RU2478644C2 - Method for preparing pharmacologically acceptable mixture of substances containing low-molecular ingredients of cell wall peptidoglycane of gram-negative bacteria and possessing immunostimulatory activity - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biochemistry. A biomass of the gram-negative bacteria Salmonella typhi fam. Enterobacteriacea is prepared. A cell wall peptidoglycane (CWP) of the bacteria is recovered by biomass extraction in 45% aqueous phenol at temperature 70-90°C or in aqueous solution of ionic or non-ionic detergents at temperature 37-100°C. That is followed by preparative enzymatic hydrolysis for insoluble CWP cleavage with the use of lysozyme at pH 4.5 - 8.9 and temperature 10 - 37°C. Simultaneously, a pharmacologically acceptable mixture of substances is removed by dialysis from the reaction mixture with the use of semipermeable membranes for ultrafiltration with cut-off size up to 5 kDa. The mixture of substances is also recovered by means of column gel-chromatography, particularly preparative gel chromatography on Sephadex or TSK gel columns. The end product yield is 320 mg.

EFFECT: method enables producing the pharmacologically acceptable mixture of substances containing the following ingredients: β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid) (GMtri); β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramoyl-(L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine) (GMtetra); and GMtetra dimer (diGMtetra) wherein a bond of monomer residues of GMtetra is enabled by a carboxyl group of terminal D-alanine of one GMtetra residue and ω-aminogroup of meso-diaminopimelic acid of the other GMtetra residue with the coupled tetrapeptide resides positioned in various polysaccharide chains.

4 cl, 2 tbl, 3 ex

Description

Field of Invention

The invention relates to the field of microbiology and can be used in immunopharmacology, in particular the creation of means of supportive therapy designed to increase the body's immune 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 problem of influenza and acute respiratory infections, which are the main reason for the decrease in the working capacity of the population and bring millions of losses to the state, continues to be one of the most important. 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 pathogens.

One of the natural 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 sufficient to note that in sterile animals (gnotobionts) the immune system is atrophied and, as a result, they are defenseless even against opportunistic microorganisms.

A powerful bacterial immunostimulant is bacterial cell wall peptidoglycan (PCB). Based on the numerous structural analysis data of fragments obtained by the action of lysozyme on the PCB, an enzyme that selectively cleaves the glycosidic bonds of muramic acid residues, it was found that the PCB is a “crosslinked” heteropolymer that contains linear polysaccharide chains constructed from alternating N-acetyl- D-glucosamine and N-acetyl-D-muramic acid, attached β- (1 → 4) -glycosidic bonds, and the lactyl groups of the residues of muramic acid carry short peptides ("antennas"). In the majority of gram-negative microorganisms studied, the “antenna” fragments are tri- and tetrapeptide residues — L-alanyl-D- from glutaminyl- meso- diaminopimelic acid and L-alanyl-D- from glutaminyl- meso- diaminopimeloyl-D-alanine, respectively. Part of the “antenna” tetrapeptide fragments located in different polysaccharide chains form “bridge” structures due to bonds between the amino group of the diamino acid residue and the carboxyl group of the terminal D-alanine residue, providing the creation of the spatial structure of the PCB. 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 conditions of its cultivation.

More than thirty years ago, a group of French researchers led by Lederer found that the minimal fragment of PKC - N-acetyl-D-muramoyl- (L-alanyl-D-glutamic acid) (muramyl dipeptide, MDP) has an adjuvant effect, the ability to stimulate anti-infection resistance, antitumor immunity, activate immunocompetent cells and induce the synthesis of a number of cytokines [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 the high pyrogenicity, it was unsuitable for clinical use. Subsequently, it was shown that the semisynthetic β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl- (L-alanyl-D-isoglutamine) (glucosaminylmuramyl dipeptide, GMDP) containing, as compared with MDP the additional N-acetyl-D-glucosamine residue has a significantly lower pyrogenicity and can be used as an immunomodulator with a wide spectrum of action. However, it must be borne in mind that GMDP is a synthetic product that only imitates the structure of natural peptidoglycan fragments in the peptide part. It, unlike the native components isolated from the PCS of gram-negative bacteria, the peptide antennas of which are built from three or four amino acid residues, includes only two. Moreover, natural peptidoglycan fragments carry negative and positive charges (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 - the molecule is neutral.

