RU2412197C2 - Polymer fragment of peptidoglican of gram-negative bacteria cell wall, method of its obtaining and its application as immunostimulator - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: claimed is biopolymer with expressed immunostimulating effect, consisting of highly molecular fragment of peptidoglican of cell wall of Gram-negative bacteria, whose repeating link (RL) represents tetrasaccharide 4-O-{4-O-[4-O-(N-acetyl-β-D-glucosaminyl)-N-acetyl-β-D-muramyl]-N-acetyl-β-D-glucosaminyl}-N-acetyl-D-muramic acid, to which on carboxyl groups of both residues of muramic acid bound is tetrapeptide residue: N-(L-alanyl-D-isoglutaminyl-meso-diaminopimelonyl-D-alanine). Binding between RL is perfomed due to octapeptide bridges (dwg.1), number of RL constituting from 5 to 20 or their combination. Also claimed are method of biopolymer obtaining, based on it pharmaceutical immunostimulating composition and its application in methods of stimulating immune system of mammals and non-specific protection against bacterial infections.

EFFECT: possibility of application in immunopharmacology, in particular, for creation of medications of supporting therapy, intended for enhancing immune resistance of organism.

11 cl, 2 dwg, 2 tbl, 5 ex

Description

Field of Invention

The invention relates to the field of microbiology and can be used in immunopharmacology, in particular, to create means of maintenance therapy, designed to increase the body's natural resistance.

State of the art

Currently, there is a decrease in the natural resistance of people to infectious agents, resulting in an increase in infectious diseases occurring in almost all countries of the world. The consequence of this is the growth of chronic infectious and inflammatory processes that are difficult to treat with traditional methods of treatment. Therefore, the urgent task of medical science is the creation of highly effective and safe immunostimulating drugs that increase the body's resistance to various infections.

One of the sources for creating immunostimulants is bacteria, which are the main activators of the immune system in humans and animals. The full functioning of the immune system completely depends on the normal microflora of the intestines, respiratory tract, skin, etc. It is enough to note that in sterile animals (gnotobionts) the immune system is atrophied and they are defenseless even against opportunistic microorganisms.

A powerful immunostimulant of a bacterial nature is the peptidoglycan of the cell wall (PCS) of the bacterium. Based on the numerous structural analysis data of fragments obtained during the hydrolysis of PCB with a lysozyme enzyme that selectively cleaves the glycosidic bonds of muramic acid residues, it has been established that PCB is a “crosslinked” heteropolymer that contains linear polysaccharide chains constructed from alternating N-acetyl-D residues -glucosamine (GlcNAc) and N-acetyl-D-muramic acid (MurNAc), attached β- (1 → 4) -glycosidic bonds, and the lactyl groups of the residues of muramic acid carry short peptides. In most studied gram-negative microorganisms, the peptide fragment is represented by the tetrapeptide residue: L-alanyl-D-isoglutaminyl-mesodiaminopimeloyl-D-alanine. Part of the "antenna" oligopeptide fragments form a "bridge" structure, providing the creation of the spatial structure of the PCB (Fig.1 and 2).

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

In 1974, a group of French researchers led by E. Lederer found that a relatively small molecular weight substance, muramoyl-L-lysyl-D-glutamic acid (muramyl dipeptide, MDP), which is a component of the cell wall peptidoglycan, has the same adjuvant effect , the ability to stimulate anti-infectious 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 requir ements for adjuvant activity of bacterial peptidoglycan subunits. Biochem. Biophys. Res. Commun. 1974. 59. 1317-1325]. However, due to the high pyrogenicity, it was not suitable for clinical use. Therefore, a number of its analogues were synthesized, which have received permission for medical use or are currently undergoing clinical trials [Werner G.H .. Jolles P. Immunostimulating agents: Europ. J. Biochem. 1996.224, 1-19]. Muramyl peptide compounds such as 6-O-tetradecylhexadecanoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (substance B30-MDP) can be attributed to such analogs; an MTP-PE preparation in which dipalmitoylphosphatidylethanolamine is covalently attached to the muramyl tripeptide, and the whole complex is incorporated into liposomes; Murabutide represented by N-acetylmuramyl-L-alanyl-D-isoglutamine-a-n-butyl ether; Romurtide - N-acetylmuramyl-L-alanyl-D-isoglutamine-stearoyl-L-lysine; as well as β-N-acetylglucosaminyl- (1 → 4) -N-acetylmuramoyl-L-alanyl-D-isoglutamine (GMDP) - a semi-synthetic drug Likopid. Depending on the bacterial source (gram-positive or gram-negative microorganisms), the method of its processing and purification, complexes of biologically active fragments of PKC with different structures can be isolated, which can become the basis for obtaining preparations with properties characteristic of each complex.

