RU2176916C1 - Selenium-containing glycoprotein element-organic substance possessing immunostimulating activity - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: method involves obtaining selenium-containing glycoprotein element-organic substance possessing immunostimulating activity. The substances have low molecular mass component like protein, selenium, carbohydrate residue and various inorganic ions introducible with aqueous solutions like calcium, strontium, iron, magnesium cations and cyanide, hydrophosphate, dihydro-orthosilicate, iodide anions. EFFECT: enhanced effectiveness of treatment; increased CD4 T-helper cells production; increased killer and phagocytic activity. 4 cl, 8 dwg

Description

 The invention relates to the field of chemical pharmacology and pharmacodynamics, biochemical medicine, cellular and molecular biology, immunopharmacology of trace elements and can be used in clinical medicine for the treatment of various diseases, accompanied by disorders of the stimulating function of the immune system, which manifests itself in the inhibition of the production of CD4 helper cells, a decrease in killer and phagocytic activity.

 Currently, in synthetic pharmacology, both synthetic and organic substances of natural origin are used for immunostimulation. These are, as a rule, preparations from extracts of the goiter, lymph nodes, connective tissue and spleen. Such medicines include substances well known in clinical practice, for example, thymogen, T-activin, myelopid, thymoptin, etc. (Mashkovsky MD "Drugs that correct immunity processes" in the book "Medicines", Kharkov, "Torsing", 1997, v. 2, pp. 199-207).

 Recently, the attention of many researchers has been attracted to selenium and its compounds, since it has been proved that cellular and humoral immune processes, especially the stimulation of cellular immune responses, are carried out using this trace element. (Kudrin A.V., Skalny A.V., Zhavoronkov A.A., Skalnaya M.G., Gromova O.A. in the book "Immunopharmacology of trace elements", ed. KMK, M., 2000, p. 222 -223, 354-357).

 In this regard, to maintain the active function of the immune system, selenium is introduced as a trace element into vitamin complexes, for example, Centrum (VIDAL Directory "Medicinal Products in Russia", Moscow: AstraFarmService, 1997, p. B-654). Selenium-containing preparations based on algae biomass (patent GB 2203043, 1988 or patent RU 2096037) are also known, having a tonic effect, the implementation of which is associated with the direct effect of selenium compounds on the immune system. However, these medicinal substances have a low enough content of organic selenium, and the technology for their preparation is complex and requires a long time.

 In the patent (RU 2138271 C1, 1999), a PSK (proteoselen complex) was proposed, the production technology of which to some extent solved the previous difficulties, however, the structure of this selenium organic substance was not identified.

 The data presented indicate that the modern technology for the production of a new class of selenium preparations that carry out immunostimulating activity should be based on the structure and topology of active biomolecules associated with selenium, especially glycoproteins, stereochemically corresponding to receptors of the body’s lymphocytic immune system. (J. Playfer, "Immunology Imaging", M. Medicine, 1999, p. 34 and 35).

 In this regard, the new class of created drugs in their structure and mechanism of action must stereochemically, i.e., biologically and chemically, correspond to those substances of the human body that directly carry out the stimulating function of the immune system.

 To solve this problem, medicinal substances with an immunostimulating effect are proposed. They include a low molecular weight component - a protein obtained by extraction from protein-containing plant materials with organic solvents; selenium, a carbohydrate residue (for example, sucrose) and various inorganic ions introduced by saline aqueous solutions: calcium, strontium, magnesium, iron cations; anions - cyanide, hydrogen phosphate, dihydroorthosilicate, iodide.

 The various substances we have obtained are salt forms of selenoglycoprotein.

 The invention is illustrated by the accompanying description of the drawings (see Fig. 1 - 8).

