PRODUCING AND USE THEREOF

Technical Field

This invention relates to organic and pharmaceutical chemistry and concerns methods for producing beta-glucans and use thereof in pharmaceuticals.

Background Art

Beta-glucans, which are natural polysaccharides, are among the most essential classes of biopolymers, since they have a broad spectrum of biological activity. However, intra- and intermolecular interactions between its polysaccharide molecules significantly impact its biological effects. These interactions can be affected by physical methods and chemical structural changes to amend biological properties in a targeted manner.

The most common methods of chemical modification of the structure of beta- glucans are carboxymethylation (Carbohydr. Polym. 2004, 57,319-325, J. Biochem. Mol. Biol. 1998, 14, 636-640), sulfation (Carbohydr. Res. 2003, 338, 2863-2870) and phosphorylation (Carbohydr. Polym. 2009, 78, 581-587., Carbohydr. Res. 1997, 299, 203-208).

The modification of the structure of beta-glucans by oxidation is closer to the proposed invention. In this case, the main directions are the oxidation of the alcohol group in the 6th position to obtain the corresponding acid and its derivatives (International Immunopharmacology 2001, 1, 539-550, J. Agric. Food Chem. 2009, 57, 439-443) and oxidation followed by obtaining conjugates with proteins and polyphenols (Planta, 1991, 185, 1, 1-8, RU 2014124783 A).

The closest to the proposed invention is WO2013183049A1, which describes antiviral compositions of glucans oxidized to the corresponding uronic acids, US3856775, dedicated to the antitumor activity of 1,3-beta-glucan oxidation products, and (Journal of Materials Chemistry, 2017, 5, 38), dedicated to the antimicrobial activity of nano fibrillar products of cellulose oxidation.

Summary of Invention

This invention relates to an antimicrobial agent that is a product of the treatment of beta-glucan with iodic acid or its salt, followed by purification and isolation, where the treatment is carried out in an aqueous or aqueous-organic medium containing organic solvents in an amount from 0.1 to 100%, at pH in the range from 1 to 14, at a temperature from 0 to 100 °C.

In a preferred embodiment, the beta-glucan is carboxymethylcellulose, baker's yeast beta-glucan, oat beta-glucan, barley beta-glucan, reishi beta-glucan, optionally after pre-purification and/or fractionation.

In a preferred embodiment, the content of carbonyl groups in the antimicrobial agent is from 0.1 to 3 mmol/g.

Another subject of the invention is an antimicrobial agent, a product of the treatment of beta-glucan with haloalkyl carboxylic acid or its salt, followed by its purification and isolation, where the treatment is carried out in an aqueous or aqueous- organic medium containing organic solvents in an amount of 0.1 to 100%, at pH in the range from 7 to 14, at a temperature from 0 to 100 °C in the presence of organic and/or inorganic bases.

In a preferred embodiment, the beta-glucan is baker's yeast beta-glucan, oat beta-glucan, barley beta-glucan, reishi beta-glucan, optionally after pre -purification and/or fractionation.

In a preferred embodiment, the degree of substitution is between 0.1 and 1.5.

Another subject of the invention is an antimicrobial agent, a product of the treatment of beta-glucan with a haloalkyl carboxylic acid or its salt, followed by purification and isolation of the semiproduct and its oxidation with iodic acid or its salt, followed by its purification and isolation.

In a preferred embodiment, the beta-glucan is baker's yeast beta-glucan, oat beta-glucan, barley beta-glucan, reishi beta-glucan, optionally after pre -purification and/or fractionation.

In a preferred embodiment, the content of carbonyl groups in the antimicrobial agent is between 0.1 and 3 mmol/g.

Another subject of the invention is a method for producing an antimicrobial agent, which comprises treating beta-glucan with iodic acid or its salt in an aqueous or aqueous-organic medium containing organic solvents in an amount from 0.1 to 100%, at a pH range from 1 to 14, preferably 1 to 7, at a temperature of 0 to 100 °C, preferably 15 to 50 °C, followed by purification and isolation. Another subject of the invention is a method for producing an antimicrobial agent, which comprises treating beta-glucan with a haloalkyl carboxylic acid or its salt in an aqueous or aqueous organic medium containing organic solvents in an amount of 0.1 to 100%, at a pH range of 7 to 14, at a temperature from 0 to 100 °C, preferably from 15 to 50 °C, in the presence of organic and/or inorganic bases, followed by purification and isolation.

Another subject of the invention is a method for producing an antimicrobial agent, which includes the steps of: treatment of beta-glucan with a haloalkyl carboxylic acid or its salt in an aqueous or aqueous -organic medium at a pH range from 7 to 14, at a temperature from 0 to 100 °C, preferably from 15 to 50 °C, followed by their purification and isolation of the semiproduct; and treatment of the obtained semiproduct with iodic acid or its salt in an aqueous or aqueous-organic medium at a pH range from 1 to 14, preferably from 1 to 7, at a temperature from 0 to 100 °C, preferably from 15 to 50 °C, followed by purification and isolation of the target product.

Another subject of the invention is an antimicrobial composition containing an effective amount of an antimicrobial agent following any one of claims 1-3 and pharmaceutically acceptable additives.

The proposed antimicrobial composition is effective against influenza viruses, herpes, respiratory viral, and bacterial infections.

Another aspect of the invention is an antimicrobial composition comprising an effective amount of any of the above antimicrobial agents and at least one other antimicrobial agent.

The proposed antimicrobial composition is effective against influenza viruses, herpes, respiratory viral, and bacterial infections.