Of the known methods for producing biologically active substances with an immunostimulating effect, the closest to the described invention is a method for producing a biologically active substance from a substance of a bacterial nature (US No. 5185321 A61K 37/18, 1993), including the preparation of biomass, the isolation of PKC bacteria, and its cleavage by enzymatic hydrolysis using lysozyme and isolating fragments of PKC as the target product.

However, the known method does not ensure the release of the reaction mixture from proteins, nucleic acids and other components of the bacterial cell that remain in the target product. Fragments of PKC obtained in a known manner, along with other bacterial biopolymers, are directly added to a number of dairy products, which can cause undesirable effects (allergization of the body, endotoxic effects, in particular pyrogenic) when it is used.

In addition, the known method involves the use of gram-positive bacteria Lactobacillus bulgaricus, in which the main polysaccharide antigens of gram-positive microorganisms - teichoic acids, are attached to the remainder of the PKC muramic acid by a strong covalent bond. Therefore, to purify the target product from teichoic acids (or fragments thereof), their preliminary chemical degradation is required, which represents significant technological difficulties at the stage of purification, and the use of chemical reagents creates environmentally unfavorable conditions for the implementation of the known method.

The presence of adverse side effects, for example, individual sensitivity of patients to synthesized drugs, the possibility of reducing the therapeutic effect with prolonged use of known immunostimulants, necessitates a directed search for new ways to obtain new, more effective and safe complexes of biologically active substances to restore natural resistance and increase resistance to infectious agents and especially to the causative agents of respiratory infections.

SUMMARY OF THE INVENTION

In a comparative analysis of the products of the treatment of PKC of various gram-negative bacteria with lysozyme, we found that in most cases, along with PCB not solubilized and low molecular weight products of enzymatic cleavage, the hydrolyzate usually contains water-soluble high molecular weight fragments of PCB, the appearance of which is obviously due to the presence in the composition PKS of more or less extensive areas with a high content of "bridge" structures, which purely spatially can impede the effect of lyso tsima, and these sites are adjacent to areas that are less "saturated" "crosslinked" and thereby more easily cleaved by lysozyme. The discovery of large regions in the PCS that are not solubilized by lysozyme, with a maximum content of peptide “bridges” between polysaccharide chains, along with sites that, under the action of lysozyme, split into water-soluble poly- and oligomeric fragments, confirms the complexity and heterogeneity of spatial PKS structure. This is due, on the one hand, to the fact that PCS makes the main contribution to ensuring the strength of a bacterial cell, and, on the other hand, to the presence of numerous channels in the cell wall for transporting compounds necessary for the cell. The available literature data allowed us to assume that the high molecular weight compounds of a glucosaminyl muramyl peptide nature that we found containing octapeptide "bridging" fragments corresponding to natural ones should have a wider spectrum and a higher level of immunostimulating characteristics than those described previously.

In this regard, the present invention discloses a biopolymer (BP) having a pronounced immunostimulating effect. This BP is isolated from gram-negative bacteria and consists of a high molecular weight fragment of the bacterial cell wall peptidoglycan (PCB), the repeating unit of which is tetrasaccharide 4-O- {4-O- [4-O- (N-acetyl-β-D -glucosaminyl) -N-acetyl-β-D-muramyl] -N-acetyl-β-D-glucosaminyl} -N-acetyl-D-muramic acid to which the tetrapeptide residue is attached at both carboxyl groups of both muramic acid residues: N - (L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine), while the connection between the PP is due to octapeptide bridges, and the carboxyl groups of the terminal residues of D-alanine and the ω-amino group of the residues of meso-diaminopimelic acid are involved in the formation of peptide bonds. The number of PS BP is in the range of values from 5 to 20.

