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

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

 

The scope of the invention

The invention relates to the field of Microbiology and can be used in immunopharmacology, in particular a maintenance therapy designed to increase immune resistance of the organism.

The level of technology

Currently, there is a decrease in the natural resistance of people to infectious agents, resulting in a growth of infectious diseases occurring in almost all countries of the world. One of the most important remains the problem of influenza and acute respiratory diseases, which is the main reason for the decrease in work capacity of the population and bringing the state multimillion-dollar losses. Therefore, an urgent task of medical science is the creation of highly effective and safe immunostimulatory drugs that increase the body's resistance to the effects of different pathogens.

One of the natural sources to create Immunostimulants are bacteria that humans and animals are the main activators of the immune system. Proper functioning of the immune system depends entirely on the normal microflora of the intestine, respiratory tract, skin, etc. is Enough to note that sterile animals (gnotobiotic) the immune system is nonfunctional, and, following the journey of this, they are vulnerable even before conditionally pathogenic microorganisms.

A powerful immune stimulator bacterial nature is a peptidoglycan cell wall (PKS) bacteria. On the basis of numerous data structural analysis of the fragments obtained by the action of the PCB lysozyme - an enzyme that selectively cleave glycosidic bonds balances maramboi acid, it was found that the PCB represents a "stitched" heteropolymer, which contains a linear polysaccharide chain, built of alternating residues of N-acetyl-D-glucosamine and N-acetyl-D-maramboi acid attached β-(1→4)-glycosidic bonds, and lacticinia group balances maramboi acid are short peptides ("antenna"). In the majority of the studied gram-negative microorganisms "antenna" fragments represent a three - and tetrapeptide remains - L-alanyl-D-fromglutaminyl-meso-diaminopimelic acid and L-alanyl-D-fromglutaminyl-meso-diaminopimelate-D-alanine, respectively. Part of the "antenna" tetrapeptide fragments located in different polysaccharide chains form a "bridge" structure at the expense of ties between the amino group of residue diaminoanisole and carboxyl groups of the terminal residue of D-alanine, ensuring that the spatial patterns of the PCB. Was neotectonically, what degree of schiesty", i.e. the number of "bridge" fragments in the PCB depends not only on the type of microorganism, but from the conditions of its cultivation.

More than thirty years ago, a group of French researchers under the leadership of Lederer found that the minimum fragment of PKS - N-acetyl-D-muramyl-(L-alanyl-D-glutamic acid) (muramyldipeptide, TIR) has an adjuvant effect, the ability to stimulate anti-infective resistance, anti-tumor immunity, activate immunocompetent cells and to induce the synthesis of several 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 progenote he proved to be unsuitable for clinical use. Subsequently, it was shown that semi-synthetic β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramyl-(L-alanyl-D-isoglutamine) (glucosaminilmuramildipeptide, GMDP)containing compared to TIR additional residue N-acetyl-D-glucosamine has a significantly lower pyrogenalum and can be used as immunomodulator wide spectrum of action. However, be aware that GMDP is a synthetic product, only mimic the peptide part of the structure of natural peptidoglycan fragments. He, unlike native components selected from the PCB gram-negative bacteria, peptide antennas are built from three or four amino acid residues, contains only two. Moreover, natural peptidoglycans fragments are negative and positive charges (contain free carboxyl and amino groups), whereas in GMDP carboxyl group residue isoglutamine acid is in the form of an amide, i.e GMDP - neutral molecule.

Analysis of recent research allows us to conclude that the interest for medical practice can imagine three groups of natural biologically active compounds isolated from the cell wall of gram-negative microorganisms.

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

The second group includes soluble high-molecular fraction of the PCB, which can be obtained from it using the powerful impact of ultrasound or by treatment with lysozyme as described in the technician level is. Upon receipt of this group of drugs is often observed irreproducibility of the parameters of the molecular-mass distribution, and content of the "antenna" and "cross-linked high-molecular fragments in the fission products of the PCB, which is caused mainly by the difficulty of standardizing the cultivation of microorganisms, as well as the fact that the process is quite lengthy decontamination of microbial cells after cultivation possible hydrolytic action of bacterial enzymes - muramidase and amides. This leads to uncontrolled variations immunobiological properties of these drugs in a fairly wide range. In addition, upon receipt of high molecular weight fractions PKS enzymatic method occurs intractable problem of the removal of large quantities of lysozyme, as the molecular weight of the enzyme and the resulting peptidoglycan factions are pretty close.

The third group of biologically active preparations of peptidoglycan nature, the most studied in the chemical and biologic respect, presents low-molecular components (800-2000 daltons)that are the end products of hydrolysis of the PCB by the action of lysozyme. In the vast majority of the PCB gram-negative pathogens as major found two "antenna" fragment - β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramyl-(L-alanyl-D- fromglutaminyl-meso-diaminopimelic acid) (Gmti) and β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramyl-(L-alanyl-D-fromglutaminyl-meso-diaminopimelate-D-alanine).(Metra)and cross-linked fragment representing a dimer Metra (digmer), in which the relationship between Monomeric residues Metra is due to the carboxyl group of the terminal D-alanine of one residue Metra and ω-aminomeso-diaminopimelic acid other residue Metra, both tetrapeptide residue localized in different polysaccharide chains.

