidin C-5 in the composition of the active pharmaceutical substance is in the form of dihydrochloride.

The subject of the present invention is also a pharmaceutical composition for the treatment of infectious and inflammatory diseases for topical use, containing the active pharmaceutical ingredient described above in a therapeutically effective amount and pharmaceutically acceptable excipients.

The subject of the present invention is also a pharmaceutical composition for the treatment of infectious and inflammatory diseases for local use, containing cyclic decapeptides from the gramicidin C family and pharmaceutically acceptable excipients, characterized in that it contains gramicidin C, gramicidin C- 4-1 and/or gramicidin C-4-2 and/or gramicidin C-5 in the following ratio (wt %):

Gramicidin C - 91.5-99.9 wt%;

Gramicidin C-4-1 and/or C-4-2 - 0-3.5% wt.;

Gramicidin C-5 - 0-5.0% of the mass.

In one of the embodiments of the present invention, gramicidin C-5 in the composition of the active pharmaceutical substance is in the form of its pharmaceutically acceptable salt.

In one embodiment of the present invention, gramicidin C-5 in the composition of the active pharmaceutical substance is in the form of dihydrochloride.

The subject of the present invention is also a pharmaceutical composition for the treatment of infectious and inflammatory diseases for topical use, containing the active pharmaceutical ingredient described above in a therapeutically effective amount and pharmaceutically acceptable excipients.

The subject of the present invention is also a pharmaceutical composition for the treatment of infectious and inflammatory diseases for local use, containing cyclic decapeptides from the gramicidin C family and pharmaceutically acceptable excipients, characterized in that it contains gramicidin C, gramicidin C- 4-2 and/or gramicidin C-5 in the following ratio (wt %):

Gramicidin C - 91.5-99.9 wt%;

Gramicidin C-4-2 - 0-3.5% wt.;

Gramicidin C-5 - 0-5.0% of the mass.

The subject of the present invention is also the active pharmaceutical substance of gramicidin C, intended for the treatment of infectious and inflammatory diseases, containing gramicidin C or a pharmaceutically acceptable salt thereof and 0-3.5% of the mass. cyclic decapeptide having the structure cyclo(-Val-Orn-Leu-D-Phe-Pro-Val-Orn-2-amino-(5-methylhexanoic acid)-D-Phe-Pro-) and/or cyclo(-Val- Orn-Leu-D-Phe-Pro-Val-Orn-β-homoleucine-D-Phe-Pro-).

The subject of the present invention is also the active pharmaceutical substance of gramicidin C, intended for the treatment of infectious and inflammatory diseases, containing gramicidin C or a pharmaceutically acceptable salt thereof and 0-5.0% of the mass. a cyclic decapeptide having the structure cyclo(-Val-α-methyl-L-ornithine-Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-).

The subject of the present invention is also the use of the pharmaceutical compositions of the present invention for the treatment or improvement of an infectious inflammatory disease of the mouth and throat.

The subject of the present invention is also a drug for the treatment of infectious and inflammatory diseases, containing gramicidin C or its pharmaceutically acceptable salt, gramicidin C-4-1 and/or C-4-2, and gramicidin C-5 (% wt. of the total content cyclic decapeptides from the gramicidin C family):

Gramicidin C - 91.5-99.9 wt%;

Gramicidin C-4-1 and/or C-4-2 - 0-3.5% wt.;

Gramicidin C-5 - 0-5.0% of the mass.

The subject of the present invention is also a drug for the treatment of infectious and inflammatory diseases, containing gramicidin C or its pharmaceutically acceptable salt gramicidin C-4-1 and/or C-4-2 in the following amount (% wt. of the total content of cyclic decapeptides from the family gramicidin C):

Gramicidin C - 96.5-99.9 wt%;

Gramicidin C-4-1 and/or C-4-2 - 0-3.5% wt.

The subject of the present invention is also a drug for the treatment of infectious and inflammatory diseases, containing gramicidin C or a pharmaceutically acceptable salt thereof and gramicidin C-5 in the following amount (% wt. of the total content of cyclic decapeptides from the gramicidin C family):

Gramicidin C - 95.0-99.9 wt%;

Gramicidin C-5 - 0-5.0% of the mass.

The subject of the present invention is also a process for the separation of cyclic decapeptides from the gramicidin C family in substances, based on the HPLC method, comprising the following steps:

- preparation of a buffer solution with pH 2.5 by mixing lithium perchlorate, water, phosphoric acid and sodium hydroxide;

- preparation of a solvent by mixing a buffer solution with pH 2.5 and acetonitrile in a ratio of 1:1 (o:o);

- preparation of the test solution by dissolving the powder of the substance in a solvent;

- separation of cyclic decapeptides from the gramicidin C family in substances by HPLC using a Kinetex® C18 column (2.6 µm; 100 x 4.6 mm, Phenomenex) at a flow rate of 0.9 ml/min, column temperature (15± 1)°C and with the following gradient mode:

Time,
min Movable
phase A (buffer solution pH 2.5), % Movable
phase B (acetonitrile for liquid chromatography), % 0 -…18 52 => 40 48 => 60 18 -…24 40 => 20 60 => 80 24 -…27 20 80 27 -…28 20 => 52 80 => 48 28 -…35 52 48

In one embodiment of the present invention, a method for separating cyclic decapeptides from the gramicidin C family in substances includes determining the content of cyclic decapeptides from the gramicidin C family in the test solution from the obtained chromatograms.

The inventors unexpectedly found that the active pharmaceutical ingredients of gramicidin C and the pharmaceutical compositions of the present invention have:

- increased antibacterial activity against pathogenic bacteria;

- reduced antibacterial activity against non-pathogenic bacteria of normal human microbiota;

- reduced antibacterial activity in relation to non-pathogenic bacteria of the normal microbiota of the human respiratory tract;

- reduced frequency of occurrence and intensity of side effects;

- increased anti-inflammatory activity;

- improved storage stability;

- reduced hygroscopicity.

Thus, the technical results of the present invention are:

- expansion of the arsenal of gramicidin C drugs;

- increased antibacterial activity against pathogenic bacteria in the active pharmaceutical ingredients and pharmaceutical compositions of the present invention;

- reduced antibacterial activity against non-pathogenic bacteria of the normal human microbiota in the active pharmaceutical ingredients and pharmaceutical compositions of the present invention;

- reduced antibacterial activity against non-pathogenic bacteria of the normal microbiota of the human respiratory tract in active pharmaceutical substances and pharmaceutical compositions of the present invention;

- reduced incidence and intensity of side effects;

- increased anti-inflammatory activity of the active pharmaceutical ingredients and pharmaceutical compositions of the present invention;

- improved storage stability of active pharmaceutical ingredients and pharmaceutical compositions of the present invention;

- reduced hygroscopicity of active pharmaceutical ingredients and pharmaceutical compositions of the present invention.