The present invention discloses a method for producing the above BP. The production method is based on the isolation of BP from PKC and includes cultivation, isolation of PKC bacteria, digestion of insoluble PKC by preparative enzymatic hydrolysis using lysozyme, and isolation of the biologically active fraction by column gel permeation chromatography.

The invention also discloses an immunostimulating composition containing the obtained BP and a pharmaceutically acceptable excipient (excipients), which can be used, in particular, sodium chloride and polyvinylpyrrolidone. The composition may be presented in dry form, which is reconstituted with water or another diluent before use.

The composition of the invention is used to stimulate the immune system of a mammal, wherein the pharmaceutical composition is administered to the mammal. The composition provides stimulation of the immune system at a level sufficient to provide protection against bacterial infections, in particular against lethal doses of staphylococcus and salmonella.

In this regard, the present invention discloses a method of nonspecific protection against bacterial infections, comprising administering to said mammal a pharmaceutical composition of claim 8 prior to the alleged infection.

The technical result that can be obtained by carrying out the invention consists in the isolation of gram-negative bacteria PS with a pronounced immunostimulating effect from PCB, consisting of a high molecular weight fragment of PCB, the PZ of which is tetrasaccharide 4-O- {4-O- [4-O- ( N-acetyl-β-D-glucosaminyl) -N-acetyl-β-D-muramyl] -N-acetyl-β-D-glucosaminyl} -N-acetyl-D-muramic acid, to which both carboxyl groups of both residues muramic acid joins the tetrapeptide residue: N- (L-alanyl-D-isoglutaminyl-meso-diamine pimeloil-D-alanine). The relationship between the PZ is due to the octapeptide bridges, and the carboxyl groups of the terminal residues of D-alanine and the ω-amino group of the residues of meso-diaminopimelic acid are involved in the formation of peptide bonds, and the amount of PZ in the BP is selected from the interval from 5 to 20 or their combinations (Fig. .one). Water-soluble high molecular weight BP is formed as a result of treatment with bacterial PKC, which contains sites with a large number of octapeptide “bridges,” such fragments non-hydrolyzable by lysozyme are adjacent to areas in which the degree of “crosslinking” is much lower and which is therefore much more susceptible to enzymatic cleavage.

The technical result from the implementation of the method of obtaining BP is to increase the degree of purification of fragments of the PCB.

The specified technical result is achieved by the fact that the following operations are included in the method for producing BP with an immunostimulating effect from a substance of bacterial nature in the following sequence: preparation of the bacterial mass, isolation of PKC bacteria, cleavage of the insoluble PKC by the action of the lysozyme enzyme that specifically cleaves the glycosidic bonds of muramic acid residues.

The unique properties of the resulting BP provide the provision of new drugs - immunostimulants, which, based on their effectiveness, provide protection against staphylococcal and salmonella infections.

Description of drawings

Figure 1 presents the formula selected biopolymer of the present invention.

Figure 2 presents the NMR spectrum of the selected BP.

Disclosure of invention

The method of producing a biopolymer is characterized by a certain combination of the stages of separation of BP from PCS, including the preparation of biomass containing substances of a bacterial nature, the isolation of PKC bacteria, the breakdown of insoluble PCB by preparative enzymatic hydrolysis using lysozyme and a buffered aqueous solution and the isolation of BP, due to the fact that as a substance Gram-negative bacteria were used of a bacterial nature, while the isolation of PKC is carried out by extraction, the splitting of insoluble PKC is carried out simultaneously by removing the products of enzymatic hydrolysis by dialysis followed by lyophilization of the dialysate; in this case, a buffer solution of lyophilized components at pH 4.5-8.9 is used as a buffer solution, and column gel chromatography is used to isolate BP.

In particular, in the method, as preferred, the following modes and operations are used:

the extraction of microbial cells is carried out with 45% aqueous phenol at a temperature of from 70 to 90 ° C; the extraction of microbial cells is carried out with aqueous solutions of ionic and nonionic detergents;

release from endotoxic LPS (detoxification) is carried out by heating the PCB with 1-3% aqueous organic acid at 100 ° C for 1-3 hours;

dialysis is carried out using dialysis tubes;

dialysis is carried out using semipermeable ultrafiltration membranes with a cut-off size of 1-10 kD;

To isolate BP, column gel permeation chromatography is used to separate PCS fragments using Sephadex and Sepharose gels during elution with buffers containing lyophilized components.