Until recently, the practical use of this group of natural low-molecular peptidoglycan derivatives was complicated, in addition to their insufficient knowledge about the nature of the induced them effects the fact that, on the one hand, these compounds were not allowed to rely on their practical use, and on the other hand, difficulties in their purification from pyrogenic, toxic and possibly allergenic substances (bacterial LPS, proteins, nucleic acids etc).

Known methods of obtaining biologically active substances immunostimulating action closest to the described invention is a method of obtaining biologically active substances of substances of bacterial origin (US No. 5185321, A61K 37/18, 1993), R is the overall preparation of the biomass, the allocation of the PCB bacteria, splitting its enzymatic hydrolysis using lysozyme and allocation of fragments of the PCB as the target product.

However, the known method does not provide the release of the reaction mixture from proteins, nucleic acids and other components of bacterial cells that remain in the target product. Obtained in a known manner fragments of the PCB along with other bacterial biopolymers directly add to a number of dairy products, causing its use undesirable effects (Allergy of the body, endotoxic effects, in particular pyrogenic).

In addition, the known method involves the use of gram-positive bacteria Lactobacillus bulgaricus, in which the main polysaccharide antigens of gram-positive microorganisms - resemble teichoic acid attached to the remainder of maramboi acid PKS strong covalent bond. Therefore, purification of the target product from the resemble teichoic acids (or their fragments) requires their prior chemical degradation, which represents a significant technological difficulties at the stage of purification.

In a recently published paper describes a method of obtaining biologically active dimer of β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramyl-(L-alanyl-D-fromglutaminyl-meso-diaminopimelic D-D-alanine), dedicated analytical quantities of cell wallSalmonella typhi. However, when analyzing the PCB virulent and vaccine strains of different genera of gram-negative microorganisms, and in particular, fromS. typhiand from bacteria of the genusShigella,described in this publication fragment was not detected. In all cases, the main (and often only) components hydrolysis CPS lysozyme were described above Hmtri, Metra and Dimetra, while peptidoglycan component with five residues of the amino acid or its dimer is unidentifiable.

The presence of unwanted side effects, for example,individual sensitivity of the patient to the synthesized drugs, the possibility of reducing therapeutic effect with long-term use is known Immunostimulants leads to the need for focused search for new ways of obtaining new, more efficient and secure complexes of biologically active substances to restore the natural resistance and resilience to infectious agents.

The invention

A comparative analysis of the treatment products PKS various gram-negative bacteria by lysozyme was found that in most cases along with not subjected to solubilize the PCB and isomolecular products fer entatives cleavage hydrolysate usually contains water-soluble nizkomolekulyarnye fragments of the PCB, detection in which the hydrolysate is due apparently to the presence in the composition of the PCB more or less extensive areas with a high content of "antenna" (but not "bridge") structures. The presence of the PCB vast areas, not solubilizing lysozyme, with a maximum content of peptide "bridges" between polysaccharide chains, along with areas, which under the action of lysozyme hydrolysis into water-soluble poly - and oligomeric fragments confirms detected in many experiments the complexity and heterogeneity of the spatial structure of the PCB.

The invention consists in developing a method of allocation of the PCB gram-negative bacteria pharmacologically acceptable mixture of substances with an immunostimulating effect and comprising as active ingredients the following three main components - Gmti, Metra and Dimetra.

The technical result of the invention is to provide circuit isolation and purification of the fragments of the PCB, including the minimum number of stages (which implies a significant reduction in the number of process impurities), each of which provides simultaneous removal of impurity compounds of different nature and identification of target preparation of high purity, as well as providing the above pharmacologically acceptable mixture of substances.

This technical result is achieved in that a method of obtaining a pharmacologically acceptable mixture of substances immunostimulating action of substances of bacterial origin include the preparation of bacterial mass, the allocation of the PCB bacteria, decomposition of insoluble DCC action of lysozyme - enzyme specifically cleave glycosidic bonds balances maramboi acid and chromatographic purification pharmacologically acceptable mixture of substances.

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

Disclosure of invention

The method involves using a combination of stages of selection pharmacologically acceptable mixture of substances of the PCB, comprising preparing a biomass containing substances of bacterial origin, highlighting the PCB bacteria, splitting insoluble DCC preparative enzymatic hydrolysis using lysozyme and buffer aqueous solution and isolation of pharmacologically acceptable mixture of substances, due to the fact that, as a matter of bacterial nature applied to gram-negative bacteria, however, the selection and cleaning the PCB carry out extraction, splitting insoluble DCC is carried out with simultaneous removal of target products of enzymatic hydrolysis of dialysis followed by lyophilization of dialysate, at the same time as the buffer solution using a buffered aqueous solution lyophilisate or reliabilitya components at pH 4,5-8,9, to highlight pharmacologically acceptable mixture of substances used preparative column gel permeation chromatography. In one of the embodiments of the invention the extraction of microbial cells carry 45%aqueous phenol at a temperature of from 70 to 90 º.