In one embodiment of the present invention, the content of cyclic decapeptides from the gramicidin C family in the composition of active pharmaceutical ingredients and/or compositions (in % wt.) decapeptide is divided by the sum of the areas of all considered peaks and multiplied by 100%. When determining the content of cyclic decapeptides from the gramicidin C family, a chromatographic system is used that is suitable for the separation of gramicidin C, gramicidins of the C-4 group and gramicidin C-5.

In one embodiment of the present invention, the minimum detectable amount of cyclic decapeptide, determined by HPLC, is 0.5% of the mass.

In one embodiment of the present invention, the gramicidin C substances may further comprise gramicidin C-2 and/or gramicidin C-3.

In one embodiment of the present invention, the gramicidin C substances contain gramicidin C in an amount of 85.0-99.9% by weight. from the total content of cyclic decapeptides from the gramicidin C family in the substance.

In one of the embodiments of the present invention, the infectious-inflammatory disease is selected from the group consisting of tonsillitis, periodontal disease, pharyngitis, tonsillitis, periodontitis, gingivitis, stomatitis, glossitis, adenoiditis, laryngitis.

In one embodiment of the present invention, the inflammatory disease refers to inflammatory diseases of the mouth and throat.

In one embodiment of the present invention, the pharmaceutical composition is in the form of a topical solution.

In one embodiment of the present invention, the pharmaceutical composition is in the form of a spray.

In one embodiment of the present invention, the pharmaceutical composition in the form of a topical solution is applied after meals, topically by irrigation or by spraying onto the mucous membranes of the mouth and throat.

In one embodiment of the present invention, when using a pharmaceutical composition in the form of a spray, for maximum therapeutic effect, rinse the throat well. Immediately after using the drug, you should refrain from eating and drinking for 1 hour.

In one embodiment of the present invention, when using a pharmaceutical composition in the form of a spray, the dosage is 3-4 injections (1 injection = 200 μl, 0.3 mg/ml gramicidin C) per application, 3-4 times a day for 3-7 days.

In one embodiment of the present invention, the pharmaceutical composition is in the form of a tablet.

In one embodiment of the present invention, the pharmaceutical composition is in the form of a buccal tablet.

In one embodiment of the present invention, the pharmaceutical composition is in the form of a lozenge.

In one embodiment of the present invention, the pharmaceutical composition in the form of a lozenge is administered after a meal by dissolving in the mouth without chewing. Immediately after using the drug, you should refrain from eating and drinking for 1-2 hours.

In one embodiment of the present invention, when using a pharmaceutical composition in the form of a lozenge, the dosage is 1 tablet (3 mg of gramicidin C) 1-4 times a day for 7 days.

In one embodiment of the present invention, the pharmaceutical composition contains an antiseptic. Cetylpyridinium, benzalkonium, benzethonium, cetalkonium chloride, cetrimonium chloride, cetrimide, chlorhexidine, didecyldimethylammonium chloride, domifene bromide, dophanium chloride, tetraethylammonium bromide, etc. are used as an antiseptic. The group is not limited to this list.

In one embodiment of the present invention, the pharmaceutical composition contains an anesthetic. As an anesthetic, lidocaine, trimecaine, ultracaine, mepivacaine, procaine, dicaine, benzocaine, oxybuprocaine, bupivacaine are used. The group is not exhausted by this list.

In one embodiment of the present invention, the pharmaceutical compositions of the present invention further comprise cetylpyridinium chloride and/or oxybuprocaine hydrochloride.

In one of the embodiments of the present invention, the pharmaceutical compositions of the present invention can be administered (but not limited to) orally, inhalation, topically.

Dosage forms of the pharmaceutical compositions of the present invention include, but are not limited to: tablets, in particular buccal tablets, effervescent tablets, orodispersible tablets, and the like; solutions, aerosols, sprays, and other dosage forms.

The therapeutic dosage of the active pharmaceutical substance or pharmaceutical composition of the present invention containing gramicidin C in a therapeutically effective amount in patients may be adjusted depending on the therapeutic efficacy, age, sex and stage of the disease of the patient.

The following are definitions of terms that are used in the description of the present invention.

Unless otherwise indicated, all technical and technical terms used in this context have the meaning generally accepted in the art.

The term "medicinal principle", "drug substance", "drug substance" means a physiologically active substance (active substance) of synthetic or other (biotechnological, plant, animal, microbial and other) origin, which has pharmacological activity and is the active principle of the pharmaceutical composition used for the production and manufacture of a medicinal product (means).

The term “drug” means a substance (or combination of substances) that comes into contact with the human or animal body, penetrates the organs and tissues of the human or animal body, and is intended to restore, correct or change physiological functions in humans and animals, as well as to treatment and prevention of diseases. Medicinal products include pharmaceutical substances and drugs.

The term "drug", "preparation" means a medicinal product in the form of a dosage form used for the treatment and prevention of a disease.

The term "dosage form" means the state of the medicinal product, corresponding to the methods of its administration and use and ensuring the achievement of the desired therapeutic effect.

The term "pharmaceutically acceptable excipient" in the context of the present invention characterizes substances of inorganic or organic origin used in the manufacturing process, the manufacture of drugs to give them the necessary physical and chemical properties.

"Pharmaceutical composition" in the context of the present invention means a composition comprising an active substance and at least one of the pharmaceutically acceptable excipients used in the art, where the "pharmaceutically acceptable excipient" in the context of the present invention is selected from the group, consisting of pharmaceutically acceptable and pharmacologically compatible carriers, adjuvants such as solvents, diluents, preservatives, stabilizers, fillers, disintegrants, humectants, emulsifiers, suspending agents, thickeners, sweeteners, flavoring agents, flavoring agents, antibacterial agents, fungicides, lubricants, slow release regulators delivery agents, antiseptics, anesthetics, solubilizers, buffer agents, the choice and ratio of which depends on their nature, method of administration of the composition and dosage. Protection against the action of microorganisms can be provided with a variety of antibacterial and antifungal agents, such as benzyl alcohol, urotropine, ethylenediaminetetraacetic acid, benzoic acid, chlorobutanol, sorbic acid, parabens, alkylpyridinium, benzethonium and their pharmaceutically acceptable salts and the like. The pharmaceutical composition may also include isotonic agents such as sugars, sodium chloride and the like. Prolonged action of the composition can be achieved with agents that slow down the absorption of the active principle, such as hydrophilic polymeric release retardants, for example, cellulose derivatives, in particular hydroxypropyl methylcellulose, polyethylene oxide, gelatin, polyvinyl alcohol, polyvinylpyrrolidone, alginates, carbomers, hydrophobic release retarders such as glyceryl behenate, aluminum monostearate. Examples of suitable carriers, solvents and diluents are water, ethanol, polyalcohols, buffer solutions and mixtures thereof, vegetable oils (such as, for example, olive oil) and organic esters (such as, for example, ethyl oleate). Examples of fillers are lactose, microcrystalline cellulose, sodium citrate, calcium carbonate, calcium phosphate and the like. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and polyethylene glycol. Examples of sliding agents are colloidal silicon dioxide, starch, talc and the like.

