RU2765270C1 - Composition of soluble monomeric and oligomeric fragments of peptidoglycan of the cell wall of gram-negative bacteria, methods for production and application thereof - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: described in the invention is a pharmaceutical composition including fragments of peptidoglycan of the cell wall of gram-negative bacteria, the main components whereof are oligomers of muramyldipeptides D, E, F, G, H with a molecular weight of 2,000 to 4,000 u, built from A, B and C blocks connected via glycosidic bonds, wherein muramylpeptide A is of β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramyl-L-alanyl-D-isoglutamine-meso-diaminopimelic acid, muramylpeptide B is of β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramyl-L-alanyl-D-isoglutamine-meso-diaminopimeloyl-D-alanine and muramylpeptide C constitutes a dimer of muramylpeptide B wherein the bond between the monomer residues of muramylpeptide B is executed by means of a carboxyl group of terminal D-alanine of one residue of B and the ω-amino group of the meso-diaminopimelic acid of another residue of B, wherein the trimeric muramyldipeptides D and E are formed due to the combination of the dimeric block C with the monomeric blocks B and A, respectively, the tetrameric muramylpeptides F and G are formed due to the combination of the dimeric block C with two monomeric blocks B or two monomeric blocks A, respectively, and the tetrameric muramylpeptide H formed due to the combination of the dimeric block C with the monomer blocks A and B. Also proposed is a method for producing the composition, including extraction of bacterial cells by a solution of a detergent at elevated temperature, dispersion of the resulting highly purified peptidoglycan under the impact of powerful ultrasound, followed by treatment with lysozyme.

EFFECT: described composition can be used in immunopharmacology, in particular, for creating supportive therapy agents intended for increasing the natural resistance of the body to infections of various natures.

11 cl, 7 dwg, 6 tbl

Description

Field of invention

The invention relates to the field of microbiology and biotechnology, and can be used in immunopharmacology, in particular, to create supportive therapy products designed to increase the body's natural resistance to infections of various nature.

State of the art

In a macroorganism, the main source of exogenous immunostimulating agents are the bacteria of the microbiota of the intestine, respiratory tract, skin, etc. and the full functioning of the immune system directly depends on its qualitative and quantitative composition. Sterile animals (gnotobionts), whose immune system is atrophied, are practically defenseless in contact even with opportunistic microorganisms.

Cell wall peptidoglycan (PKC) of bacteria is known to be a powerful immunostimulator of bacterial nature. Depending on the bacterial source (gram-positive or gram-negative microorganisms), the method of its processing and purification, complexes of biologically active PKC fragments (muramilpetides) with different structures can be isolated, which can become the basis for obtaining preparations with properties characteristic of each complex.

On the basis of numerous data on the structural analysis of fragments obtained during the hydrolysis of PCS by lysocm, an enzyme that selectively cleaves glycosidic bonds of muramic acid residues, it has been established that PCS is a “cross-linked” heteropolymer that contains linear polysaccharide chains built from alternating N-acetyl-D residues. -glucosamine (GlcNAc) and N-acetyl-D-muramic acid (MurNAc) linked by β-(1→4)-glycosidic linkages, with lactyl groups of muramic acid residues bearing short peptides. In most of the studied gram-negative microorganisms, the peptide fragment is represented by a tetrapeptide residue: L-alanyl-D-isoglutaminyl-mesodiaminopimeloyl-D-alanine. Part of the "antenna" oligopeptide fragments form "bridge" structures, providing the creation of a network-like spatial structure of the PCS.

It has been repeatedly noted that the degree of "crosslinking", i.e. the number of "bridge" fragments in PCD depends not only on the type of microorganism, but also on the type and conditions of cultivation.

It was previously found that muramoyl-L-lysyl-D-glutamic acid (muramyl dipeptide, MDP), which is the minimum component of cell wall peptidoglycan, has the same adjuvant effect, the ability to stimulate anti-infective resistance, antitumor immunity, activate immunocompetent cells and induce the synthesis of a number of cytokines. , as well as whole cells of mycobacteria [Ellouz AF, Ciorubaru R, Lederer E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan subunits. Biochem. Biophys. Res. commun. 1974. 59. 1317-1325]. However, due to its high pyrogenicity, it was not suitable for clinical use. Subsequently, it was shown that semi-synthetic β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutamine) (glucosaminylmuramyl dipeptide, GMDP) containing compared to MDP an additional residue of N-acetyl-D-glucosamine, has a significantly lower pyrogenicity and can be used as a broad-spectrum immunomodulator. However, GMDP is a semi-synthetic product that only imitates the structure of natural muramylpetides in the peptide part. It, in contrast to the native components isolated from the PCD of Gram-negative bacteria, whose peptide antennae are built from three or four amino acid residues, includes only two amino acid residues. Moreover, natural peptidoglycan fragments carry negative and positive charges (they contain free carboxyl and amino groups), while in GMDP the carboxyl group of the isoglutamic acid residue is in the form of an amide, i.e. GMDP is a neutral molecule. GMDP is the active component of the immunomodulatory drug Likopid.

Patents RU 2441906 and RU 2478644 describe a method for the production and use of a natural composition consisting of three muramylpetides of Gram-negative bacteria with a molecular weight of not more than 2 kDa: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D -muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid) (muramylpeptide A), β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl -D-isoglutaminyl-meso-diaminopimeloyl-D-alanine) (muramylpeptide B), as well as dimer B (muramylpeptide C), in which the bond between monomer residues B is carried out due to the carboxyl group of the terminal D-alanine of one residue B and ω-amino group meso-diaminopimelic acid of another residue B.

A - β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid,

B - β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine,

C - dimer of muramylpeptide B, where the connection between the monomeric residues of muramylpeptide B is carried out due to the carboxyl group of the terminal D-alanine of one residue of muramylpeptide B and the ω-amino group of meso-diaminopimelic acid of another residue of muramylpeptide B,

In 2013, this composition was registered in the Russian Federation as an immunomodulator Polymuramil ^® .

Immunostimulants that activate the innate immune system and provide rapid protection against a wide range of pathogens have many applications in modern medicine. They can be used, for example, for the treatment or prevention of chronic infectious and inflammatory diseases, HIV infection, in case of potential infection with pathogenic bacteria, for the activation of immune system cells suppressed by the tumor process. The search for new, effective immunostimulants with a minimum of side effects is an urgent task of immunopharmacology.

The essence of the invention

Patents RU 2478644 and RU 2441906 relate to the method of obtaining and properties of the substance Polymuramil (hereinafter, PM), which is a combination of muramyl peptides obtained by treating PKC of various gram-negative bacteria with lysozyme. It was found that a significant part of PCS is resistant to enzymatic degradation and remains in an insoluble form. It is mainly low molecular weight products of enzymatic cleavage that pass into the solution - antenna muramyl peptides A and B, which are repeating units of the linear PKC polymer, as well as “cross-linked” dimeric muramyl peptide C, characteristic of the “bridge” sections of these linear polymers. Along with them, the hydrolyzate usually contains a small amount of muramylpetides with a higher molecular weight (2–5 kDa), which are built from residues of low molecular weight muramylpetides A and B.

