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WO2021171225A1 - Agents antimicrobiens à base de bêta-glucanes, leurs procédés de production et d'utilisation - Google Patents

Agents antimicrobiens à base de bêta-glucanes, leurs procédés de production et d'utilisation Download PDF

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WO2021171225A1
WO2021171225A1 PCT/IB2021/051588 IB2021051588W WO2021171225A1 WO 2021171225 A1 WO2021171225 A1 WO 2021171225A1 IB 2021051588 W IB2021051588 W IB 2021051588W WO 2021171225 A1 WO2021171225 A1 WO 2021171225A1
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beta
glucan
acetone
stir
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Vladimir Georglevich Nesterenko
Anatoly Petrovich SUSLOV
Sergey Alexandrovich TSYRULNIKOV
Irina Vladislavovna KISELEVA
Maria Veniaminovna KONOPLEVA
Maria Sergeevna BLYAKHER
Vitaly Vladimirovich KUZNETSOV
Elena Yurievna SHPORTA
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Nearmedic International Ltd
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
    • C08L1/08—Cellulose derivatives
    • C08L1/26—Cellulose ethers
    • C08L1/28—Alkyl ethers
    • C08L1/286—Alkyl ethers substituted with acid radicals, e.g. carboxymethyl cellulose [CMC]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • A61K36/064—Saccharomycetales, e.g. baker's yeast
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/899—Poaceae or Gramineae (Grass family), e.g. bamboo, corn or sugar cane
    • A61K36/8998—Hordeum (barley)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B11/00—Preparation of cellulose ethers
    • C08B11/02—Alkyl or cycloalkyl ethers
    • C08B11/04—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals
    • C08B11/10—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals substituted with acid radicals
    • C08B11/12—Alkyl or cycloalkyl ethers with substituted hydrocarbon radicals substituted with acid radicals substituted with carboxylic radicals, e.g. carboxymethylcellulose [CMC]
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00

Definitions

  • This invention relates to organic and pharmaceutical chemistry and concerns methods for producing beta-glucans and use thereof in pharmaceuticals.
  • 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 modification of the structure of beta-glucans by oxidation is closer to the proposed invention.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • beta-glucan means a polysaccharide of beta-D-glucose monomers linked by glycosidic bonds.
  • 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.
  • baker's yeast beta-glucan means beta-glucan extracted from baker's yeast (Saccharomyces cerevisiae).
  • oat beta-glucan means a beta-l,3-l,4-D-glucan polysaccharide extracted from oats.
  • barley beta-glucan means a beta-l,3-l,4-D-glucan polysaccharide extracted from barley.
  • reishi beta-glucan means a polysaccharide of beta- 1,3-1, 6-D-glucan extracted from reishi mushrooms.
  • iodic acid means hydrogen tetraoxoiodate HI04 or iodic acid dihydrate - hydrogen hexaoxoiodate H5IO6, molecular weight 227.941 g/mol.
  • iodic acid salts means a compound consisting of an iodic acid anion (periodate) and an alkali metal cation, preferably sodium or potassium.
  • 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.).
  • 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.
  • purification means desalting, rinsing with a solvent, ultrafiltration (dialysis), and combinations thereof.
  • 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
  • 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.
  • haloalkyl carboxylic acid is monochloroacetic acid, monobromoacetic acid, 2-chloropropionic acid.
  • 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.
  • the haloalkyl carboxylic acid is the sodium salt of monochloroacetic acid, monobromoacetic acid, 2-chloropropionic acid.
  • 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.
  • antimicrobial agent means a substance used to combat viruses, bacteria, lower fungi, and protozoa or to suppress their activity, for example, oseltamivir, arbidol.
  • semiproduct means a semiproduct that is used for further synthesis.
  • pre-purification means the purification of the starting materials to remove impurities before processing.
  • fractionation means the separation of the starting materials into fractions with a specific molecular weight.
  • 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;
  • 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;
  • 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.
  • degree of substitution means the number of functional groups introduced into the molecule, preferably carboxymethyl.
  • 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.
  • mass spectrometric detection was 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.
  • SIM selected ion mode
  • Mw weight average molecular weights
  • 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.
  • 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.
  • the alkali also reacts with iodine to form hypoiodite, which oxidizes the carbonyl group to carboxyl.
  • 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.
  • 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.
  • 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.
  • Eagle's MEM medium PanEco
  • HyClone fetal calf serum
  • normocin normocin
  • 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.
  • Eagle's MEM medium PanEko
  • HyClone fetal calf serum
  • chymopsin trypsin-chymotrypsin
  • normocin normocin
  • 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.
  • CC50 50% cytotoxic concentration
  • 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.
  • MDCK ECACC cells 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.
  • 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.
  • group O human erythrocytes
  • the viral inhibitory effect of the test substances 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.
  • HELF Human embryo lung fibroblasts cells
  • the titration of the substance solutions was carried out by double dilutions in the wells with HELF.
  • 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.
  • 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).
  • 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 m ⁇ (in the case of ICR).
  • 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.
  • mice 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.
  • 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-BlueTM hDectin-la Cells, a reporter cell line based on HEK 293 cells expressing the human dectin-la gene, and HEK-BlueTM 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.
  • SEAP secreted alkaline phosphatase
  • 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.
  • 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 (%).
  • CD69+ cells 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).
  • Blanose 7 MF Na-carboxymethylcellulose manufactured by Ashland. Fight yellow powder. Degree of carboxymethylation: 0.80. Assay: 99.5% (min.).
  • 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.
  • sample 10 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
  • HINT antiviral activity against influenza A/California/7/09 pdm
  • 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).
  • 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.
  • 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 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.
  • 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).
  • substances 12 and 13 also highly produce IL-Ib, in contrast to CMC 7 MF, which is incapable of producing this cytokine.
  • 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.
  • 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.
  • 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.
  • compositions containing pharmaceutically acceptable additives comprising 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 are provided.
  • 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.
  • compositions with antiviral activity when added the substance of oseltamivir phosphate to the modified beta-glucans (Tables 11, 12).
  • compositions with oseltamivir Table 12 Compositions with oseltamivir Table 12.

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Abstract

La présente invention a trait à la chimie organique et pharmaceutique et concerne la production de bêta-glucanes et leur utilisation dans des produits pharmaceutiques.
PCT/IB2021/051588 2020-02-27 2021-02-26 Agents antimicrobiens à base de bêta-glucanes, leurs procédés de production et d'utilisation Ceased WO2021171225A1 (fr)

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