Detailed Description of the Invention

In the context of this description:

The term "beta-glucan" means a polysaccharide of beta-D-glucose monomers linked by glycosidic bonds. The term "carboxymethylcellulose" means a polysaccharide obtained by reacting cellulose with haloacetic acid or its salts, in which the carboxymethyl group (-CH2- COOH) in acidic or salt form is combined with hydroxyl groups of glucose monomers.

The term "baker's yeast beta-glucan" means beta-glucan extracted from baker's yeast (Saccharomyces cerevisiae).

The term "oat beta-glucan" means a beta-l,3-l,4-D-glucan polysaccharide extracted from oats.

The term "barley beta-glucan" means a beta-l,3-l,4-D-glucan polysaccharide extracted from barley.

The term "reishi beta-glucan" means a polysaccharide of beta- 1,3-1, 6-D-glucan extracted from reishi mushrooms.

The term "iodic acid" means hydrogen tetraoxoiodate HI04 or iodic acid dihydrate - hydrogen hexaoxoiodate H5IO6, molecular weight 227.941 g/mol.

The term "iodic acid salts" means a compound consisting of an iodic acid anion (periodate) and an alkali metal cation, preferably sodium or potassium.

The term "treatment" means the impact of chemical reagents, carrying out a chemical reaction under certain conditions (temperature, pH of the solution, the concentration of reagents, etc.).

The term "isolation" means the process of precipitation in a solvent, decantation, ultrafiltration (dialysis), centrifugation, filtration, neutralization, dissolution, drying at ambient or elevated temperature, drying by lyophilization, and combination thereof.

The term "purification" means desalting, rinsing with a solvent, ultrafiltration (dialysis), and combinations thereof.

The term "haloalkyl carboxylic acid" means an aliphatic carboxylic acid in which one or more hydrogen atoms in the radical are replaced by halogen atoms of the general formula R2R3(CH) _n COORi; where

Ri is H;

R2 is H, linear or branched C1-C10 alkyl, preferably H; n is from 0 to 10, preferably n is from 1 to 2;

R3 is halogen F, Cl, Br, I, preferably Cl, Br.

Preferably, the haloalkyl carboxylic acid is monochloroacetic acid, monobromoacetic acid, 2-chloropropionic acid. The term "haloalkyl carboxylic acid salt" means a compound of the general formula R ₂ R ₃ (CH) _n COORi;

Ri is Li, Na, K, preferably Na;

R ₂ is H, linear or branched Ci-Cio alkyl, preferably H; n is from 0 to 10, preferably n is from 1 to 2;

R ₃ is halogen F, Cl, Br, I, preferably Cl, Br.

Preferably, the haloalkyl carboxylic acid is the sodium salt of monochloroacetic acid, monobromoacetic acid, 2-chloropropionic acid.

The term "pharmaceutically acceptable additive" means a substance that is approved for use in the pharmaceuticals to create finished dosage forms and is not an active substance but can affect both the biological effectiveness of the active substance and the physical properties of the finished dosage form, e.g., sodium carbonate, sodium bicarbonate, sodium ascorbate, lactose, calcium stearate, starch.

The term "other antimicrobial agent" means a substance used to combat viruses, bacteria, lower fungi, and protozoa or to suppress their activity, for example, oseltamivir, arbidol.

The term "semiproduct" means a semiproduct that is used for further synthesis.

The term "pre-purification" means the purification of the starting materials to remove impurities before processing.

The term "fractionation" means the separation of the starting materials into fractions with a specific molecular weight.

The term "aqueous-organic medium" means a solution with a solute concentration from 0.00001% to 99.999% m/m, in which a homogeneous mixture of water and an organic solvent in a mass ratio from 100:0.1 to 0.1:100 acts as a solvent, in particular, mixtures of water with ethanol, water with acetone, water with isopropanol, water with dioxane;

The term "inorganic base" means any inorganic compound capable of accepting positively charged ions, in particular sodium carbonate, potassium hydroxide, sodium acetate, sodium hydroxide, sodium bicarbonate, cesium carbonate, potassium carbonate, lithium hydroxide; The term "organic base" means any organic compound capable of accepting positively charged ions, in particular triethylamine, 4-methyl morpholine, N-ethyl diisopropylamine, potassium tert-butylate, sodium ethylate.

The term "degree of substitution" means the number of functional groups introduced into the molecule, preferably carboxymethyl.

Methods of study

High-performance liquid chromatography with UV spectrophotometric and mass spectrometric detection (HPLC-UV-MS )

The method was used for the assay of oseltamivir in samples. The analysis was carried out on an Agilent 1260 liquid chromatograph with sequential detection on diode array and mass selective detectors. The separation was carried out on Purospher STAR RP-18e chromatographic column, 125 x 4.6 mm in size with a particle size of 3 pm, equipped with a 12.5 x 4.6 mm protective guard column with the same sorbent manufactured by Agilent, using a composition, containing 50% V/V of 100 mM ammonium acetate pH 6.9 and 50% V/V of acetonitrile as an eluent in isocratic elution mode.

The column was maintained at a constant temperature of 40 °C and a flow rate of 0.5 mL/min. In the used single quadrupole mass spectrometric detector, ionization is carried out by electro spraying. Mass spectrometric detection was carried out in the mode of registration of positive ions, the pressure on the nebulizer was 35 psi, the voltage on the fragmentor was 100 V, the gas flow of the desiccant was 10 L/min, the voltage on the capillary was 4000 V, scan mode by mass range - 300-400 Da, and selected ion mode (SIM) - 313.