Patent RU 2478644 describes a method for obtaining Polymuramil by cleavage of PCS with lysozyme in a buffer with pH 7.2 at 10°C for three days using dialysis tubes or semi-permeable membranes for ultrafiltration with a cut-off size of up to 5 kDa.

In the course of an extended study of the influence of the conditions for obtaining PCS and the parameters of the process of its enzymatic treatment on the composition of the resulting composition of muramyl peptides, it turned out that when using powerful ultrasound at the stage of obtaining PCS and changing the conditions of enzymatic processing, the main products of the resulting composition of muramyl peptides are higher molecular weight trimeric muramyl peptides D and E with a molecular with a mass of 2779.2 m/z and 2708.2 m/z, as well as tetrameric muramyl peptides F, G and H with a molecular mass of 3557.6 m/z, 3699.6 m/z and 3628.6 m/z, which are built from the "primary" blocks A, B and C, connected by glycosidic bonds between the residues of D-GlcNAc and D-MurNAc.

Based on mass spectrometry data, it was found that muramylpeptide D is built from C and B blocks and is represented by four position isomers, two of which contain a β-glycosidic bond between C1 of one of the N-acetyl-D-muramic acid residues from muramylpeptide C and C4 of the terminal N-acetyl-D-glucosamine residue from muramylpeptide B, while the other two contain a β-glycosidic bond between C1 of the N-acetyl-D-muramic acid residue of muramylpeptide B and C4 of one of the terminal residues of N-acetyl-D -glucosamine from muramylpeptide C (arrows in the diagrams below indicate one of the possible options for attaching muramylpeptide; attachment is possible at any of the four potential sites, forming four potential isomers).

Muramylpeptide E is built from blocks C and A and is represented by four position isomers, two of which contain a β-glycosidic bond between C1 of one of the N-acetyl-D-muramic acid residues from muramylpeptide C and C4 of the terminal N-acetyl-D residue -glucosamine from muramylpeptide A, and the other two have a β-glycosidic bond between C1 of the N-acetyl-D-muramic acid residue from muramylpeptide A and C4 of one of the terminal residues of N-acetyl-D-glucosamine from muramylpeptide C.

The composition may also contain tetrameric position F isomers, built (by analogy with trimeric muramyl peptides D and E) from block C and two blocks B, tetramer position G isomers, built from block C and two blocks A, as well as tetrameric isomers of position H, built from blocks C, A and B (also referred to hereinafter as BCB, ACA, DIA). It should be noted that the designations BCB, ACA, DIA do not imply a specific sequence of blocks: BCB does not imply that blocks B are located to the left and right of C; on the contrary, in this description, the designation BCB also includes, for example, variants B2C or CB2, that is, it combines various variants of position isomers (attachment of two blocks B to block C). The whole complex of such position isomers of muramyl peptides is also designated as F.

In some embodiments of the invention, a composition is claimed for stimulating the immune response of a mammalian organism, which is formed as a result of enzymatic cleavage of highly purified peptidoglycan of the cell wall of gram-negative bacteria, which is a high-molecular spatial polymer built from repeating muramylpeptide units, containing an effective amount of a mixture of soluble muramylpeptide oligomers BC, AC, or a mixture of soluble muramylpeptide oligomers BCB, ACA, ACB, while A, B and C are interconnected via a β-glycosidic bond and represent muramylpeptides of the following structure:

muramylpeptide A: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid;

muramylpeptide B: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine;

muramyl peptide C: a dimer of muramyl peptide B, where the bond between two muramyl peptides B is carried out by the carboxyl group of the terminal D-alanine of one molecule B and the ω-amino group of meso-diaminopimelic acid of the other molecule B; or consisting of an effective amount of a mixture of salts of the above soluble muramyl peptide oligomers.

Various variants of pharmaceutically acceptable salts of muramylpeptide oligomers are possible, for example, sodium salts (Na + is a traditional counterion for carboxyl groups). Solicalium or ammonium are also possible

In some embodiments of the invention, the composition contains an effective amount of a mixture of soluble muramyl peptide oligomers BC, AC, BCB, ACA, ACB, or an effective amount of a mixture of salts of the above soluble muramyl peptide oligomers.

In some embodiments of the invention, the composition additionally contains a mixture of soluble muramyl peptides A, B and C, or salts of the above muramyl peptides, while the total mass fraction of muramyl peptides A, B and C in the composition is not more than 50%. At the same time, in some preferred embodiments of the invention, the total mass fraction of muramylpeptides A, B and C in the composition is not more than 10%. In other embodiments, the total mass fraction of muramylpeptides A, B and C in the composition may be no more than 3%, no more than 5%, no more than 7%, no more than 15%, no more than 20%, no more than 30%, no more than 40 %, not more than 50%.

In some embodiments of the invention, the composition further comprises a pharmaceutically acceptable carrier or excipient.

In some preferred embodiments of the invention, the composition contains a mixture of muramyl peptides, including muramyl peptides D, E, F, G, H with molecular weights (amu) 2779.2; 2708.2; 3557.6; 3699.6 and 3628.6, respectively, as well as up to 10% of three muramyl peptides A, B and C with molecular masses (a.m.u.) 867.4, 938.4 and 1858.8, respectively (hereinafter this option composition is designated as FKM).

In some embodiments of the invention, a method is disclosed for obtaining the above compositions containing muramylpeptide oligomers from cell wall peptidoglycan (PKC) of gram-negative bacteria, which includes the following steps: preparing the obtained bacterial biomass for destruction by treatment with an ultrasonic disperser with a power of 400 - 800 W;

the prepared biomass is treated with a buffer containing an ionic detergent at a concentration of 0.1-1%, at a temperature of 40 - 90°C;

clean the resulting biomass from impurities and detergent;

mechanically crush the resulting biomass using a mechanical mill; produce enzymatic hydrolysis of biomass using lysozyme at a pH of 4.5-7.0 and a temperature of 40 to 60°C;

the target products of enzymatic hydrolysis are isolated by ultrafiltration with a cut-off size of semipermeable membranes up to 10 kDa.

In some embodiments of the invention, the method further includes the step of separating the target enzymatic hydrolysis products using size exclusion chromatography, low pressure chromatography, or high performance liquid chromatography.

In some embodiments of the invention, the method further includes the step of lyophilization of the selected enzymatic hydrolysis target products.

Also disclosed is the use of compositions containing muramyl peptide oligomers for the treatment and prevention of diseases in mammals that require stimulation of the immune system.

