BG65084B1 - New class of bioactive glycoproteins - Google Patents

Abstract

The invention relates to a bioactive chemical composition, more specifically to proteins and can be used in medicine, veterinary and cell biology. The inventive glycoproteins are extracted from an intercellular space of the tissue of various organs, blood serum and bile of vertebrates (human beings and animals). Said glycoproteins have a high biological activity in super low doses at a concentration ranging from 10-12 to 10-29 mol/liter and lower. It is thus possible to produce a pharmaceutical form including glycoprotein in an effective quantity and a pharmaceutically acceptable carrier. The glycoprotein protein produced finds application as a medicamentous form.

Description

(54) NEW CLASS BIOACTIVE Glycoproteins

Technical field

The invention relates to the preparation of a biologically active compound, applicable in applied and technological biochemistry, in cell biology, practical medicine and veterinary medicine.

BACKGROUND OF THE INVENTION

Glycoproteins are complex proteins or proteins containing, in addition to a protein moiety, a non-protein organic or inorganic component that can be linked to the polypeptide chain by a covalent, heteropoly or coordination bond, and which together with the amino acids are present in the hydrolysis. The prosthetic group of glycoproteins can be represented by neutral sugars (galactose, mannose, fucose) or by amino sugars (N-acetylglucosamine, N-acetylgalactosamine or acid derivatives of monosaccharides) (X. D. Jakubke, X. Eshkigini, Amiqiigini, Amiqiigini, Amykid, , proteins ”, M., Mir, 1985, p. 345).

Glycoproteins are widespread in nature. These include the most important components of blood serum (immunoglobulins, transferrin, etc.), group substances in the blood, antigens of many viruses (influenza, measles, etc.), some hormones, lectins, enzymes, etc.

Pectins are a large group of glycoproteins whose protein part of the molecule is characterized by the absence of serine and threonine residues by which the carbohydrate component in the lectin molecule is linked to the polypeptide chain. The average carbohydrate content of pectins is about 5%, and their composition is restricted mainly to galactose, mannose, fucose, N-acetylglucosamine residues (X. D. Jakubke, X. Ashkayt, "Amino acids, peptides, proteins", M., World, 1985, pp. 428-429).

From GB 2078229, 1982, a glycoprotein having immunosuppressive properties is known. Glycoprotein is obtained by isoelectric focusing of blood serum or ascitic fluid from a human or warm-blooded life but further separating the isoelectric point (pl) fractions in the range pH = 2.6-3.6. This glycoprotein is used to suppress the response in transplant patients.

According to GB 2095260,1982, a biologically active substance is obtained - a glycoprotein of molecular weight 3000-5000, which is able to inhibit the reproduction of toxoplasma in homologous and heterologous cells.

From GB 2117385, 1982, a glycoprotein having anti-cancer activity is known, with a molecular weight of 7000-90000, a sugar content of 8-45%, 6-28% of hexose, 1-11% of hexosamine and 1-6% of sialic acids; stable in aqueous solution at pH = 2.0; 7.0 or 11.0 at 4 ° C for 24 hours or more and in aqueous solution with pH = 7.0 at 60 ° C for 3 hours or more; selectively hits cancer cells without affecting normal ones.

From CH 634334,1983, an immunological glycoprotein containing protein part 89 ± 4%, carbohydrate part 11.1 ± 2.2%, hexose 5.3 ± 1.1%, N-acetyl hexosamine residue 2.8+ is known 0.5%, N-acetyl neuramic acid residue 2.9 ± 0.5%, molecular weight 32000 ± 6000, p1 = 4.3 + 0.3, sedimentation factor S _Mn '3.2 + 0, 3.

From RU 2136695,1999, a serum glycoprotein with a molecular weight of 12.5 kDa is known, having low-dose biological activity and containing about 50% carbohydrate residues, with isoelectric point values in the pH range 4.6-4.7.

EP 000134, 1978, discloses a novel glycoprotein containing alpha-amino acid 75 + 6%, carbohydrate 24,6 + 5,2%, hexosan 2,9 + 2%, N-acetyl hexosamine residue 7,1 ± 1 , 5%, fucose 0.2 ± 0.2%, N-acetyl neuramic acid residue 8.4 ± 1.5%; with a molecular weight of 65000 + 10000, p1 = 3.4 + 0.4.

From FVO and Geneva P. falciparum a homogeneous glycoprotein with a molecular weight of 56 kDa, an isoelectric point value around pH 5.5 containing N-acetylgalactosamine and mannose (US 4835259, class С07К15 / 15, 1989, was extracted and studied) ).

Closest to the present invention is a glycoprotein with a molecular weight of 5-300 kDa, an isoelectric point value in the pH range of 2.5-5.0, a protein-to-carbohydrate moiety by weight of 50:50 - 80:20 containing in its carbohydrate fucose, ribose, arabinose, xylose, mannose, galactose, glucose and glucosamine residues, and aspartic and glutamic acid, threonine, serine, proline, glycine, alanine, cysteine, valine, methionine, cyanin, valine, methionine isoleucine, leucine, tyrosine, phenylalanine, tryptophan, ornithine, lysine, hist dia, arginine (US 4683438, class C07K15 / 14.1987).

SUMMARY OF THE INVENTION

The invention relates to glycoproteins having biological activity in ultra-small doses suitable for use in the medical and pharmaceutical industries.

On the other hand, the invention relates to the preparation of a glycoprotein having biological activity in ultra-small doses and to its use as a medicament.

Thus, it is an object of the invention to provide a glycoprotein having biological activity in ultra-small doses (10 ^'12 - 10' ²⁹ mol / l and less).

This technical result is achieved by novel glycoproteins extracted by isoelectric focusing from the intracellular space of blood serum, bile, tissues of various organs of the vertebrate (human and animal) soluble in saturated (100%) ammonium sulphate solution having a presumed ammonium sulphate implying mass 10-45 kDa and having biological activity in ultra-small doses. Solutions of glycoproteins at a concentration of 10 ¹² -10 ¹⁸ mol / Ι completely retain their biological activity upon repeated freezing and thawing as well as after heating at 100 ° C for 10 min.

The molecular weight of glycoproteins was determined by the PAGE electrophoresis method in the presence of sodium dodecyl sulfate by the Lemly method. A LKB kit comprising 6 proteins with a molecular weight of 94 kDa to 14.4 kDa was used as molecular weight markers. The estimated molecular weight for blood serum glycoprotein with pl in the pH range of 4.65-5.1 (SG) is 35-37 kDa according to PAGE phoresis and 25-27 kDa according to gel chromatography data. For the neutral liver glycoprotein (pl in the pH range 6.8-7.2) (NGD), these values are 15 kDa and 22 kDa, respectively. For the acidic liver glycoprotein (KGCD), the estimated molecular weight is determined by the gay filtration method only and is equal to 17 kDa (the molecular weight value is not determined by the PAGE electrophoresis method due to the inability to stain the acid glycoprotein fraction ).

Polyacrylamide gel electrophoresis was performed using an AVGZ-1 vertical electrophoresis apparatus (Himifil, USSR) under denaturing conditions with detergent supplementation (DDSNa) by the Lemly method. The gel thickness is 0.75 mm, the size is -115x115 mm, the maximum number of stripes is -13.

The separating gel (12.5%) was prepared from the following components: distilled water (6.7 ml), 1.5M Tris-HCl with pH 8.8 (5.0 ml), 10% DDS-Na solution (0 , 2 ml), 30% acrylamide / N-bis-methylene acrylamide solution (8.0 ml), TEMED (10 mcl), 10% ammonium persulfate solution (100 mcl).