An analysis of recent studies allows us to conclude that three groups of natural biologically active drugs isolated from the cell wall of gram-negative microorganisms may be of interest for medical practice.

The first is highly purified, mainly from trace amounts of highly endotoxic bacterial lipopolysaccharide (LPS), insoluble in water (including buffer solutions and detergents and chaotropic compounds) of PCB, the potential scope of which is due to its pronounced adjuvant and immunostimulating properties, and in the case of subcutaneous the introduction of a possibly prolonged effect of PKC (effect "depot").

The second group includes soluble high molecular weight fractions of PCB, which can be obtained from it by the action of powerful ultrasound or by treatment with lysozyme, as described in the prior art. Upon receipt of this group of preparations, it is often observed that the parameters of molecular weight distribution and the content of “antenna” and “crosslinked” fragments in high molecular weight PCS cleavage products are irreproducible, which is mainly due to the difficulty in standardizing the processes of cultivation of microorganisms, as well as the fact that in the process rather long-term deactivation of microbial cells after completion of cultivation, the action of hydrolytic bacterial enzymes - muramidases and amidases is possible. This leads to uncontrolled variations in the immunobiological properties of these drugs over a fairly wide range. In addition, when obtaining high molecular weight fractions of PCB by the enzymatic method, the intractable problem of removing large amounts of lysozyme arises, since the molecular weights of the enzyme and the resulting peptidoglycan fractions are quite close.

The third group of biologically active preparations of peptidoglycan nature, the most studied chemically and immunobiologically, is represented by low molecular weight components (800-2000 daltons), which are the final products of PKC hydrolysis by lysozyme. In the overwhelming majority of PCD of gram-negative microorganisms, two “antenna” fragments are found as major fragments - β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl- (L-alanyl-D- from glutaminyl- Meso- diaminopimelic acid) (GMtree) and β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl- (L-alanyl-D- iso glutaminyl- meso- diaminopimeloyl-D-alanine ). (GMetter), as well as a “crosslinked” fragment, which is a GMetter dimer (diHMetter), in which the connection between the monomer residues of the GMetter is carried out terminally by the carboxyl group D-alanine of one GM tetra residue and the ω-amino group of the meso- diaminopimelic acid of another GM tetra residue, both tetrapeptide residues are localized in different polysaccharide chains.

Until recently, the practical use of this group of natural low molecular weight peptidoglycan derivatives was complicated, in addition to their insufficient study of the nature of the effects induced by them, because, on the one hand, the yield of these compounds did not allow to rely on their practical use, and on the other hand, difficulties in their purification from pyrogenic, toxic and possibly allergenic impurities (bacterial LPS, proteins, nucleic acids, etc.).

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, isolation of PKC bacteria, and its splitting with enzymatic hydrolysis using lysozyme and the allocation of 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. The fragments of PCB obtained in a known manner, along with other bacterial biopolymers, are directly added to a number of fermented milk products, which causes 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 their fragments), their preliminary chemical degradation is required, which represents significant technological difficulties at the stage of purification.

A recently published work describes a method for producing a biologically active dimer of β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl- (L-alanyl-D- from glutaminyl- meso- diaminopimeloyl D-alanyl -D-alanine) isolated in analytical amounts from the cell wall of Salmonella typhi . However, when analyzing the PCS of virulent and vaccine strains of various genera of gram-negative microorganisms, and in particular, from S. typhi and bacteria of the genus Shigella, the fragment described in this publication was not found. In all cases, the main (and, often, the only) components of KPS hydrolysis by lysozyme were the above described GMtri, GMtetra and diHMtetra, while the peptidoglycan component with five amino acid residues or its dimer could not be identified.

Presence of unwanted side effects, such as individual patient sensitivity to synthesized drugs, the possibility of reducing the therapeutic effect with prolonged use of known immunostimulants leads to the need for 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.

SUMMARY OF THE INVENTION

In a comparative analysis of the products of the treatment of PCB of various gram-negative bacteria with lysozyme, it was found that in most cases, along with the PCB that has not been solubilized and high molecular weight products of enzymatic cleavage, the hydrolyzate usually contains water-soluble low molecular weight fragments of PCB, the detection of which in the hydrolyzate is evidently due to the presence of more or less extensive areas with a high content of “antenna” (but not “bridge”) structures. The presence in the PCS of vast areas that are not solubilized by lysozyme, with a maximum content of peptide "bridges" between polysaccharide chains, along with sites that, when exposed to lysozyme, break down into water-soluble poly- and oligomeric fragments, confirms the complexity and heterogeneity of spatial PKS structure.