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

The dialysis is carried out with the use of dialysis tubing, in particular with the use of semi-permeable membranes for ultrafiltration with a size cutoff of 1-5 kDa.

To highlight pharmacologically acceptable mixture of substances used column gel permeation chromatography using a gel-type Sephadex and TSK at the elution buffers containing liabilities or reliabilities components, and at this stage there is a release of the pharmacologically acceptable mixture of substances from impurities (1-3%) of tri - and tetramer cross-linked and/or linear peptidoglycan fragment the data from mass spectrometry).

For carrying out the process according to this invention is prepared biomass containing substances of bacterial origin, which are applied to gram-negative bacteria of the familyEnterobacteriacea(for example,Salmonella typhi, Salmonella typhimurium, Shigella flexneri, Shigella sonnei) by culturing in a nutrient medium, the subsequent selection of bacterial cells by centrifugation or filtration and thorough washing of bacterial cells from the nutrient medium components.

Using different bacterial sources have the ability to obtain pharmacologically acceptable mixture of substances with different content in them peptidoglycan components Hmtri, Metra and Dimetra (usually markedly different ratio Gmti and Metra).

Thus obtained biomass containing gram-negative bacteria, is a source for further PCB. The content of PCB in gram-negative bacteria although lower than in gram-positive, however, their use eliminates considerable difficulties at the stage of separation of the target product from the resemble teichoic acids (or their fragments), the major polysaccharide antigens of gram-positive microorganisms, covalently attached to the remainder of maramboi acid PKS. When using gram-negative bacteria stage before kiteley chemical treatment is not required, since the main polysaccharide-containing antigen of gram-negative bacteria - endotoxins FSC easily eliminated from the outer membrane of the cell wall as a result of extraction, and it is at this stage it is freed from proteins, nucleic acids and other ecovalence related PCB components of the bacterial cell. As extractant often use 45%aqueous phenol at the temperature of 65-68°C (extraction by Westphal), since at this temperature there is a complete homogenization of the mixture of phenol and water. In addition, for the extraction of bacterial cells can be used aqueous solutions chaotropic compounds and detergents, which are able to destroy the aggregates formed amphiphilic polymer molecules.

In the framework of the present invention is most effective using the 45%aqueous phenol at a temperature that is higher than is generally used in the extraction of LPS by Westphal, namely at a temperature of 70-90ºC, as in the specified temperature range in addition to a complete homogenization of the mixture of phenol and water is dissolved in the mixture of cell walls and, as a consequence, the maximum extraction of "outsiders" biopolymers. Thus, a comparative analysis of PKS obtained by the traditional method (temperature extraction 65-68aboutC) and when bolivianos temperature (70-90 about(C) shows that the content of protein impurities varies by 2-3 times. At lower protein content in PKC significantly (10-50%) increases the output BAFA under the action of lysozyme, which is obviously related to the disscreening the PCB in the removal of bulk protein biopolymers.

The suspension of the PCB in the buffer aqueous solution is subjected to preparative enzymatic hydrolysis at a temperature 10-37aboutWith the use of endo-N-acetylmuramic, such as lysozyme. The use of lysozyme due to the selective cleavage of glycosidic linkages between remnants of maramboi acid and glucosamine in the polysaccharide portion of the PCB in combination with relatively low cost, high efficiency and stability in a wide range of pH values and temperatures.

One of the main problems when carrying out preparative enzymatic hydrolysis is slow reaction by increasing the concentration of the hydrolysis products. Therefore, to improve yield and increase the rate of enzymatic hydrolysis of the PCB in accordance with the present invention, the splitting of the PCB is carried out with simultaneous removal of the products of enzymatic hydrolysis of dialysis. Dialysis should be performed within 1-5 days when deleting a buffer solution containing the products of hydrolysis, and subsequent addition of fresh tialization the th buffer solution, and change the buffer spend 1-3 times during the day.

During dialysis as a buffer solution using liofilizados water buffer solution with a pH of 4,5-8,9 containing volatile amines (e.g. triethylamine, ammonia, pyridine), and volatile organic acids (e.g. formic or acetic), can also be used buffer solutions based on reliabilitya components (for example, chloride, and sodium phosphate or TRIS-Hcl); can also be used commercially available dialysis tubes with different diameter porous or semi-permeable ultrafiltration membranes with a size cutoff to 5 kDa. In buffer solution, against which the dialysis pass low-molecular products of enzymatic hydrolysis, containing mainly pharmacologically acceptable mixture of substances,which leads to a significant increase in the speed of the process due to the continuous removal of the products of hydrolysis of the reaction mixture. The use of semi-permeable membranes, in particular devices and membranes for ultrafiltration (as in "deadlock", and the tangential mode)allows you to continuously dilute hydrolysate fresh buffer solution and at the same speed to remove the buffer solution containing pharmacologically acceptable mixture of substances.