It is obvious that specific modes can vary, based on the equipment, reagents and materials used, taking into account the recommendations of suppliers and manufacturers.

Obvious modifications and embodiments of the present invention are included in the scope of the claims defined by the claims.

To implement the method according to this invention, a biomass is prepared containing substances of a bacterial nature, which are used gram-negative bacteria of the Enterobacteriacea family (Salmonella typhi, Salmonella typhimurium, Shigella flexneri and others) by culturing them in a nutrient medium, followed by isolation of bacterial cells by centrifugation or microfiltration, and thoroughly washing bacterial cells from the components of the nutrient medium.

Thus obtained biomass containing gram-negative bacteria is a source for further production of PCS. The content of PCS in gram-negative bacteria, although lower than in gram-positive ones, however, their use eliminates significant difficulties at the stage of separation of the target product from teichoic acids (or their fragments) - the main polysaccharide antigens of gram-positive microorganisms covalently attached to the remainder of muramic acid PCS. When using gram-negative bacteria, preliminary chemical degradation is not required, since the main polysaccharide-containing antigen of gram-negative bacteria - endotoxic lipopolysaccharide (LPS) - is easily eliminated from the outer membrane of the cell wall as a result of extraction, and at the same stage liberation from proteins, nucleic acids and other non-covalently occurs PKC-related components of the bacterial cell. 45% aqueous phenol is most often used as an extractant at a temperature of 65-68 ° C (Westphalle extraction), since at this temperature the mixture of phenol and water is completely homogenized. In addition, aqueous solutions of chaotropic compounds, as well as detergents with the ability to destroy aggregates formed by amphiphilic polymer molecules, can be used to extract bacterial cells.

In the framework of this invention, it is most effective to use 45% aqueous phenol at a temperature higher than previously used, namely at a temperature of 70-90 ° C, since in the indicated temperature range the maximum extraction of “extraneous” biopolymers is observed.

The extraction product (actually the cell walls) is then boiled with a dilute aqueous solution of an organic acid (formic, acetic, trichloroacetic) to free from trace amounts of LPS: under these conditions, the ketoside bond of the residues of the ketodesoxy octulosonic acid located between the polysaccharide and lipid components of L is extremely easily broken.

A suspension of PKC in a buffer aqueous solution is subjected to preparative enzymatic hydrolysis using endo-N-acetylmuramidases, for example lysozyme. The use of lysozyme is due to the selectivity of the cleavage of glycosidic bonds of the residues of muramic acid in the polysaccharide part of PCB in combination with a relatively low cost, high efficiency and stability at fairly high temperatures and in a wide range of pH values.

One of the main problems during preparative enzymatic hydrolysis is the slowing down of the reaction as the concentration of hydrolysis products increases. Therefore, to increase the yield and increase the rate of enzymatic hydrolysis of PKC in accordance with the present invention, the cleavage of PKC is carried out while removing the products of enzymatic hydrolysis by dialysis. Dialysis is carried out for 1-5 days when removing the buffer solution containing the hydrolysis products, and then adding fresh dialysis buffer solution, and changing the buffer solution is carried out 1-3 times during the day.

For dialysis, a buffer solution of lyophilized components at pH 4.5-8.9, for example triethylamine, ammonia, pyridine, as well as formic and acetic acids, is used as a buffer solution, and commercially available dialysis tubes with different pore diameters can also be used or semipermeable ultrafiltration membranes with a cut-off size of 1-10 kD. In this case, low molecular weight products of enzymatic hydrolysis pass into the buffer solution against which dialysis is performed, which leads to a significant increase in the speed of the process due to the continuous removal of hydrolysis products from the reaction mixture. The use of semi-permeable membranes, in particular devices and membranes for ultrafiltration (both in the "dead end" and tangential modes), allows you to continuously dilute the hydrolyzate with a "fresh" buffer solution and remove the buffer solution containing hydrolysis products at the same speed. The use of membranes with different pore sizes allows the removal of low molecular weight PCS hydrolysis products, for example, with a molecular weight of 1-2 kD.