Size-exclusion chromatography (SEC)

Size-exclusion chromatography was used to determine the molecular weight and molecular weight characteristics of the resulting 1,4 and initial beta-glucans and raw materials. The analysis was carried out on a Waters liquid chromatograph with sequential refractometric and diode array detectors. The separation was carried out on series-connected Ultrahydrogel 1000 and Ultrahydrogel 120 columns 300 x 7.8 mm filled with a hydroxylated polymethacrylate-based gel with pore sizes of 1000 and 120 A respectively (Waters), using 0.05 M phosphate buffer solution as eluent (pH = 7.0) at isocratic elution mode. Dextran standards with weight average molecular weights (Mw) 9900, 16230, 41100, 60300, 129000, 214800, 456800 were used to calibrate the column.

The calibration curves were fitted with a 3rd order polynomial. The calculation of the molecular weight characteristics of the polymer was carried out using universal calibration and the Breeze 2 software.

Iodometric titration

The iodometric titration method was used to determine the number of carbonyl groups in the samples.

This method is based on the oxidation of carbonyl groups with iodine in an alkaline medium. Alkali was added to convert the resulting acids into salts due to the reversibility of this reaction. In this case, the alkali also reacts with iodine to form hypoiodite, which oxidizes the carbonyl group to carboxyl. After the oxidation of the carbonyl, acid is added to release iodine from the remaining sodium hypoiodite. The liberated iodine is titrated with sodium thiosulfate. The difference in the amount of thiosulfate that used for the titration of the added iodine and the excess left after the reaction determines the amount of iodine that used for the oxidation of carbonyl groups.

Potentiometric acid-base titration

This method is used to determine the degree of substitution and determine the content of carbonates and hydrocarbons. It is based on the combustion of the salt form of 1,4-beta-glucan to form sodium carbonate, followed by determination of its amount by acidimetric titration with hydrochloric acid.

Determination of the cytotoxic effect of substances in cell culture

The studies were carried out on the MDCK ECACC cell line (Sigma, Cat. No. 85011435) at 67-70 passages. The line was cultured in Eagle's MEM medium (PanEco) containing 10% of fetal calf serum (HyClone), 300 pg/mL of L-glutamine, and 0.1 mg/mL of normocin.

MDCK ECACC cells were added to 96-well plates in Eagle's MEM medium (PanEco) containing 10% of fetal calf serum (HyClone), 300 pg/mL of L-glutamine, and 0.1 mg/mL of normocin at 18000 cells per well, cultured for 24 h and rinsed with serum- free medium once before adding substances.

An essential medium of the following composition was used to dilute the tested substances: Eagle's MEM medium (PanEko) containing 2% of fetal calf serum (HyClone), 300 pg/mL of L-glutamine, 12 pg/mL of trypsin-chymotrypsin (chymopsin), and 0.1 mg/mL of normocin.

Each experimental point was tested in 4 parallel wells. The last dilution was placed in the first 2 out of 4 wells, the essential medium was added in the last two out of 4 wells (control of the cultivation medium).

The cells were incubated with the test substances for 48 h in a CO2 incubator at 37 °C, after which the culture medium was removed and 100 pL of the essential medium and 20 pL of a solution of vital dye MTS (Promega, Cat. No. G3581) were added to each well. After incubation for 3 h at 37 °C, the absorbance was determined at a wavelength of 492 nm and a reference wavelength of 620 nm using a BIO-RAD plate spectrophotometer. The concentration of the test substance, which reduces the absorbance by 50% compared to the cell control, was taken as the 50% cytotoxic concentration (CC50). The range from 1 to 30 mg/mL is considered low toxic.

Study of the effect of drugs on the infectious titer of the influenza virus in MDCK cell culture

The studies used the influenza A/Puerto Rico/8/34 (H1N1) virus adapted to the MDCK line. The infectious and hemagglutinating activity of the virus was determined according to the methods recommended by the WHO.

Studies of the virus -specific action of the tested substances against strain A/Puerto Rico/8/34 were carried out on MDCK ECACC cells, using Eagle's MEM medium (PanEco) containing 2% of fetal calf serum (HyClone), 300 pg/mL of L- glutamine, 12 pg/mL of trypsin-chymotrypsin (chymopsin) and 0.1 mg/mL normocin as an essential medium. The culture of MDCK ECACC cells was prepared in the same way as in experiments to determine the cytotoxic effect of the substances examined. Before infection with the virus, MDCK cells were rinsed once with serum-free Eagle's MEM medium, previously prepared dilutions of the substances were added in 100 pL of the essential medium, and incubated for 1 h at 37 °C. Then added 10 pL of each previously prepared 10-fold dilutions of the virus. Virus and cell controls were cultured in the same medium. The results were recorded 48 h later in terms of cytopathic effect and haemagglutination reaction (HAR). In the HAR, a 0.75% suspension of human erythrocytes (group O) in saline was used.

The calculation of the values of 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50) for each of the studied compounds was performed using the Excel software package and GraphPad Prism 6.01. The 4-parameter equation of the logistic curve (menu items "Nonlinear regression" - "Sigmoidal dose-response (variable slope)") was used as a working model for the analysis of CC50. For the analysis of IC50, a 4-parameter equation of the logistic curve was used (menu items "Nonlinear regression" - "log (inhibitor) vs. response (variable slope)"). The viral inhibitory effect of the test substances (AlgTCTDso) was assessed by the decrease in the infectious viral dose in the experiment compared with the control, calculated by the Reed and Muench method. Based on all the data obtained, the chemotherapeutic index (SI) was calculated using the equation:

SI = CC50/IC50.

Activation of HELF culture by substances

Human embryo lung fibroblasts cells (HELF) were used at a concentration of 2 x 10 ⁵ in 1 mL of growth medium containing FBS. The cell suspension was added into 96-well flat-bottomed plates (Costar/Coming, USA), 100 pL per well. The plates were incubated at 37 °C under an atmosphere with 5% CO2. The incubation lasted 24 h.