Further along before agemay combination stages combined solutions of low-molecular products of enzymatic hydrolysis lyophilized and the remainder either subjected to repeated freeze-drying (to remove trace amounts of the components of the buffer), if lyophilisate dualization buffers, or absoluut using preparative gel chromatography on a column of Sephadex G10 or TSK-gel in deionized water.

The proposed combined approach, including enzymatic hydrolysis with simultaneous removal from the reaction mixture of the target product significantly reduces the time splitting the PCB and increases the yield of a pharmacologically acceptable mixture of substances compared to the traditional approach, in which stage of hydrolysis and the allocation of its products are separated. Analysis of the product remaining inside the dialysis tube or namebrand space after dialysis, shows that there was virtually no biopolymers peptidoglycans nature. Thus, the proposed combination of stages provides a fairly complete extraction of the target pharmacologically acceptable mixture of substances from the PCB.

Selection pharmacologically acceptable mixture of substances carried out also without the use of stage dialysis using column gel permeation chromatography, in particular, gel-chromatography using a gel-type Sephadex (Pharmacia) by elution with water or aqueous buffer solutions containing liabilities or reliabilities components, followed by repeated lyophilization and desalting by gel-chromatog is the her, as explained above. At this stage the Department also found in most cases krakhmalopatochnyi spare polysaccharides of bacteria.

Pharmacologically acceptable mixture of substances obtained by the proposed method contain no more than 1% protein and no more than 1% of nucleic acids, have an acceptable level of progenote test on rabbits and endotoxicosis in limulus-lysate test and do not contain extraneous signals compounds in NMR spectra and mass spectra.

Thus, the proposed method first obtained preparative (g) the amount of the mixture of natural native fragments of peptidoglycan nature with immunostimulatory activity, which are formed mainly in the intestines of warm-blooded animals and humans from the PCB gram-negative bacteria and play an important role in the maintenance of their immune status. Immunological analysis of the pharmacologically acceptable mixture of substances isolated from various microorganisms and sharply differing in value included in their components, have shown that their activities are similar. From these data it appeared that in the course of the study all the components of the pharmacologically acceptable mixture of substances have similar level of immunological activity. The results of chemical and physico-chemical studies showed that farmacologicas acceptable mixture of substances contains minimal amounts of impurities and that, therefore, the proposed technological scheme allows to remove from the target product almost all of the components, which could provide a pharmacologically acceptable mixture of substances undesirable properties.

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

EXAMPLES

The following examples are given for the purpose of detail and illustration of the invention, do not have the goal of limiting the claims of the present invention.

Example 1

Selection PKS

20 g dried with acetone and ether (or 120-180 g of wet bacterial cells S.typhi suspended under vigorous stirring in 350 ml of distilled water. The resulting suspension was heated to a temperature of 68-70°C and was added 350 ml heated to the same temperature 90%aqueous phenol. 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 min at 5000 rpm/min Residue after decanting, the aqueous and phenol layer was subjected to re-extraction according to the same scheme (possibly 2-3 fold reduction in the volume of extractants). The resulting extraction residue washed with 0.2m ammonium bicarbonate buffer to the lack poglosheniya 260-280 nm in the UV spectrum of the supernatant (region specific absorption of nucleic acids and proteins), and then 2-3 times with water.

Output washed and freeze-dried sediment, representing untreated PCB was 20-40% by weight of dry cells. Amino acid analysis showed that along with the Monomeric components to the PCB (murakawa acid, glucosamine, alanine, diaminopimelate and glutamic acid), the product usually contains a significant amount of bound protein.

The number of extraction with hot water phenol may be reduced to one or increased to 3-4, the volume of the extractant may be reduced or increased in 1,5-2 times, when carrying out the extraction at a temperature higher than 65-68aboutWith the time of extraction is reduced to 10-15 minutes.

Enzymatic hydrolysis of the PCB lysozyme

5 g of DCC was suspensively in 100 ml of 0.2m triethylamine-acetate buffer pH of 7.2. To stir at a temperature (hereinafter) 10aboutTo the suspension was added 300 mg of lysozyme and after an hour of stirring, the reaction mixture was transferred into a dialysis tube, which was placed in a vessel with 300 ml of the above buffer. Dialysis was performed for 3 days, occasionally (1-3 times daily) removing dialysate and adding in an external vesselfresh buffer solution. At the end of the process the United dialysates was evaporated to a volume of 200 ml at a temperature of less than 40aboutWith, the rest was liofilizovane and then was dissolved in 200 ml of d is ionized water and the solution was re liofilizovane. Output pharmacologically acceptable mixture of substances amounted to 320 mg.