The concentrating gel (4%) was prepared from the following components: distilled water (6.1 ml), 0.5M Tris-HCl with pH 6.8 (2.5 ml), 10% VAT-Na solution (0.1 ml), 30% acrylamide / N'N'-bis-methylene acrylamide solution (1.3 ml), TEMED (10 mcl), 10% ammonium persulfate solution (50 mcl).

Electrode buffer: Tris-glycine with pH 8.3 and 0.1% VAT-Na. The lyophilized sample was dissolved (about 0.1 mg / ml) in the following solution: distilled water (4.0 ml), 0.5M Tris-HCl with pH 6.8 (1.0 ml), glycerol (0.8 ml) ), 10% DDS-Na solution (1.6 ml), 2-mercaptoethanol (0.4 ml), 0.05% bromophenol blue solution (0.2 ml). Then, before applying to the gel, the samples were incubated for 3-5 min at 100 ° C. 10-15 mcl of sample were applied to one strip. The electrophoresis was carried out at a constant voltage of 200 V. Upon passage of the concentrating gel, a voltage of 100 V. was applied.

The LKB kit comprising: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), egg albumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) was used as molecular weight markers. ), chicken lysozyme (14.4 kDa).

The staining of the gels is done in colloidal silver or with Kumasi G250 dye. In colloidal silver staining, the gels were prewashed in distilled water and then in aqueous solution with 5% ethanol and 5% acetic acid for at least 3 hours. Further, the body was rapidly (for 5 min) washed with distilled water and placed in 10% glutaraldehyde solution. The unreacted glutaraldehyde was removed by washing three or five times with water (30 min for each wash). The body was then incubated for 30 minutes in a solution of dithiothreitol (5 mg / ί) and washed thoroughly with water. The washed gel was placed for 30 minutes in a solution of AgNO ₃ (0.1 g / l), washed again with water and placed in the developer until clear bands emerged. The developer is a 37% formalin solution in 3% aqueous Na ₂ CO _{3 solution} (50100 mcl formalin / 100 ml solution). 3-5 ml of citric acid were added to stop the development.

When stained with Kumasi G-250, the body is fixed with a mixture of trichloroacetic acid, methanol and water (10:40:50). The staining was carried out with a 0.04% solution of Kumasi G-250 in 3.5% perchloric acid. Typically, blue protein bands show up within 5 minutes. To reduce the background color, the body can be placed in a solution of acetic acid, methanol and water (10:40:50) for 1-3 hours.

The molecular weight of glycoproteins was determined by the gel-permeable HPLC method on a TSK G3000SW column (300x7.5 mm), eluting with 100 mM NaH ₂ PO ₄ at pH = 7. The following marker proteins were used for calibration: ovotransferrin (78 kDa), bovine serum albumin (67 kDa), egg albumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), myoglobin (17.2 kDa), chicken lysozyme (14.3 kDa) - preparations of the companies Serva (Germany) and Sigma (USA).

The amino acid and carbohydrate composition of the glycoproteins as well as the presence of glycosylation are also determined according to the invention.

Determination of amino acid composition

The determination of the amino acid composition was accomplished using a Hitachi 835 amino acid analyzer at the Institute of Physicochemical Biology “A. N. Belozersky ”.

Separation was performed on a chromatographic column with sulfopolystyrene cations, brand 2613, detection was spectrophotometric, in ninhydrin derivatives (wavelength 570 and 440 nm). Protein hydrolysis was carried out before analysis with a mixture of 12N HCl and concentrated trifluoroacetic acid (2: 1) with the addition of 0.005% mercaptoethanol for 1 h at 155 ° C.

Determination of carbohydrate composition

Determination was performed with a Biotronic LC 2000 carbohydrate analyzer (FRG) on a Durrum 8-11 (USA) sorbent column at 70 ° C in 0.4 M borate buffer pH 8.0. The size of the column is 3.7x75 mm. Detection was carried out at 570 nm with a solution of copper 2,2'bincinonate. Prior to determination, hydrolysis of 1 ml of the preparation (0.1 mg / ml) in 10 ml of 2 M trifluoroacetic acid was carried out for 3 h at 100 ° C.

Determination of glycosylation

To determine the presence of glycosylation, 50 mcl aqueous protein solution (0.1 mg / ml) was added 5 mcl 0.1 M acetate buffer with pH 4.6, then a solution (510 mcl) of sodium periodate was added thereto. buffer so that the concentration of periodate in the reaction mixture is 1-2 mM, then allowed to stand at room temperature for 30 min. The unreacted periodate was neutralized with 0.5 M aqueous sodium thiosulphate solution until discoloration disappeared. Then, a solution of dinitrophenylhydrazine (DNPH) was added to dimethyl sulfoxide (DMSO) until the concentration of DNPH in the reaction mixture reached 2.5-5 mM, and was allowed to stand at room temperature for 30 min. The Schiff bases formed were then recovered using sodium borohydride solution in DMSO (0.1 mg / ml).

Product analysis was performed by gel permeation chromatography on a TSK 2000 PW column (5.6x300 mm) using a high pressure liquid chromatography apparatus, eluent - 50 mM acetate buffer at pH 4.5, speed - 0, 5 ml / min, UV detection at 365 nm. The retention time of DNPH, intact glycoprotein (at 280 nm absorption), and DNPH modified glycoprotein (in case of an absorption peak at 365 nm with a retention time approximately equal to the retention time of unmodified glycoprotein ).

The following were used as modeling glycoproteins: egg albumin, ovvukoid, S-carboxymethylated mucin, rhodopsin-binding egg yolk glycoprotein.

Determination of glycosylation of glycoproteins according to the invention

Following experiments on modeling glycoproteins of known structure, the present method is applied to the extracted proteins. The results show that all these substances are glycoproteins. The chromatogram shows the peak of the studied glycoprotein as well as the peak corresponding to unreacted DNPH. The timing of the modified glycoprotein is slightly different from the natural one that might be expected.

For example, analysis of the carbohydrate composition of a purified sample of SG shows that the protein in question is highly glycosylated - it contains 40-55% by weight of carbohydrates. Of the carbohydrates, N-acetylglucosamine and mannose were found to be 2: 5. Knowledge of the biological pathways for glycoprotein synthesis suggests that SG contains predominantly mannose-rich N-linked oligosaccharide chains.

Another aspect of the invention is also the use of the glycoproteins described in the invention for the preparation of various pharmaceutical compositions (drops, ointments, therapeutic creams, gels, etc.).

The pharmaceutical composition according to the invention contains an effective amount of glycoproteins having biological activity in ultra-small doses, which is one aspect of the invention, and a pharmaceutically acceptable carrier (Examples 15-17).

The pharmaceutically acceptable carrier may be an organic, polymeric (carbohydrate, cellulose) or inorganic carrier, the choice of which is determined primarily by the method of administration (purpose) of the pharmaceutical composition.

In the foregoing examples, and in particular, Example 15 describes a pharmaceutical composition which is a bioregulator of reparative processes in epithelial and connective tissues.

The glycoproteins of the invention are derived from the following tissues: liver, lung, thymus, spleen, heart, kidney, pancreas, mammary and thyroid gut, testes, ovaries, brain, bone marrow, tissues of the eye, as well as blood serum and bile.