Further, according to the proposed combination of stages, the lysozyme-insoluble PCB is removed by centrifugation and subsequent lyophilization of the supernatant containing BP. Isolation (fractionation) of BP is carried out using column gel permeation chromatography, in particular gel chromatography using Sephadex (Pharmacy) gels, eluting with water or aqueous buffer solutions containing lyophilized components. At this stage, the starch-like storage bacterial polysaccharides found in most cases are also separated.

The structure of the obtained biopolymer is proved by traditional methods of analysis.

Its biological activity was determined in immunological studies, after confirming its safety in use (toxicity and pyrogenicity control).

Based on the biopolymer, pharmaceutical compositions were prepared in liquid and dry forms using traditional components and methods for preparing pharmaceutical compositions.

Buffering agents, tonicity agents, preservatives, and the like were used as pharmaceutically acceptable additives.

The pharmaceutical composition was packaged in bottles, either hermetically sealed under sterile conditions, or subjected to lyophilization and capping.

It has been experimentally established that the drug can be administered at a dose of approximately 1.5 μg of active ingredient per kilogram of patient weight per day. This dosage can vary from 0.1 to 10 mcg, depending on the patient's condition and drug tolerance.

The duration of use of the drug is usually 7-10 days, although in the case of a chronic infection, the duration of use can be significantly increased, since the development of antibodies to the drug or a decrease in its effectiveness during the period of use up to one month has not been identified. In some cases, determined in practice, a single use of the drug may be sufficient.

It was established that the drug has the ability to enhance the absorption of bacteria by human peripheral blood leukocytes and increase the ability of these cells to kill microbes. This increases the ability of human blood monocytes to produce immunity activating cytokines. It enhances the synthesis of antibodies and increases the functional activity of natural killers, which play an important role in antiviral immunity.

In this regard, the drug is used in complex therapy with antimicrobial drugs for the treatment of secondary immunodeficiency states, manifested in increased infectious diseases.

These conditions and diseases include

surgical infections

- chronic recurrent furunculosis and other purulent-inflammatory diseases of the skin and soft tissues;

- chronic recurrent herpes of any localization;

- chronic infections of the bronchopulmonary apparatus;

- chronic infections of the genitourinary tract.

This list of diseases is not restrictive. The choice of conditions in which an increase in immune status is required is within the competence of a specialist.

The drug is administered, as a rule, intramuscularly. The drug is compatible with all antimicrobial and anti-inflammatory drugs that were available to the authors, among those traditionally used in the treatment of the above.

EXAMPLES

The following examples, given in order to detail and illustrate the invention, are not intended to limit the claims of the present invention.

Example 1

I. Isolation of PCB

20 g of S. typhi dried acetone and ether (or 120-180 g wet) bacterial cells were suspended with vigorous stirring in 350 ml of distilled water. The resulting suspension was heated to a temperature of 68-70 ° C and 350 ml of 90% aqueous phenol heated to the same temperature was added. The mixture was stirred for 20-30 minutes at a temperature of 85-87 ° C, after which it was cooled to a temperature of 10-12 ° C and centrifuged for 40 minutes at 5000 rpm. The precipitate after decantation of the aqueous and phenolic layers was subjected to repeated extraction according to the same scheme (2-3-fold reduction of extractants is possible). The precipitate obtained as a result of extraction was washed with 0.2 M ammonium bicarbonate buffer until absorbance at 260-280 nm in the UV spectrum of the supernatant (the region of specific absorption of nucleic acids and proteins), and then 2-3 times with water.

The yield of the washed and lyophilized dried pellet, which is crude PKC, was 20–40% of the weight of the dry starting cells. Amino acid analysis showed that, along with the monomeric components of PCS (muramic acid, glucoseamine, alanine, diaminpimelic and glutamic acids), the resulting product, as a rule, contains a significant amount of bound protein.