The essence of the invention consists in the development of a method for isolating gram-negative bacteria from a PCS of a pharmacologically acceptable mixture of substances with an immunostimulating effect and including three main components as active substances - GMtri, GMtetra and diHMtetra.

The technical result of the invention is to create a scheme for the separation and purification of fragments of PCB, including a minimum number of stages (which implies a significant reduction in the number of process impurities), each of which provides the simultaneous removal of impurity compounds of various nature and the selection of the target drug of high purity, as well as providing the specified above a pharmacologically acceptable mixture of substances.

The specified technical result is achieved in that the method of obtaining a pharmacologically acceptable mixture of substances with an immunostimulating effect from substances of a bacterial nature includes the preparation of a bacterial mass, the isolation of PKC bacteria, the breakdown of insoluble PKC by the action of lysozyme, an enzyme specifically cleaving glycosidic bonds of muramic acid residues, and chromatographic purification of a pharmacologically acceptable mixture of substances .

The difference of the proposed method, as will be described below, is the use of a combination and sequence of operations, each of which is characterized by a unique set of conditions for their implementation, which leads to a product with a high level of purity and yield.

Disclosure of invention

The method involves the use of a combination of stages for isolating a pharmacologically acceptable mixture of substances from PCS, including the preparation of biomass containing substances of a bacterial nature, the isolation of PCS bacteria, the breakdown of insoluble PCS by preparative enzymatic hydrolysis using lysozyme and a buffered aqueous solution, and the isolation of a pharmacologically acceptable mixture of substances, due to that gram-negative bacteria were used as a substance of bacterial nature, while the isolation and purification of PCB carried out They are removed by extraction, the insoluble PCB is cleaved with the simultaneous removal of the target products of enzymatic hydrolysis by dialysis followed by lyophilization of the dialysate, and a buffer solution of lyophilized or non-lyophilized components at pH 4.5-8.9 is used as a buffer solution to isolate a pharmacologically acceptable mixture of substances preparative column gel permeation chromatography is used. Moreover, in one embodiment, the extraction of microbial cells is carried out with 45% aqueous phenol at a temperature of from 70 to 90 ° C.

In another preferred embodiment, the extraction of microbial cells is carried out with aqueous solutions of ionic and nonionic detergents.

Dialysis is carried out using dialysis tubes, in particular using semipermeable ultrafiltration membranes with a cut-off size of 1-5 kDa.

Column gel permeation chromatography using Sephadex and TSK gels is used to isolate a pharmacologically acceptable mixture of substances using elution with buffers containing lyophilized or non-lyophilized components, and at this stage, a pharmacologically acceptable mixture of substances from impurities (1-3%) tri- and tetrameric "crosslinked" and / or linear peptidoglycan fragments (mass spectrometry data).

To carry out the process according to this invention, a biomass containing substances of a bacterial nature is prepared, using gram-negative bacteria of the Enterobacteriacea family (e.g., Salmonella typhi, Salmonella typhimurium, Shigella flexneri, Shigella sonnei ) 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 substances with different contents of peptidoglycan components in them - GMtri, GMtetra and diHMtetra (the ratio of GMtri and GMtetre usually differs noticeably).

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, a preliminary chemical treatment step is not required, since the main polysaccharide-containing antigen of gram-negative bacteria, endotoxic LPS, is easily eliminated from the outer membrane of the cell wall as a result of extraction, and at the same stage, proteins, nucleic acids and other non-covalently bound with PKC 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.

Within the framework of the present invention, it is most efficient to use 45% aqueous phenol at a temperature higher than that commonly used in the extraction of LPS according to Westphal, namely at a temperature of 70-90 ° C, since in the indicated temperature range, in addition to the complete homogenization of the phenol-water mixture dissolution of cell walls in this mixture and, as a result, maximum extraction of “extraneous” biopolymers from them. Thus, a comparative analysis of PKC obtained in conventional manner (extraction temperature 65-68 ° ^C) and at higher temperature (70-90 ° ^C) shows that the level of content of protein contaminants varies 2-3. At a lower protein content in the PCB, the BAF yield increases significantly under the action of lysozyme (10–50%), which is obviously associated with de-screening of the PCB as a result of the removal of bulk protein biopolymers.

