Pharmaceutical compositions containing glutathione disulfide and glutathione disulfide s-oxide

Abstract

A pharmaceutical composition comprising glutathione disulfide or pharmaceutically acceptable organic or inorganic salts thereof and glutathione disulfide S-oxide of the structure (I) or pharmaceutically acceptable organic or inorganic salts thereof for eliminating dose-related toxicity of pharmacologically active compounds and enhancing therapeutic activity of pharmacologically active compounds in the treatment of infectious and non-infectious diseases. Typically, the composition contains 0.01-10% of glutathione disulfide S-oxide by weight of the total composition, in addition to a metal (Me) in the form of a complexing compound containing Me-S-glutathione bond, said metal being selected from platinum group elements, typically platinum. The amount of d-metal complexing compound administered to a patient can be 10 ‑3 to 10 ‑15 mol / kg of body weight. The composition can be used in combination with pharmacologically active compounds, which are anticoagulants, factor Xa inhibitors, antibacterial or antiviral agents, to increase their therapeutic activity and eliminate dose-related toxicity.

Description

Technical Field

[0001] This invention relates to the pharmaceutical industry and medicine, specifically to the field of drug preparation, and can be used in pharmacology, medicine, and veterinary medicine. Background Technology

[0002] Enhancing the therapeutic effects of pharmacological molecules by optimizing them pharmacokineticly and / or pharmacodynamically, and / or reducing toxicity by chemically modifying drug molecules and / or combining drug molecules with one or more other compounds, is one direction for developing next-generation drugs that exhibit physiological activity at more ideal doses.

[0003] Currently, one known substance is oxidized glutathione (oxidized glutathione, glutathione disulfide, GSSG), which is a dimer of the glutathione tripeptide (γ-glutamylcysteine ​​glycine), wherein the two molecules of the tripeptide are linked together by a covalent disulfide bond between cysteine ​​residues. Both the tripeptide glutathione (reduced glutathione, GSH) and its dimer GSSG are natural metabolites and are found in human and animal tissues and bodily fluids [Isabella Dalle-Donne et al. S-glutathionylation in protein redox regulation / Free Radical Biology & Medicine, 2007, V.43, pp.883-898; 2014, Т.54, с.299-348.

[0004] It is known in the art that oxidized glutathione (GSSG) possesses a variety of pharmacological activities. In particular, oxidized glutathione enhances the production of various cytokines that control a range of protective responses in the body, exhibiting effects including antiviral, antibacterial, antitumor, and antifibrotic activity.

[0005] Therefore, patent RU 2089179 C1 (publication date: September 10, 1997) and patent WO 9721444 A1 (publication date: June 19, 1997) disclose the use of oxidized glutathione and pharmaceutical compositions thereof in the treatment of tumors, infectious diseases, immunological diseases, tumorigenetic diseases and hematological diseases, wherein the endogenous secretion of cytokines and hematopoietic factors is appropriately stimulated.

[0006] Patents RU 2206334 C1 (publication date: June 20, 2003), RU 2208452 C1 (publication date: July 20, 2003) and RU 2208453 C1 (publication date: July 20, 2003) respectively disclose pharmaceutical compositions containing oxidized glutathione for increasing the body's resistance (tolerance) to the thermal effects of the environment, increased respiratory gas pressure, and motion sickness.

[0007] Oxidized glutathione dosage forms are certified for use and have pharmacological effects such as immunomodulation, hepatoprotection, hematopoiesis, and regulation of redox processes in the body [http: / / www.rlsnet.ru / tn_index_id_10764.htm].

[0008] It is known in the art to develop compositions of oxidized glutathione or its pharmaceutically acceptable salts with platinum or palladium compounds (particularly compositions consisting of disodium salts of oxidized glutathione and cisplatin) to provide regulation of the endogenous production of cytokines and / or hematopoietic factors, as well as regulation of metabolic, proliferation, differentiation, and apoptosis processes in normal and transformed cells, and for the treatment of cancer, infectious diseases, immunological diseases, hematological diseases, ischemic diseases, neurotrophic disorders, and metabolic diseases. (Patent RU 2144374 C1, published January 20, 2000; Patent RU 2153350 C1, published July 27, 2000; US Patent 6,312,734 B1, published November 6, 2001).