Unlike the semi-synthetic drug Licopid, as well as its synthetic analogues (Romurtide, Mifamurtide, Murabutide, B30-MDP substance), a composition containing muramyl peptide oligomers, such as, for example, FCM, is a completely natural immunostimulant. Since the immune system of humans and mammals is “tuned” to recognize substances of natural origin, the effect on the immune system of such a combination of natural muramilpetides as FCM will be more balanced in terms of the ratio of therapeutic and side effects.

The technical result achieved in the implementation of the invention is to more effectively stimulate the immune response of the mammalian body in response to a composition containing muramyl peptide oligomers, such as, for example, FCM, as well as to simplify the procedure for obtaining and increase the yield of this composition from the PKC of gram-negative bacteria.

The specified technical result is achieved by an increased content of oligomeric structures of soluble muramyl peptides in the composition, as well as an improved method for its isolation from the PKC of gram-negative bacteria.

Brief description of the figures.

Fig. 1. Analytical HPLC picture of the FCM preparation.

Fig. 2. Analytical HPLC picture of Polymuramil preparation.

Fig. 3. Picture of the preparative separation of the FCM preparation.

Fig. 4. ¹ H-NMR spectrum of FCM (D ₂ O).

Fig. 5. ¹ H-NMR spectrum of the substance PM (D ₂ O).

Fig. 6. HPLC pattern of preparation 2 (for preparation 1 the picture is similar).

Fig. 7. HPLC picture of preparation 3.

DISCLOSURE OF THE INVENTION

In the description of the present invention, the terms "comprises" and "comprising" are interpreted to mean "includes, among other things." These terms are not intended to be construed as "consisting only of". Unless otherwise defined, the technical and scientific terms in this application have the standard meanings generally accepted in the scientific and technical literature.

The invention describes a pharmaceutical composition comprising cell wall peptidoglycan fragments (muramyl peptide oligomers) of gram-negative bacteria, the main components of which are muramyl peptides D, E, F, G, H with a molecular weight of 2000 - 4000 amu, which are built from blocks A , B and C, where muramylpeptide A is β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid, muramylpeptide B is β -N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine and muramylpeptide C, which is a dimer of muramylpeptide B, in in which the connection between the monomeric residues of muramylpeptide B is carried out due to the carboxyl group of the terminal D-alanine of one residue B and the ω-amino group of meso-diaminopimelic acid of another residue B, and the trimeric muramylpeptides D and E are formed by connecting the dimeric block C with the monomeric blocks B and A , respectively, tetrameric muramyl peptides F and G are formed by the connection of the dimer block C with two monomer blocks B or with two monomer blocks A, respectively, and the tetrameric muramyl peptide H is formed by the connection of the dimer block C with monomer blocks A and B. In muramyl peptides D, E, F , G and H connection between the dimeric block C and blocks A and B is due to glycosidic bonds between the residues of D-GlcNAc and D-MurNAc. Trimeric muramyl peptides D and E are a set of position isomers with a molecular weight of 2779.2 m/z and 2708.2 m/z, respectively, tetrameric muramyl peptides F, G and H are a set of position isomers with a molecular weight of 3557.6 m/z , 3699.6 m/z and 3628.6 m/z, respectively.

The method for obtaining this composition involves the use of a combination of steps for isolating pharmacologically acceptable FKM from PKM of gram-negative bacteria, including the preparation of biomass containing substances of a bacterial nature, the isolation of PKM of bacteria, the cleavage of insoluble PKM by preparative enzymatic hydrolysis using lysozyme, and the isolation of pharmacologically acceptable FKM, while preparing the biomass for extraction is carried out using sonication, the isolation of PCS is carried out using the extraction of biomass with buffers containing detergents, the mechanical grinding of the resulting PCS is carried out using a mechanical mill, the splitting of insoluble PCS is carried out using the enzyme lysozyme, which cleaves the glycosidic bonds of muramic acid residues, at a temperature above 40 ° C followed by isolation of FCM by ultrafiltration.

To implement the process according to this invention, a biomass is prepared containing substances of a bacterial nature, which are Gram-negative bacteria of the Enterobacteriacea family (for example, Salmonella typhi, Salmonella typhimurium, Escherichia coli ) by culturing them in a nutrient medium, followed by isolation of bacterial cells by centrifugation or microfiltration and thorough washing of bacterial cells from the components of the nutrient medium.

When using various bacterial sources, it is possible to obtain a pharmacologically acceptable mixture of muramyl peptide oligomers with different contents of individual components. Also, the relative content of individual oligomeric components (such as BC, AC, BCB, ACA, ACB) can be changed during the preparation of the composition by chromatographic separation of the resulting mixture and subsequent separation of fractions containing various oligomeric components (see below, for example, Fig. 3).

The somatic antigen of gram-negative bacteria, a highly endotoxic lipopolysaccharide (LPS), located on the outer membrane of the bacterial cell wall, can be removed using various extraction procedures, and proteins, nucleic acids, and other non-covalently associated with PKC components of the bacterial cell are released at the same stage. . As an extractant, 45% aqueous phenol is most often used at a temperature of 65-68°C, since at this temperature the mixture of phenol and water is completely homogenized. In addition, aqueous solutions of chaotropic compounds, as well as detergents capable of destroying aggregates formed by amphiphilic polymer molecules, can be used to extract bacterial cells.

The method for producing Polymuramil described in patent RU 2478644 has a number of disadvantages: the use of a toxic and aggressive 45% aqueous solution of phenol as an extractant at a temperature of 68-70 ° C, as a result of extraction with aqueous phenol, PCS is formed in a rubber-like form, which is difficult to be further purified from impurities of various nature. Within the scope of the present invention, buffered solutions of ionic detergents have proven to be safer in place of hot aqueous phenol. To increase the efficiency of extraction, pretreatment of the bacterial cell mass with ultrasound was used. This treatment makes it possible to significantly reduce the size of the extracted PCS particles, which leads to a significant increase in the efficiency of the extraction process.

Ultrasonic treatment of an aqueous suspension of biomass can be carried out at a temperature of 10 - 50°C, ultrasonic disperser power 400 - 800 W, for 30 - 90 minutes, preferably at a temperature of 30 - 40°C, ultrasonic disperser power 500 - 600 W, for 40 - 60 min. The optimization of the sonication process can be carried out by determining the particle size by light microscopy and dynamic light scattering.

Within the framework of this method for obtaining PCS, the extraction of sonicated biomass in order to remove impurities of LPS, proteins and nucleic acids can be carried out using ionic detergents (preferably using a 0.1-1% solution of sodium deoxycholate in a buffer with a molarity of 0.2 - 2 0, better 0.5 - 1.0 M and pH 8.0 - 9.0, at a temperature of 40 - 90 ° C, better at a temperature of 40 - 60 ° C, for 0.5 - 2 hours. variant, sodium decyl sulfate, sodium dodecyl sulfate or N-lauryl sarcosinate can be used as ionic detergents.