The term "filler" or "diluent" means excipients used to give solid dosage forms a given volume or mass. Starch, glucose, sucrose, lactose (milk sugar), basic magnesium carbonate, magnesium oxide, sodium chloride, sodium bicarbonate, white clay (kaolin), gelatin, microcrystalline cellulose (MCC), methylcellulose (MC) can be used as fillers. sodium carboxymethyl cellulose (Na CMC), calcium carbonate, dibasic calcium phosphate, glycine (aminoacetic acid), dextrin, amylopectin, ultraamyl pectin, sorbitol, mannitol, pectin, etc. The group is not limited to this list.

The term "binders" means substances that are part of the tableting mass to give it the necessary viscosity. Water, ethyl alcohol, starch paste, sugar syrup, solutions are used as binders: carboxymethylcellulose (CMC), hydroxyethylcellulose (OEC), hydroxypropylmethylcellulose (OPMC); polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), alginic acid, sodium alginate, gelatin, etc. The group is not limited to this list.

The term "disintegrant" means substances used to improve disintegration or dissolution by providing mechanical disintegration of tablets in a liquid medium, which is necessary for the rapid release of the active substance. The disintegrant may be one or more of microcrystalline cellulose, croscarmellose sodium, crospovidone, sodium starch glycolate, starch, pectin, gelatin, amylopectin, ultramylopectin, agar-agar, alginic acid, potassium and sodium alginate, tween-80, and others. The group is not exhausted by this list.

The term "glidant" means adjuvants used in the tablet manufacturing process at the compression stage to improve the flow of granules or powder by reducing friction between particles. The lubricant may be one or more of starch, talc, polyethylene oxide-4000, stearic acid, calcium and magnesium stearate, and others. The group is not limited to this list.

The term "lubricant" or "lubricant" means excipients that help reduce the friction force between the tablet surface and the walls of the punch cell in which the tablet is formed, used in the technological process of tablet production at the pressing stage. The lubricant may be one or more of magnesium stearate, calcium stearate, sodium stearyl fumarate, polyethylene glycol (over 3350 molecular weight), sodium lauryl sulfate, talc, mineral oil, leucine and poloxamer, etc. The group is not limited to this list.

The term "antiseptic" means quaternary ammonium salts, as well as other compounds: cetylpyridinium, benzalkonium, benzethonium, cetalkonium chloride, cetrimonium chloride, cetrimide, chlorhexidine, didecyldimethylammonium chloride, domifene bromide, dophanium chloride, tetraethylammonium bromide. The group is not exhausted by this list.

The term "anesthetic" means lidocaine, trimecaine, ultracaine, mepivacaine, procaine, dicaine, benzocaine, oxybuprocaine, bupivacaine. The group is not exhausted by this list.

The term "flavoring" means aromatic substances related to flavorings artificial, identical to natural or natural origin.

The term "moisturizer" also means a thickener to slow down the hydrolysis of components; as a moisturizer, you can use: propylene glycol, hexylene glycol, butylene glycol, glyceryl triacetate (aka triacetin, if I'm not mistaken), neoagarobiose, sorbitol, xylitol, maltitol, maltol. The group is not exhausted by this list.

The term "sweetener" means sucralose, acesulfame potassium, sodium saccharinate. The group is not exhausted by this list.

The term "preservative" means propyl paraben, methyl paraben, benzoic acid, ethyl paraben, sodium benzoate, potassium. The group is not exhausted by this list.

The term "solubilizer" means polysorbate, a surfactant, in the presence of which sparingly soluble antibiotics dissolve better. The group includes pluronics, polyethylglycols. The group is not exhausted by this list.

The term "pharmaceutically acceptable" in the context of the present invention means that the substance or composition to which this term is applied must be compatible from the point of view of chemistry and / or toxicology with other ingredients that make up the drug, and are safe for the person who treat (relieve symptoms) with the substance or composition.

The expression "is in solid form" in this context characterizes pharmaceutical compositions and medicines of a solid, dense or viscous consistency, in which the particles cannot move freely, but can only make small vibrations around the equilibrium position, while the particles actively interact with each other.

The term active pharmaceutical ingredient (API) in the context of the present invention means a synthesis product containing any substance or mixture of substances of synthetic or other (biotechnological, plant, animal, microbial and other) origin, intended for the production of medicines, which in the process of manufacturing a medicine ( drug) becomes the active ingredient of that drug. Such products are intended to exhibit pharmacological activity or other direct effect in the diagnosis, treatment, symptomatic relief or prevention of disease, or to affect the structure or function of the body.

The term "pharmaceutically acceptable salts" or "salts" includes salts of active compounds that are prepared with relatively non-toxic acids. Examples of pharmaceutically acceptable non-toxic salts are salts formed with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids, or with organic acids such as acetic, oxalic, maleic, tartaric, succinic, citric or malonic acids, or obtained by others. methods used in this area. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanate, hexanate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate (mesylate), 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, hemi-fumarate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate (tosylate), undecanate, valeriate, and the like.

The terms "comprising", "comprises" in the context of the present invention means that the specified pharmaceutical compositions and medicinal products include the listed components, but do not exclude the inclusion of other components.

The term "effective amount" in the context of the present invention refers to the amount of a pharmaceutical composition or drug that, when administered to a subject for the treatment of a disease or at least one of the clinical symptoms of the disease or disorder, is sufficient to effect such treatment on the disease, disorder or symptom. An "effective amount" may vary depending on the disease, disorder and/or symptoms of the disease or disorder, the severity of the disease, disorder and/or symptoms of the disease, the age of the subject being treated, and/or the weight of the subject being treated. The proper amount in any particular case will be apparent to one of skill in the art, or can be determined by routine experimentation.

The term "dosage" used here characterizes the content of one or more active substances in quantitative terms per dose unit, or volume unit, or mass unit in accordance with the dosage form, or for some types of dosage forms, the amount of active substance released from the dosage form per unit time .

The terms "treatment", "therapy" cover the treatment of pathological conditions in mammals, preferably in humans, and include: a) reduction, b) blocking (suspension) of the course of the disease, c) alleviation of the severity of the disease, i. induction of regression of the disease, d) reversal of the disease or condition to which the term is applied, or one or more symptoms of the disease or condition.