The titration of the substance solutions was carried out by double dilutions in the wells with HELF. As a result, the concentration of the drugs ranged from 2000 to 15 pg/mL.

Each dose of substances was added to 2 wells of the HELF cell plate to obtain a sufficient supernatant volume. To control the influence of the culture medium, in the plate 1 vertical row was allocated (a total of 8 wells).

The incubation was carried out at a temperature of 37 °C under an atmosphere of 5% CO2. After 4 h, the supernatants were selected for further studies.

Murine encephalomyocarditis virus (EMC) in a cytopathogenic dose (100 CPE of the virus) in a serum-free Eagle's MEM medium was added to the wells with the HELF monolayer. The account of the cytopathic effect of the virus on the fibroblast monolayer was carried out 24 hours later under an inverted microscope, according to the procedure for the interferon status assessment (F.I. Ershov, 1984).

Determination of the antiviral efficacy of a substance against the influenza A/California/7/09 pdm (H1N1) virus in a model of influenza pneumonia in mice with an emergency prophylactic regimen of administration Pre- weighed mice of the B ALB/c and ICR (CD-I) lines from the "Nursery for laboratory animals" of the IBCh RAS (Moscow region, Pushchino) (100% of males, average weight 12-16 g) were infected intranasally under light anesthesia by influenza A/California/7/09 pdm (H1N1) vims at a dose 10 MLDso/50 pL (in the case of BALB/c) or 1 MLDso/50 mΐ (in the case of ICR). In a preliminary experiment, the determination of doses was carried out by titration of the allantoic vims on the same mice, which were then used in the main experiment.

The study of the effect of the substances to be examined using an emergency prophylactic regimen of administration was carried out by oral administration (intragastrically, using a gavage needles) of the substance to infected animals 1 h, 25 h, 49 h, 73 h, and 97 h after infection in a volume of 100 pL in five doses (0.1 mg/kg/mouse, 1 mg/kg/mouse, 12 mg/kg/mouse, 100 mg/kg/mouse, and 400 mg/kg/mouse) in the case of using BALB/c mice and in four doses (0.1 mg/kg/mouse, 1 mg/kg/mouse, 12 mg/kg/mouse, 100 mg/kg/mouse) in the case of using ICR mice.

The comparison drug Oseltamivir phosphate was administered to infected animals intragastrically using a gavage needles once a day after 1 h, 25 h, 49 h, 73 h, and 97 h after infection at a dose of 25 mg/kg/mouse. The mice of the control group were administrated a placebo (saline) under the same conditions. The animals were observed for 14 days after infection, taking into account the death of mice from influenza pneumonia in the groups of treated animals and in the control group. The specificity of the death of animals from influenza pneumonia was confirmed by the registration of pathological changes in the lungs of the dead animals of the experimental and control groups.

Each group consisted of 15 animals. The treated and control animals were monitored daily. The chemotherapeutic activity of the compounds in the model of influenza pneumonia in mice was assessed according to three criteria: an indicator of protection against a fatal viral infection, an increase in the average life expectancy, and an increase in the weight of animals as compared to the control group (intact mice).

The activity of the substances was assessed by comparing the lethality in treated and control animals. The decrease in the lethality of the treated animals relative to the control was expressed as a percentage. All results were statistically processed using the EXCEL program and are presented as mean values and mean deviations.

Assessment of the ability of substances to activate immune receptors

The study of the ability of samples to activate the human dectin-la receptor was carried out by the gene-reporter method using specialized transgenic cell lines manufactured by Invivogen: HEK-Blue™ hDectin-la Cells, a reporter cell line based on HEK 293 cells expressing the human dectin-la gene, and HEK-Blue™ Nulll Cells, a control cell line that is parental to a line expressing dectin-la. Receptor activation was assessed by the color reaction with secreted alkaline phosphatase (SEAP). We took into account while assessing the specificity of the reaction, that the parental cell line expresses low levels of TLR3, TLR5, and NODI receptors.

Similarly, using the gene reporter lines THP-1 CD 14 and HEK293-hTLR4 CD14/MD2, the ability of the samples to activate the CD 14 receptor was evaluated.

The studies were carried out according to the instructions of the manufacturer of cell lines in the range of non-toxic concentrations of b-glucans, which was controlled in a parallel experiment using the vital dye MTS.

The stimulation of blood lymphocytes with substances

From peripheral blood taken into BD VACUTAINER tubes with heparin, lymphocytes were isolated in a HISTOPAQUE-1077 density gradient by centrifugation at 400 g for 15 min.

Isolated and rinsed lymphocytes in a complete culture medium (working medium RPMI-1640 with 10% of fetal calf serum) were introduced into the wells of a 96-well round-bottomed plate at a concentration of 2.5-3 x 10 ⁶ cells per 1 mL (in a volume of 100 pL). The number of lymphocytes in the well is 2.5-3 x 10 ⁵ .

The working concentration of solutions of the substances to be examined for stimulation was 2 mg/mL (the concentration in the well was 1 mg/mL, i.e., 100 pL of the indicated solution of the substance was added).

The incubation was carried out at a temperature of 37 °C under an atmosphere of 5% CO2. After 20 h, the supernatants were collected for further studies and stored in portions at -40 °C, and lymphocytes collected from the same wells were examined for the content of CD69+ T-cells among them. The toxicity of the substances at this concentration did not appear since lymphocytes during cytofluorometry looked standard and were in a specific gate for them.