In some embodiments of the invention, the thus obtained suspension is further subjected to concentration by centrifugation, preferably flow centrifugation (centrifuge HSTC GQ75, 12000 rpm). The concentrated suspension of PCS is again subjected to extraction under the conditions described above. To increase the degree of purification of PCS, the extraction of biomass is preferably carried out 2 to 3 times.

In some embodiments of the invention, the isolation and purification of PKC from impurities of a bacterial nature and from the components of the extraction buffer, in particular sodium deoxycholate, is carried out using centrifugation, preferably flow centrifugation, washing the PKC suspension with a buffer that does not contain detergent, and then with water. If necessary, the aqueous suspension of PCS can be lyophilized or dried with acetone and then with ether.

In some embodiments of the invention, in order to increase the yield of FCM during the subsequent enzymatic treatment with lysozyme, the PCS powder is subjected to grinding using a planetary ball mill (RETSCH PM 200) in dry form or as a suspension in water or preferably in a buffer, which is used for subsequent treatment with lysozyme. The efficiency of PKC grinding is controlled by determining the particle size using light microscopy and dynamic light scattering methods.

In some embodiments of the invention, to obtain FKM, a suspension of PKC in a buffered aqueous solution is subjected to enzymatic hydrolysis using endo-N-acetylmuramidases, preferably lysozyme. The use of lysozyme is due to the selective cleavage of glycosidic bonds of muramic acid residues in the polysaccharide part of PCD, combined with a relatively low cost, high efficiency in a wide range of pH values.

In order to find conditions for the enzymatic hydrolysis of PKC, which would increase the yield of muramyl peptides, we studied the effect of temperature and reaction time on the quantitative and qualitative composition of the cleavage products using high performance liquid chromatography (column G2000PW, eluent 0.05 M NaOAc). It turned out that, in contrast to the standard reaction interval (10–40°C), at temperatures from 40 to 60°C, along with the usually observed low molecular weight muramyl peptides (peaks III and IV), their higher molecular weight homologues (peaks I and II), and it is this composition that is further designated as FCM. In the course of optimization, it was found that carrying out the process at 45 - 55°C, preferably at 50°C for 15 - 60 minutes, preferably for 30 minutes, provides the maximum yield of FCM. A typical picture of FCM separation by high performance liquid chromatography (HPLC) on a G2000SW column is shown in FIG. one.

Comparative HPLC analysis of FCM and Polymuramil (PM) showed that the substances of peaks III and IV are Polymuramyl (Fig. 2).

In some embodiments of the invention, when carrying out enzymatic hydrolysis, a freeze-dried buffer aqueous solution with a pH of 4.5-7.0, preferably 4.5-5.0, (for example, pyridine acetate buffer) is used as a solvent.

In some embodiments of the invention, after treatment with lysozyme, not subjected to degradation, the insoluble fraction of PKC is removed by centrifugation (9000 rpm, 30 min) and the resulting supernatant is subjected to ultrafiltration, preferably tangential ultrafiltration, using a membrane with a cut-off size of 10 kDa to remove polymer fragments PCS and lysozyme. The filtrate containing FCM is then lyophilized, and the product is re-lyophilized to remove trace amounts of buffer components.

The structure of all FCM components was confirmed by electrospray mass spectrometry and ¹³ C-NMR spectroscopy. To do this, the FCM sample was subjected to preparative gel chromatography using successively connected columns (0.9x60 cm) filled with Toyopearl TSK40(SF) and TSK50(SF) gel. The elution was carried out with 0.05 M ammonium bicarbonate buffer, detection - using a flow UV detector, wavelength 220 nm. Pictures preparative and analytical separation (Fig. 1) almost coincided.

As a result of preparative separation, 4 fractions were obtained (see Fig. 3), each of which was subjected to lyophilization. Data on the composition of each fraction and the molecular weight of the components are given in Table 1.

Table 1. Molecular weights of FCM components.

Fraction number Factions Molecular weights of the components I F, G, N 3557.6*, 3699.6*, 3628.6* II D and E 2779.2 and 2708.2 III WITH 1858.8 IV A and B 867.4 and 938.4

*value calculated from the m/z of the corresponding multiply charged ions

From the obtained data, it followed that FCM contains homologous muramyl peptides, which differ only in the number of A and B monomeric units.

A similar conclusion followed from the data of a comparative analysis of 13C1H-NMR spectra of FCM (Fig. 4) and five isolated fractions, since all spectra contained almost the same set of signals (Fig. 4). Almost complete agreement of the signals was also established by comparing the 13C-NMR spectra of FCM and the substance of the drug Polymuramil (Fig. 5), the components of which are muramylpeptides A, B and B 2 are part of the FCM.

The specialist knows that the relative content of individual oligomeric components (such as BC, AC, BCB, ACA, ACB) in the composition can be changed during the preparation of the composition by chromatographic separation of the resulting mixture and subsequent separation of fractions containing various oligomeric components (see Fig. .3). Various well-known methods for separating components can be used. In particular, preparative low pressure gel permeation column chromatography using Sephadex, TSK gels or Bio gels and ion exchange chromatography using both anion and cation exchange gels, as well as preparative high performance liquid chromatography can be used. Alternatively, the sequential ultrafiltration method using 1, 3 and 5 kDa membranes (Millipore) can be used.

Such compositions with a changed relative content of individual oligomeric components (BC, AC, BCB, ACA, ACB) are referred to in the present description as compositions similar to FCM. The relative content of each of the individual oligomeric components in compositions like FCM can vary over a wide range from 0 to 99%.

The biological activity of compositions similar to FCM was determined in immunological studies, after confirming its safety in use (control of toxicity and pyrogenicity).

Thus, a composition of PKC fragments with a pronounced immunostimulating activity is proposed, including instead of low molecular weight muramyl peptides, their higher molecular weight analogs. As part of the study, it was shown that all components of a pharmacologically acceptable mixture of substances have a similar level of immunological activity. The results of chemical and physico-chemical studies have shown that a pharmacologically acceptable mixture of substances contains minimal amounts of impurities and that, therefore, the proposed technological scheme makes it possible to remove almost all components from the target product, the presence of which could impart undesirable properties to a pharmacologically acceptable mixture of substances.

The proposed method for isolating FCM is distinguished by an increased safety level of extraction by replacing concentrated aqueous phenol with buffers containing ionic detergents, as well as by increasing the yield of muramyl peptide components by reducing the particle size of the bacterial mass before extraction using ultrasonic treatment, as well as by grinding PKC particles mechanically before treatment with lysozyme.

The proposed method can be implemented in large-scale production, because each step described in the examples below is an adequate industrial model.