The term "prophylaxis", "prevention" encompasses the elimination of risk factors as well as the prophylactic treatment of subclinical stages of the disease in mammals, preferably in humans, aimed at reducing the likelihood of occurrence of clinical stages of the disease. Patients for prophylactic therapy are selected on the basis of factors that, based on known data, entail an increased risk of clinical stages of the disease compared with the general population. Preventive therapy includes: a) primary prevention and b) secondary prevention. Primary prevention is defined as prophylactic treatment in patients who have not yet reached the clinical stage of the disease. Secondary prevention is the prevention of the recurrence of the same or a closely related clinical condition or disease.

The term "HPLC" refers to a high-performance liquid chromatography method, one of the effective methods for separating complex mixtures of substances, which is widely used in both analytical chemistry and chemical technology.

The term "PPHS solution" refers to a chromatographic system suitability test solution used in the process of determining the content of cyclic decapeptides from the gramicidin C family in substances by HPLC.

The term "SPChS solution" refers to a solution for assessing the sensitivity of a chromatographic system used in the process of determining the content of cyclic decapeptides from the gramicidin C family in substances by HPLC.

Unless otherwise indicated, all technical and technical terms used in this context have the meaning generally accepted in the art.

Implementation of the invention

The present invention is further illustrated, but not limited, by the following examples, which illustrate the present invention.

Example 1 Preparation and determination of new cyclic decapeptides from the gramicidin C family

New cyclic decapeptides from the gramicidin C family (gramicidins C-4-1, C-4-2 and C-5) were isolated and identified from the gramicidin C substance obtained by cultivating a mutant strain derived from the Aneurinibacillus migulanus strain VKPMV-10212 (RU2627182, 08/03/2017).

The mutant strain was obtained as follows.

The original strain of Aneurinibacillus migulanus VKPMV-10212, producing gramicidin C, was grown on a standard liquid semi-synthetic medium containing yeast autolysate in shaking flasks with a capacity of 0.75 l (0.1 l of the medium) on a circular shaker (speed 220 rpm, amplitude 1 m) at 37°C for 48 hours. The cells were then sequentially treated with rubomycin and acridine orange for 20 minutes each by sequentially adding the indicated mutagens to the cells at final concentrations of 100 and 40 μg/ml, respectively.

The cells treated with mutagens were placed on agarized Gauze-Brazhnikova medium in Petri dishes (50 µl of cell suspension per 1 Petri dish). The cells were then incubated at 37°C for 24 hours. At the end of the incubation, the resulting cell colonies were examined and those that were morphologically different from the original strain were selected.

As a result of the mutagenesis, seven cell colonies were selected. Next, for each selected mutant colony, the yield of gramicidin C was determined.

Gramicidin C was extracted with ethanol:HCl (2:1 ratio, respectively). For the quantitative determination of gramicidin C in the obtained substances of gramicidin C, the HPLC method was used.

The analysis of HPFA showed that in the chromatogram of the gramicidin C substance obtained by cultivating one of the obtained mutant strains, peaks are detected that differ from the peaks of gramicidins C, C-2 and C-3 known from the literature.

Further, HPLC technique compatible with mass spectrometric detection was used for analysis. A 0.1% formic acid solution (pH 2.7) was chosen as the aqueous phase compatible with mass spectrometry. Substances with chromatographic peaks other than those of gramicidins C, C-2 and C-3 were extracted and the collected samples were analyzed by chromatography-mass spectrometry to obtain MS and MS/MS spectra for the collected substances. To obtain the spectra, ions were first determined, the release time of which coincided with the release time of the substances of interest (MS1). Then these ions were subjected to fragmentation and the resulting fragments were detected (MS/MS). Based on the analysis of the fragments, the isolated substances were identified, which belonged to cyclic decapeptides not previously described in the literature. To determine the structure of the isolated cyclic decapeptides, their hydrolysis was also carried out, followed by a chromato-mass-spectrometric analysis of the hydrolysis products. To determine the amino acid substitutions found in the isolated decapeptides compared to the structure of gramicidin C, extracted ion chromatograms were constructed for amino acids and their homologues.

As a result of the analysis, the following amino acid substitutions in the isolated decapeptides were determined in comparison with the structure of gramicidin C:

1) the leucine residue was replaced by a 2-amino-5-methylhexanoic acid residue (the decapeptide was given the name gramicidin C-4-1);

2) the leucine residue was replaced by a β-homoleucine residue (the decapeptide was given the name gramicidin C-4-2);

3) the ornithine residue was replaced by the α-methyl-L-ornithine residue (the decapeptide was given the name gramicidin C-5).

To check the correctness of determining the structures of the obtained decapeptides, three reference substances were synthesized by peptide synthesis:

- substance No. 1, having the proposed structure of gramicidin C-4-1: cyclo (-Val-Orn-Leu-D-Phe-Pro-Val-Orn-2-amino-(5-methylhexanoic acid)-D-Phe-Pro -);

- substance No. 2, having the proposed structure of gramicidin C-4-2: cyclo (-Val-Orn-Leu-D-Phe-Pro-Val-Orn-β-homoleucine-D-Phe-Pro-);

- substance No. 3, having the proposed structure of gramicidin C-5: cyclo(-Val-α-methyl-L-ornithine-Leu-D-Phe-Pro-Val-Orn-Leu-D-Phe-Pro-).

Chromatography-mass-spectrometric analysis of the reference substances confirmed the identity of the structures of the reference substances and new cyclic decapeptides - gramicidins C-4-1, C-4-2, C-5, isolated from the gramicidin C substance of the mutant strain. The results obtained confirmed the correctness of the determination of the structures of gramicidins C-4-1, C-4-2, C-5.

When studying the obtained strain, the quantitative content of gramicidins C-4-1, C-4-2, C-5 in the substance of gramicidin C obtained during its cultivation was also measured by HPLC using a chromatographic system suitable for separating gramicidin C, gramicidins of the group C-4 and gramicidin C-5.

Before HPLC analysis, the gramicidin C substance was separated from the culture liquid and biomass.

The results obtained showed that, depending on the conditions of cultivation of the strain (variation of the optical density of the culture, temperature, illumination, composition and type of medium, cultivation duration, fermenter volume, pH, oxygen level, etc.) strain, the following amounts of gramicidin group C-4 and gramicidin C-5 are found:

1) gramicidin C-4-1 or C-4-2 - 3.5% of the mass. or less;

2) gramicidin C-5 - 5.0% of the mass. or less.