Isolation and purification of pharmaceutically acceptable mixture of substances

Preparative isolation of pharmacologically acceptable mixture of substances was performed using gel-chromatography on a column of Sephadex G-50 in 0,05M ammonium bicarbonate buffer pH 8.3. Pharmacologically acceptable mixture of substances eluted as two similar square peaks, with Kd 0.42 and 0.53 per share, and with less time eluted Dimetra, and with a great mixture Gmti and Metra. Combined fractions lyophilized and then re-lyophilized of deionized water. Alternatively, the combined fractions may be subjected to desalting on a column of Sephadex G-10 (eluent - deionized water).

Analysis of the hydrolyzate pharmacologically acceptable mixture of substances (4M Hcl, 100°C, 16 h) using amino acid analyzer showed that it consists of close to equimolar amounts of glucosamine, maramboi, glutamic and diaminopimelic acids, as well as twice the number of alanine, which corresponds to the set and the ratio of components in the PCB S.tyhhi.

From the data of mass spectrometry (MALDI-TOF) showed that pharmacologically acceptable mixture of substances contains three components - Gmti, Metra and Dimetra mol. mass (calculated values in brackets) 868,4 (868,8), 939,4 (939,8 and 1860,8 (1861.7), respectively.

Analysis of the spectrum of the 13C-NMR confirmed the structure of the pharmacologically acceptable mixture of substances and the absence of appreciable quantities of impurity substances.

Thus obtained the drug in a dose of 0.1 μg/ml did not cause pyrogenic effect in the test on rabbits. Bacterial endotoxin content in the solution preparation with a concentration of 100 μg/ml filed LAL-test did not exceed 1.5 EE/ml Single intramuscular and intraperitoneal administration of the drug to mice BALB/C mice and Wistar rats in the range of tested doses of 1-500 mg/kg does not cause any signs of intoxication and death. The highest tested on mice and rats, the dose of 500 mg/kg more than 300000 times higher therapeutic dose (100 µg/person or 1.5 µg/kg), recommended for humans.

Using the above examples, the specialists able to carry out an adequate scale to create large-scale production.

Immunostimulirutuyu activity muramylpeptide connections

Definition immunostimulating activity pharmacologically acceptable mixture of substances was carried out in accordance with the "guidelines for the study of immunotropic activity of pharmacological substances" Rmitv with co-authors approved the State Pharmacological Committee of the Ministry of Health, the surveillance of the Russian Federation (In Russian). Manual on experimental (preclinical) study of new pharmacological agents. M. 2000. 257-262). In accordance with these instructions the test substance, which is presumably immune stimulator, should increase the number of parameters of the immune system, among which the Central place belongs to the study of the humoral immune response and 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 were used for comparison polyacrylic acid - PAK (production Petrovax PHARM, Russia), stimulating mice in optimal doses, the formation of antibody productive cells (AFC) 4-6 times; in the study of the synthesis of TNF-α were used for comparison lipopolysaccharide (LPS) from E. coli (Sigma, USA), which is a strong inducer of this cytokine.

Example2

Evaluation of humoral immune response

In this example, the humoral immune response was assessed by the ability to form antibody productive cells (AFC) in response to the introduction of thymusdependent antigen, sheep red blood cells (EB). The KLA was determined by the method of local hemolysis agar on the NRN and Nordin. The study was conducted on mice, the females (C57Bl/6)F1weighing 22±0.6 grams. Each group was 8-10 mice.

The prep is at diluted with saline in a volume of 200 µl was injected into mice subcutaneously in the shoulder at doses of 0.01, 0.1 and 1.0 microgram 3 hours prior to intraperitoneal immunization of EB dose of 5x106. This dose of EB is suboptimal for the development of humoral immune response. The control animals instead of the drug was administered saline and also were immunized their DL. PAK was administered subcutaneously at a dose of 100 μg/mouse. On the 4th day determined the amount of the KLA in the spleens. The results were expressed as the number of AFC in the spleen and in the form of a stimulation index (IP) of the immune response compared with the control group (table 1).

Table 1
The effect of pharmacologically acceptable mixture of substances on the humoral immune response AOK/sales. (M+m)
Treatment of animalsPharmacologically acceptable mixture of substancesPAK
MedicationAntigenAbs.THEMAbs.THEM
SFR+ DL343,6±40,81,00±0,12343,6±40,81,00±0,12
mcg Polyaramid + DL1080,9±167,63,15±0,49*NN
10 µg Polyaramide+ DL1141,1±182,73,30±0,50*980±1282,8±0,65*
100 mcg Polyaramide+ DL868,1+190,4of 2.51±0,55*1290±2103,7±0,9*
*p<0,05

The table shows that pharmacologically acceptable mixture of substances has a pronounced ability to stimulate humoral immune response. This effect depends on the dose. The maximum stimulatory effect was observed at a dose of 10 mcg. At the dose of 100 μg/mouse future growth stimulating effect does not occur. The stimulating effect of the pharmacologically acceptable mixture of substances is comparable with the action of the classical activator of humoral immunity PAK and the dose of 10 μg/mouse even slightly exceeds it.