The present invention relates to the use of compositions like FCM for the treatment and prevention of diseases that require stimulation of the immune system. The list of these diseases is determined based on the types of FCM activity established by the authors. According to these studies, FCM has the following activities: 1) induces the production of activation cytokines (interleukin [IL]-1β, IL-6, IL-17, IFN-γ, TNF-α) and inflammatory chemokines (IP-10, IL- 8, MIP-1α, MIP-1β, RANTES) by cells of the innate immune system, which leads to a rapid increase in the body's anti-infective resistance; 2) enhances the ability of blood leukocytes to kill bacteria; 3) enhances the production of natural antibodies to common antigenic determinants of bacteria; 4) protects experimental animals from lethal infections caused by gram-positive and gram-negative bacteria; 5) induces the production of myelopoietins and enhances myelopoiesis; 6) induces the production of chemokines, which are natural antagonists of the human immunodeficiency virus; 7) increases the functional activity of NK cells, which play one of the key roles in protecting the body from viral infections and malignant neoplasms; 8) slows down the growth of transplantable malignant tumors in experimental animals. At the same time, FCM does not have pyrogenic and other serious side effects when administered parenterally to a person at a dose of up to 10 μg/kg/day.

Compositions like FCM, being a mixture of amphoteric compounds, can be used in neutral, anionic and cationic forms. In the anionic form, all or part of the protons in the terminal carboxyl groups are replaced by other cations, which can be used, for example, sodium, potassium, ammonium, triethylammonium cations. In the cationic form, all or part of the free amino groups are converted to the ammonium form, and as a counterion, for example, chloride, sulfate or acetate anions can be used.

The unique properties of the resulting compositions provide the provision of new drugs - immunostimulants, which, based on their effectiveness, provide an increase in the humoral immune response, determined by the number of antibody-producing cells (AFC) and the level of specific antibodies, as well as the ability to stimulate innate immunity factors (non-specific resistance) , which is determined by the protection of experimental animals stimulated with the test substance from a fatal infection caused by pathogenic bacteria.

Previously, it was found that muramilpetide preparations, which include FCM, are able to penetrate through epithelial barriers into the bloodstream [Andronova T.M., Pinegin B.V. Licopid (GMDP) is a modern domestic highly effective immunomodulator. Russia, 2005]. This makes it possible to release compositions similar to FCM in the form of pharmaceutical compositions for transmucosal and transdermal use. Thus, compositions like FCM can be presented as pharmaceutical compositions for oral and oral administration, in particular in the form of tablets, capsules, oral solutions, lozenges and buccal forms. Capsules may contain mixtures of FCM-like compositions with excipients and/or diluents such as pharmaceutically acceptable starches, sugars, synthetic sweeteners, powdered celluloses, various types of flours, gelatins, gums. Suitable tablet formulations may be prepared by conventional compression, wet granulation or dry granulation processes using pharmaceutically acceptable excipients, diluents, binders, lubricants, disintegrants, surface modifiers (including surfactants), suspending or stabilizing agents. The oral preparation may also consist of the active ingredient to be administered in water or fruit juice containing, if desired, suitable solvents or emulsifiers. The packaging of the drug can vary from 0.01 to 20 mg per tablet or capsule, or from 0.01 to 20 mg per 1 ml of solution.

FCM can be produced as a solution for intranasal use with an active substance concentration of 0.01 to 20 mg/ml in combination with pharmacologically acceptable additives.

Pharmaceutical compositions similar to FCM for external use include ointments, creams and gels containing FCM at a concentration of from 0.01 to 20 mg/ml, optionally in combination with traditional substances that improve the penetration of the drug through the skin barrier (penetrators).

Another form of presentation of compositions similar to FCM, can be suppositories for rectal or vaginal use, containing the composition at a dose of from 0.01 to 20 mg per suppository, in combination with pharmacologically acceptable additives. The composition of carrier compositions for suppositories is known from the prior art.

Another variant of the pharmaceutical composition may be a ready-made sterile solution for parenteral administration. The concentration of FCM in this solution can vary from 0.01 to 20 mg/ml. The solution may optionally contain pharmacologically acceptable excipients, including drug stability enhancers, pH adjusters, tonicity adjusters. The solution is packaged in sterile ampoules or ready-to-use injectors containing FCM in an amount of 0.01 to 20 mg per ampoule/injector. Injectors for parenteral administration are described in the prior art and are commercially available from various companies. These injectors are filled with an injection solution, sterilized if necessary, and packed in boxes, containers, etc. The advantage of the injector is that it is a ready-to-use device that even an untrained person can use.

Finished forms of preparations and compositions based on FCM are packed in boxes, bags, tubes, vials, etc. depending on tradition and market needs.

For example, tablets may be blister packed for one or more course doses. The blister may contain instructions for use printed on it and be packed in a container or box, in which instructions for use are separately enclosed.

Various packaging options are known in the art and require no further description.

The appointment of compositions similar to FCM, as well as monitoring the patient's condition during treatment, are carried out by the attending physician. Based on the mechanism of action of the drug discovered by the authors, compositions similar to FCM can be indicated for the following groups of diseases.

I. Treatment and prevention of diseases caused by bacteria:

1) treatment of acute and chronic diseases of the skin, soft tissues, respiratory tract and lungs, gastrointestinal tract, kidneys, urinary tract, female genital organs and prostate caused by bacteria;

2) prevention and treatment of purulent complications of surgical operations;

3) prevention of contagious infectious diseases caused by pathogenic bacteria and manifested in the form of epidemics.

The use of compositions similar to FCM for the treatment and prevention of diseases of bacterial origin is justified by the following types of their activity: 1) enhancing the ability of phagocytes to kill bacteria; 2) induction of the production of cytokines (IL-1β, IL-6, IL-17, IFN-γ, TNF-α) and chemokines (IP-10, IL-8, MIP-1α, MIP-1β, RANTES) necessary for development of an optimal innate immune response against bacteria; 3) induction of the production of antimicrobial peptides - defensins; 4) increased production of natural antibodies to common antigenic determinants of bacteria; 5) induction of the production of myelopoietins (G-CSF, GM-CSF) and increased output of myeloid cells (monocytes, granulocytes) from the bone marrow; 6) protection of experimental animals from infection with lethal doses of pathogenic gram-positive and gram-negative bacteria. As will be demonstrated in the Examples, the effect of FCM develops rapidly, appearing already on the 1st - 3rd day of daily administration, which is important when using the drug for prophylactic purposes in risk groups.

II. Treatment of diseases of the skin, soft tissues, respiratory tract and lungs, gastrointestinal tract, kidneys, urinary tract and female genital organs caused by pathogenic and opportunistic fungi.

The use of compositions similar to FCM for the treatment and prevention of diseases of fungal origin is justified by the following types of its activity: immune response against fungi; 2) induction of the production of antimicrobial peptides - defensins; 3) induction of the production of myelopoietins (G-CSF, GM-CSF) and increased release of myeloid cells (monocytes, granulocytes) from the bone marrow.