Obtaining selenoglycoprotein (SeGP)
As the starting products use the green mass of protein-containing plants, such as cereals, legumes, corn leaves. Protein is extracted from the plant mass. The extraction is carried out with ether, ethyl alcohol, dimexide. The extract obtained is evaporated and lyophilized. The powder is dissolved in a 60-90% ethanol solution. Selenium obtained by thermolysis of selenium urea is introduced into the solution (at the rate of 6-12 μg per 1 ml of solution). Then a certain amount of carbohydrate, such as sucrose, is added to the mixture and placed in an electromagnetic high-frequency field (power 5 • 10 ^-3 W / cm, frequency 20-30 MHz). The field can be created, for example, in SQUID magnetometers used in magnetoencephalography.

 The Selenoglycoprotein – SeHP molecule (Fig. 1) is two polypeptoid alpha and beta chains of a protein connected by a disulfide bridge at cysteine residues (the alpha chain consists of residues of 20 amino acids, beta of 29 amino acids). Selenium obtained by thermolysis of selenourea, enters the lateral alkyl radicals of amino acids, connecting some units of the alpha and / or beta chains of one or two neighboring protein molecules, thereby additionally "linking" them. Sucrose is attached to selenoprotein via the basic amino acid residues of serine and / or tyrosine via O-glycosidic bonds. The molecular weight of SeГП is 6835. The molar ratios of protein, selenium, and carbohydrate are 1: 4: 5.

At all physiological pH values, selenoglycoprotein is ionized. Charged are the C- and N-terminal groups and the side chains bonded to the α-carbon atoms containing a carboxyl or amino group (—COO ^- , —NH ₃ ⁺ ) (FIG. 1).

 When selenoglycoprotein interacts with aqueous solutions of salts, salt bonds arise between unlike charged groups of selenoglycoprotein and various cations and anions of aqueous solutions.

1. Calcium substance dihydroorthosilicate-selenoglycoprotein (CaH ₂ SiO ₄ -SeHP).

The substance includes ionized selenoglycoprotein (Fig. 1), to which calcium cations (Ca ²⁺ ) are attached at the carboxyl groups (-COO ^- ) of glutamic acid (and possibly proline) and the C-terminal groups, and histidine at the amino groups (-NH ₃ ⁺ ) , arginine and the N-terminal group are attached dihydroorthosilicate anions (H ₂ SiO ₄ ^2- ) to the α and / or β chains of one molecule or neighboring molecules of selenoglycoprotein (figure 2). The molecular weight of CaH ₂ SiO ₄ -SeHP is 7531, the molar ratios of SeHP: Ca ²⁺ : H ₂ SiO ₄ ^2- - 1: 8: 4.

2. Calcium substance cyanide-selenoglycoprotein (Ca (CN) ₂ -SeGP).

The substance includes ionized selenoglycoprotein (Fig. 1), to which calcium cations are attached to the carboxyl groups (-COO ^- ) of glutamic acid (and possibly proline) and to the C-terminal groups, and histidine, arginine and N- at the amino groups (-NH ₃ ^- ) the end group is attached cyanide anions (CN ^- ) to the α and / or β chains of selenoglycoprotein (Fig. 3). The molecular weight of Ca (CN) _2- SeGP is 7363, the molar ratios of SeGP: Ca ²⁺ : CN ^- are 1: 8: 8.

3. Calcium substance hydrophosphate-selenoglycoprotein (CaHPO ₄ -SeGP).

The substance includes ionized selenoglycoprotein (Fig. 1), to which calcium cations are attached at the carboxyl groups (-COO ^- ) of glutamic acid (and possibly proline) and terminal α-carbon; at the amino groups (-NH ₃ ⁺ ) of histidine, arginine and the N-terminal group, hydrophosphate anions (HPO ₄ ^{-2 are} attached to the α and / or β chain of one molecule or neighboring molecules of selenoglycoprotein (figure 4). Molecular mass of CaHPO ₄ -SeGP - 7539. The molar ratios of SeGP: Ca ²⁺ : HPO ₄ ^2- - 1: 8: 4.

4. Calcium substance iodide-selenoglycoprotein (CaI ₂ -SeGP).

The substance includes ionized selenoglycoprotein (Fig. 1), to which calcium cations are attached at the carboxyl groups (-COO ^- ) of glutamic acid (and possibly proline) and terminal α-carbon; at the amino groups (-NH ₃ ^- ) of histidine, arginine and the N-terminal group, iodide anions (I ^- ) are attached to the α and / or β chain (Fig. 5). The molecular weight (CaI ₂ -SeHP) is 7409. The molar ratios of SeHP: Ca ²⁺ : I ^- are 1: 8: 8.