The actual process of extracting said glycoproteins according to the invention involves the following steps:

(a) the preparation of tissue extracts from various human and animal organs;

b) salting the proteins from the tissue extract;

c) actual extraction (separation and purification) of glycoproteins by the isoelectric focusing method;

d) collection of fractions and identification of glycoproteins, determination of their biological activity and their biological effect.

The glycoproteins we identify can be divided into three groups:

acid proteins migrating to the anode; proteins with pl in the pH range from 4.6 to 8.5 (weakly acidic, neutral and weakly basic);

basic proteins migrating to the cathode.

The method of isoelectric focusing is one of the traditional methods for the extraction and purification (separation) of biopolymers, including the glycoproteins of the invention, and is used in physical and analytical chemistry.

It is based on the following: a stable pH gradient is created under the influence of an external electric field, increasing the pH from the anode to the cathode. In such a system, each protein moves in one direction or another according to its charge sign until it reaches a region where the pH value coincides with its isoelectric point (pl). In this region, its further movement under the influence of the electric field is terminated because its charge becomes 0.

The applied electric field, maintaining a stable pH gradient, prevents diffuse erosion of the zone. Special substances called ampholines are used to create a stable pH gradient.

Isoelectric focusing was performed in a sucrose gradient using a LKB-440 column (LKB, Sweden) and ampholines (Serva, Sweden) with a pH interval of 3.5-10.0 at 4 ° C for 100 h and at a voltage of 500-1500 V. In heavy gradient solution, the supernatant is introduced in the form of a solution in an amount not exceeding 100 mg total protein. Fraction detection was performed by spectrophotometry at 280 nm. Dialysis of fractions after isoelectric focusing was carried out in cellulose film bags in distilled water. The following fractions were collected:

1. acid glycoproteins migrating to the positively charged electrode;

2. glycoproteins with a value of pl in the pH range 4.6-8.5 (weakly acidic, neutral and weakly basic);

3. basic glycoproteins migrating to the negatively charged electrode.

The glycoproteins of the three groups mentioned are derived from the tissues of the following organs: liver, lung, thymus, spleen, heart, kidney, pancreas, mammary and thyroid gut, intestines, testes, ovaries, brain, bone, eye lens, cornea of the eye, pigment epithelium of the eye, retina of the eye, as well as blood serum and bile.

All identified glycoproteins exhibit biological activity in ultra-small doses corresponding to concentrations of ΙΟ ¹² - 10 ^'29 mol / Ι and less.

The biological effect (E _a ) of the recovered glycoproteins according to the invention is determined by, for example, the formula:

E = 200% -N / Νχ100% a where k:

Well _a is the biological effect caused by glycoproteins,

N _on and N _k are the amount of cell nuclei separated from 1 mg of tissue respectively in the assay (organ cultures in the presence of glycoprotein) and in the control (organ cultures without the addition of glycoproteins).

The cell nucleus count was performed in a certain volume by Goryaev's chamber. Statistical processing of the data was performed by the method of variational statistics using the Student criterion. The procedure for the preparation of organic culture is as follows. After decapitation of the animal (CBA / C57BI hybrid mice, male, 16-18 g), the liver was placed in medium 199 at room temperature. A tissue fragment of 1-1.5 mm ² is excised from the central part of the large liver lobule, the ends of the large forehead and areas with large blood vessels not being used. 5 organ cultures were placed in penicillin vials with a nutrient medium consisting of: 1 ml medium 199 + 0.2 ml bovine or equine serum + 0.1 ml of the test glycoprotein solution at a given concentration; 0.1 ml Ringer's solution was added to the control vials instead of the glycoprotein solution. All vials were incubated at 37 ° C for 2 h.

To evaluate the parameter characterizing the visco-elastic properties of the hepatocyte membrane, each tissue fragment was dried with filter paper, placed in a special 50 mcm glass homogenizer, 0.1 ml 0.1% trypan blue solution was prepared, prepared with Ringer's solution, and dispersed, rotating the pestle 25-30 times. The single cell nuclei separated by dispersion are then counted. The biological effect of the glycoprotein is calculated using the formula above.

The glycoproteins of the three groups that have affected the visco-elastic properties of the hepatocyte membrane are extracted from the tissues of the following organs: liver, lung, thymus, spleen, heart, kidney, pancreas, mammary and thyroid, glandular, gut, , ovaries, brain, bone marrow, lens of the eye, cornea, pigment epithelium of the eye, retina of the eye, as well as blood serum and bile.

For the glycoproteins we identified, the value of E _a was 125 to 150% at concentrations of 1 (Y ¹² - ²⁹ mol / Ι and less (Fig. 1,2).

Examples of carrying out the invention

Glycoproteins of the invention exhibiting biological activity in ultra-small doses are extracted from the intracellular space, from blood serum and bile, from tissues of various organs: liver, lung, thymus, spleen, heart, kidney, gastrointestinal, mammary and thyroid , intestines, testes, ovaries, brain, bone marrow, lens of the eye, cornea, pigment epithelium of the eye, retina of the eye, using the steps above.

The examples below illustrate, but in no way limit the invention.

Example 1. Mammalian liver glycoproteins

1.1. Preparation of liver extract

The experiments were performed on rats of the Wistar line, of both sexes, weighing 150-180 g. Animals are decapitated. Through the portal vein, the liver of each animal for 30-40 s was flushed out of the blood with a perfusion solution (0.15 mol / 1 NaCl; 0.04 mol / 1 KCl; 0.001 mol / Ι CaCl ₂ ) at a rate of flow 5-7 ml / min, cut into fragments weighing 1.52.0 g and place in solution the indicated composition at 4 ° C for 2 h (1520 ml solution for 1 liver).

The resulting extract was collected. Pour the tissue with fresh saline and extract for 1 hour. The extracts obtained were combined. For removal of blood and damaged liver cells, the tissue extract was centrifuged at 5000 g for 20 min, then decanted and used for further purification.

1.2. Salting of proteins from tissue extract

With vigorous stirring, dry ammonium sulfate was gradually added to the tissue extract to give a saturated saline solution (4 ° C, pH 8.0-8.5). The resulting impurity protein precipitate was precipitated by centrifugation at 35,000 g for 30 min, the supernatant collected and subjected to continuous dialysis in distilled water using a cellulose film conforming to GOST 7730-89. During dialysis, distilled water is repeatedly changed to fresh water. After complete removal of the ammonium ions, the supernatant was concentrated to 100 ml volume by the lyophilic drying method and the salting procedure was repeated under the same conditions. The second supernatant thus obtained is also dialyzed in distilled water until complete removal of the ammonium ions and further separated by isoelectric focusing method.

Isoelectric focusing of the supernatant from the tissue extract is carried out according to the procedure described above.

Example 2. Mammalian thymus glycoproteins

The experiments were performed on rats of the Wistar line, of both sexes, weighing 150-180 g. The animals were decapitated, the thymus was excised, washed thoroughly in saline (0.15 mol / 1 NaCl; 0.04 mol / 1 KCl; 0.001 mol / 1 CaCl ₂ ) and extracted into solution with the indicated composition at 4 ° C. C for 2 h (3-4 ml extraction solution for 1 thymus). The resulting extract was collected. The thymus is then filled with fresh saline and extracted for an additional 1 h. The extracts obtained were combined. To remove blood and damaged thymic cells, the tissue extract was centrifuged at 5000 g for 20 min, then decanted and used for further purification.

Salting and pH-isoelectric focusing of the tissue extract is carried out according to the above procedure. After pH-isoelectric focusing, as in the separation of the liver extract, three protein fractions were collected: acid glycoproteins, glycoproteins with pl in the pH range 4.6-8.5 and basic glycoproteins.