Example 3

Evaluation of the synthesis of TNF-α

In this example, the comparison muramylpeptide compounds was performed is a model of TNF-α - one of the Central mediators of the immune system. To do this, mononuclear cells from peripheral blood of 7 healthy donors aged 20-40 years, a dedicated sterile on the gradient ficoll-urografin, the density of 1.077, incubated for 2 days in CO2-incubator in complete culture medium consisting of RPMI-1640 medium with gentamicin 150 µg/ml, with 10% inactivated fetal calf serum, 15 mm glutamine (Sigma). In the experimental wells was added pharmacologically acceptable mixture of substances in doses of 100 μg/ml, 10 μg/ml and 1 μg/ml in the control wells - nutrient medium. As a comparison, used LPS of enterobacteria in the same doses. The content of TNF-α in supernatant cultured cells was determined using solid-phase ELISA using commercial test systems Cytelisa company Cytimmune (USA).

The results are presented in Table 2.

Table 2
The level of TNF-α in supernatant stimulated mononuclear
The drug concentrationTNF-α
Restimulate cell-47±44
pharmacologically acceptable mixture of substances 0.1 mg/ml2±2
1 mcg/ml1552±1757
10 mg/ml4269±2422
100 µg/ml8326±1665
FSC0.1 mg/ml1504±1092
1 mcg/ml2462±1721
10 mg/ml4248±1002
150 mg/ml3559±636

The table shows that LPS at low concentrations significantly greater than the pharmacologically acceptable mixture of substances according to their ability to induce TNF, but in large doses of the pharmacologically acceptable mixture of substances approximates or slightly exceeds the immunostimulating effect of LPS.

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

1. The method of obtaining pharmacologic is viable mixture of substances, having immunostimulatory activity, where the mixture of substances includes the following components:
β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramyl-(L-alanyl-D-isoglutamine-meso-diaminopimelic acid) (Hmtri);
β-N-acetyl-D-glucosaminyl-(1→4)-N-acetyl-D-muramyl-(L-alanyl-D-isoglutamine-meso-diaminopimelate-D-alanine) (Metra);
and dimer Metra (digmer), in which the relationship between Monomeric residues Metra is due to the carboxyl group of the terminal D-alanine of one residue Metra and ω-amino meso-diaminopimelic acid other residue Metra, and interconnected tetrapeptide residues are located in different polysaccharide chains, including
a) preparation of the biomass of gram-negative bacteria of the family Enterobacteriacea,
b) isolation of peptidoglycan cell wall (in pixels) of bacteria in the form of water insoluble material by extraction with a specified biomass 45% aqueous phenol at a temperature of 70-90°C or extraction is carried out with aqueous solutions of ionic or non-ionic detergent at a temperature of 37 stop 100°C,
c) cleavage of insoluble DCC preparative enzymatic hydrolysis with the use of lysozyme at pH 4,5-8,9 and temperature 10-37°C with simultaneous removal of the pharmacologically acceptable mixture of substances from the reaction mixture by dialysis, using polyphonic is controlled membranes for ultrafiltration, for separation of low molecular weight products of enzymatic degradation of the PCB;
d) isolation of the pharmacologically acceptable mixtures 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, wherein the dialysis is carried out with the use of dialysis tubing or semi-permeable membranes for ultrafiltration with a size cutoff to 5 kDa.

4. The method according to claim 1, characterized in that the selection pharmacologically acceptable mixture of substances is carried out using preparative gel chromatography on a column of Sephadex or TSK-gel.



 

Same patents:

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

FIELD: medicine, veterinary science.

SUBSTANCE: invention concerns veterinary medicine. Currently, for differentiating nonspecific tuberculin reactions in cattle, dried purified tuberculin is used for mammals and "КАМ" with considering the sensitisation pattern by the reaction intensity. A common complex allergen is produced by protein settling from M scrofulaceum No. 12-C and M intracellulare No. 13-H strain cultures with added allergen produced from Corynebacterium xerosis N1911, in amount 1350 units of activity. The presence of coryneformic bacteria allergen in the "КАМ" composition improves the efficacy of a simultaneous dried purified tuberculin test for mammals in differentiating the nonspecific coryneformic bacteria reactions.

EFFECT: use of the declared allergen allows to prevent unreasonable slaughter, as well as further diagnostic finding expenses.

2 tbl

FIELD: medicine.

SUBSTANCE: method involves deactivation of definite VGC2 DNA sequence of Salmonella typhimurium positioned between ydhE and pykF genes or its part containing at least 50 nucleotides, or the DNA version of at least 85% identity, representing VGC2 DNA of any microbe out of Salmonella aberdeen, Salmonella gallinarum, Salmonella cubana and Salmonella typhi.

EFFECT: obtainment of microbe with reduced adaptability to specific environmental conditions.