A suspension of PCB in a buffer aqueous solution is subjected to preparative enzymatic hydrolysis at a temperature of 10-37 ^about With the use of endo-N-acetylmuramidases, for example lysozyme. The use of lysozyme is due to the selectivity of cleavage of glycosidic bonds between the residues of muramic acid and glucosamine in the polysaccharide part of PCB in combination with a relatively low cost, high efficiency and stability over a wide range of pH and temperatures.

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.

During dialysis, a lyophilized buffer aqueous solution with a pH of 4.5-8.9 containing volatile amines (e.g. triethylamine, ammonia, pyridine), as well as volatile organic acids (e.g. formic or acetic) can also be used as a buffer solution. buffer solutions based on non-lyophilizing components (for example, chloride and sodium phosphates or TRIS-Hcl) should be used; commercially available dialysis tubes with different pore diameters or semipermeable ultrafiltration membranes with cut-off sizes up to 5 kDa can also be used. In this case, low molecular weight enzymatic hydrolysis products are transferred to the buffer solution against which dialysis is performed, containing mainly a pharmacologically acceptable mixture of substances , which leads to a significant increase in the process speed 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 a pharmacologically acceptable mixture of substances with the same speed.

Further, according to the proposed combination of steps, the combined solutions of low molecular weight enzymatic hydrolysis products are lyophilized and the residue is either re-lyophilized (to remove trace amounts of buffer components), in the case of lyophilized dialysis buffers, or desalted using preparative gel chromatography on a G10 or TSK Sephadex gel column in deionized water.

The proposed combined approach, including enzymatic hydrolysis with simultaneous removal of the target product from the reaction mixture, significantly reduces the breakdown time of PCB and increases the yield of a pharmacologically acceptable mixture of substances compared to the traditional approach in which the hydrolysis stage and the isolation of its products are separated. An analysis of the products remaining inside the dialysis tube or in the supmembrane space after dialysis shows that among them there are practically no biopolymers of peptidoglycan nature. Thus, the proposed combination of stages provides a fairly complete extraction of the target pharmacologically acceptable mixture of substances from PCS.

Isolation of a pharmacologically acceptable mixture of substances is also carried out without using the dialysis step using column gel permeation chromatography, in particular gel chromatography using Sephadex (Pharmacy) gels, eluting with water or aqueous buffer solutions containing lyophilized or non-lyophilized components, followed by lyophilization and desalting using gel chromatography as described above. At this stage, the starch-like storage bacterial polysaccharides found in most cases are also separated.

Pharmacologically acceptable mixtures of substances obtained by the proposed method contain no more than 1% proteins and no more than 1% nucleic acids, have an acceptable level of pyrogenicity in the rabbit test and endotoxicity in the limulus-lysate test, and do not contain signals of foreign compounds in the NMR and mass spectra -spectra.

Thus, according to the proposed method, preparative (gram) amounts of a mixture of natural native fragments of peptidoglycan nature with immunostimulating activity, which are formed mainly in the intestines of warm-blooded and human from PCS gram-negative bacteria and play a crucial role in maintaining their immune status, were first obtained. Immunological analysis of a pharmacologically acceptable mixture of substances isolated from various microorganisms and dramatically different in the ratio of their constituent components showed that their activities are close. From these data it follows that in the framework of the study, 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 allows 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 can be implemented in large-scale production, because Each stage described in the examples below is an adequate industrial model.

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

PCS isolation

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 65-68 ° C and then 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 (a 2-3-fold decrease in the volume of extractants is possible). The precipitate obtained by extraction was washed with 0.2 M ammonium bicarbonate buffer until absorbance at 260-280 nm in the UV spectrum of the supernatant (region of specific absorption of nucleic acids and proteins), and then 2-3 times with water.

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

The number of extractions hot aqueous phenol may be reduced to one or increased to 3-4, the amount of extractant can be reduced or increased by 1.5-2 times, during the extraction at a temperature higher than 65-68 ^{° C,} the extraction is reduced to 10 -15 minutes.

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. To mix at temperature (hereinafter) 10^about300 mg of lysozyme was added from the 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 for 3 days, periodically (1-3 times a day) removing the dialysate and adding to the external vessel fresh buffer solution. At the end of the process, the combined dialysates were evaporated to a volume of 200 ml at a temperature of less than 40^aboutC, the residue was lyophilized and then dissolved in 200 ml of deionized water, and the solution was re-lyophilized. The yield of a pharmacologically acceptable mixture of substances was 320 mg.