All identified thymus glycoproteins exhibit biological activity in ultra-small doses corresponding to concentrations of 10 ¹² - 10 ^'29 mol / Ι and less.

Example 3. Mammalian tissue glycoproteins

The following tissues are separated from the fresh eyes of a bull (30 pcs.): Lentils, cornea, pigment epithelium, retina. The tissues were washed thoroughly in saline (0.15 mol / 1 NaCl; 0.04 mol / 1 KCl; 0.001 mol / 1 CaCl ₂ ), cut into large pieces and extracted individually into a solution of the indicated composition at 4 ° C. C for 2 h. The resulting extracts were centrifuged for 15 min at 3000 g, then decanted and used for further purification. Expression and pH-isoelectric focusing of the tissue extract is performed by the above procedure. After pH-isoelectric focusing, three protein fractions from each tissue were collected: acidic glycoproteins, glycoproteins with pl in the pH range 4,6-8,5 and basic glycoproteins from the lens of the eye, the cornea, the pigment epithelium of the eye, the retina the eye.

All identified glycoproteins from the tissues of the eye exhibit biological activity in ultra-small doses corresponding to concentrations of 10 ¹² -10 ^'29 mol / Ι and less.

Example 4. Extraction of glycoprotein is pl in the pH range 4.65-5.1 from bovine blood serum

To the 51 bovine serum was gradually added dry ammonium sulfate to form a saturated saline solution (4 ° C, pH 8.0-8.5). The resulting precipitate was removed by filtration and the filtrate was centrifuged at 10,000 g for 15 min. The protein sediment was then removed, the supernatant collected and subjected to continuous dialysis in distilled water until complete removal of ammonium ions, then lyophilized and re-dissolved in distilled water to a volume of 0.5 1. To the resulting stirred solution was added dry ammonium sulfate to saturation, leave the mixture for 4 days at 4 ° C, then centrifuge at 10000 g for 15 min. The supernatant was dialyzed in distilled water until complete removal of the ammonium ions, then lyophilized and redissolved in 50 ml of distilled water. Further separation is performed by the pH-isoelectric focusing method according to the procedure described above. After pH-isoelectric focusing, the fraction was collected in the pH range of 4.65-5.1, dialyzed in distilled water and lyophilized. 0.55 mg is obtained. The resulting preparation is a fine powder of homogeneous glycoprotein, slightly colored in cream. The glycoprotein contains 40-55% carbohydrates. The molar ratio of D-mannose to Acetyl-O-glucosamine is 5: 2 to 3: 2; biological activity (the effect on the visco-elastic properties of the hepatocyte membrane in vitro) at a glycoprotein concentration of 10 ¹² 10 ^'29 mol / ско and less than 125% relative to the control.

Similarly, glycope rotein is extracted from the blood serum of rats, dogs, horses and humans. All blood serum glycoproteins have an isoelectric point in the pH range of 4.65-5.1, an estimated molecular weight of 35-37 kDa according to SDS polyacrylamide gel electrophoresis and 25-27 kDa of gel chromatatherapy and have ultra-small biological activity at concentrations of 10 ^'12 -10' ²⁹ mol / Ι or less.

Industrial application of the invention

The examples (5-17) and the figures (1-29) and tables (1-8) below illustrate the biological activity and biological effect of the glycoproteins claimed in the invention.

Example 5. Effect of glycoproteins on the visco-elastic properties of the hepatocyte membrane in vitro

The study was performed using multiple organ cultivation of liver tissue fragments weighing 1-1.5 mg in culture medium to which a certain volume of the glycoprotein test solution was added. For each glycoprotein, the concentration range at which its biological action is manifested is determined. For this purpose, consecutive dilutions of the parent preparation (concentration 0.1 mg protein / ml) are performed 10, 100, 1000 times, etc. up to 10 * ¹⁵ mg protein / ml. A similar volume of Ringer's solution was added to the control vials instead of the test preparation. All explants examined in one experiment were obtained from the liver of one animal. In each individual experiment, at least 5 tissue fragments were taken for each experimental point (corresponding concentration of glycoprotein solution). At least 3-4 trials were performed to determine the biological activity of each glycoprotein.

The biological effect is calculated by the formula above and is illustrated in FIG. 1-2.

Example 6. Effect of a Thymic Basic Glycoprotein on the Delayed Type of Hypersensitivity Reaction

Thymus Basic glycoprotein (BGT) was administered to four groups of 5 C57BL / 6 spf status mice weighing 20 g intraperitoneally three times at doses of 2x1ο · ⁶ , 2x10 ¹² , 2x10 ¹⁶ and

2xl0 ¹⁸ g per animal for three days starting from the time of intraperitoneal immunization of mice with sheep erythrocytes at a dose of 2x10 ⁸ per animal. Five days after the immunization of the recipients, 1x10 sheep erythrocytes in a volume of 50 microl subcutaneously in the foot of the hind foot were applied and after one more day the intensity of the edema was compared with the contralateral limb by the Kitamura method and the number of ant5-forming cells in the lacrimal body.

Jerne. The results are presented in Table 1.

Table 1. Effect of basic thymic glycoprotein on antibody formation and delayed-type hypersensitivity in C57BL / 6 mice.

Experimental 2E £ pi Delayed type hypersensitivity reaction (%) Antibody-forming cells in the spleen Control 25.2 ± 1.61 5900 ± 1500 BHT, 2x10 ' ⁶ g 25.22 ± 2.5 3350 ± 1600 BHT, 2x10 ^'12 g 32.88 ± 1.31 ** 7600 ± 1500 BGT, 2x10 ^'16 g 29.5 ± 2.59 7360 ± 440 BHT, 2x10 ^'18 g 30.1 ± 0.8 * 4450 ± 900

Results are presented as arithmetic mean ± mean error. ** - Significant control differences, P <0.001 * -P <0.05

As the table shows, the preparation does not significantly affect the formation of antibody-forming cells in the spleen. While application of the preparation at a dose of 2x10 ^"12 g of an animal results in highly reliably enhance the reaction of delayed hypersensitivity.

Thus, we demonstrate the ability of the BGT preparation at a dose of 2x10 ¹² g per animal to stimulate a delayed-type hypersensitivity response conditioned by the activity of type I helper cells, on which the development of a specific antitumor immune response depends.

Jerne N., Nordin A. (1963) Plaque formation in agar by single antibody production cells. Science, V. 140, P. 405.

Kitamura K. (1980) A footpad weight assay to evaluate delayed type hypersensitivity in the mice. J. Immunol. Meth., V. 39, P. 277-283.

Example 7. Effect of Thymic Basic Glycoprotein on the Survival of EL4 Thymic Mice

BGT has been tested for antitumor immunity stimulating activity. To this end, mice from line C57BL / 6 (K ^b I ^b D ^b ) were given a single injection with cells from the EL4 thymoma derived from mice in the same line at 1 × 10 ³ , 1x10 ⁴ and 1x10 ⁵ doses. The composition is administered daily for a week in doses of 2x10 ^, 2x10 ^-12 and 2x10 ¹⁶ g per animal, starting the day of immunization (nine treatment groups and three control groups of 5-7 animals each). The effect of the preparation was evaluated according to the life expectancy of the recipients of the different doses of tumor cells compared to the respective control recipient groups. The results are presented in Figures 3-5. Figure 3 shows the protective effect of the preparation at a dose of 2x10 ¹² g per animal (thick line) compared to the cont40 roll (dotted line). In this experimental group of 10 recipients, 1 remain alive, while in the control, all animals die as a result of tumor growth in ascites. Figure 4 shows negligible za45 shield effect of the preparation at a dose of 2 x 10 ^-6 g per animal (thick line) compared to the control (dashed line). At a dose of tumor cells of 1x10 ^{s it} is impossible to notice the effect of the preparation (Fig. 5). From the data presented, it follows that BGT at a dose of 2x10 ¹² g per mouse results in a statistically significant increase in the life span of the experimental animals - the group receiving the minimum dose of tumor cells (1x10 ³ ).