5. Substance of strontium dihydroorthosilicate-selenoglycoprotein (SrH ₂ SiO ₄ -SeHP).

The substance includes ionized selenoglycoprotein (Fig. 1), to which strontium cations (Sr ²⁺ ) are attached at the carboxyl groups (-COO ^- ) of glutamic acid (and possibly proline) and terminal α-carbon; at the amino groups (-NH ₃ ⁺ ) of histidine, arginine and the terminal amino group, the dihydroorthosilicate anions (H ₂ SiO ₄ ^2- ) are attached to the α and / or β chain of one molecule or neighboring selenoglycoprotein molecules (Fig. 6). The molecular weight of SrH ₂ SiO ₄ -SeHP is 7915. The molar ratios of SeHP: Sr ²⁺ : H ₂ SiO ₄ ^2- - 1: 8: 4.

6. The substance is magnesium hydrophosphate-selenoglycoprotein (MgHPO ₄ - SeGP).

The substance includes ionized selenoglycoprotein (Fig. 1), to which magnesium cations (Mg ²⁺ ) are attached at the carboxyl groups (-COO ^- ) of glutamic acid and the C-terminal groups; at the amino groups of histidine, arginine, and the N-terminal group, hydrophosphate anions (HPO ₄ ^2- ) are attached to the alpha and / or beta chain of one molecule or neighboring selenoglycoprotein molecules (Fig. 7). The molecular weight of MgHPO _4- SeGP is 7411. The molar ratios of SeGP: Mg ²⁺ : HPO ₄ ^2- are 1: 8: 4.

7. The substance of iron is hydrogen phosphate-selenoglycoprotein (FeHPO ₄ -SeGP).

The substance includes ionized selenoglycoprotein (Fig. 1), to which iron (II) cations (Fe ²⁺ ) are attached to the carboxyl groups (-COO ^- ) of glutamic acid and the C-terminal groups; at the amino groups of histidine, arginine and the N-terminal group, hydrophosphate anions are attached to the alpha and / or beta chain of one molecule or neighboring molecules of selenoglycoprotein (Fig. 8). The molecular weight of FeHPO _4- SeGP is 7667. The molar ratios of SeGP: Fe ²⁺ : HPO ₄ ^2- - 1: 8: 4.

Examples of the production of selenium glycoprotein organoelement substances - Se-GPEOV (salt forms)
Example 1. A method of producing calcium dihydroorthosilicate-selenoglycoprotein (CaH ₂ SiO ₄ -SeHP).

To the selenoglycoprotein add the necessary amount of an aqueous solution of calcium orthosilicate, previously subjected to electrolysis and hydrolysis. The mixture is kept in an electromagnetic high-frequency field (power 5 • 10 ^-3 W / cm ² , frequency 20-30 MHz).

Example 2. A method of producing calcium cyanide-selenoglycoprotein (Ca (CN) ₂ -SeGP).

To the selenoglycoprotein add the necessary amount of an aqueous solution of calcium cyanide, previously subjected to electrolysis. The mixture is kept in an electromagnetic high-frequency field (power 5 • 10 ^-3 W / cm ² , frequency 20-30 MHz).

Example 3. A method of producing calcium hydrogen phosphate-selenoglycoprotein (CaHPO ₄ -SeGP).

The necessary amount of calcium hydrophosphate, previously subjected to electrolysis, is added to selenoglycoprotein. The mixture is kept in an electromagnetic high-frequency field (power 5 • 10 ^-3 W / cm, frequency 20-30 MHz).

Example 4. A method of producing calcium iodide-selenoglycoprotein (CaI ₂ -SeGP).

To selenoglycoprotein add a certain amount of an aqueous solution of calcium iodide previously subjected to electrolysis. A similar mixture is kept in an electromagnetic high-frequency field (power 5 • 10 ^-3 W / cm ² , frequency 20-30 MHz).