Isolation and purification of a pharmacologically acceptable mixture of substances

Preparative isolation of a pharmacologically acceptable mixture of substances was carried out using gel chromatography on a Sephadex G-50 column in 0.05 M ammonium bicarbonate buffer, pH 8.3. A pharmacologically acceptable mixture of substances elutes in the form of two peaks close in terms of area, with Kd 0.42 and 0.53, with diGMtetra eluting with shorter time, and a mixture of GMtri and GMteter with longer time. The combined fractions are lyophilized and then re-lyophilized from deionized water. Alternatively, the combined fractions may be desalted on a Sephadex G-10 column (eluent is deionized water).

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

From the data of mass spectrometry (MALDI-TOF) it followed that a pharmacologically acceptable mixture of substances contains three components - GMtri, GMtetra and diHMtetra with mol. in masses (calculated values in brackets) 868.4 (868.8), 939.4 (939.8) and 1860.8 (1861.7), respectively.

Analysis of the 13C-NMR spectrum confirmed the structure of a pharmacologically acceptable mixture of substances and the absence of noticeable amounts of impurity substances.

Thus obtained drug at a dose of 0.1 μg / ml did not cause a pyrogenic effect in the test on rabbits. The content of bacterial endotoxins in the drug solution with a concentration of 100 μg / ml filed by the LAL test did not exceed 1.5 EU / ml. A single intramuscular and intraperitoneal administration of the drug to BALB / C mice and Wistar rats in the tested dose range of 1-500 mg / kg does not cause any signs of intoxication and death of animals. The highest dose tested in mice and rats at a dose of 500 mg / kg was more than 300,000 times higher than the highest therapeutic dose (100 μg / person or 1.5 μg / kg) recommended for humans.

Using the examples described above, specialists are able to carry out adequate scaling in order to create large-scale production.

Immunostimulating activity of muramyl peptide compounds

The determination of the immunostimulating activity of a pharmacologically acceptable mixture of substances was carried out in accordance with the “Methodological guidelines for the study of the immunotropic activity of pharmacological substances” R.M.Khaitova et al., Approved by the Pharmacological State Committee of the Ministry of Health of the Russian Federation (In the book Guide on the experimental (preclinical) study of new pharmacological funds. M. 2000. 257-262). In accordance with these instructions, the test substance, which is supposedly an immunostimulant, should enhance a number of parameters of the immune system, among which the central place belongs to the study of the humoral immune response and the synthesis of tumor necrosis factor-α (TNF-α). These parameters of the immune system play a central role in protecting the body from infection.

In the study of humoral immunity, polyacrylic acid - PAA (manufactured by Petrovax PHARM, Russia) was used for comparison, which stimulates the formation of antibody-forming cells (AOK) 4-6 times in mice at optimal doses; when studying the synthesis of TNF-α, E. coli lipopolysaccharide (LPS) (Sigma, USA), which is a strong inducer of this cytokine, was used for comparison.

Example 2

Evaluation of the humoral immune response

In this example, the humoral immune response was assessed by the ability to form antibody-forming cells (AOK) in response to the administration of a thymus-dependent antigen, sheep erythrocytes (EB). AOK was determined by local hemolysis in agar according to Yern and Nordin. The study was conducted on mice, females (CBAxC57Bl / 6) F ₁ weighing 22 ± 0.6 grams. In each group there were 8-10 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 0.01, 0.1 and 1.0 μg 3 hours before the intraperitoneal immunization of EB with a dose of ⁵ × ^{10 6} . This 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. PAA was administered subcutaneously at a dose of 100 μg / mouse. On day 4, the amount of AOK in the spleens was determined. The results were expressed as the number of AOK per spleen and as a stimulation index (IS) of the immune response compared to the control group (Table 1).