II. PKS cleaning

6 g of crude PCB containing impurities of nucleic acids and water-soluble proteins was suspended in 120 ml of buffer containing 0.2 M NaCl, 0.05 M Tris-HCl and 0.001 M MgCl ₂ and CaCl ₂ , pH 7.2-7.6 . The resulting suspension was stirred for 2 hours at a temperature of 37 ° C with ribonuclease A (activity of 50-100 units / mg, 50 μg / ml of solution) and deoxyribonuclease (activity of 400-800 units / mg, 5 μg / ml of solution), then was added to the solution disodium salt of ethylenediaminetetraacetic acid (EDTA) to a concentration of 0.01 M and then proteinase K (activity of 10-20 units / mg, 20 μg / ml of solution). After 1 hour of stirring at a temperature of 50 ° C, an insoluble precipitate was separated from the products of enzymatic hydrolysis by centrifugation (3000 rpm, 30 minutes) and then washed several times with water (until no absorption in the UV spectrum at 260-280 nm) and lyophilized, yield about 5 g

Amino acid analysis showed an almost complete absence in the hydrolyzate of amino acids purified in this way as appropriate for impurity proteins.

III. Depyrogenation of PKS

The product obtained in the previous step was stirred with 100 ml of 2% acetic acid at 100 ° C for 2 hours, the precipitate after cooling was separated by centrifugation, washed three times with water and lyophilized. The resulting product was extracted three times (stirring for 30 minutes) with a mixture of chloroform-methanol 2: 1, the precipitate of PCB was separated by centrifugation and dried in vacuum. The yield of PCB at this stage is quantitative.

IV. Enzymatic hydrolysis of PKC with lysozyme

5 g of PCB was suspended in 100 ml of 0.2 M triethylammonium acetate buffer pH 7.2. 300 mg of lysozyme was added to the stirred suspension, and after an hour of stirring, the reaction mixture was transferred to a dialysis tube, which was placed in a vessel with 300 ml of the buffer described above. Dialysis was carried out at a temperature of 37 ° C, periodically (1-3 times a day) for 3 days removing the dialysate and adding a fresh buffer solution to the external vessel. At the end of the process, the suspension contained in the dialysis tube was centrifuged, the supernatant was lyophilized, and the residue was lyophilized 2-3 times from water. The yield of crude BP at this stage was 7-8%.

V. Isolation and purification of BP

PD was isolated by gel chromatography on a Sephadex G-50 or G-75 column in 0.05 M ammonium bicarbonate buffer pH 8.3. Fractions eluting near the free volume of the column and containing starch-like spare glucan (NMR data) are discarded. Then eluting fractions containing BP are eluted, which are combined and lyophilized. Alternatively, the combined fractions could be desalted on a Sephadex G-15 column (eluent — deionized water) or dialyzed using ultrafiltration membranes with a cut-off size of kDa.

Analysis of the BP hydrolyzate (4M HCl, 100 ° C, 16 hours) using an amino acid analyzer showed that it contains equimolar amounts of glucosamine, muramic, glutamic and diaminopimelic acids, as well as twice the amount of alanine, which corresponds to the set and ratio of components in the PCB S.typhi.

An analysis of the 13C-NMR spectrum made it possible to confirm the structure of BP built from tetrasaccharide PZs bound by octapeptide “bridges” and the absence of noticeable amounts of impurity substances.

Example 2

5 g of dried cells with acetone and ether were suspended in 500 ml of water and subjected to ultrasound treatment (processor company Cole-Parmer, 500 watts, 20 kHz) at a temperature of less than 10 ° C for 30-40 minutes. Sodium chloride was added to the resulting suspension to 0.1 M concentration, TRIS to 0.1 M concentration, EDTA to 0.01 M concentration and sodium dodecyl sulfate to 2% concentration. The pH of the mixture was adjusted to 7.6 by the addition of concentrated HCl. After 2 hours of stirring at 100 ° C, 5-7 mg of proteinase K was added to the cooled reaction mixture.

The enzymatic treatment of PKC with proteinase K was carried out without preliminary action of nucleases (in the presence of detergents they lose activity) at a temperature of 56 ° C for 3 hours. The PCB precipitate was washed sequentially with a buffer containing no detergent, and then with water.

To obtain the target products of PCB cleavage with lysozyme, simultaneously with enzyme treatment, dialysis was performed using an ultrafiltration unit Pellikon or Minitan (Millipor company) using cartridges with a cut-off size of 10 kD. The feed rate of the “fresh” buffer and the selection of the filtrate containing low molecular weight fragments of PCB was 0.1–0.3 ml / min. The process takes 20-30 hours. Received thus the product (yield 5-6%) in its structure and properties is similar to BP, the isolation of which is described in Example 1.