Although the biological effect exhibited by BHT is not large, it should be taken into account that the doubling time of thymoma cells exceeds several times the doubling time of T lymphocytes with an angitumor effector function. In addition, complete 10 rejection of cells of the introduced tumor is generally observed only with the introduction of tumor variants modified with genetic constructs for the production of cellular immunity-stimulating cytokines or in combination with cytostatic therapy.

Sumimoto N., Tani K., Nakazaki Y, Tanabe T., Hibino N., Wu M. S., Izawa K., Hamada H., Asano S. 1998 Superiority of interleukin-12transdused murine lung cancer cells to GM-CSF 20 or B7 -1 (CD 80) transfectants for therapeutic antitumor immunity in syngeneic immunocompetent mice. Cancer Gene Ther v.5, N 1, p. 29-37.

Ehrke M.J., Verstovsek S., Pocchari S.K., Krawczyk C.M., Ujhazy P., Zaleskis G., 25 Maccubbin D.L., Meer J.M., Mihich E. (1998) Thymic anti-tumor effectors in mouse cured lymphoma by cyclophosphamide plus TNF -alpha therapy: phenotypic and functional characterization up to 20 months after initial tumor inoculation. Int. 30 J. Cancer, V. 76, N 4, P. 579-586.

Example 8. Effect of Thymic Acid Glycoprotein on the Delayed Type of Hypersensitivity Reaction

Thymic acid glycoprotein (CGT) was administered to four groups of 5 C57BL / 6 spf status mice weighing 20 g intraperitoneally three times at 2x10 ' ⁶ , 2x10 · ¹² , 2x10' ¹⁶ and 2x1ο ⁴⁸ g per animal for one three days, starting from the time of intraperitoneal immunization of sheep erythrocyte mice at a dose of 2x10 ⁸ per animal. Five days after immunization, recipients received 1x10 ⁸ sheep erythrocytes in a volume of 50 µl subcutaneously in the foot of the hind foot and after another day the intensity of edema was determined in comparison with the contralateral limb by the Kitamura method and the number of antibody-forming cells in the spleen . The results are presented in Table 2.

As the table shows, the preparation does not significantly affect the formation of antibody-forming cells in the spleen. At the same time, administration of the preparation at a dose of 2x10 ¹² g per animal results in a highly reliable inhibition of the delayed-type hypersensitivity reaction.

In this way, we demonstrate the ability of the CGT preparation at a dose of 2x10 ¹² g per animal to retard the delayed-type hypersensitivity reaction.

Table 2. Effect of Thymic Acid Glycoprotein on Antibody Formation and Delayed Type Hypersensitivity in C57BL / 6 Mice.

Experimental groups Delayed type hypersensitivity reaction (%) Antibody-forming cells in the spleen Control 25,211.61 590011500 CGT, 2x10 ⁶ g 25,2212,5 485011200 KGT, 2x10 ^'12 g 17,6411,52 * 413011400 TAG, 2xl0 ^'l6 g 22.5 ± 1.44 51501780 KGT, 2x10 ^'18 g 26,411.2 55501600

Results are presented as arithmetic mean ± mean error.

* - Significant control differences, P <0.001

Example 9, Effect of Mammalian Thymus Glycoproteins on Excitatory Conductivity in Demyelination of Nerve Fiber

Myelin nerves of the grass frog (Rana temporaria) were used as the object of study. To achieve focal demyelination of the nerves in vivo, lysolecithin (LL) is used, which is inserted into the myelin sheath of the sciatic nerve of the frog by surgery (intracavitary injection). This effect is known to cause nerve demyelination, many stages of which correspond to autoimmune demyelination. The first changes in the state of myelin are observed one day after surgery, and after 6-12 days myelin completely "strips" the internodal sections of the nerve fiber. Three to four weeks after surgery, new myelin formation is observed.

The study of the effect of basic and acid glycoprotein on the thymus on the processes of demyelination and recovery in demyelination was performed by two experiments: in vitro on isolated nerve and in vivo one week after administration of LL to animals. LL and the thymus glycoprotein under study are administered to animals simultaneously. The following series of experiments are performed when working in vivo:

1. The effect of LL and thymus glycoprotein on the electrophysiological parameters of the nerve is evaluated.

2. The effect of the combined effect of LL and the corresponding thymus glycoprotein is determined.

Control animals were not exposed to LL and thymus glycoproteins, but were found under the same conditions throughout the experiment. When working in vitro, the standard method of extracellular registration of membrane potential and action potential (AP) is used to determine:

1. Dynamics of changes in AP amplitude, threshold, rate of AP, and maximum frequency of rhythmic response of the isolated nerve under the action of LL, of thymus glycoproteins and their combined action.

2. Dynamics of changes in the amplitude of the AP date, the threshold, the rate of AP, and the maximum frequency of rhythmic response one week after the combined administration of LL and glycoproteins to the animal.

The glycoproteins of the thymus were tested in concentrations of 10 ⁴⁴ mol / ί, 10 ^'18 to1 / 1 and 10' ²⁴ mol / l.

The isolated nerves pre-conditioned for at least 30 min in saline composition (mM): NaCl - 111.2; KC1 1.88; CaCl ₂ -1.08; pH - 7.2; 18-20 ° C. Redistillate is used for the preparation of saline and glycoprotein solutions prepared on the basis of saline with the above composition.

The method of fluorescence spectroscopy, using a localized Ca ²⁺ -binding probe - chlortetracycline capable of complexing with the calcium ion, was used to evaluate changes in the level of membrane-bound calcium in the study. Nerve experiments are performed in a specially designed chamber, allowing both the level of nerve fluorescence and the amplitude of the AP to be recorded simultaneously. Throughout the experiment, the luminescence was recorded in the same nerve section or isolated fiber. Fluorescence of the nerves was recorded using a Lumam 1-3 fluorescence microscope (LOMO). The excitation of the fluorescence of chlortetracycline was induced by using a KGM 9x70 halogen lamp and a combination of FS-1-6 filters and CZS 21-2. The registration is performed by means of a photometric tip and interference light filters with a maximum wavelength of light transmission at 490 and 550 nm. The diameter of the photometric nerve section is 50 mcm with a lens x 10.

Substantial changes in some electrophysiological parameters of rhythmic excitation (PB) are observed under the influence of LL and nerve demyelination. In the present study, we found that the in vivo thymic basic and acid glycoprotein compensated for changes in PB parameters during nerve demyelination (Tables 3, 4, and 5). It should be noted that injection of glycoprotein solutions alone does not lead to significant changes in myelin nerve excitability.

Table 3.

Investigation of the combined effect of lysolecithin and thymus glycoproteins on the PB of the demyelinated nerve (glycoprotein concentration is 10 ¹⁴ M)

In vivo Time of the AP (% vs. control) AP amplitude (% vs. control, 100 Hz) LL 160 30 Thymic acid glycoprotein 130 66 Basic thymus glycoprotein 140 40

In a separate experiment, concentrations of thymus glycoproteins were investigated (10 ¹⁸ M excitation of nerve excitability of the ci and 10 ²⁴ M). The study results are the anterior animal after injection of lower joints in Tables 4 and 5.