At the same time, regardless of the cultivation conditions, the substance of gramicidin C obtained by cultivating the obtained mutant strain always contained amounts of gramicidin C-5 and/or one of the gramicidins of the C-4 group: C-4-1 or C-4-2 detected by HPLC. . The minimum detectable amount of cyclic decapeptide using the HPLC method was determined as 0.5% of the mass.

Also, regardless of the cultivation conditions, the substance of gramicidin C, obtained by cultivating the obtained mutant strain, always contained no more than 3.5% of the mass. gramicidin C-4-1 or C-4-2 and not more than 5.0% of the mass. gramicidin C-5.

Example 2 HPLC Method Using a Chromatographic System Suitable for the Separation of Gramicidin C, Group C-4 Gramicidins, and Gramicidin C-5

To separate and determine the content of cyclic decapeptides from the gramicidin C family by HPLC, the following reagents were used: acetonitrile for liquid chromatography; phosphoric acid concentrated; lithium perchlorate; sodium hydroxide.

As a buffer solution, a solution with pH 2.5 was used, which was prepared as follows: 6.00 g of lithium perchlorate was placed in a volumetric flask with a capacity of 1000 ml, 900 ml of water for chromatography was added, stirred until the salt was completely dissolved, 1.0 ml of phosphoric acid was added concentrated acid, stirred, adjusted the pH of the resulting solution to 2.5 ± 0.05 using a 30% sodium hydroxide solution, brought the volume of the solution with water for chromatography to 1000 ml, stirred and filtered through a membrane filter with a pore diameter of 0.45 μm.

The solvent used was a solution prepared as follows: sufficient amounts of pH 2.5 buffer solution and acetonitrile were mixed in a 1:1 (v/v) ratio.

The test solution was prepared as follows: 25.0 mg of the powder of the substance was placed in a volumetric flask with a capacity of 25 ml, dissolved in 20 ml of the solvent, the volume of the solution was brought up to the mark with the solvent and mixed. 1.0 ml of the obtained solution was transferred into a volumetric flask with a capacity of 10 ml, the volume of the solution was adjusted to the mark with the solvent and stirred.

As a standard solution, a solution was used that was prepared as follows: 25.0 mg of a standard sample of gramicidin C dihydrochloride (a sample containing detectable amounts of gramicidin C, gramicidin C-4-1 or C-4-2, gramicidin C-5 ) was placed in a volumetric flask with a capacity of 25 ml, dissolved in 20 ml of the solvent, the volume of the solution was brought up to the mark with the solvent, and mixed. 1.0 ml of the obtained solution was transferred into a volumetric flask with a capacity of 10 ml, the volume of the solution was adjusted to the mark with the solvent and stirred.

As a solution for checking the suitability of the chromatographic system (solution for PPHS), a solution was used, which was prepared as follows: 1.0 ml of the standard solution was placed in a 20.0 ml volumetric flask, the volume of the solution was brought up to the mark with the solvent and stirred.

As a solution for assessing the sensitivity of the chromatographic system (solution for SPChS), a solution was used, which was prepared as follows: 1.0 ml of a solution for PPHS was transferred into a volumetric flask with a capacity of 50.0 ml, the volume of the solution was brought up to the mark with a solvent and stirred.

To separate and determine the content of cyclic decapeptides from the gramicidin C family by HPLC, the following chromatographic conditions were used:

Column - "Kinetex ^® C18", 2.6 microns; 100 × 4.6 mm Phenomenex, mobile phase - A: buffer solution pH 2.5,
B: acetonitrile for liquid chromatography; gradient mode Flow rate - 0.9 ml/min; Column temperature - (15±1)°С; Sample compartment temperature - (20±3)°С; Detector
Injection volume - spectrophotometric or diode-matrix, 210 nm;
- 20 µl.

To separate and determine the content of cyclic decapeptides from the gramicidin C family by HPLC, the solvent (2 injections), solution for SPChS (1 injection), solution for PPCS (1 injection), standard solution (3 injections) and the test solution (3 injections) were sequentially chromatographed .

The retention time of the gramicidin C peak was determined to be about 17 min, the relative retention time of the C-4 group gramicidin peak relative to the gramicidin C peak was about 1.20, and the relative retention time of the gramicidin C-5 peak was about 0.95.

The exact retention times of the peaks of gramicidins C, C-4-1, C-4-2, C-5 were determined from the chromatogram of the solution for PPHS. A typical chromatogram of a solution for PPHS (a solution containing detectable amounts of gramicidin C, gramicidin C-4-1 or C-4-2, gramicidin C-5) is obtained by HPLC using a chromatographic system suitable for separating gramicidin C, gramicidins of the C-4 group and gramicidin C-5.

To determine the content of cyclic decapeptides from the gramicidin C family, HPLC did not take into account peaks from the solvent, peaks up to 7.0 min (substances not related to cyclic decapeptides from the gramicidin C family), peaks after 25 min (return of the system to its original state) and peaks with an area less than the area of the peak of gramicidin C on the chromatogram of the solution for PTCS (less than 0.10%).

To determine the content of cyclic decapeptides from the gramicidin C family by HPLC, the content of the target decapeptide (i) in percent (Xi) was calculated by the formula:

where S _i is the peak area of the target decapeptide i on the chromatogram of the test solution (average value over three chromatograms);

ΣS _i is the sum of the areas of all peaks of decapeptides from the gramicidin C family on the chromatogram of the test solution (average value over three chromatograms).

Example 3 Active Pharmaceutical Ingredients of the Present Invention

To study the properties of new substances of gramicidin C, compositions 1-14 (Table 1) were obtained by adding a certain amount of gramicidins C-4-1, C-4-2, C-5, synthesized by peptide synthesis, to the purified substance of gramicidin C, containing only gramicidin C-1.

The content of gramicidins C, C-4-1, C-4-2 and C-5 in the obtained compositions of substances are shown in Table 1.

Table 1.
Compositions of active pharmaceutical ingredients according to the present invention indicating the ratio of gramicidins C (in wt %) composition number Gramicidin C,
% wt. Gramicidin C-4-1, % wt. Gramicidin C-4-2, % wt. Gramicidin C-5, % wt. Composition 1 99.0 0.5 0 0.5 Composition 2 96.0 3.5 0 0.5 Composition 3 94.5 0.5 0 5.0 Composition 4 91.5 3.5 0 5.0 Composition 5 99.0 0 0.5 0.5 Composition 6 96.0 0 3.5 0.5 Composition 7 94.5 0 0.5 5.0 Composition 8 91.5 0 3.5 5.0 Composition 9 99.5 0 0 0.5 Composition 10 95.0 0 0 5.0 Composition 11 99.5 0.5 0 0 Composition 12 96.5 3.5 0 0 Composition 13 99.5 0 0.5 0 Composition 14 96.5 0 3.5 0

Example 4 In Vitro Antibacterial Activity of the Active Pharmaceutical Substances of the Present Invention

For the active pharmaceutical ingredients obtained in example 3 (compositions 1-14), their antibacterial activity in vitro was determined according to the following method: the minimum inhibitory concentration (in μg/ml) of each of the compositions 1-14, which completely inhibited the growth of the test bacterial strain on agarized meat-peptone medium.