Based on BP isolated by the described technology, a pharmaceutical preparation was prepared (Example 3) and its immunostimulating properties were evaluated.

Example 3

Pharmaceutical product

The composition of the drug per 1 ampoule (bottle):

1. Biopolymer (active principle) 0.1 mg 2. Table salt (NaCl) 9 mg 3. Polyvinylpyrrolidone low molecular weight medical 1.5 mg

The corresponding weighed portions of these ingredients were dissolved in a certain volume of pyrogen-free water, sterilized through filters with a pore size of less than 0.2 μm, poured into ampoules (vials) of 1 ml, freeze-dried and sterile sealed.

Before use, the drug was diluted in 1 ml of sterile water.

Example 4

Evaluation of the humoral immune response

An important property of immunostimulating drugs is the ability to enhance the humoral immune response, determined by the number of antibody-producing cells (AOKs) and the level of specific antibodies. In this example, the humoral immune response was evaluated by the ability to form AOK in response to the administration of a thymus-dependent antigen - ram erythrocytes (EB). AOK was determined by local hemolysis in agar according to Yern and Nordin (1965). The study was conducted on mice, females (CBA × C57Bl / 6) F ₁ weighing 22 ± 0.6 grams. In each group there were 10-12 mice.

The preparation was diluted with physiological saline and in a volume of 200 μl was administered to mice subcutaneously at the withers at doses of 1, 10 and 100 μg 3 hours before the intraperitoneal immunization of EB at a dose of 5 × 10 ⁶ . The indicated dose of EB is suboptimal for the development of a humoral immune response. Instead of the preparation, control animals were injected with saline and their EBs were also immunized. On the 4th day, the number of antibody-forming cells in the spleens was determined by local hemolysis in an agarose gel. The results were expressed as the number of AOK per spleen, per 1 million nucleated cells (SSC) of the spleen, and as the modulation index (IM) of the immune response compared to the control group (Table 1).

Table 1 The effect of the drug on the humoral immune response Animal processing AOK / spleen (M ± m) AOK / 10 ⁶ YASK villages. (M ± m) Dose of the drug Antigen amount THEM amount THEM Control (PFR) + EB 343.6 ± 40.8 1.00 ± 0.12 1.70 ± 0.20 1.0 ± 0.12 1 mcg + EB 1080.9 ± 167.6 3.15 ± 0.49 4.50 ± 0.70 2.7 ± 0.41 10 mcg + EB 1141.1 ± 182.7 3.30 ± 0.50 4.70 ± 0.75 2.8 ± 0.44 100 mcg + EB 868.1 ± 190.4 2.51 ± 0.55 3.80 ± 0.84 2.3 ± 0.44

It was found that the introduction of the drug in doses of 1, 10 and 100 μg causes a pronounced stimulation of the humoral immune response. The number of AOK to EB with the introduction of 1 μg, 10 μg and 100 μg increases in 3.15; 3.3 and 2.5 times, respectively. The optimal dose is a dose of 10 μg / mouse. At a dose of 100 μg, there is no further increase in the number of AOKs.

Thus, the experiments show the ability of the drug to stimulate antibody formation in mice.

Example 5

One of the most important indicators of the immunotropic activity of a substance is its ability to stimulate factors of innate immunity (nonspecific resistance), which is determined by the protection of experimental animals stimulated by the test substance from a deadly infection caused by pathogenic bacteria. In this example, the study of the influence of the biopolymer according to the invention on the non-specific resistance of mice was performed on outbred animals weighing 12-14 grams. Salmonella typhimurium (strain No. 415) and Staphylococcus aureus (strain Wood 46) were used as infectious agents. Infection was performed at a dose of 1 DCL (absolutely lethal dose) intraperitoneally 24 hours after drug administration. The drug was administered once, subcutaneously, at doses of 100 μg, 10 μg, 1 and 0.1 μg / mouse. As a control, animals were injected with sterile saline. In each group, there were 10 mice.