Co- stimulation of blood cells with substances and PHA

Whole blood samples from donors were treated as follows: 20 pL of blood, 20 pL of PHA (12.5 pg/mL), 100 pL of a solution of the test preparations (2 mg/mL) were added to the wells of a 96-well round-bottom culture plate with 60 pL of the working culture medium RPMI-1640. The total volume of the contents of each well is 200 pL, the final concentration of the experimental substances is 1 mg/mL.

After 24 hours of cultivation at 37 °C with 5% CO2, culture supernatants were collected, which were poured in portions and stored at -40 °C until ELISA was performed.

Determination of the activation of T-lymphocytes that occurred during stimulation by an increase in the percentage of cells expressing the CD69 marker

A supernatant in a volume of 100 pL was collected from lymphocytes that passed the stimulation stage, and the lymphocytes were resuspended in the remaining volume of the culture medium (100 pL). Then, monoclonal antibodies, labeled with fluorochromes CD3 (FITC), CD69 (PE), CD8 (PC5) were added to them, all antibodies were manufactured by Beckman Coulter (USA). Sample preparation was carried out on an automatic station TQprep (Beckman Coulter) using a no-wash procedure. The measurements were carried out on a Cytomics FC500 flow cytometer (Beckman Coulter, USA) in a measurement protocol for a 3-color marker. Thus, we found out which T-cells were activated by substances and in what quantity (%).

The per cent increase of CD69+ cells by 2% was significant, compared to their content in a culture not stimulated with substances (i.e., with spontaneous expression).

Reagents

Blanose 7 MF (Na-carboxymethylcellulose) manufactured by Ashland. Fight yellow powder. Degree of carboxymethylation: 0.80. Assay: 99.5% (min.).

Reishi, baker's yeast, oat, barley beta-glucans manufactured by Purest-R.

Oseltamivir phosphate manufactured by Sigma Aldrich. White powder. Assay:

99.2%.

Monochloroacetic acid manufactured by IRea 2000 EEC. Grade: pure. Manufactured in accordance with GOST 5836-85. Iodic acid manufactured by Panreac. White crystalline powder. Assay: 98%.

Claim 1.

Sample 1.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 7.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry.

Sample 2.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 7.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in a sodium carbonate solution, adjust the pH to 3-4, precipitate the product in acetone, filter the precipitate, rinse with aqueous acetone, pure acetone, and dry.

Sample 3.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 7.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in a sodium carbonate solution, adjust the pH to 3-4, precipitate the product in acetone, filter the precipitate, rinse with aqueous acetone, pure acetone, and dry. Dissolve the resulting product in a solution of sodium bicarbonate, adjust the pH to 6-7, stir for 30 minutes. Dry the resulting product.

Sample 4.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 7.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in a sodium carbonate solution, adjust the pH to 3-4, precipitate the product in acetone, filter the precipitate, rinse with aqueous acetone, pure acetone, and dry. Dissolve the resulting product in sodium carbonate solution, adjust the pH to 9-10, stir for 30 minutes. Dry the resulting product.

Sample 5.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 22.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry.

Sample 6.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 22.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in 5% sodium carbonate, fractionate through a 10 kDa membrane, dry the resulting product.

Sample 7.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 22.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in a sodium carbonate solution, adjust the pH to 3-4, precipitate the product in acetone, filter the precipitate, rinse with aqueous acetone, pure acetone, and dry.

Sample 8.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 22.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in a sodium carbonate solution, adjust the pH to 3-4, precipitate the product in acetone, filter, rinse with aqueous acetone, pure acetone, and dry. Dissolve the resulting product in a solution of sodium bicarbonate, adjust the pH to 6-7, stir for 30 minutes. Dry the resulting product.

Sample 9.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 22.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in a sodium carbonate solution, adjust the pH to 3-4, precipitate the product in acetone, filter the precipitate, rinse with aqueous acetone, pure acetone, and dry. Dissolve the resulting product in sodium carbonate solution, adjust the pH to 9-10, stir for 30 minutes. Dry the resulting product.

Sample 10.

Add 1800 mL of water and 75 g of CMC to a 5000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 15 g of iodic acid in 150 g of water to the solution, and stir for several hours at 40 °C and 20 hours at 25 °C. Then add sodium carbonate to the reaction mass, adjust the pH to 8-9, fractionate through a 10 kDa membrane, adjust the pH to 3-4. Dry the resulting product.

Sample 11.

Add 1800 mL of water and 75 g of CMC to a 5000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 30 g of iodic acid in 150 g of water and stir for several hours at 40 °C and 20 hours at 25 °C. Then dialyze against water through dialysis bags 12-14 kDa for 48 hours. Dry the resulting product.

Sample 12.

Add 1800 mL of water and 75 g of CMC to a 5000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 30 g of iodic acid in 150 g of water and stir for several hours at 40 °C and 20 hours at 25 °C. Then dialyze against water through dialysis bags 12-14 kDa for 48 hours. Dry the resulting product. Dissolve the resulting product in water, add a solution of sodium ascorbate in 0.1M sodium hydroxide, stir for 30 minutes. Dry the resulting product. Sample 13.

Add 1800 mL of water and 75 g of CMC to a 5000 mL beaker. Stir the reaction mixture for 3 hours, after which add a solution of 30 g of iodic acid in 150 g of water to the solution, and stir the mixture for several hours at 40 °C and 20 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in 5% sodium carbonate, fractionate through a 10 kDa membrane, dry the resulting product.

Sample 14. Add 62.5 mL of water and 5 g of barley beta-glucan to a 150 mL beaker. Stir the reaction mixture for 1 hour, then add a solution of 2.03 g of iodic acid in 6.9 ml of water to the suspension, and stir the mixture for 18 hours at 25 °C. Separate the precipitate by centrifugation and rinse first with water, then with pure acetone, and dry.