6 cl, 12 dwg, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing a peptide, which is characterised by that it involves conversion of the -SH group of the peptide which contains an amino acid residue having an -SH group to an -OH group, said method comprising the following steps from (a) to (c): (a) reaction of the -SH group in the peptide with a methylating agent to convert the -SH group to an -SMe group; (b) reaction of the -SMe group formed at step (a) with a cyanating agent to obtain an intermediate reaction product in form of an ester; (c) converting the intermediate reaction product obtained at step (b) to a peptide which contains an amino acid residue having an -OH group in more basic conditions than conditions at step (b).

EFFECT: improved method.

20 cl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to crystalline modifications: 1 (polymorphous form F), 2 (polymorphous form I) and 3 (polymorphous form X) of monosodium salt of D-isoglytamyl-D-tryptophan (1:1) characterised by powder X-ray pattern peaks presented in the application materials, as well as to pharmaceutical compositions containing them. The invention describes their use for treating various diseases and body conditions of at least one autoimmune diseases specified in a group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis.

EFFECT: present invention describes the methods for producing the declared crystalline modifications of monosodium salt of D-isoglytamyl-D-tryptophan (1:1).

42 cl, 4 ex, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to crystalline modifications: 1 (polymorphous form F), 2 (polymorphous form I) and 3 (polymorphous form X) of monosodium salt of D-isoglytamyl-D-tryptophan (1:1) characterised by powder X-ray pattern peaks presented in the application materials, as well as to pharmaceutical compositions containing them. The invention describes their use for treating various diseases and body conditions of at least one autoimmune diseases specified in a group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis.

EFFECT: present invention describes the methods for producing the declared crystalline modifications of monosodium salt of D-isoglytamyl-D-tryptophan (1:1).

42 cl, 4 ex, 9 dwg

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of purifying mono-pegylated erythropoietin using two cation-exchange chromatography steps, where the same type of cationite is used at both cation-exchange chromatography steps.

EFFECT: method of producing mono-pegylated erythropoietin in a substantially homogeneous form.

17 cl, 3 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of purifying antibodies by adding a negatively charged polyelectrolyte such as polyvinyl sulphonic acid, polyvinyl sulphonate, polystyrene sulphonic acid or polyacrylic acid, to a mixture containing an antibody and extracting the obtained precipitate containing the antibody. The invention can further be used in extracting antibodies from body fluids and purification thereof.

EFFECT: improved method.

13 cl, 13 ex, 13 tbl, 38 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a method for production and purification of human recombinant growth hormone (rHGH). The presented method for production and purification of rHGH involves dissolution of inclusion bodies in 2 M urea at pH=11.0 followed by renaturation in a buffer solution 20 mM "Трис"-HCl at pH=8.0. Hydrogen peroxide is used as an rHGH sulphhydryl group oxidiser. N-terminal methionine is split from leucinic aminopeptidase. Chromatographic purification of rHGH is enabled with two-stage ion-exchange chromatography on the sorbent Q Sepharose FF and the stage of purification by hydrophobic chromatography on the sorbent Butyl Sepharose 4 FF. Gel filtration on Sephadex G-25 and ion-exchange chromatography on Q Sepharose FF, pH=6.5. It is followed by ion-exchange chromatography on Sephacryl S-100HR.

EFFECT: invention enables producing a preparation of high-purity rHGH protein of high compendial grade applicable for preparing drug preparations.

3 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns biochemistry and medicine area. What is presented is a visualisation agent representing a conjugate of structure I as described in the patent claim. The visualisation agent refers to marked cMet-binding peptides. These peptides include a mark with an optical reporter group applicable for visualisation in vivo with the use of light within the range of wave length in the spectrum of 600-1200 nm. There are also presented a pharmaceutical composition for optical visualisation, containing the visualisation agent, a kit for preparing it and a method for optical visualization of a mammalian body in vivo. What is also presented is a method for managing the patients suffering colorectal cancer, involving the stage of optical visualisation in vivo.

EFFECT: presented visualisation agent possess higher cMet-binding affinity and selective cell targeting in vivo.

28 cl, 2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention discloses versions of a method of cleaning a liquid composition which contains recombinant factor VII from viruses. The composition to be cleaned contains at least 5% recombinant polypeptide of factor VII in activated form. Cleaning is carried out using a nanofilter with pore size of at most 80 nm.

EFFECT: methods enable to obtain a clean recombinant polypeptide of factor VII without breakdown of its active form.

34 cl, 1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method of preparative recovery of basic proteins from supramolecular structures of Escherichia coli growing population. The Escherichia coli cells are preserved in buffered 80-90% glycerol at -25°C. Then the sediment cells are washed in 3% triton X-100. It is followed by sediment extraction in salts of increasing concentrations: 0.14 M, 0.35 M; 2 M NaCl, 6 M in guanidine hydrochloride with 0.1% β-mercaptoethanol. Basic proteins are recovered from the prepared fractions by means of ion-exchange chromatography with amberlite resin IRC-50 of discontinuous gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% on 0.1 M potassium phosphate buffer pH 6.8.