The minimum inhibitory concentration was determined by the method of serial microdilutions of the starting substances and the addition of each microdilution to the tested bacterial strain. The minimum inhibitory concentration was determined as the last dilution in which there was no growth of the test bacterial strain.

As a test bacterial strain, a strain of Bacillus cereus var. Mycoides 537.

As reference compositions, substances were prepared that either contain gramicidin C-4-1 or C-4-2 in an amount of more than 3.5% of the mass. and/or gramicidin C-5 more than 5% of the mass. (compositions 15-21), or do not contain gramicidins C-4-1, C-4-2 and C-5 (compositions 22, 23). Comparison compositions are shown in Table 2.

Gramicidins C-2 and C-3 in composition 22 were obtained according to the method described in [Nozaki, S., & Muramatsu, I. (1985). Natural Homologs of Gramicidin S. II. Synthesis of Gramicidin S-2 and S-3. Bulletin of the Chemical Society of Japan, 58(1), 331-335].

Gramicidin C in formulation 23 was prepared according to the procedure described in [Bu, X., Wu, X., Ng, N. L. J., Mak, C. K., Qin, C., & Guo, Z. (2004). Synthesis of Gramicidin S and Its Analogues via an On-Resin Macrolactamization Assisted by a Predisposed Conformation of the Linear Precursors. The Journal of Organic Chemistry, 69(8), 2681-2685].

Table 2.
Comparison compositions indicating the ratio of gramicidins C (in wt %) Compound
No. Gramicidin C,
% Gramicidin C-2, % wt. Gramicidin C-3, % wt. Gramicidin C-4-1, % wt. Gramicidin C-4-2, % wt. Gramicidin C-5, % wt. Composition 15 90.5 0 0 2.5 0 7 Composition 16 92.5 0 0 0 1.5 6 Composition 17 89 0 0 1 0 10 Composition 18 91.5 0 0 7 0 1.5 Composition 19 90.5 0 0 0 8.5 1 Composition 20 94 0 0 4 0 2 Composition 21 90.5 0 0 0 4 5.5 Composition 22 95 3 2 0 0 0 Composition 23 100 0 0 0 0 0

The obtained antibacterial activity of the pharmaceutical substances of the present invention and reference formulations is shown in Table 3.

Table 3
In vitro antibacterial activity of the pharmaceutical substances of the present invention (compositions 1-14) and reference compounds (compositions 15-23) (minimum inhibitory concentration (in µg/ml) against Bacillus cereus var. Mycoides 537 is given) Compound Antibacterial activity (minimum inhibitory concentration in µg/ml) 1 6.5 2 4.3 3 5.8 4 6.3 5 5.0 6 3.8 7 5.5 8 6.0 9 6.8 10 7.0 eleven 6.8 12 6.5 13 6.5 14 6.3 15 8.0 16 8.5 17 8.0 18 8.8 19 8.3 20 9.0 21 8.8 22 13.5 23 14.3

Table 3 shows that the active pharmaceutical ingredients of the present invention have lower minimum inhibitory concentration values than the reference formulations, i.e. the substances of the present invention have higher antibacterial activity than the reference formulations.

Thus, the content of gramicidin C-4-1 or C-4-2 in an amount of not more than 3.5% of the mass. and gramicidin C-5 in an amount of not more than 5.0% of the mass. in substance gramicidin C leads to an unexpected improvement in their antibacterial properties against pathogenic bacteria in vitro. At the same time, reference substances that do not contain gramicidins C-4 and C-5 are characterized by reduced antibacterial activity against pathogenic bacteria in vitro compared to the substances of the present invention.

The active pharmaceutical ingredients of the present invention also have surprisingly higher minimum inhibitory concentration values with respect to strains of non-pathogenic bacteria in the microbiota of healthy people than the reference formulations, i.e. the substances of the present invention have lower antibacterial activity against non-pathogenic bacteria than the reference formulations. The found selectivity of the antibacterial action of the substances according to the present invention is a great advantage of the claimed invention, since it provides:

(1) enhanced antibacterial effect against pathogenic microorganisms,

(2) a simultaneous reduction in the incidence and intensity of side effects associated with a negative effect on the normal microbiota of the digestive and respiratory systems.

For the substances of the present invention obtained by culturing the mutant strain from Example 1, the results of antibacterial activity were similar to those indicated in Table 3.

In addition, gramicidin compounds C-4-1, C-4-2, C-5 showed independent antibacterial activity against pathogenic bacteria.

Example 5 Hemolytic Activity of Active Pharmaceutical Ingredients of the Present Invention

For the active pharmaceutical ingredients of the present invention obtained in example 3 (compositions 1-14) and the comparative compositions obtained in example 4 (compositions 15-23), their hemolytic activity was determined.

The study of hemolytic activity was carried out in the following way.

Freshly collected human blood with heparin was centrifuged to remove leukocytes, the resulting erythrocytes were washed three times with saline and stored at 4°C. Serial dilutions of formulations 1-23 in saline were prepared in round bottom microtiter plates using 100 μl volumes. Erythrocytes were diluted with saline at a ratio of 1:25 (cell volume: volume of saline). The resulting suspensions were added 50 μl to each well with dilutions of compositions 1-23. The plates were incubated by shaking at 37° C. and the concentration required for complete lysis was determined visually after 4 hours. The results obtained are shown in Table 4.

Table 4
Hemolytic activity of pharmaceutical substances of the present invention (compositions 1-14) and reference compounds (compositions 15-23) Compound Hemolytic activity (concentration in mcg / ml required for 100% lysis of red blood cells) 1 15.5 2 18.0 3 17.5 4 17.0 5 16.5 6 17.5 7 15.0 8 16.0 9 18.0 10 16.5 eleven 18.0 12 17.5 13 18.0 14 16.0 15 11.5 16 11.5 17 10.0 18 11.0 19 10.5 20 10.5 21 11.0 22 13.0 23 12.5

It can be seen from Table 4 that the substances of the present invention (compositions 1-14) unexpectedly have higher concentrations required for lysis of 100% of red blood cells, that is, they have reduced hemolytic activity compared to the reference compounds (compositions 15-23). Reduced hemolytic activity is a great advantage of the substances of the present invention, as it reduces the frequency and intensity of side effects.