III. Prevention and treatment of diseases caused by viruses:

prevention and treatment of HIV infection;

treatment of chronic recurrent herpes virus infection (HRVI) caused by herpes simplex virus types 1 and 2 (HSV-1 and -2).

The use of compositions similar to FCM in infections with these viruses is due to the ability of the drug to increase the activity of NK cells, which are one of the key effectors of antiviral immunity, to induce the production of chemokine IP-10, which is a chemoattractant for natural interferon-producing cells - the most important effectors of antiviral immunity, and also to induce the production of defensins - antimicrobial peptides that can inhibit the penetration of HSV and HIV into target cells and/or intracellular replication of these viruses [Mackewicz C et al. alpha-Defensins can have anti-HIV activity but are not CD8 cell anti-HIV factors. AIDS 2003; 17: F23-32. Hazrati E et al. Human α- and β-defensins block multiple steps in herpes simplex virus infection. J Immunol 2006; 177: 8658-66]. In addition, FCM induces the secretion of the chemokines RANTES, MIP-1α and MIP-1β, which block cellular co-receptors for HIV and prevent HIV from entering cells.

IV. Adjuvant immunotherapy of solid malignant neoplasms of various localization. The use of compositions similar to FCM for the treatment of malignant tumors is due to the following activities: 1) suppression of the growth of solid malignant tumors in experimental animals; 2) induction of the production of cytokines involved in tumor rejection, in particular TNF-α; 3) increased activity of NK cells.

V. Treatment of conditions accompanied by suppression of myelopoiesis (acute and chronic radiation sickness, poisoning with myelotoxic substances, conditions during or after radiation therapy or chemotherapy of malignant neoplasms). The use of FCM in this group of diseases is due to the ability of the drug to cause the production of colony-stimulating factors (G-CSF, GM-CSF) necessary for the differentiation of myeloid cells in the bone marrow, which leads to an increase in the levels of neutrophils and monocytes in the bloodstream.

However, the above list of diseases is not restrictive. The choice of specific conditions in which the appointment of compositions like FCM is required is within the competence of a specialist, based on the identified and described biological activity of these compositions.

The preferred route of administration of FCM-like compositions in most nosological forms is parenteral. As a rule, FCM is administered intramuscularly, however, depending on the nosological form, the severity of the patient's condition and other factors, intradermal, subcutaneous and intravenous administration of the drug is possible. The dosage may vary from approximately 0.1 to 100 mcg / kg / day, depending on the condition of the patient and the individual tolerability of FKM.

In some cases, for example, for the treatment of inflammatory infectious diseases and malignant neoplasms of the gastrointestinal tract, FCM can be used in the form of tablets, capsules or oral solutions. The dosage of the drug for oral administration is, as a rule, from 0.1 to 300 mcg / kg / day, depending on the patient's condition and the individual tolerance of FKM.

For the treatment of diseases of the rectum, prostate and female genital organs, as well as for the prevention of HIV infection, FCM can be used in the form of rectal and vaginal suppositories.

For the treatment of skin diseases, FCM can be applied externally in the form of creams and ointments.

For the treatment of diseases of the upper respiratory tract, FKM can be used in the form of drops for intranasal use.

When prescribing compositions like FCM, traditional dose selection tactics are followed. Factors influencing the choice of treatment parameters are the type and severity of the underlying disease, individual susceptibility to the action of these compositions, the presence of concomitant diseases. The duration of the use of the drug for medicinal purposes is, as a rule, 5-7 days, however, depending on the nosological form, condition and reactivity of the patient, it can vary from 1 to 30 days.

EXAMPLES

The following examples are given for the purpose of disclosing the characteristics of the present invention and should not be construed as limiting the scope of the invention in any way.

Example I. Obtaining compositions similar to FCM.

50 g of acetone-dried S.typhi bacterial cells were suspended with vigorous stirring in 500 ml of distilled water. The resulting suspension was subjected to ultrasonic treatment for 60 min using a laboratory ultrasonic disperser LUZD-1.5-1P (power 500 W, temperature 10°C). To the resulting suspension was added 500 ml of water, and then DOC to a concentration of 0.5%, sodium chloride to a concentration of 0.2 M, trishydroxymethylaminomethane to a concentration of 0.05 M, disodium salt of ethylenediaminetetraacetic acid to a concentration of 0.01 M and sodium azide to a concentration of 0.01% and adjusted the pH to 8.3. The suspension was stirred for 1 hour at a temperature of 60°C, centrifuged (8000 rpm, 30 min, 14°C), the resulting PKC precipitate was again extracted under the conditions described above with a buffer containing DOC (1000 ml), the precipitate obtained after centrifugation 3 - It was washed 4 times with 1000 ml of the buffer described above, which did not contain DOC, centrifuged, the described procedure was repeated 4 more times, and then the precipitate was washed from the buffer components with water until there was no Cl-ion in the supernatant. The resulting precipitate of purified PCS was suspended in 1000 ml of pyridine acetate buffer (pH 5.2) and subjected to grinding using a planetary ball mill (RETSCH PM 200) for 30 min at room temperature. The crushed mass was removed, the mill glasses were washed with pyridine-acetate buffer, brought to a total volume of 1200 ml, after adding 50 mg of lysozyme, the resulting suspension was divided into three parts of 400 ml and stirred at 50°C for 15, 30 and 45 min, insoluble fractions PKC was removed by centrifugation (9000 rpm, 30 min) and the resulting supernatants were subjected to tangential ultrafiltration using membranes with a cut-off size of 10 kDa to remove polymer fragments of PKC, the filtrates were further lyophilized. After treatment with lysozyme for 15, 30 and 45 minutes, FKM preparations with a yield of 150, 730 and 860 mg, respectively. The obtained preparations were analyzed by HPLC on a Beckman Coulter instrument using a G2000SW column eluting with 0.05 M sodium acetate buffer pH 8.0 at a rate of 1.0 ml/min. Detection was carried out using a UV detector at a wavelength of 220 nm. Analysis of preparations 1 - 3 showed that the HPLC patterns of preparations 1 and 2 obtained by treatment with lysozyme for 15 and 30 minutes practically coincide (Fig. 6), and according to the integration of analytical chromatograms of preparations 1 and 2, the content of PM impurity in them was 5.0 - 7.5%, while the yield of preparation 3 with a longer treatment with lysozyme is significantly higher, but according to the integration of the analytical chromatogram, the content of the PM impurity in preparation 3 was 48.7%.

An increase in the duration of the enzymatic reaction leads to a slight increase in the yield of muramylpeptide oligomers, but the amount of low molecular weight muramylpeptides (PM) increases, see Fig.7.

Example 2 FCM induces the production of a wide range of activation cytokines and chemokines by human dendritic cells (DC) and macrophages (MF) in vitro.