[0009] In addition, combination agents containing glutathione disulfide are known.

[0010] Therefore, the document (patent application WO 1998030228 A1, publication date: July 16, 1998) discloses the use of oxidized glutathione (GSSG) alone or in combination with reduced glutathione (GSH), or in combination with ascorbic acid-2-phosphate, or in combination with N-acetyl-L-cysteine ​​for the treatment of influenza virus infection.

[0011] Patent RU 2482868 C1 (publication date: May 27, 2013) describes a combination of glutathione disulfide (GSSG) in the form of disodium salt, lipoic acid in the form of sodium salt, and a coordination compound formed by palladium, copper, and reduced glutathione (GSH), which has hypoglycemic, cholesterol-lowering, lipid-lowering, and / or antioxidant activities.

[0012] The closest analogue is a pharmaceutical composition disclosed in patent RU 2153351 C2 (publication date: July 27, 2000), comprising a combination of oxidized glutathione (GSSG) and its pharmaceutically acceptable salts and prolonging agents, the components of which regulate the endogenous production of cytokines and hematopoietic factors. Ascorbic acid, dimethyl sulfoxide, inosine (hypoxanthine-9-D-furanoside), cystamine (2,2'-dithiobis(ethylamine)), and platinum compounds (e.g., platinum chloride) are used as prolonging agents for the action of oxidized glutathione.

[0013] The disadvantages of the known drugs and all the aforementioned agents are that their medical use is limited by a variety of factors. In particular, GSSG has a very short half-life ranging from 5 to 10 seconds after administration, which requires some training and appropriately qualified healthcare professionals to determine the exact administration site to achieve the desired therapeutic effect, or to increase the dosage and administer multiple doses. This problem has been partially addressed by using a large number of extended-actuation compounds as described in RU 2153351, but this also increases the potential risk to patients and requires careful selection of combinations of GSSG with extended-actuation agents. In combination therapy with other drugs, the use of GSSG with extended-actuation agents in combination or sequentially requires additional consideration of pharmacokinetic similarity to achieve the desired therapeutic effect without negative changes to the toxicity profile of the treated drug. Developing combinations of GSSG with any extended-actuation agent requires additional processing equipment, including one or more additional steps in the production of the active pharmaceutical ingredient and the corresponding dosage form, as well as an expanded list of excipients. Despite the limitations of oxidized glutathione-based drugs, GSSG is undoubtedly of interest in pharmacological solutions due to its biological activity. Metabolic characteristics during pathological processes can adversely affect the therapeutic effect of drugs, thereby reducing the effectiveness and safety of treatment. Summary of the Invention

[0014] The object of this invention is to provide a novel pharmaceutical composition that exhibits high efficacy and enhanced activity for pharmacologically active molecules from a variety of different drug therapy combinations. In particular, the object is to optimize pharmacodynamics, and ultimately optimize the pharmacodynamics of GSSG, so that when administered to patients in need, the necessary therapeutic effect can be achieved with a smaller dose via inhalation, enteral, parenteral administration, external use alone, and external use in combination with other pharmacologically active substances in a single dosage form. The pharmacologically active substances can be selected from any drug therapy combination, including antibacterial and antiviral drugs, anticoagulants, factor Xa inhibitors; modulators of cell membrane ion channel activity; and other drugs that achieve pharmacodynamic and / or pharmacokinetic optimization and / or reduced toxicity.

[0015] Both drug components and drug combinations can be used as drugs containing other excipients.

[0016] The technical effect of this invention is to reduce the single dose or course dose, and thus reduce dose-related toxicity at a determined therapeutic dose of the pharmacologically active substance; to enhance the efficacy of therapeutic agents from various drug treatment groups, and accordingly reduce their single dose or course dose, and thereby reduce dose-related toxicity.