Table 1
The effect of a pharmacologically acceptable mixture of substances on the humoral immune response of AOK / spleen. (M + m) Animal processing Pharmacologically acceptable mixture of substances PACK A drug Antigen Abs. THEM Abs. THEM SFR + EB 343.6 ± 40.8 1.00 ± 0.12 343.6 ± 40.8 1.00 ± 0.12 1 mcg Polimuramil + EB 1080.9 ± 167.6 3.15 ± 0.49 * N.D. N.D. 10 mcg Polimuramila + EB 1141.1 ± 182.7 3.30 ± 0.50 * 980 ± 128 2.8 ± 0.65 * 100 mcg Polimuramila + EB 868.1 + 190.4 2.51 ± 0.55 * 1290 ± 210 3.7 ± 0.9 * * p <0.05

It can be seen from the table that a pharmacologically acceptable mixture of substances has a pronounced ability to stimulate a humoral immune response. This effect is dose dependent. The maximum stimulating effect is observed when applying a dose of 10 μg. At a dose of 100 μg / mouse, a further increase in the stimulating effect does not occur. The stimulating effect of a pharmacologically acceptable mixture of substances is comparable to the action of the classical activator of humoral immunity PAA and at a dose of 10 μg / mouse it even slightly exceeds it.

Example 3

Assessment of the synthesis of TNF-α

In this example, the comparison of muramyl peptide compounds was carried out on a model of TNF-α, one of the central mediators of the immune system. For this, the peripheral blood mononuclear cells of 7 healthy donors aged 20-40 years old, sterile isolated on a ficoll-verographin gradient, density 1.077, were incubated for 2 days in a CO ₂ incubator in a complete culture medium consisting of RPMI-1640 medium with 150 g gentamicin / ml, with 10% inactivated fetal calf serum, 15 mM glutamine (Sigma). A pharmacologically acceptable mixture of substances in doses of 100 μg / ml, 10 μg / ml and 1 μg / ml was added to the test wells, and culture medium was added to the control wells. LPS of enterobacteria in the same doses was used as a comparison. The content of TNF-α in the supernatants of cultured cells was determined using solid-phase ELISA using commercial Cytelisa test systems from Cytimmune (USA).

The results are presented in Table 2.

table 2
TNF-α level in supernatants of stimulated mononuclear cells Drug concentration TNF-α Unstimulated cells - 47 ± 44 pharmacologically acceptable mixture of substances 0.1 μg / ml 2 ± 2 1 mcg / ml 1552 ± 1757 10 mcg / ml 4269 ± 2422 100 mcg / ml 8326 ± 1665 LPS 0.1 μg / ml 1504 ± 1092 1 mcg / ml 2462 ± 1721 10 mcg / ml 4248 ± 1002 150 mcg / ml 3559 ± 636

It can be seen from the table that LPS in low concentrations significantly exceeds the pharmacologically acceptable mixture of substances in the ability to induce TNF, but in large doses, the pharmacologically acceptable mixture of substances approaches or somewhat exceeds the immunostimulating effect of LPS.

The data obtained allow us to conclude that a pharmacologically acceptable mixture of substances according to the two studied parameters of the immune system has pronounced immunostimulating properties.

Claims (4)

1. A method of obtaining a pharmacologically acceptable mixture of substances having immunostimulating activity, where the mixture of substances includes the following components:
β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl- (L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid) (GMtri);
β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl- (L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine) (GM tetra);
and a GMetter dimer (diHMetter), in which the connection between the monomer residues of the GMetter is due to the carboxyl group of the terminal D-alanine of one GMetetra residue and the ω-amino group of the meso-diaminopimelic acid of the other GMtetra residue, the tetrapeptide residues connected to each other located in different polypeptide chains including
a) preparation of biomass of gram-negative bacteria of the Enterobacteriacea family,
b) the isolation of bacterial cell wall peptidoglycan (PCB) as a water-insoluble material by extraction of said biomass with 45% aqueous phenol at a temperature of 70-90 ° C or extraction is carried out with aqueous solutions of ionic or non-ionic detergents at a temperature of 37-100 ° C,
c) cleavage of insoluble PCB by preparative enzymatic hydrolysis using lysozyme at pH 4.5-8.9 and a temperature of 10-37 ° C while removing a pharmacologically acceptable mixture of substances from the reaction mixture by dialysis using semipermeable ultrafiltration membranes to isolate low molecular weight products enzymatic cleavage of PKC;
d) isolation of a pharmacologically acceptable mixture by column gel permeation chromatography. 2. The method according to claim 1, characterized in that the gram-negative bacterium is Salmonella typhi. 3. The method according to claim 1, characterized in that the dialysis is carried out using dialysis tubes or semipermeable membranes for ultrafiltration with a cut-off size of up to 5 kDa. 4. The method according to claim 1, characterized in that the selection of a pharmacologically acceptable mixture of substances is carried out using preparative gel chromatography on columns with Sephadex or TSK gel.