Patent RU 2441906 disclosed the ability of the substance Polymuramil (PM) to cause the production of a set of cytokines and chemokines by dendritic cells and macrophages, which transmit an activation signal to different parts of the immune system. In this example, we compared PM (contamination of high molecular weight muramyl peptides 4.0%) and FCM (contamination of low molecular weight muramyl peptides 4.8%) in terms of their ability to induce the synthesis of cytokines and chemokines. DC and MF were obtained from MNCs of healthy donors in vitro using the generally accepted method [Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994; 179: 1109-18]. DC and MF were cultivated for 24 h without adding PM and FCM to the medium or with the addition of PM and FCM at a concentration of 10 μg/mL, after which supernatants were collected and the levels of cytokines in them were analyzed by multiplex analysis (Table 2).

Table 2. Levels of cytokines and chemokines (pg/ml) in DC and MF supernatants after 24 hours of incubation with PM/FCM (M±σ, n=4)

DC MF PM/FCM, mcg/ml 0/0 10/10 0/0 10/10 Cytokines Interleukin-1β 12±7 20±2/22±2 42±38 113±110*/135±111* Interleukin-6 10±6 59±23*/83±21* 675±306 4537±1103*/5376±1013* Interleukin-17 11±8 34±9*/42±11* 38±21 74±28*/110±49* Interferon-γ 6±5 64±29*/67±41* 221±127 805±228*/953±321* TNF-α 91±51 247±132*/287±134* 531±223 20351±8987*/22539±9547* Interferon-α 7±2 6±1/75±19 5±4 6±4/68±41 Chemokines Interleukin-8 1142±621 12637±6723*/14865±6836* 24312±9043 28523±5034*/31593±5672* IP-10 53±32 468±283*/582±256* 4942±2705 6581±5692*/8201±6523*

*p<0.05 compared with cultivation of the same cells in the absence of PM/FCM (paired t-test)

Data in Table. 2 show that already in the first 24 hours of contact with the cells of the immune system, both PM and FCM induce the production of a set of cytokines and chemokines by dendritic cells and/or macrophages. These cytokines are essential for a successful immune system response to bacterial, fungal, and some viral infections.

FCM more actively than PM induced the production of the entire set of studied cytokines and chemokines, including the production of interferon-α, the level of which did not increase when DC and MF were cultivated in a medium supplemented with PM. In this regard, it can be assumed that FCM will be effective in the treatment/prevention of viral infections.

Example 3. Comparison of the induction of TNF-α production by dendritic cells and macrophages during their cultivation with FCM and PM.

The intensity of induction of TNF-α production by dendritic cells and macrophages in the presence of FCM : PM mixtures of various compositions was studied in the experiment. FCM was obtained as described in Example 1, and according to the integration of an analytical chromatogram similar to Fig. 1 contained 7.3% PM impurities. PM was obtained as described in patent RU 2478644, and according to the integration of an analytical chromatogram similar to Fig. 2, contained about 3.3% impurities of high molecular weight muramyl peptides. Isolation of DC and MF from MNCs of healthy donors and their subsequent cultivation with FCM and PM was carried out as described in Example 2. The results of the experiment are presented in Table. 3.

Table 3. TNF-α levels (pg/ml) in DC and MF supernatants after 24 hours of incubation in the presence of FCM : PM mixtures (M±σ, n=4) at a total concentration of muramyl peptides of 10 μg/ml.

FKM:PM DC MF 100:0 387±134* 32539±9547* 75:25 358±133* 28735±9326* 50:50 324±125* 26359±9127* 25:75 283±128* 24647±8547* 0:100 247±132* 20351±8987*

*p<0.05 compared with cultivation of the same cells in the absence of PM/FCM (paired t-test)

Example 4. FCM stimulates myelopoiesis

The ability for absorption and intracellular killing of microbes is mainly possessed by neutrophils, monocytes and macrophages. In particular, a drop in the levels of neutrophils in the blood below 1000/mm ³ (neutropenia) dramatically increases the risk of local and systemic bacterial and fungal infections, which is higher, the more pronounced and longer the neutropenia. In the absence of treatment, this condition leads to the death of the patient. The causes of neutropenia are radiation sickness, poisoning with myelotoxic substances, as well as radiation or chemotherapy of malignant neoplasms. For the treatment of neutropenia, the growth factors necessary for the differentiation of myelocytes in the bone marrow are relatively successfully used: granulocyte-macrophage colony-stimulating factor (GM-CSF, molgrastim) and granulocyte colony-stimulating factor (G-CSF, filgrastim). Both drugs are extremely expensive, because. their production is based on recombinant technology. The induction of the same factors in vivo seems to be an adequate and less expensive replacement for recombinant proteins.

In the experiment, FKM samples with an impurity content of low molecular weight muramyl peptides of 2.4% and PM with an impurity content of high molecular weight muramyl peptides of 2.8% were used.

We found that FCM, like PM, causes significant production of G-CSF and GM-CSF by DC and MF in vitro (Table 3). The experiments were carried out as in Example 2. The results are presented in Table. 4.

Table 4. Levels of G-CSF and GM-CSF (pg/ml) in supernatants of DC and MF after 24 hours of cultivation in the presence of PM/FCM (M±σ, n=4).

DC MF PM/FCM, mcg/ml 0/0 10/10 0/0 10/10 G-CSF 11±3/10±4 326±98/576±113 71±32/85±44 3286±2745/5127±3142 GM-CSF 13±5/18±5 136±29/221±73 23±9/28±7 1845±841/2741±959

* p<0.05 compared to incubation in the absence of PM and FCM (t-test)

Example 5. FCM increases the resistance of mice to lethal infection with gram-positive and gram-negative bacteria.

An integral indicator of the immunostimulatory activity of a substance is its ability to increase the resistance of a macroorganism to lethal infections caused by various types of pathogenic microorganisms. The increase in resistance to infection may be due to several mechanisms, including those described in Examples 1-4. Bacterial immunostimulants, such as those in the present invention, act primarily on the innate component of the immune system and therefore induce immediate, relatively indiscriminate, but transient resistance to pathogens.

In this example, the study of the effect of FCM on nonspecific resistance and comparison with the effect of PM on this indicator of immunostimulatory activity was carried out on outbred mice weighing 12–14 g (the FCM preparation contained 3.5% impurities of low molecular weight muramyl peptides, the admixture of high molecular weight muramyl peptides in the PM preparation was 4.2 %) . Groups of 10 mice were injected with FCM/PM once, subcutaneously, at doses of 100, 10, and 1 μg/mouse. The control group was injected with sterile saline. 24 hours after injection, mice were challenged intraperitoneally with 1 DCL (absolutely lethal dose) of Salmonella typhimurium (strain 415) or Staphylococcus aureus (strain Wood 46). These microorganisms are typical representatives of gram-negative and gram-positive pathogenic bacteria, respectively, and are traditionally used in such experiments.