[0017] The aforementioned technical effects are achieved by providing a novel pharmaceutical composition comprising a therapeutically effective amount of glutathione disulfide (GSSG) or a pharmaceutically acceptable organic or inorganic salt thereof, a combination of glutathione disulfide S-oxide (GS(O)SG) or a pharmaceutically acceptable organic or inorganic salt thereof, a pharmaceutically acceptable excipient, and a pharmacologically active molecule from any therapeutic group of drugs, for which a reduction in single-dose or course-of-treatment dose has been determined, and thus a reduction in dose-related toxicity.

[0018] Typically, pharmacologically active compounds are selected from the following drug therapy combinations:

[0019] —Anticoagulants, Factor Xa inhibitors, especially amidine hydrochloride;

[0020] —Antibacterial and antiviral drugs, especially moxifloxacin, antigenic substances in rabies vaccines, and interferon-alpha;

[0021] —Regulators of cell membrane calcium channel activity, particularly nifedipine; thereby enabling optimization of pharmacodynamics and / or pharmacokinetics, and / or reduction of toxicity.

[0022] Typically, the amount of glutathione disulfide S-oxide is 0.01-10% of the total weight of the composition.

[0023] Furthermore, the composition may further contain a d-metal (Me), preferably derived from platinum group elements, or even more preferably platinum, which exists in the form of a coordination compound containing a Me-S-glutathione bond.

[0024] The amount of d-metal added to the composition as a coordination compound shall not exceed the physiologically acceptable value for a given d-metal. However, this value may be exceeded in cases where a large amount of metal in the form of a coordination compound is required to achieve a therapeutic effect.

[0025] The amount of d-metal in the composition ranges from 1 × 10⁻⁶ per 1 kg of composition. -10 mol to 1×10 -3 mol change, preferably, 1×10 mol per 1 kg of composition -5 mol.

[0026] The provided composition can be prepared for external, inhalation, enteral or parenteral administration.

[0027] Compositional characteristics

[0028] Glutathione disulfide (or oxidized glutathione, GSSG) is a dimer of the glutathione tripeptide (γ-glutamylcysteine ​​glycine), wherein the two molecules of the tripeptide are linked together by a covalent disulfide bond between cysteine ​​residues. According to the present invention, glutathione disulfide in the form of an alkali salt or an alkaline earth metal salt can be prepared by any method known in the art (Patent RU2144374C1, published January 20, 2000).

[0029] Glutathione disulfide S-oxide (also known as glutathione thiosulfinate or GS(O)SG) has the following structure:

[0030]

[0031] Glutathione disulfide S-oxide possesses similar pharmacokinetic characteristics to oxidized glutathione and is therefore a negative regulator of oxidized glutathione degrading enzymes. Consequently, it can act as a prolonger of GSSG, optimizing its pharmacokinetics, enhancing the biological effects of oxidized glutathione, and pharmacodynamically optimizing GSSG, allowing for the use of lower doses of GSSG to achieve the desired therapeutic effect. Transitioning to lower levels is one of the key conditions for reducing the toxicity of the active pharmaceutical ingredient. Therefore, glutathione disulfide S-oxide optimizes pharmacokinetics and pharmacodynamics, increases the safety of GSSG use, and all of these factors contribute to the pharmacoeconomic advantages of combining glutathione disulfide S-oxide and GSSG in therapeutic practice.

[0032] The molecules of glutathione disulfide and glutathione disulfide S-oxide can form weak intermolecular interactions, such as van der Waals interactions, which have the mechanism of drug activity and optimize their therapeutic properties by influencing their pharmacokinetics and / or pharmacodynamics and / or toxicity.

[0033] "Coordination compound" refers to a compound containing a group of ions or neutral molecules called ligands, which are arranged in a certain order (coordination) around a central atom (ion) called a complexing agent.

[0034] "d-metals," "transition metals," and "transition elements" are all the same; they are chemical elements in the periodic system in which electrons fill the d-sublevel.