Sample 15. Add 50 mL of water and 5 g of oat beta-glucan to a 250 mL beaker. Stir the reaction mixture for 1 hour, then add a solution of 2.5 g of iodic acid in 10 ml of water to the suspension, adjust the pH to 3-4, and stir for 20 hours at 25 °C. Then dialyze the reaction mixture against water through 1 kDa dialysis bags for 120 hours, then lyophilize it. Sample 16.

Add 300 ml of water and 5 g of oat beta-glucan to a 600 mL beaker. Stir the reaction mixture for 1 hour, then add a solution of 7.6 g of iodic acid in 30 ml of water to the suspension, and stir the mixture for 18 hours at 25 °C. Separate the precipitate by centrifugation and rinse first with water, then with pure acetone, and dry. Sample 17.

Add 365 mL of 0.5M aqueous sodium hydroxide solution to a 500 mL beaker and dissolve 7.3 g of barley beta-glucan, after which fractionate it through a 10 kDa membrane, dry the resulting product. Dissolve the resulting product is in 62.5 ml of water, then add a solution of 1.42 g of iodic acid in 4.7 mL of water and stir the mixture for 20 hours at 25 ° C. Neutralize the excess of the oxidizing agent, precipitate the product into acetone, filter the precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Sample 18.

Add 70 ml of water and 5 g of Reishi mushroom b-glucan to a beaker. Stir the reaction mixture for 1 hour, then add a solution of 2.1 g of iodic acid in 10 mL of water and stir the mixture for 72 hours at 25 °C. Neutralize the excess oxidant, precipitate the product into acetone, filter the precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry.

The number of samples, which are shown in Table 1, was obtained by periodate oxidation of the original beta-glucans by the Malaprad reaction. Natural (baker's yeast, oat, barley, reishi) beta-glucans and synthetic 1,4-beta-glucan (carboxymethylcellulose) were used for work.

According to the literature, oxidation proceeds predominantly at the C2 and C3 with the rupture of the pyranose ring. In our case, the reaction was accompanied by a decrease in the molecular weight and an increase in the number of carbonyl groups in the product as compared to the initial glucan (Table 1). Table 1

Samples obtained by periodate oxidation of beta-glucans

All products of the treatment of beta-glucan with iodic acid under the specified process conditions (pH and temperature ranges, solvent composition) in the indicated range of carbonyl group content, had antiviral activity against influenza virus (see Table 2) and mouse encephalomyocarditis virus (see Table 3), which is absent in the original beta-glucans. In this case, the activity increased in proportion to the increase of carbonyl groups number.

Table 2.

Antiviral activity of samples against influenza A/Puerto Rico/8/34 (H1N1) virus in vitro

* Substances with pronounced antiviral activity have AlgTCIDsoavg. > 1.50.

Table 3.

Resistance of the HELF culture to the cytopathic effect of the murine encephalomyocarditis virus (EMC), arising after stimulation with the substances to be examined (% protection of the HELF monolayer) for 4 h protecting against the cytopathic effect of EMC on the monolayer of HELF cells (% of monolayer protection is 20-40%), however, substances 12 and 13 have higher capacity. Antiviral activity of sample 10 in vivo against influenza A/California/7/09 pdm (H1N1 ) virus in a model of influenza pneumonia in mice with an emergency prophylactic regimen of administration The study showed that sample 10 exhibits antiviral activity against influenza A/California/7/09 pdm (HINT) virus dose-dependently, in both white outbred ICR mice and linear BALB/c mice, at an infecting dose of 1 to 10 MLD50. At the same time, a dose of 0.1 mg/kg/mouse in the tested range has the highest activity, which reliably causes protection of influenza-infected animals and significantly increases their life expectancy compared to viral control, and slightly concedes to the control direct-acting drug (oseltamivir phosphate) (see Table 4).

Table 4.

*N/D - no data The ability of substance 13 to activate important immune receptors

The introduction of carbonyl groups into the structure of beta-glucan by treatment with iodic acid under the specified conditions for 1,4-beta-glucan (carboxymethylcellulose) resulted in the appearance of the ability to activate the CD 14 receptor, which was absent in the original beta-glucan. Studies showed that sample 13, in contrast to 1,3- and I,ό-b-glucans, does not activate the dectin-la receptor but acquires the ability to activate the CD14 receptor. Thus, sample 13 has unique immunological properties that distinguish it from other b-glucans. Activation of T-lymphocytes by samples 12 and 13, recorded by the change in the number of cells expressing CD69 among T-helpers and cytotoxic lymphocytes (CTL)

The CD69 molecule is a marker of early activation (in the first 4-24 hours of exposure) of T-lymphocytes and a convenient object for quick assessment of various substances in terms of their ability to activate lymphocytes. During the cultivation of lymphocytes, the percentage of CD3+CD69+ cells usually slightly increases even in the absence of stimulating additives, but it rarely exceeds 5-8% in healthy people. The nature of the influence of substances added to the culture to stimulate lymphocytes is assessed by D = (% CD3+CD69+ in a stimulated test) - (% CD3+CD69+ in a spontaneous test). In our study, the activating effect of the substances to be examined was assessed separately on T-helpers (CD3+CD4+CD69+) and CTL (CD3+CD8+CD69+). See the results of our research in Table 5.