Thus, the content of gramicidin C-4-1 or C-4-2 in an amount of not more than 3.5% of the mass. and/or gramicidin C-5 in an amount of not more than 5.0% of the mass. in the substance of gramicidin C leads to an unexpected improvement in its properties, namely, to a decrease in hemolytic activity. At the same time, reference substances that do not contain gramicidin C-4 and C-5 or contain gramicidin C-4-1 or C-4-2 in an amount of more than 3.5% of the mass. or gramicidin C-5 in an amount of more than 5.0% wt., are characterized by increased hemolytic activity compared to the substances of the present invention.

For the substances of the present invention, obtained by culturing the mutant strain from Example 1, the results of hemolytic activity were similar to those indicated in Table 4.

Example 6 Pharmaceutical compositions of the present invention

Based on the active pharmaceutical ingredients of gramicidin C obtained in example 3 (compositions 1-14, table 1), a pharmaceutical composition was prepared in the form of a spray of the following composition:

Gramicidin C (any of Formulations 1-14 of Table 1) 0.027-0.033% wt. Ethanol 96% 7.3-10.9% wt. Sucralose 0.09-0.11% wt. Glycerol 14.9-18.2% wt. Mint flavor 0.37-0.44% wt. Citric acid monohydrate 0.026-0.032% wt. sodium citrate 0.0099-0.012% wt. Polysorbate 80 0.18-0.22% wt. Methyl parahydroxybenzoate (methylparaben) 0.0736-0.1104% wt. Propyl parahydroxybenzoate (propylparaben) 0.0080-0.0120% wt. Purified water rest

The specified pharmaceutical composition in the form of a spray was prepared in the following way: glycerol was added to purified water, stirred, then citric acid monohydrate, sodium citrate and sucralose were added, stirred until complete dissolution. Separately, a solution of gramicidin C was prepared by mixing purified water, ethanol 96% and polysorbate 80, adding gramicidin C, methyl parahydroxybenzoate, propyl parahydroxybenzoate and stirring until complete dissolution, followed by the addition of mint flavor and stirring. The resulting solutions were stirred until complete homogenization and filtered.

Based on the active pharmaceutical ingredients of gramicidin C obtained in example 3 (compositions 1-14, table 1), a pharmaceutical composition was prepared in the form of a tablet for buccal administration of the following composition:

Gramicidin C (any of Formulations 1-14 of Table 1) 0.2-0.4% wt. Powdered sugar 48.1-48.4% wt. milk sugar 48.1-48.4% wt. Methyl cellulose MTs-100 0.2-0.4% wt. calcium stearate 2.8-3% wt.

The specified pharmaceutical composition in the form of a tablet for buccal administration was prepared in the following way: refined sugar in the form of powder and lactose at a ratio of 1:1 were sieved through a 38 sieve, mixed, moistened with a mixture of solutions from a 5% aqueous solution of methylcellulose and an alcoholic solution of gramicidin C. The moistened mass was granulated through a sieve with D-1.2 mm, dried in air or in a dryer at a temperature not exceeding 40°C to a moisture content of 1.5-2.5%. Upon completion of drying, the mass was passed through a granulator with a drum opening diameter of 1–1.2 mm. The resulting granulate was powdered with calcium stearate and tableted.

The pharmaceutical compositions of the present invention are by no means limited to the examples presented, which are provided merely as an illustration of the present invention.

The present invention is by no means limited to the examples presented, which are provided merely as an illustration of the present invention.

List of cited literature

RF patent No. 2213558, publ. 10.10.2003.

RF patent No. 2633635, publ. 10/16/2017.

RF patent No. 2604576, publ. 12/10/2016.

RF patent No. 2627423, publ. 08/08/2017.

RF patent No. 2749902, publ. 06/18/2021.

RF patent No. 2627182, publ. 08/03/2017.

Morozova S.V. Pain in the throat: causes and possibilities of drug therapy. BC, Vol. 13, No. 21, 2005, p. 1447-1452.

Polin A.N., Egorov N.S. Structural and functional features of gramicidin C in connection with its antibiotic activity // Antibiotics and Chemotherapy. - 2003. - No. 48 (12). - S. 29-32.

Bu, X., Wu, X., Ng, N. L. J., Mak, C. K., Qin, C., & Guo, Z. (2004). Synthesis of Gramicidin S and Its Analogues via an On-Resin Macrolactamization Assisted by a Predisposed Conformation of the Linear Precursors. The Journal of Organic Chemistry, 69(8), 2681-2685.

Gause, G. G.; Brazhnikova, M. G. Gramicidin S and Its Use in the Treatment of Infected Wounds. Nature 1944, 154, 703.

Kondejewski, L. H.; Farmer, S.W.; Wishart, D. S.; Hancock, R. E. W.; Hodges, R. S. Gramicidin S Is Active against Both GramPositive and Gram-Negative Bacteria. Int. J. Pept. Protein Res. 1996, 47(6), 460-466.

Nozaki, S., & Muramatsu, I. (1985). Natural Homologs of Gramicidin S. II. Synthesis of Gramicidin S-2 and S-3. Bulletin of the Chemical Society of Japan, 58(1), 331-335.

Swingle K., Hamilton R., Harrington J., Kvam D. “3-Benzoyldifluoromethanesulfonanilide, sodium salt (diflumidone sodium, MBR 4164-8): a new anti-inflammatory agent” // Arch. Int. Pharmacodyn, 1971, vol. 189, pp. 129-144.

Claims (44)