[0035] "Pharmaceutically acceptable excipients" are substances known to those skilled in the art and are applicable to obtaining medicaments comprising the compositions described in this invention for external, inhalation, enteric, parenteral, or other routes of administration. For example, any known pharmaceutically acceptable inorganic or organic carrier, preservative, solubilizer, stabilizer, humectant, emulsifier, sweetener, colorant, flavoring agent, salt for adjusting osmotic pressure, buffer, masking agent, or antioxidant, as well as other necessary components, can be used as excipients.

[0036] "Pharmaceutical acceptable" means a compound that will not cause poisoning or other adverse effects when administered to a patient.

[0037] "Therapeutic efficacy" refers to any substance used for therapeutic purposes.

[0038] "Patient" means a human or other mammal, bird, amphibian or fish, whose body is administered a drug in one or another manner by a composition or a combination of the composition with a compound of known pharmacological activity, particularly in combination with the factor Xa inhibitor amitine hydrochloride, the antibacterial agent moxifloxacin, the antigenic substance of a rabies vaccine, the antiviral agent interferon alpha, and the calcium channel inhibitor nifedipine. Attached Figure Description

[0039] Figure 1 —Electrophoresis images of monoclonal antibody preparations dissolved in various solutions and stored at 37°C, lane 1—size standard (Fermentas PageRuler) TM (Pre-stained protein); Lane 2—Sample 2; Lane 3—Sample 3; Lane 4—Sample 4; Lane 5—Sample 1.

[0040] Figure 2 —HPLC data of monoclonal antibody structural intermediates stored in serum samples under simulated physiological conditions ((1)—Sample 1; (2)—Sample 2; (3)—Sample 3; (4)—Sample 4).

[0041] Figure 3 —Average INR (1) —Amidine hydrochloride obtained in Table 1, and (2) —A mixture of amidine hydrochloride and excipients obtained in Table 2 (the excipients are compositions containing a combination of glutathione disulfide and glutathione disulfide S-oxide).

[0042] Figure 4 —in the presence of Ca 2+ In ion-free culture media (1), (2) and calcium-free culture media (3), (4), the calcium oxidase in peritoneal macrophages induced by ATP (1), (3) and carotenoids (2), (4) 2+ Signal. Vertical axis—Ca in the cytoplasm 2+The concentration, nM. Horizontal axis—time in minutes.

[0043] Figure 5 —Glutathione disulfide on the resting state [Ca 2+ ] i The effects of glutathione disulfide on 200 μM ATP (1, 2) and 0.5 μM carotenoid (TG) (3) on the induced calcium levels in macrophages in physiological saline (1) or nominally calcium-free medium (2, 3) 2+ The influence of the signal.

[0044] Figure 6 —The composition (formulation) of Example 3 affects the intracellular calcium concentration [Ca] at rest. 2+ ] i and ATP-induced Ca 2+ The effect of the signal. This formulation eliminates the inhibitory effect of the selective calcium channel inhibitor nifedipine (1), in which the effect of the drug itself is inhibited by the reducing agent dithiothreitol (DTT) (2). Detailed Implementation

[0045] The invention is illustrated by specific embodiments thereof, which are illustrative in nature and do not limit the scope of the claims in any way.

[0046] Abbreviations:

[0047] GSH—Glutathione (reduced glutathione);

[0048] GSSG—oxidized glutathione (glutathione disulfide);

[0049] GSO3H—glutathione sulfonic acid;

[0050] GS(O)SG—Glutathione disulfide S-oxide or sulfoxide;

[0051] GS(O2)SG—Glutathione disulfide S-dioxide;

[0052] HPLC—High Performance Liquid Chromatography;

[0053] PAAG—Polyacrylamide gel;

[0054] SDS—Sodium dodecyl sulfate.

[0055] Preparation method of the composition

[0056] Method A

[0057] To a sodium salt solution of glutathione disulfide derived from L-glutathione (Example 2), glutathione disulfide S-oxide synthesized according to step (Example 1) is added. The amount of glutathione disulfide S-oxide can be 0.1-10% of the total composition weight. In practical embodiments, particularly Examples 3 and 4, the amount of glutathione disulfide S-oxide is 2% and 4% respectively, based on the total composition weight. The provided method allows for precise control of the glutathione disulfide S-oxide content.