Example 5. A method for producing strontium dihydroorthosilicate-selenoglycoprotein (SrH ₂ SiO ₄ -SeHP).

To selenoglycoprotein add a certain amount of an aqueous solution of strontium orthosilicate, previously subjected to electrolysis and hydrolysis. The mixture is kept in an electromagnetic high-frequency field (power 5 • 10 ^-3 W / cm ² , frequency 20-30 MHz).

Example 6. A method of producing magnesium hydrophosphate-selenoglycoprotene (MgHPO ₄ -SeGP).

The necessary amount of magnesium ions obtained by electrolysis of magnesium glycerophosphate and the necessary amount of hydrogen phosphate anions obtained by electrolysis of calcium hydrogen phosphate are added to selenoglycoprotein. The mixture is kept in an electromagnetic high-frequency field (power 5 • 10 ² W / cm ² , frequency 20-30 MHz).

Example 7. A method of producing iron hydrogen phosphate-selenoglycoprotein (FeHPO ₄ -SeGP).

The necessary amount of iron ions obtained by electrolysis of iron acetate tetrahydrate and the necessary amount of hydrogen phosphate anions obtained by electrolysis of calcium hydrogen phosphate are added to selenoglycoprotein. The mixture is kept in an electromagnetic field (power 5 • 10 ^-3 W / cm ² , frequency 20-30 MHz).

 Se-GPEOVs include an asymmetric protein molecule with an alpha helix and a beta structure. The hydrophobic end of the Se-GPEOV molecule transforms into an alpha helix, the beta structure of the Se-GPEOV molecule is crimped accordingly and on the outside of the amino acid chain, as well as on the inside, there are both selenide and disulfide bonds providing spatial stability and at the same time flexibility this molecule.

 The entire Se-GPEOV molecule contains 15 amino acids. Accordingly, the above technology can be obtained molecules of a variety of selenium glycoprotein organoelement substances with different numbers of amino acids and relatively low molecular weight.

 The acute toxicity of the drug was studied with intraperitoneal administration of Se-GPEOV at a dose of 250 mg / kg to mice (age 6 weeks, weight 28-30 g). The LD value was 700 mg / kg.

 In the study of general toxicity, mice were injected with Se-GPEOV at a dose of 100 mg / kg per day intraperitoneally continuously for 10 days. No weight loss or any other abnormalities were observed, and subsequently, when these mice were observed for three months, no abnormalities were detected.

 As a result of the studies, it was found that Se-GPEOV are organic compounds, have immunostimulating properties, are non-toxic, completely soluble in water and biological fluids - blood, lymph and cerebrospinal fluid. In 1 ml of Se-GPEOV, depending on the amount of amino acids in the ligand, from 20 to 30 mg of the substance and 6 μg of selenium are contained.

 The dose of the drug administered depends on the clinical condition of the patient, his age, weight, as well as the route of administration. An effective therapeutic daily dose for a patient is from 6 to 150 mg of the active substance, which is administered simultaneously or fractionally.

 The method of activation and stimulation of the immune system is as follows.

 The obtained preparation Se-GPEOV is a solution of the active substance in 60 - 90% ethanol, 1 ml of which contains 20-30 mg of the drug substance. Se-GPEOV is administered orally, intramuscularly, intravenously, intraarterially, and also externally as a suspension in an oil solution or as an ointment with an oil-alcohol excipient.

When administered orally, the required dose is calculated as follows:
20 drops of the solution contain 20-30 mg of the drug substance;
10 drops (1/2 ml) contain 10-15 mg of the drug;
5 drops (1/4 ml) contain 5-7.5 mg of the drug;
3 drops (1/7 ml) contain 3.3 mg of the drug.

 When administered intramuscularly, intravenously or intraarterially, the drug is dissolved in physiological saline for better dissociation in a ratio of 1:10 when using 60% alcohol or 1:15 in 90% alcohol.