Table 5. The influence of the substances to be examined on the level of activation of T- helpers and CTLs in culture in 12 people (individual data)

In this series of experiments, it turned out that substances 12 and 13 activate both T-helpers and CTLs in almost all people, while the CMC 7 MF substance - in 1 donor. Therefore, substances 12 and 13 have a more pronounced activating effect on lymphocytes to increase the percentage of CD69+ T-lymphocytes than CMC 7 MF. Substances 12, 13, and CMC 7 MF highly stimulate the production of IL-Ib by blood cells under conditions of co-stimulation with PHA in blood cells of people with a "reduced ratio of CD4/CD8 cells," while 12 and 13 - significantly higher than CMC 7 MF (determined by ELISA using a test system manufactured by Vector-Best). In healthy donors, substances 12 and 13 also highly produce IL-Ib, in contrast to CMC 7 MF, which is incapable of producing this cytokine.

Thus, the introduction of carbonyl groups into the structure of beta-glucan by treatment with iodic acid under the specified conditions for 1,4-beta-glucan (carboxymethylcellulose) led to an increase in the ability to induce the production of cytokines and the activation of immunocompetent cells as compared to the original beta-glucan.

Claim 2.

Sample 19.

Add 100 mL of isopropyl alcohol and 5 g of oat beta-glucan to a 250 mL beaker. Stir the reaction mixture overnight, then add 25 g of 30% m/m aqueous sodium hydroxide solution, and stir for 1 hour. Then add a solution of 3.5 g of monochloroacetic acid in 25 mL of isopropyl alcohol to the resulting suspension and stir for 3 hours at 72 °C. Decant the solution, dissolve the precipitate in water, adjust the pH to 2-3, precipitate the product into acetone, filter the precipitate, and rinse with acetone. After that, dissolve the precipitate in water, adjust the pH to 8, and precipitate the product into acetone, rinse with pure acetone, and dry.

Sample 20.

Add 100 mL of isopropyl alcohol and 5 g of baker's yeast beta-glucan to a 250 mL beaker. Stir the reaction mixture overnight, then add 25 g of 30% m/m aqueous sodium hydroxide solution, and stir for 1 hour. Then add a solution of 3.5 g of monochloroacetic acid in 25 ml of isopropyl alcohol to the resulting suspension and stir for 5 hours at 72 °C. Decant the solution, dissolve the precipitate in water, adjust the pH to 2-3, precipitate the product into acetone, filter the precipitate, and rinse with acetone. After that, dissolve the precipitate in water, adjust the pH to 8, and precipitate the product into acetone, rinse with pure acetone, and dry.

Sample 21. Add 200 mL of isopropyl alcohol and 15 g of baker's yeast beta-glucan to a 600 mL beaker. Stir the reaction mixture for 1 hour, then add 75 g of 10% m/m aqueous sodium hydroxide solution, and stir for another 1 hour. Then add a solution of 10.70 g of monochloroacetic acid in 55 mL of isopropyl alcohol to the resulting suspension and stir for 5 hours at 72 °C. Decant the solution, dissolved the precipitate in water, adjust the pH to 2-3, precipitate the product into acetone, filter the precipitate, and rinse with acetone. After that, dissolve the precipitate in water, adjust the pH to 8, and precipitate the product into acetone, rinse with pure acetone, and dry.

The number of samples was obtained by alkylation of the original beta-glucans with monochloroacetic acid (Table 6). This type of structural modification results in an increase in the content of carboxyl groups in beta-glucan, as it is indicated by the degree of substitution.

Table 6

Samples obtained by alkylation of beta-glucans

The obtained products of the treatment of beta-glucan with haloalkyl carboxylic acid had antiviral activity against influenza virus (see Table 7) under the specified process conditions (pH and temperature ranges, solvent composition) in the indicated range of substitution degree, which was absent in the original beta-glucans. However, unlike the oxidized samples, there was no clear link between the degree of substitution value and biological effectiveness. Table 7.

Antiviral activity of samples against influenza A/Puerto Rico/8/34 (H1N1) virus in vitro

Claim 3.

Sample 22.

Add 173 mL of isopropyl alcohol and 10.5 g of oat beta-glucan to a 600 mL beaker. Stir the reaction mixture overnight, then add 65 g of a 10% m/m aqueous solution of sodium hydroxide, and stir for 1 hour. Then add a solution of 9.13 g of monochloroacetic acid in 47 mL of isopropyl alcohol to the resulting suspension and stir for 5 hours at 72 °C. Decant the solution, dissolve the precipitate in water, adjust the pH to 2-3, precipitate the product into acetone, filter the precipitate, and rinse with acetone. After that, dissolve the precipitate in water, adjust the pH to 8, and precipitate the product into acetone, rinse with pure acetone, and dry. Dissolve the resulting product in 162.5 ml of water, then add a solution of 1.77 g of iodic acid in 6.32 mL of water, and stir the mixture for 18 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product into acetone. Filter the formed precipitate, rinse with acetone. Dissolve the resulting product in water, adjust the pH to 8, and precipitate into acetone, rinse with an aqueous solution of acetone and pure acetone, and dry.

Sample 23.

Add 160 mL of isopropyl alcohol and 12.0 g of baker's yeast beta-glucan to a 600 mL beaker. Stir the reaction mixture overnight, then add 60 g of a 10% m/m aqueous solution of sodium hydroxide, and stir for 1 hour. Then add a solution of 8.56 g of monochloroacetic acid in 44 mL of isopropyl alcohol to the resulting suspension and stir for 5 hours at 72 °C. Decant the solution, dissolve the precipitate in water, adjust the pH to 2-3, precipitate the product into acetone, filter the precipitate, and rinse with acetone. After that, dissolve the precipitate in water, adjust the pH to 8, and precipitate the product into acetone, rinse with pure acetone, and dry. Dissolve the resulting product in 175 ml of water, then add a solution of 1.45 g of iodic acid in 4.8 mL of water, and stir the mixture for 18 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product into acetone. Filter the formed precipitate, rinse with acetone. Dissolve the resulting product in water, adjust the pH to 8, and precipitate into acetone, rinse with an aqueous solution of acetone and pure acetone, and dry.