[0058] Method B

[0059] Excess hydrogen peroxide is added to a sodium glutamate disulfide solution at a typically reduced temperature of 0-5°C to generate glutamate disulfide S-oxide in situ. The sodium glutamate disulfide solution is obtained by dissolving sodium glutamate disulfide in an aqueous sodium hydroxide solution. In one embodiment (Example 5), 127 g of 6% hydrogen peroxide is added at a temperature not exceeding +3°C. The amount of glutamate disulfide S-oxide is 5% of the total composition weight.

[0060] The compositions obtained by method A or B are characterized by their ability to influence the formation and stability of disulfide bonds in proteins (Examples 5 and 12), and thus affect protein folding, thereby allowing the formation and stabilization of the native conformation of the protein, in which the protein has functional activity, particularly the conformation of a drug expressed by a protein product, which is a monoclonal antibody composed of two heavy chains and two light chains linked by disulfide bonds to form a functionally active molecule with therapeutic activity (Example 5).

[0061] The composition obtained by method A or B is characterized by its ability to increase the expression of enzymes in the second stage of heterologous biomass detoxification (Example 13), which makes it possible to use it alone as a toxicity modifier, i.e., an agent to reduce the toxic effects of various chemical molecules, including a range of dose-dependent toxic side effects of therapeutic agents used.

[0062] Compositions obtained by method A or B can be used in combination with other known pharmacologically active and widely used therapeutic molecules to prepare drugs: in particular, anticoagulants, factor Xa inhibitors such as amitine hydrochloride (Example 8); antibiotics such as moxifloxacin; antiviral agents, antigenic substances of anti-rabies vaccines, and interferon α (Examples 9, 10, 14); calcium channel inhibitors such as nifedipine (Example 11), which can reduce the dosage and, therefore, a reduction in dose-related toxicity has been determined.

[0063] Example 1: Preparation method of glutathione disulfide S-oxide (GS(O)SG).

[0064] Over 30-40 minutes, at 0-5°C, 150 ml of an acetic acid solution containing 30% peracetic acid was added dropwise while stirring to an aqueous solution of 100 g of reduced L-glutathione (GSH) dissolved in 100 ml of water. After the addition, the reaction mixture was stirred at a temperature not exceeding +5°C for 1 hour, then frozen and lyophilized for 24 hours. 110 g of a white, foamy substance was obtained, which, according to HPLC analysis (40% GSO3H, 55% GS(O)SG, 5% GS(O2)SG), was a mixture of multiple components.

[0065] The lyophilized product was dissolved in 400 mL of water and purified using preparative HPLC (YMC-Actus Triart Prep C18-S50×250 mm column, water as eluent). Fractions containing the target compound with a purity higher than 95% were combined, evaporated to 700 mL, and lyophilized. 42 g of the desired glutathione disulfide S-oxide compound (as a mixture of diastereomers) with a purity of 95+% (HPLC) was obtained.

[0066] Example 2 Preparation of oxidized glutathione (glutathione disulfide).

[0067] 2760 g of reduced L-glutathione was suspended in 7 L of water, and 2245 g of 16% sodium hydroxide solution was added with stirring at a temperature not exceeding 17 °C. After the glutathione was completely dissolved, the mixture was cooled and 2546 g of 6% hydrogen peroxide was added with stirring at a rate of 30-50 ml / min at a reaction material temperature not exceeding +15 °C. After the addition of the peroxide, the resulting solution was stirred for another hour at the predetermined temperature. After the reaction was completed (HPLC control), a solution of 2.95 kg of disodium glutathione disulfide dissolved in 11.5 L of water was obtained and cooled to 3 °C. The chemical purity of the product, disodium glutathione disulfide, exceeded 98.5% (HPLC control), and no additional procedure was required to separate the product.

[0068] Example 3: Preparation of glutathione disulfide composition (pharmaceutical) having a given content of glutathione disulfide S-oxide.