EFFECT: method enables producing fractions enriched by basic proteins with the use of a microamount of protein of Escherichia coli cell suprastructures.

7 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry. A method of cleaning thrombin solution from infectious particles is provided. Macromolecules are added to the starting thrombin solution. The obtained solution is then passed through a nanofilter to obtain a thrombin solution free from infectious particles. Said macromolecule is not a nonionic surfactant, is different from thrombin and can be selected from a polymer containing at least 3 monomers of sugar, amino acids, glycols, alcohols, lipids or phospholipids. A thrombin-containing solution obtained using said method is also provided.

EFFECT: invention increases efficiency of removing infectious particles from thrombin solution; thrombin output reaches 93% using said method.

20 cl, 2 dwg, 13 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing a peptide, which is characterised by that it involves conversion of the -SH group of the peptide which contains an amino acid residue having an -SH group to an -OH group, said method comprising the following steps from (a) to (c): (a) reaction of the -SH group in the peptide with a methylating agent to convert the -SH group to an -SMe group; (b) reaction of the -SMe group formed at step (a) with a cyanating agent to obtain an intermediate reaction product in form of an ester; (c) converting the intermediate reaction product obtained at step (b) to a peptide which contains an amino acid residue having an -OH group in more basic conditions than conditions at step (b).

EFFECT: improved method.

20 cl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to crystalline modifications: 1 (polymorphous form F), 2 (polymorphous form I) and 3 (polymorphous form X) of monosodium salt of D-isoglytamyl-D-tryptophan (1:1) characterised by powder X-ray pattern peaks presented in the application materials, as well as to pharmaceutical compositions containing them. The invention describes their use for treating various diseases and body conditions of at least one autoimmune diseases specified in a group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis.

EFFECT: present invention describes the methods for producing the declared crystalline modifications of monosodium salt of D-isoglytamyl-D-tryptophan (1:1).

42 cl, 4 ex, 9 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to crystalline modifications: 1 (polymorphous form F), 2 (polymorphous form I) and 3 (polymorphous form X) of monosodium salt of D-isoglytamyl-D-tryptophan (1:1) characterised by powder X-ray pattern peaks presented in the application materials, as well as to pharmaceutical compositions containing them. The invention describes their use for treating various diseases and body conditions of at least one autoimmune diseases specified in a group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis.

EFFECT: present invention describes the methods for producing the declared crystalline modifications of monosodium salt of D-isoglytamyl-D-tryptophan (1:1).

42 cl, 4 ex, 9 dwg

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of purifying mono-pegylated erythropoietin using two cation-exchange chromatography steps, where the same type of cationite is used at both cation-exchange chromatography steps.

EFFECT: method of producing mono-pegylated erythropoietin in a substantially homogeneous form.

17 cl, 3 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of purifying antibodies by adding a negatively charged polyelectrolyte such as polyvinyl sulphonic acid, polyvinyl sulphonate, polystyrene sulphonic acid or polyacrylic acid, to a mixture containing an antibody and extracting the obtained precipitate containing the antibody. The invention can further be used in extracting antibodies from body fluids and purification thereof.

EFFECT: improved method.

13 cl, 13 ex, 13 tbl, 38 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a method for production and purification of human recombinant growth hormone (rHGH). The presented method for production and purification of rHGH involves dissolution of inclusion bodies in 2 M urea at pH=11.0 followed by renaturation in a buffer solution 20 mM "Трис"-HCl at pH=8.0. Hydrogen peroxide is used as an rHGH sulphhydryl group oxidiser. N-terminal methionine is split from leucinic aminopeptidase. Chromatographic purification of rHGH is enabled with two-stage ion-exchange chromatography on the sorbent Q Sepharose FF and the stage of purification by hydrophobic chromatography on the sorbent Butyl Sepharose 4 FF. Gel filtration on Sephadex G-25 and ion-exchange chromatography on Q Sepharose FF, pH=6.5. It is followed by ion-exchange chromatography on Sephacryl S-100HR.

EFFECT: invention enables producing a preparation of high-purity rHGH protein of high compendial grade applicable for preparing drug preparations.

3 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns biochemistry and medicine area. What is presented is a visualisation agent representing a conjugate of structure I as described in the patent claim. The visualisation agent refers to marked cMet-binding peptides. These peptides include a mark with an optical reporter group applicable for visualisation in vivo with the use of light within the range of wave length in the spectrum of 600-1200 nm. There are also presented a pharmaceutical composition for optical visualisation, containing the visualisation agent, a kit for preparing it and a method for optical visualization of a mammalian body in vivo. What is also presented is a method for managing the patients suffering colorectal cancer, involving the stage of optical visualisation in vivo.

EFFECT: presented visualisation agent possess higher cMet-binding affinity and selective cell targeting in vivo.

28 cl, 2 tbl, 7 ex

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