Table 5 shows that FCM, as well as PM at a dose of 100 μg/mouse, increases the resistance of mice to infections caused by S. typhimurium and St. aureus , protecting against death by 70% and 60%, respectively (protection with PM in the experiment was 60% and 50%). Smaller doses have a weaker protective effect, but the survival rate in the groups that received FCM was higher than in the groups that received PM. Thus, FCM has a pronounced ability to protect experimental animals from a deadly infection caused by gram-negative and gram-positive bacteria.

Table 5. Effect of FCM and PM on the survival of mice after infection with lethal doses of pathogenic bacteria.

a drug Dose (µg/mouse) bacteria The death of mice (days) Survived,% 1st 2nd 3rd 4th PM one hundred S. typhimurium


0 0 3 one 60 10 0 5 2 2 10 one 0 eight one one 0 physical rr - one 9 0 0 0 PM one hundred St. aureus


3 2 0 0 50 10 5 2 one 2 0 one 5 4 one 0 0 physical rr - eight one 0 one 0 FKM one hundred S. typhimurium


0 0 2 one 70 10 0 4 2 one thirty one 0 6 one one twenty physical rr - 0 eight one one 0 FKM one hundred St. aureus 3 one 0 0 60 10 5 2 0 0 thirty one 5 2 2 0 10 physical rr - eight one one 0 0

Example 6 FCM is effective in the treatment of disruptive folliculitis in mice.

The effect of FKM (an impurity content of low molecular weight muramyl peptides 8.7%) was studied in the infection caused by S. aureus . BALB/c mice in the back region were shaved a skin area of 1.5×1.5 cm, and a suspension of the pathogen strain was injected subcutaneously in a volume of 200 μl. The therapy was carried out by applying a cream containing FCM in various concentrations to the shaved area.

To prepare the inoculum, the colonies of bacteria from the second passage were suspended in a sterile isotonic solution to a density of 5.6 according to the McFarland turbidity standard. The culture was prepared immediately before the infection procedure. To control the viability of bacteria from the working dilution, 0.1 ml was inoculated per plate with a nutrient medium before the start of the infection procedure. By seeding from serial dilutions, it was experimentally established that a suspension of S. aureus bacteria with an optical density of 5.6 contains about 20 billion CFU/ml.

The results were evaluated within 21 days according to the effect of FCM on the development of the inflammatory process in the skin (assessment of the size of the abscess, the outcome of local inflammation, restoration of the hairline). The results are presented in Table. 6.

The experiment showed the highest efficiency of therapy with a cream containing FCM 5.0%.

Table 6 Effect of FCM cream therapy on follicle healing and hairline restoration.

It was experimentally shown that when using a cream with FCM, an abscess developed relatively small in size and resolved much faster relative to similar indicators in the comparison groups. At the same time, complete restoration of the hairline was observed in more than 67% of laboratory animals (in groups using a cream with FKM 1% and 5%). In the control groups, incomplete recovery was observed, or the hairline was not restored - in 83%. The composition of the cream and 5% FCM showed the greatest activity regarding the healing of follicles and the restoration of the hairline. Thus, as a result of the experiments performed on a mouse model, the high efficiency of FCM in the treatment of a purulent process in soft tissues was shown.

Claims (23)

1. A composition for stimulating the immune response of a mammalian body, resulting from the enzymatic cleavage of highly purified peptidoglycan of the cell wall of gram-negative bacteria, which is a high-molecular spatial polymer built from repeating muramylpeptide units, comprising an effective amount of a mixture of soluble muramyl peptide oligomers BCB, ACA, ACB and / or their pharmaceutically acceptable salts, in which A, B and C are interconnected via a β-glycosidic bond and are muramyl peptides of the following structure: muramylpeptide A: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid; muramylpeptide B: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine; muramyl peptide C: dimer of muramyl peptide B, where the connection between two muramyl peptides B is carried out by the carboxyl group of the terminal D-alanine of one molecule B and the ω-amino group of meso-diaminopimelic acid of the other molecule B; in this case, the residues of muramylpeptides A, B, and C within the oligomers can be combined in any order, forming different position isomers. 2. The composition according to claim 1, further comprising a mixture of soluble muramyl peptide oligomers BC and AC and/or their pharmaceutically acceptable salts, wherein the residues of muramyl peptides A, B and C within the oligomers can be combined in any order, forming different position isomers. 3. Composition according to claim 1 or 2, additionally including soluble muramylpeptides A, B and C and / or their pharmaceutically acceptable salts, while the total mass fraction of muramylpeptides A, B and C and / or their pharmaceutically acceptable salts in the composition is not more than 50%. 4. Composition according to claim 3, in which the total mass fraction of muramyl peptides A, B and C in the composition is not more than 10%. 5. Composition according to claim 4, including muramyl peptide oligomers BCB, ACA, DIA, BC and AC, as well as muramyl peptides A, B and C, and the total mass fraction of muramyl peptides A, B and C in the composition is not more than 10%. 6. Composition according to claim 1, additionally containing a pharmaceutically acceptable carrier and/or excipient. 7. The method of obtaining a composition according to any one of paragraphs. 1-6 from cell wall peptidoglycan (PKC) of gram-negative bacteria, including the following steps: - prepare the resulting bacterial biomass for destruction by treatment with an ultrasonic disperser with a power of 400-800 W; - the prepared biomass is treated with a buffer having a pH of 8.0-9.0 and containing an ionic detergent at a concentration of 0.1-1%, at a temperature of 40-90°C, while the ionic detergent is sodium deoxycholate, sodium decyl sulfate, sodium dodecyl sulfate or N-lauryl sarcosinate; - clean the resulting biomass from impurities and detergent; - mechanically grind the resulting biomass; - produce enzymatic hydrolysis of biomass using lysozyme at a pH of 4.5-7.0 and a temperature of 40 to 60°C; - allocate the target products of enzymatic hydrolysis using ultrafiltration with a cut-off size of semi-permeable membranes up to 10 kDa; - if necessary, separate the target products of enzymatic hydrolysis with separation of the mixture, including at least soluble muramylpeptide oligomers BCB, ACA, ACB and/or their pharmaceutically acceptable salts. 8. The method according to p. 7, in which the enzymatic hydrolysis of biomass with lysozyme is carried out at a temperature of 45-55°C. 9. The method according to claim 7, in which the separation of the target products of enzymatic hydrolysis is carried out using low pressure gel permeation or ion exchange chromatography or using high performance liquid chromatography. 10. The method according to p. 7, in which the mechanical grinding of the resulting biomass is carried out using a mechanical mill 11. The method according to p. 7, further comprising the step of lyophilizing the isolated target products of enzymatic hydrolysis.

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