[0069] At a temperature of 3-5°C, 60g of glutathione disulfide S-oxide obtained according to Example 1 was added to the disodium glutathione disulfide (2.95kg in 11.5L of water) prepared in Example 2, and the mixture was thoroughly mixed for 5 minutes. The solution was then placed at 5°C for 120 minutes and then freeze-dried.

[0070] Example 4: Preparation of glutathione disulfide composition (pharmaceutical) having a given content of glutathione disulfide S-oxide.

[0071] 120 g of glutathione disulfide S-oxide prepared according to Example 1 was added to disodium glutathione disulfide (2.95 kg in 11.5 L of water) prepared in Example 2 and mixed thoroughly for 5 minutes. The solution was then placed at 5 °C for 120 minutes and freeze-dried.

[0072] Example 5: Preparation of a disodium glutamate disulfide salt composition having a given content of glutathione disulfide S-oxide.

[0073] 2760 g of reduced L-glutathione was suspended in 7 L of water, and 2245 g of 16% sodium hydroxide solution was added with stirring at a temperature not exceeding 17 °C. After the glutathione was completely dissolved, the mixture was cooled, and 2546 g of 6% hydrogen peroxide was added with stirring at a rate of 30-50 ml / min at a reaction temperature not exceeding +15 °C. After the addition of the peroxide, the resulting solution was stirred for another hour at the predetermined temperature. After the reaction was complete (HPLC control), the reaction mixture was cooled to 3 °C. The chemical purity of the product, disodium glutathione disulfide, was greater than 98.5% (HPLC control).

[0074] Then, add 127g of 6% hydrogen peroxide at a rate of 30-50ml / min at a temperature not exceeding +3℃. Allow the reactants to stand at +3℃ for 1 hour and then freeze-dry them.

[0075] The resulting composition contains 95% disodium glutathione disulfide and 4.5-5.0% disodium glutathione disulfide S-oxide (HPLC control), requiring no additional product purification procedures.

[0076] Example 6: Preparation of glutathione disulfide compositions having given amounts of glutathione disulfide S-oxide and Pt-S.

[0077] 2760 g of reduced L-glutathione was suspended in 7 L of water, and 2245 g of 16% sodium hydroxide solution was added at a temperature not exceeding 17 °C. After the glutathione was completely dissolved, the mixture was cooled, and 0.5 g of cisplatin and 2546 g of 6% hydrogen peroxide were added with stirring at a rate of 30-50 ml / min while the temperature of the reactants did not exceed +15 °C. At the end of the peroxide addition, the resulting solution was stirred for another hour at a predetermined temperature. After the reaction was complete (HPLC control), a solution of 2.95 kg of disodium glutathione disulfide dissolved in 11.5 L of water was obtained, cooled to 3 °C. The chemical purity of the product, disodium glutathione disulfide, was greater than 98.5% (HPLC control), and no further extraction process was required. The solution was cooled to 3 °C, and 60 g of glutathione disulfide S-oxide was added. The mixture was thoroughly mixed for 5 minutes, and the solution was placed at 5 °C for 120 minutes, then lyophilized.

[0078] Example 7 analyzes the folding activity of the composition obtained in Example 5.

[0079] Composition of monoclonal antibody preparations:

[0080] Monoclonal antibody—10 mg / ml;

[0081] Glycine — 2 mg / ml;

[0082] Polysorbate 80—0.05 mg / ml;

[0083] Sodium chloride — 7 mg / ml;

[0084] Citric acid monohydrate — 2101 mg / ml;

[0085] Water for injection.

[0086] Human serum was obtained with written voluntary consent in order to replicate physiological conditions. The serum in the serum repository is numbered O-17-1002.

[0087] The following were used in the refolding experiment:

[0088] Sample 1 consists of 50 μl of monoclonal antibody preparation + 1 ml of serum O-17-1002.

[0089] Sample 2 consists of a total of 50 μl of monoclonal antibody + 0.2 mM of the formulation of the composition obtained in Example 3 + 1 ml of serum O-17-1002.

[0090] Sample 3 consists of a total of 50 μl of monoclonal antibody + 0.2 mM of the formulation of the composition obtained in Example 4 + 1 ml of serum O-17-1002.