Table 4,

Investigation of the combined effect of lysolecithin and thymus glycoproteins on the PB of the demyelinated nerve (glycoprotein concentration is 10 ¹⁸ M)

In vivo Time of the AP (% vs. control) AP amplitude (% vs. control, 50 Hz) Л LL 160 30 Thymic acid glycoprotein 150 38 Basic thymus glycoprotein 155 • 35

It is known that the destruction of myelin results in a decrease in the rate of spread of PB. In our study, it was found that under the action of LL the rate of conduction of AP on the isolated nerve decreases, and under the combined action of LL and the investigated thymus glycoproteins on the nerve the nature of these changes weakens (Figs. 6, 7). At maximum dilution of thymus glycoprotein solutions (up to 1 Ο * ²⁴ M), only a tendency (but no significant differences) is observed for recovery within the first 7-15 min of incubation in LL and glycoprotein solutions. Thus, prolonged incubation of the nerves in solutions of thymus glycoproteins results in a statistically significant restoration of PD rate only at a concentration of 10 ¹⁴ mol / l.

Table 5.

Investigation of the combined effect of lysolecithin and thymus glycoproteins on the PB of the demyelinated nerve (glycoprotein concentration is ΙΟ ^'24 M)

In vivo Time of the AP (% vs. control) AP amplitude (% vs. control, 50 Hz) LL 160 30 Thymic acid glycoprotein 155 35 Basic thymus glycoprotein 158 32

In the next series of experiments, changes in AP amplitude under the action of LL and thymus glycoproteins were investigated (Fig. 8). It was found that significant changes in this parameter of PB (at 100 Hz frequency) were observed during the experiment (LL action). With the combined action of LL and thymus glycoproteins (10 ⁴⁴ M), the detected changes in AP amplitude were reversed.

In the next series of experiments, the redistribution of intracellular calcium was studied by recording the fluorescence of the probe - chlortetracycline, which allowed to reveal the location and changes in the level of membrane bound calcium. The fluorescence of nerves, nerve fibers incubated in LL solution, and fibers under the combined action of LL and thymus glycoproteins was investigated.

Earlier, it was shown that upon incubation of a nerve with chlortetracycline, the maximum fluorescence value, a parameter proportional to the level of membrane bound Ca ²⁺ , was detected by the membrane structures of the nerve (plasma membranes of axon and Schwann cells, as well as myelin) . Under the action of LL, the level of membrane bound calcium increases, but with the combined action of LL and thymus glycoproteins the binding value increases even more (Figs. 9-11). As follows from the results obtained, the maximum value of calcium binding is significantly altered by the action of LL and thymus glycoproteins at a concentration of 10 ⁴⁴ M. When the concentration of thymus glycoproteins is reduced to 10 ⁴⁸ M, the maximum calcium binding by the membrane structures of the nerve decreases but exceeds control (LL action).

Reducing the concentration of thymus glycoproteins to ΙΟ ^'24 M over 90 min leads to small changes in the level of bound calcium. The data obtained demonstrate the ability of the glycoproteins under study in "imaginary" solutions to alter the level of membrane bound calcium.

The study reveals the changes in a number of parameters characterizing PB under conditions of nerve demyelination, as well as the presence of characteristic reorganizations of PB under the action of thymus glycoproteins both in vivo and in vitro.

Kols OR, Maksimov GV, Radenovich Ch. N. Biophysics of rhythmic neurility, Moscow, Moscow State University, 206, 1993

Maksimov G. V., Orlov S. N. Transport of calcium ions while nerve fiber functioning: mechanisms and regulation. Moscow, Moscow State University, 88,1994

Waxman S. G., Kocsis J. D., Stys P. K. The axon. Structure, function and pathophysiology. Oxford Univ. Press., NY-Oxford, 325,1995

Example 10. Effect of Mammalian Serum Glycoprotein (pl in pH 4,655,1) on Excitatory Conductivity in Demyelination of Nerve Fiber

The experiment was performed according to the procedure described in Example 9.

Substantial changes in some electrophysiological parameters of rhythmic excitation (PB) are observed with the action of LL and nerve demyelination. Serum glycoprotein (SG) at a dose of 10 ⁴⁴ mol / l was found to compensate for changes in PB parameters in nerve demyelination (Table 6). It should be noted that the injection of glycoprotein solutions alone does not lead to a significant change in the excitability of the myelin nerve.

Table 6.

Investigation of the combined action of lysolecigin and serum glycoproteins on PB of demyelinated nerve in vivo

In vivo Time of the AP (% vs. control) AP amplitude (% vs. control) LL (at 50 and 100 Hz) 160 30 Serum glycoprotein at a dose of 10 ^'14 mol / l (at 100 Hz) 80 55 Serum glycoprotein at a dose of 10 ^'18 mol / I (at 50 Hz) 145 45 Serum glycoprotein at a dose of 10 ^'24 mol / l (at 50 Hz) 145 32

It was found that in the combined action of LL and SG, the nature of the changes caused by the administration of LL attenuates (Fig. 12,13). When diluting the solution of SG to a concentration of 10 ¹⁸ mol / Ι, the magnitude of the biological effect is insignificant, but the data have significant differences compared to the control ones. Thus, the prolonged incubation of the nerves in solutions of SG and LL results in a statistically significant 25 restoration of the rate of conduction of AP at concentrations of IO ¹⁴ moVl and IO ¹⁸ mol / l.

In the next series of experiments the changes in the amplitude of the AP under the action of LL and SG were investigated (Fig. 14). It was found that significant changes in this parameter of PB (at 100 Hz frequency) were observed during the experiment (LL action). With the combined action of LL and SG (10 ¹⁴ M), the detected changes in the amplitude of the AP are restored.

In the next series of experiments, the redistribution of intracellular calcium was studied by recording the fluorescence of the probe - chlortetracycline, which allowed to reveal the location and changes in the level of membrane bound calcium. The fluorescence of nerves, nerve fibers incubated in LL solution, and fibers under the combined action of LL and SG were investigated.

With the action of LL, the level of membrane bound calcium increases, but with the combined action of LL and SG the binding value increases even more (Figs. 15-17).

As is apparent from the results, the maximum value of the binding of calcium is significantly altered by the action of LL and Cl at a concentration of ¹⁴ M. In ΙΟ decrease in the concentration of Cl- to ΙΟ ^'24 M maximal binding of calcium from the membrane structures of the nerve is reduced. The data obtained demonstrate the ability of SGs in imaginary solutions to alter the level of membrane-bound calcium.

The study reveals changes in a number of parameters characterizing PB under conditions of nerve demyelination, as well as the presence of characteristic reorganizations of PB under the action of serum glycoprotein both in vivo and in vitro.

Example 11. Effect of Basic Glycoprotein from the Retina of a Bull's Eye on the Conduction of Excitation in Demyelination of Nerve Fiber

The experiment was performed according to the procedure described in Example 9.

Substantial changes in some electrophysiological parameters of rhythmic excitation (PB) are observed with the action of LL and nerve demyelination. It was found that in vivo basic retinal glycoprotein of the bull's eye retina (BGR) at doses of 10 ¹⁴ mol / Ι and 10 ^'18 mol / Ι compensates for changes in PB parameters in nerve demyelination (Table 7). It should be noted that the injection of a glycoprotein solution alone does not lead to a significant change in the excitability of the myelin nerve.