1. Cyclic decapeptide with the following structure: Cyclo - (X1–X2-Leu–D-Phe–Pro-X6-Orn-X8-D-Phe–Pro–), where X1 - valine or 2-aminobutyric acid, X2 - ornithine, α-methyl-L-ornithine or 2-aminobutyric acid, X6 - valine or 2-aminobutyric acid, X8 - 2-amino-(5-methylhexanoic acid), β-homoleucine or leucine, or a pharmaceutically acceptable salt thereof, while the cyclic decapeptide is not gramicidin C-1 - cyclo(–Val–Orn–Leu–D-Phe–Pro–Val–Orn–Leu–D-Phe–Pro–); or gramicidin C-2 - cyclo(–Val–Orn–Leu–D-Phe––Pro–Abu–Orn–Leu–D-Phe–Pro–); or gramicidin С-3 - cyclo(–Abu–Orn–Leu–D-Phe––Pro–Abu–Orn–Leu–D-Phe–Pro–). 2. A cyclic decapeptide or a pharmaceutically acceptable salt thereof according to claim 1, having the following structure: cyclo(-Val-Orn-Leu-D-Phe-Pro-Val-Orn-2-amino-(5-methylhexanoic acid)-D- Phe-Pro-). 3. A cyclic decapeptide or a pharmaceutically acceptable salt thereof according to claim 1, having the following structure: cyclo(–Val–Orn–Leu–D-Phe––Pro–Val–Orn–β-homoleucine–D-Phe–Pro–). 4. A cyclic decapeptide or a pharmaceutically acceptable salt thereof according to claim 1, having the following structure: cyclo(–Val–α-methyl-L-ornithine–Leu–D-Phe––Pro–Val–Orn–Leu–D-Phe– Pro–). 5. Active pharmaceutical ingredient intended for the treatment of infectious and inflammatory diseases, containing at least one decapeptide according to any one of paragraphs. 1-4 or a pharmaceutically acceptable salt thereof. 6. An active pharmaceutical ingredient intended for the treatment of infectious and inflammatory diseases, containing gramicidin C or a pharmaceutically acceptable salt thereof and one or more cyclic decapeptides according to paragraphs. 2-4 or their pharmaceutically acceptable salts. 7. The active pharmaceutical ingredient according to claim 6, containing gramicidin C or a pharmaceutically acceptable salt thereof in an amount of 80-99.9% of the mass. 8. Active pharmaceutical substance according to claim 6, containing a cyclic decapeptide or its pharmaceutically acceptable salt according to claim 2 in an amount of not more than 3.5% of the mass. 9. The active pharmaceutical ingredient according to claim 6, containing a cyclic decapeptide or its pharmaceutically acceptable salt according to claim 3 in an amount of not more than 3.5% of the mass. 10. The active pharmaceutical ingredient according to claim 6, containing a cyclic decapeptide or its pharmaceutically acceptable salt according to claim 4 in an amount of not more than 5.0% of the mass. 11. Active pharmaceutical ingredient according to claim 6, additionally containing gramicidin C-2 and/or gramicidin C-3 or their pharmaceutically acceptable salts. 12. Active pharmaceutical ingredient according to claim 6, where gramicidin C is in the form of dihydrochloride. 13. The active pharmaceutical substance according to claim 6, where the infectious and inflammatory disease is selected from the group consisting of tonsillitis, periodontal disease, pharyngitis, tonsillitis, periodontitis, gingivitis, stomatitis, glossitis, adenoiditis, laryngitis. 14. The active pharmaceutical ingredient according to claim 6, containing gramicidin C or its pharmaceutically acceptable salt and cyclic decapeptides or their pharmaceutically acceptable salts in the following amount: Gramicidin C - 91.5-99.9% wt.; a cyclic decapeptide or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 2, 3 - in an amount of not more than 3.5% wt.; And cyclic decapeptide or its pharmaceutically acceptable salt according to claim 4 - in an amount of not more than 5.0% of the mass. 15. Pharmaceutical composition for the treatment of infectious and inflammatory diseases for local use, containing the active pharmaceutical ingredient according to any one of paragraphs. 5-14 in a therapeutically effective amount and pharmaceutically acceptable excipients. 16. Pharmaceutical composition according to claim 15, additionally containing cetylpyridinium chloride and/or oxybuprocaine hydrochloride. 17. The pharmaceutical composition according to claim 15, where the infectious-inflammatory disease is selected from the group consisting of tonsillitis, periodontal disease, pharyngitis, tonsillitis, periodontitis, gingivitis, stomatitis, glossitis, adenoiditis, laryngitis. 18. Pharmaceutical composition according to claim 15, where the composition is in the form of a spray or tablet. 19. The use of a pharmaceutical composition according to paragraphs. 15-18 for the treatment or improvement of an infectious and inflammatory disease of the mouth and throat. 20. The use according to claim 19, where the infectious and inflammatory disease of the oral cavity and throat is selected from the group consisting of tonsillitis, periodontal disease, pharyngitis, tonsillitis, periodontitis, gingivitis, stomatitis, glossitis, adenoiditis, laryngitis. 21. The active pharmaceutical substance of gramicidin C, intended for the treatment of infectious and inflammatory diseases, containing gramicidin C or a pharmaceutically acceptable salt thereof and a cyclic decapeptide in an amount of not more than 3.5 wt%, having the structure cyclo(-Val-Orn-Leu-D -Phe-Pro-Val-Orn-2-amino-(5-methylhexanoic acid)-D-Phe-Pro-) or cyclo(–Val–Orn–Leu–D-Phe––Pro–Val–Orn–β- homoleucine-D-Phe-Pro-) or their pharmaceutically acceptable salts. 22. The active pharmaceutical substance of gramicidin C, intended for the treatment of infectious and inflammatory diseases, containing gramicidin C or its pharmaceutically acceptable salt and a cyclic decapeptide in an amount of not more than 5.0 wt%, having the structure cyclo(–Val–α-methyl-L -ornithine-Leu-D-Phe--Pro-Val-Orn-Leu-D-Phe-Pro-) or a pharmaceutically acceptable salt thereof. 23. A drug for the treatment of infectious and inflammatory diseases, containing at least one decapeptide or a pharmaceutically acceptable salt according to any one of paragraphs. 1-4. 24. A drug for the treatment of infectious and inflammatory diseases, containing at least gramicidin C or a pharmaceutically acceptable salt thereof and a cyclic decapeptide or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 2, 3 and/or a cyclic decapeptide or a pharmaceutically acceptable salt thereof according to claim 4. 25. A drug for the treatment of infectious and inflammatory diseases, containing gramicidin C or its pharmaceutically acceptable salt and cyclic decapeptides or their pharmaceutically acceptable salts in the following amount (% wt. of the total content of cyclic decapeptides from the gramicidin C family): Gramicidin C - 91.5-99.9% wt.; a cyclic decapeptide according to any one of paragraphs. 2, 3 - in an amount of not more than 3.5% wt.; And cyclic decapeptide according to claim 4 - in an amount of not more than 5.0% of the mass. 26. A drug for the treatment of infectious and inflammatory diseases, containing gramicidin C or a pharmaceutically acceptable salt thereof and a cyclic decapeptide or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 2, 3 in the following amount (% wt. of the total content of cyclic decapeptides from the gramicidin C family): Gramicidin C - 96.5-99.9% wt.; a cyclic decapeptide according to any one of paragraphs. 2, 3 - in an amount of not more than 3.5% of the mass. 27. A drug for the treatment of infectious and inflammatory diseases, containing gramicidin C or a pharmaceutically acceptable salt thereof and a cyclic decapeptide or a pharmaceutically acceptable salt thereof according to claim 4 in the following amount (% wt. of the total content of cyclic decapeptides from the gramicidin C family): Gramicidin C - 95.0-99.9% wt.; cyclic decapeptide according to claim 4 - in an amount of not more than 5.0% of the mass.