Sample 24. In a 300 mL beaker, add 118 mL of isopropyl alcohol and 8.85 g of barley beta- glucan. Stir the reaction mixture overnight, then add 44.25 g of a 10% m/m aqueous solution of sodium hydroxide, and stir for 1 hour. Then add a solution of 3.88 g of monochloroacetic acid in 25 ml of isopropyl alcohol to the resulting suspension and stir for 5 hours at 72 °C. Decant the solution, dissolve the precipitate in water, adjust the pH to 2-3, precipitate the product into acetone, filter the precipitate, and rinse with acetone. After that, dissolve the precipitate in water, adjust the pH to 8, and precipitate the product into acetone, rinse with pure acetone, and dry. Dissolve the resulting product in 131 ml of water, then add a solution of 1.77 g of iodic acid in 6.2 mL of water, and stir the mixture for 18 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product into acetone. Filter the formed precipitate, rinse with acetone. Dissolve the resulting product in water, adjust the pH to 8, and precipitate into acetone, rinse with an aqueous solution of acetone and pure acetone, and dry.

With the combination of alkylation with monochloroacetic acid followed by Malaprade oxidation of carboxymethylated beta-glucans, samples were obtained that differed from the original beta-glucans in the content of carbonyl groups and the degree of substitution (Table 8).

Table 8

Samples obtained by oxidation and alkylation of beta-glucans

The obtained products of the treatment of beta-glucan with haloalkyl carboxylic acid followed by treatment of the semiproduct with iodic acid had antiviral activity against influenza virus (see Table 9) under the specified process conditions (pH and temperature ranges, solvent composition) in the specified range of carbonyl group content, which was absent in original beta-glucans.

Table 9.

Antiviral activity of samples against influenza A/Puerto Rico/8/34 (H1N1) virus in vitro

Compositions containing pharmaceutically acceptable additives. The above methods for modification of the beta-glucans structure allow to add pharmaceutically acceptable additives to the resulting products. This possibility is illustrated by the examples below. Table 10.

Compositions with antimicrobial substances.

Sample 25.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 7.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in 5% sodium carbonate, fractionate through a 10 kDa membrane, adjust the pH to 3-4, dry the product obtained. Dissolve the resulting product in water, add 0.02% m/m oseltamivir phosphate, stir for 30 minutes. Dry the resulting product.

Sample 26.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 7.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in 5% sodium carbonate, fractionate through a 10 kDa membrane, adjust the pH to 3-4, dry the product obtained. Dissolve the resulting product in water, add 0.2% m/m of oseltamivir phosphate, stir for 30 minutes. Dry the resulting product.

Sample 27.

Add 960 mL of water and 80 g of CMC to a 2000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 7.5 g of iodic acid in 50 g of water to the solution, and stir the mixture for 24 hours at 25 °C. Neutralize the excess of the oxidizing agent, precipitate the product in acetone. Filter the formed precipitate, rinse with an aqueous solution of acetone and pure acetone, and dry. Dissolve the resulting product in 5% sodium carbonate, fractionate through a 10 kDa membrane, adjust the pH to 3-4, dry the product obtained. Dissolve the resulting product in water, add 2% m/m oseltamivir phosphate, stir for 30 minutes. Dry the resulting product.

Sample 28.

Add 1800 mL of water and 75 g of CMC to a 5000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 15 g of iodic acid in 150 g of water to the solution, and stir for several hours at 40 °C and 20 hours at 25 °C. Then add sodium carbonate to the reaction mass, adjust the pH to 8-9, fractionate through a 10 kDa membrane, adjust the pH to 3-4. Dry the resulting product. Dissolve the resulting product in water, add 0.02% m/m oseltamivir phosphate, stir for 30 minutes. Dry the resulting product.

Sample 29. Add 1800 mL of water and 75 g of CMC to a 5000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 15 g of iodic acid in 150 g of water to the solution, and stir for several hours at 40 °C and 20 hours at 25 °C. Then add sodium carbonate to the reaction mass, adjust the pH to 8-9, fractionate through a 10 kDa membrane, adjust the pH to 3-4. Dry the resulting product. Dissolve the resulting product in water, add 0.2% m/m of oseltamivir phosphate, stir for 30 minutes. Dry the resulting product.

Sample 30.

Add 1800 mL of water and 75 g of CMC to a 5000 mL beaker. Stir the reaction mixture for 3 hours, then add a solution of 15 g of iodic acid in 150 g of water to the solution, and stir for several hours at 40 °C and 20 hours at 25 °C. Then add sodium carbonate to the reaction mass, adjust the pH to 8-9, fractionate through a 10 kDa membrane, adjust the pH to 3-4. Dry the resulting product. Dissolve the resulting product in water, add 2% m/m oseltamivir phosphate, stir for 30 minutes. Dry the resulting product.

We obtained compositions with antiviral activity when added the substance of oseltamivir phosphate to the modified beta-glucans (Tables 11, 12).

Table 11

Compositions with oseltamivir Table 12.

Antiviral activity of samples against influenza A/Puerto Rico/8/34 (H1N1) virus in vitro

Obtaining beta-glucans combining antiviral and immunoactive properties

Table 13.

Emergence of antiviral activity in immunologically active samples of beta- glucans

*1imit of substance solubility.

The experimental results showed that the tested variants of b-glucans, excluding CMC, had the ability to specifically activate the dectin-la receptor, not stimulating the parental cell line. At the same time, differences were observed in the ability of b- glucans to activate dectin-la, depending on their structure (see Table 13).