Table 7.

Investigation of the combined action of lysolecithin and basic glycoprotein from the retina of a bull's eye on the PB of demyelinated nerve in vivo

In vivo Time of the AP (% vs. control) AP amplitude (% vs. control) LL (at 50 and 100 Hz) 160 30 | BGR at a dose of 10 ^'14 mol / 1 (at 100 Hz) 145 «1 BGR at a dose of 10 ^'18 mol / l (at 50 Hz) 148 35 BGR at a dose of 10 ^'24 mol / 1 (at 50 Hz) 156 30

It was found that in the combined action of LL and BGR the nature of the changes caused by the application of LL weakens (Fig. 18,19). When diluting the BGR solution to a concentration of 10 ¹⁸ mol / l, the magnitude of the biological effect was significantly different from controls. Thus, prolonged incubation of the nerves in solutions of BGR and LL results in a statistically significant restoration of the rate of conduction of AP at concentrations of IO ¹⁴ mol / l and 10 ^'18 mol / l.

In the next series of experiments the changes in the amplitude of the AP under the action of LL and BGR were investigated (Fig. 20). It was found that significant changes in this parameter of PB (at 100 Hz frequency) were observed during the experiment (LL action). With the combined action of LL and BGR (10 ¹⁴ M), the detected changes in the amplitude of the AP are restored.

In the next series of experiments, the redistribution of intracellular calcium was studied by recording the fluorescence of the probe - chlortetracycline, which allowed to reveal the location and changes in the level of membrane bound calcium. The fluorescence of nerves, nerve fibers incubated in LL solution, and fibers under the combined action of LL and BGR was investigated.

With the action of LL, the level of membrane bound calcium increases, but with the combined action of LL and BGR the binding value increases even more (Fig. 21

23). As follows from the obtained results, the maximum value of calcium binding significantly changes under the action of LL and BGR at a concentration of М. ¹⁴ M. When the BGG concentration is reduced to 10 ^'24 M, the maximum binding of calcium by the nerve membrane structures decreases. The data obtained demonstrate the ability of BGR in "imaginary" solutions to alter the level of membrane bound calcium.

The study reveals changes in a number of parameters characterizing PB under conditions of nerve demyelination, as well as the presence of characteristic PB reorganizations under the action of a basic retina glycoprotein in the bull's eye both in vivo and in vitro.

Example 12. Effect of Basic Glycoprotein by the Pigment Epithelium of a Bull's Eye on the Conduction of Nerve Fiber Demyelination Excitation

The experiment was performed according to the procedure described in Example 9.

Substantial changes in some electrophysiological parameters of rhythmic excitation (PB) are observed with the action of LL and nerve demyelination. It was found that in vivo basic glycoprotein from the pigment epithelium of the bull's eye (BGPE) at a dose of 10 ⁴⁴ mol / 1 compensates for changes in PB parameters in nerve demyelination (Table 8). It should be noted that the injection of a glycoprotein solution alone does not lead to a significant change in the excitability of the myelin nerve.

Table 8.

Investigation of the combined action of lysolecithin and basic glycoprotein from the pigment epithelium of a bull's eye on the PB of demyelinated nerve in vivo

In vivo Time of the AP (% over control) AP amplitude (% vs. control) LL (at 50 and 100 Hz) 160 30 BGPE at dose 1O ' ^And mol / ί (at 100 Hz) 145 40 BGPE at dose 10 ^'18 mol / ί (at 50 Hz) 155 38 BGPE at a dose of 10 ^'24 mol / ί (at 50 Hz) 160 30 1

It was found that in the combined action of LL and BGPE the nature of the changes caused by LL administration weakened (Figs. 24, 25). Continuous incubation of the nerves in BGPE and LL solutions results in a statistically significant recovery of PD at a concentration of 10 ^'14 mol / l.

In the next series of experiments, the changes in the amplitude of the AP under the action of LL and BGPE were investigated (Fig. 26). It was found that significant changes in this parameter of PB (at 100 Hz frequency) were observed during the experiment (LL action). With the combined action of LL and BGPE (ΙΟ ¹⁴ M), the detected changes in the amplitude of the PD are restored.

In the next series of experiments, the redistribution of intracellular calcium was studied by recording the fluorescence of the probe - chlortetracycline, which allowed to reveal the location and changes in the level of membrane bound calcium. The fluorescence of nerves, nerve fibers incubated in LL solution, and fibers under the combined action of LL and BGPE was investigated.

With the action of LL, the level of membrane bound calcium increases, but with the combined action of LL and BGPE the binding value increases even more (Figs. 27-29). As follows from the results obtained, the maximum value of calcium binding significantly changes under the action of LL and BGPE at a concentration of ΙΟ ¹⁴ M. When the concentration of BGR is reduced to ΙΟ ¹⁸ M and 10 ′ ²⁴ M, the maximum calcium binding of membrane structures. the nerve goes down. The data obtained indicate the ability of BGPE in 'imaginary' (10 ^'24 mol / ί) solutions to alter the level of membrane bound calcium.

The study reveals changes in a number of parameters characterizing PB under nerve demyelination conditions, as well as the presence of characteristic PB reorganizations under the action of a basic glycoprotein from the pigment epithelium of the bull's eye both in vivo and in vitro.

Example 13. Effect of Liver Acid Glycoprotein on Plasma Membrane Permeability of Hepatocytes and Intensity of Protein Synthesis

The experiment was performed on multiple organ cultures of liver of rats from the Wistar line, male, weighing 180 g. A total of 5 experiments were performed. 25-30 pieces of gland are used in each of them. All explants tested in one experiment were obtained from the liver of one animal. The explants were cultured at the interface between the liquid culture medium and the air using millipore filters (Synpor, 0.6 mm diameter). The cultivation was carried out for 14-16 h at 37 ° C. After cultivation, the explant group was transferred for 10 min into the culture medium containing ³ H-leucine labeled at 25 microCu. The intensity of protein synthesis was evaluated by the incorporation of leucine into the proteins compared to the pool of labeled leucine in the same sample. The cell permeability to the labeled precursor was determined by the pool of free labeled amino acid. The influence of the acid glycoprotein from the liver (HGDB) was found to be its solution at a concentration of 10 ¹⁴ mol / ί. The HCGD solution was added to the culture medium in the test vials 2 h before incubation with labeled leucine, and then transferred for 10 min to medium containing HCGD and labeled amino acid at the indicated concentrations.

It was found that the intensity of protein synthesis under the influence of CPDD significantly decreased in comparison with the control approximately twice (in the control the ratio of inclusion of labeled leucine to the pool of this precursor is approximately 0.42 ± 0.09, in the experiment - 0 , 21 ± 0.04). The permeability of the hepatocyte membrane is increased too significantly in the presence of HSCD (in the control, for example, the pool of free amino acids is 1120 ± 50 bpm and in the test vials, respectively, is 2180 ± 30).

The morphology of the explants from the test organ culture did not change compared to the control.

The results of the study show that CPGD influences the intensity of protein synthesis in hepatocytes and the permeability of their plasma membranes in an extremely small dose while preserving the structure of liver tissue.