table 2 The effect of the drug on nonspecific resistance of mice to pathogenic bacteria BP dose Bacteria The death of mice (days) Survived% Continued of life 1st 2nd 3rd 4th one hundred S. typhimurium - - 3 one 60 5.7 10 - 5 2 2 10 2.7 1,0 - 8 one one 0 2.2 0.1 - 8 one one 0 2.2 physical rr one 9 * * 0 1.8 one hundred St. aureus 3 2 0 0 fifty 2.5 10 5 2 one 2 0 1,5 1,0 5 four one * 0 1.4 0.1 7 3 * * 0 1,2 physical rr 8 one 0 one 0 1,1

From table 2 it is seen that the biopolymer according to the invention at a dose of 100 μg / mouse increases the resistance of mice to infections caused by S. typhimurium and S. aureus, protecting against death in 50% and 60%, respectively. Smaller doses have a weaker protective effect. It was also shown that the drug at a dose of 100 μg increases the life expectancy of experimental animals by about 3 times when infected with S. typhimurium and 2 times when infected with S. aureus.

Thus, the biopolymer according to the invention has a pronounced ability to protect experimental animals from fatal infections caused by pathogenic bacteria.

Claims (11)

1. Biopolymer (BP) with a pronounced immunostimulating effect of a substance of bacterial nature, consisting of a high molecular weight fragment of the peptidoglycan of the cell wall (PCB) of bacteria, the repeating unit (PZ) of which is 4-O- {4-O- [4-O- tetrasaccharide (N-acetyl-β-D-glucosaminyl) -N-acetyl-β-D-muramyl] -N-acetyl-β-D-glucosaminyl} -N-acetyl-D-muramic acid, to which both carboxyl groups of both of residues of muramic acid, a tetrapeptide residue is attached: N- (L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine), wherein tidnaya communication between PP carried out due to the carboxyl groups of terminal residues alanine and D-ω-amino residues of meso-diaminopimelic acid to form the octapeptide "bridging" moieties and the amount of PP in the BS selected from a range of values from 5 to 20 or combinations thereof. 2. The method of obtaining BP according to claim 1, including the preparation of biomass containing substances of a bacterial nature, the isolation of PKC bacteria, depyrogenation in a dilute aqueous solution of an organic acid by boiling, the breakdown of insoluble PCS by preparative enzymatic hydrolysis using lysozyme and a buffered aqueous solution, and the isolation of BP, where gram-negative bacteria are used as a substance of bacterial nature, while the separation of PCB is carried out by extraction with 45% aqueous phenol at a temperature of from 70 to 90 ° C, p sscheplenie insoluble PKC performed with simultaneous removal of enzymatic hydrolysis products by dialysis followed by lyophilization of the dialysate, wherein a buffer solution is used as a buffered aqueous solution was lyophilized components at pH 4,5-8,9, and is used for isolating PD column by gel permeation chromatography. 3. The method of obtaining BP according to claim 2, characterized in that Salmonella typhi cells are used as a bacterial substance. 4. The method of obtaining BP according to claim 2, characterized in that the extraction of microbial cells is carried out with aqueous solutions of ionic and nonionic detergents at a temperature of 37-100 ° C. 5. The method of obtaining BP according to claim 2, characterized in that the depyrogenation for the degradation of the bacterial lipopolysaccharide is carried out by heating the PCB with 1-3% aqueous acetic acid at 100 ° C for 1-3 hours 6. The method of obtaining BP according to claim 2, characterized in that the dialysis is carried out using dialysis tubes with a cut-off size of up to 12 kD or using semipermeable membranes for ultrafiltration with a cut-off size of more than 10 kD. 7. A pharmaceutical immunostimulatory composition comprising the biopolymer according to claim 1 and a pharmaceutically acceptable excipient (excipients). 8. The composition according to claim 7, containing sodium chloride and polyvinylpyrrolidone as excipients. 9. The composition according to claim 7, which is a dry preparation, which is restored with water before use. 10. A method of stimulating the mammalian immune system, comprising administering to said mammal a pharmaceutical composition according to claim 7. 11. A method for nonspecific protection against bacterial infections, comprising administering to the mammal the pharmaceutical composition of claim 7 prior to the alleged infection.

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