For example: for children under 6 years of age with a moderate clinical pathology, the drug is administered once as an injection from 0.1 to 0.5 ml per day, with a severe degree of pathology, the specified dose is administered twice a day. When conducting intensive therapy, the drug is administered intramuscularly or intravenously (intraarterially) in 1.0-2.0 ml, respectively, with the addition of 10 or 20 ml of physiological saline. In the case of oral administration, depending on the age, weight and degree of damage, from 0.5 to 3.0 ml, moreover, the drug is dissolved in 1/3 cup boiled slightly warm water (36-38 ^o C). The duration of treatment can vary from 1 to 3 months, in case of a severe course of pathology, the treatment is repeated with an interval of 1-2 months until a pronounced clinical effect. During the treatment, the immunological status of the patient is monitored according to the detailed clinical formula and immunogram.

 When using immunostimulating selenium glycoprotein organoelement substances (salt forms), active stimulation of the immune system is observed, which manifests itself as an improvement in the patients' clinical well-being and normalization of immunity - an increase in the production of CD4 T helper cells, as well as an increase in killer and phagocytic activity.

Claims (4)

1. Selenoprotein drug having an immunostimulating effect based on proteoselen, characterized in that it contains selenoprotein, which is two polypeptide α and β chains of the protein, in the side alkyl radicals of which selenium is introduced, connecting some links of the α and β chains of one or of two neighboring protein molecules, the polypeptide chains are also connected by a disulfide bridge at the cysteine residues (the α-chain consists of 20 amino acid residues, the β-chain of 29 amino acids), while selenoprotein is connected to sucrose via the volume of O-glycosidic bonds through amino acid residues of serine and / or tyrosine with a molar ratio of protein: selenium: carbohydrate = 1: 4: 5 (Fig. 1) and obtained under the action of a sinusoidal electromagnetic field with a power flux density of 5 • 10 ^-3 W / cm ² at a frequency of 20-30 MHz. 2. Selenoprotein preparation having an immunostimulating effect on the basis of selenoprotein, characterized in that the preparation contains selenoglycoprotein described in claim 1, to which calcium cations (Ca ²⁺ ) are attached to the carboxyl groups (-COO ^- ) of glutamic acid and the C-terminal groups ) or strontium (Sr ²⁺ ), at the amino groups (NH ₃ ⁺ ) of histidine, arginine, and the N-terminal group, the dihydroorthosilicate anions (H ₂ SiO ₄ ^2- ) are attached to the α and / or β chains of selenoglycoprotein at a molar ratio of selenoglycoprotein : calcium cation or pp ntsiya: H ₂ SiO ₄ ^2- - 1: 8: 4 (Figures 2 and 6). 3. Selenoprotein preparation having an immunostimulating effect on the basis of selenoprotein, characterized in that the preparation contains selenoglycoprotein, characterized in paragraph 1, to which calcium cations (Ca ²⁺ ) are attached to the carboxyl groups (-COO ^- ) of glutamic acid and the C-terminal groups ), at the amino groups (NH ₃ ⁺ ) of histidine, arginine, and the N-terminal group, cyanide anions (CN ^- ) or iodide anions (J ^- ) are attached to the α or β chains of selenoglycoprotein at a molar ratio of protein: Ca ²⁺ : CN ^- or J ^- equal to 1: 8: 8 (Figs. 3 and 5). 4. Selenoprotein preparation having an immunostimulating effect based on selenoprotein, characterized in that the preparation contains selenoglycoprotein, characterized in paragraph 1, to which calcium cations (Ca ²⁺ ) are attached to the carboxyl groups (-COO ^- ) of glutamic acid and the C-terminal groups ), or magnesium (Mg ²⁺ ), or iron (Fe ²⁺ ), at the amino groups (NH ₃ ⁺ ) of histidine, arginine and the N-terminal group are attached hydrophosphate anions (НРО ₄ ^2- ) to α- and / or β - chains of selenoglycoprotein with a molar ratio of protein: Ca ²⁺ or: Mg ²⁺ or: Fe ²⁺ : NRA ₄ ^2- equal to 1: 8: 4 (Figs. 4, 7, 8).

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