Example 14. Effect of Mammalian Serum Glycoprotein (pl in pH 4,655,1) on Hepatocyte Plasma Membrane Permeability and Intensity of Protein Synthesis

The experiment was performed on multiple organ cultures of liver of rats from the Wistar line, male, weighing 180 g. A total of 5 experiments were performed. 25-30 pieces of gland are used in each of them. All explants tested in one experiment were obtained from the liver of one animal. The explants were cultured at the interface between the liquid culture medium and the air using millipore filters (Synpor, 0.6 mm diameter). The cultivation was carried out for 14-16 h at 37 ° C. After cultivation, the group of explants was transferred for 10 min in culture medium containing ³ H-leucine labeled at 25 microCu. The intensity of protein synthesis was evaluated by the incorporation of leucine into proteins, relative to the pool of labeled leucine in the same sample. The cell permeability to the labeled precursor was determined according to the pool of free labeled amino acid. The effect of serum glycoprotein (SG) was found to be its solution at a concentration of 10 ¹⁴ mol / ί. The SG solution was added to the culture medium in the test vials 2 h before incubation with labeled leucine and then transferred to media containing SG and labeled amino acid at the indicated concentrations for 10 min.

It was found that the intensity of protein synthesis under the influence of SG significantly decreased compared to the control approximately twice (in the control the ratio of inclusion of labeled leucine to the pool of this precursor is approximately 0.42 ± 0.09, in the experiment - 0 , 17 ± 0.05). The permeability of the hepatocyte membrane increases too significantly in the presence of SG (in the control, for example, the pool of free amino acids is 1120 ± 50 bpm, and in the test vials is 2980 ± 30, respectively).

The morphology of the explants from the test organ culture did not change compared to the control.

The results of the study show that SG influences the intensity of protein synthesis in hepatocytes and the permeability of their plasma membranes in ultra-small doses while preserving the structure of liver tissue.

Example 15. Mammalian serum glycoprotein composition (pl in pH range 4.65-5.1) in ultra-small doses having pharmacological action

Composition with composition:

Serum glycoprotein 1 x 10 ¹⁰ g

Sodium chloride 8.8 g

Calcium chloride 0.001 g

Water up to 11 is a bioregulator of reparative processes in epithelial and connective tissue.

When applied in the form of eye drops, the composition of said composition assists the healing of the cornea after mechanical trauma or burning, causes the formation of fine cicatrix, while limiting the excessive expansion of the cicatricial tissue. It is especially effective in corneal grafting, treating keratitis and some conjunctivitis. The application of the composition in the treatment of penetrating corneal injuries leads to a shortened duration of the inflammatory reaction, rapid elimination of diastasis of the wound edges, accelerated epithelization of the wound surface, earlier recovery of the anterior chamber, reducing the incidence of complications fibrin and hypopyon), accelerating the processes of reparative regeneration and reconstruction of the newly formed cicatricial tissue, resulting in a more compact and structured cicatrix hp with a predominance of proliferative component without active vascularization of the cornea. At the cellular level, the migration of epithelium, macrophages, keratoblasts and reduction of leukocyte infiltration, early formation of a continuous layer of endothelium without signs of desquamation in the area of the wound canal are observed, which eventually leads to its rapid filling with epithelium. component, early and accurate closure of wound ends, active fibrin resorption and replacement with keratoblastic proliferate synthesizing new collagen fibers. When applying the composition, a more compact arrangement of the collagen plates is observed, the predominance of the fibrous component in the proliferator over the cellular, which determines the quality of the cicatricial tissue. The formation of finer, compact, non-vascular cicatrixes results in a smaller change in the transparency and refractive index of the cornea. In the case of burns of the eye, the effective therapeutic composition of said composition manifests itself by the 14th day and is expressed in significant proliferation of fibroblastic elements infiltrating the injured corneal tissue, and the vector orientation of the proliferating cells determines the layered structure of the morphology. tissue structure.

When administered in injectable formulation, the composition of said composition stimulates bone regeneration in limb fractures, including fracture of the femoral neck, and is effective in treating a number of severe joint pathologies associated with disorders in the structure and function of the cartilage tissue, in the treatment of mechanical damage to the joint cartilage of the knee joint, in the treatment of arthrosis and synovitis. Injection of the composition inhibits the degenerative processes in the damaged cartilage tissue. In the application of the composition for treating articular cartilage damage, young cartilage cells accumulate rapidly and differentiate, resulting in faster than control controls, the formation of cartilage replacement tissue and the restoration of smooth joint surface in the damaged area, which in its own right order plays a major role in restoring joint mobility. The damaged area is filled with young cartilage cells and cartilage tissue is definitively identified, as it is differentiated into layers inherent in the original hyaline cartilage. Intravenous administration of the composition results in a decrease in pain syndrome after 2-3 injections of the preparation. If we compare the effectiveness of the application of the composition with the above composition and of the most famous choproprotectors, for example "Zeel", in traumatic injuries of the articular cartilage of the knee joint in athletes, it can be noted that the pain syndrome and synovitis disappear on average by 7- 10th day of application of the composition and 14-17th day of application of Zeel. Therefore, the restoration of sports performance to the previous level is 22.5 times faster than with Zeel.

Compositions of different composition containing as active ingredient serum glycoprotein at a concentration of 1x10 · 10 g glycoprotein / 1 (or kg) composition, in various dosage forms (gels, ointments, suppositories, solution) have a healing effect on wounds and are effective in healing skin lesions, including the treatment of burns, decubitus and their prevention; stimulate reparative processes in the skin after radiation lesions occurring in cancer patients after radiotherapy. The use of such compositions is effective in gastroenterology (ulcer, gastritis, gastroduodenitis), in proctology (diseases of the colon), in gynecology (erosion of the cervix), in cardiology (rehabilitation period after myocardial infarction).

Example 16. Composition on the basis of acidic glycoprotein from the retina of the bovine eye in ultra-small doses having pharmacological action

Composition with composition:

Retinal acid glycoprotein 1 x 10 ¹⁰ g

Sodium chloride 8.8 g

Calcium chloride 0.001 g

Water up to 11 is a bioregulator of reparative processes that helps to restore impaired retinal function and prevents retinal detachment during surgery.

The composition of said composition stimulates the functioning of the main enzymatic systems in the retina of the eye responsible for the visual process, inhibiting lipid peroxidation in the retinal cell membranes. The composition is an effective bioregulator responsible for the positioning of cells in the histological structure of the retina and for cell division; it assists in the restoration of the spatial-functional organization of the retina after injury or development of a pathological process. The present composition has a curative effect on myopia, retinal degeneration with different etiologies and conditions after penetrating eye injuries.

Example 17. Acid glycoprotein composition from pigment epithelium of bovine in ultra-small doses having pharmacological action

Composition with composition: Acid glycoprotein from the pigment epithelium of the eye 1x10 ¹⁰ g Sodium chloride 8,8 g Calcium chloride 0,001 g Water to 11 is a bioregulator that helps restore-

dysfunction of the pigment epithelium of the eye.

Pharmacological agents are currently lacking, the use of which will suppress the pathological process in the early stages of development of retinitis with different etiology and maculopathies. The composition of said composition is effective for treating maculopathies and retinitis with different etiology.

Claims

Claims (3)

Glycoproteins, characterized in that they are extracted by isoelectric focusing from the intercellular space of tissues from different organs, serum, bile of man and animals soluble in saturated (100%) ammonium sulphate solution having an estimated molecular weight of 10- 45 kDa and having biological activity at ultra-small doses 10 ¹² 10 ^'29 mol / Ι and below. A pharmaceutical composition comprising a glycoprotein according to claim 1 in an effective amount of 10 ^'10 to 10 ²⁴ and a pharmaceutically acceptable carrier. Use of a glycoprotein according to claim 1 as a medicament. Attachment: 29 figures