[0091] Sample 4 consists of a total of 50 μl of monoclonal antibody + 0.2 mM of the formulation of the composition obtained in Example 5 + 1 ml of serum O-17-1002.

[0092] Vials containing samples 1 and 2 were stored at 37°C. After 24 hours, the vials were removed from the incubator and the stability of the monoclonal antibodies stored at different temperatures was analyzed under conditions simulating human physiological environment.

[0093] First, the results of the stability study were analyzed by polyacrylamide gel electrophoresis (PAAG) under reducing conditions.

[0094] In the sample concentration step, isotachophoresis (ITP) was performed in a heterogeneous (gradient) buffer system (disk electrophoresis) under denaturing conditions in 15% PAAG. Samples were prepared by centrifuging to precipitate cells and resuspending them in 200 μL of buffer (0.2 M Tris-HCl pH 7.5; 0.2 M NaCl; 0.01 M sodium acetate; 0.01 M β-mercaptoethanol and 5% glycerol) and then boiling for 2 minutes.

[0095] For electrophoresis, a system of several buffer solutions was used: the cathode buffer consisted of 0.1 M Tris base, 0.1 M N-tris(hydroxymethyl)methylglycine, and 0.1% SDS (terminal anion -N-tris(hydroxymethyl)methylglycine); the anode buffer consisted of 0.2 M Tris base at pH 8.9 (leading ion -Cl). - Stacking gels with T = 2.5-3%, separating gels with T = 5-15% and C = 2-5% (where T is the relative content of monomers in the gel, and C is the content of crosslinking agent in the total amount of crosslinking agent and monomers). Electrophoresis of cell lysates was performed under denaturing conditions of 2% SDS.

[0096] Analyzing the electrophoretic images of protein preparations using the ImageJ program ( Figure 1 This program is designed for the optical density analysis of various experimental data. Lanes are labeled in manual mode, and then bands corresponding to proteins are labeled within each lane. The program evaluates the concentration of each band (subtracting background) to calculate the purity of the target protein.

[0097] HPLC conditions used to study structural intermediates of monoclonal antibodies produced under simulated virological conditions.

[0098] Shimadzu LC-20 "Prominence" Chromatograph

[0099] The chromatographic column used by Fenoxamel is a Jupiter C18, 5μm, 300A, 250×4.6 mm.

[0100] Detection wavelength = 210nm

[0101] Injection volume = 25 μl

[0102] Flow rate = 1.0 ml / min

[0103] Column temperature = 35℃

[0104] Cell detector temperature = 35℃

[0105] Mobile phase:

[0106] Eluent A: 30% acetonitrile + 0.1% trifluoroacetic acid aqueous solution

[0107] Eluent B: 70% acetonitrile + 0.1% trifluoroacetic acid aqueous solution

[0108] Running time = 47 minutes

[0109] Gradient procedure:

[0110]

[0111]

[0112] The data obtained is as follows Figure 2 As shown,

[0113] No monoclonal antibody fragments with damaged structures that cannot recognize antigens were found in serum samples containing the compositions obtained according to Examples 3, 4, and 5. Figure 2 (2), Figure 2 (3), Figure 2 (4)) in contrast to a sample that does not contain the composition. Figure 2 (1)).

[0114] Example 8: The combined use of a composition containing glutathione disulfide and glutathione disulfide S-oxide with the anticoagulant factor Xa inhibitor amitine hydrochloride.

[0115] The ability of the composition obtained according to Example 3 of this application to enhance the therapeutic efficacy of the pharmacologically active agent amitine hydrochloride, an anticoagulant and factor Xa inhibitor, was investigated. The test substance amitine hydrochloride (e.g., according to patent EA015918B1, published on December 30, 2011, in the region near the base of the tail) or a mixture of amitine hydrochloride and an excipient (the excipient being the composition obtained in Example 3 of this application) was injected intravenously into the caudal vein in the region near the base of the tail using a 1 ml insulin syringe equipped with a 30G needle. Individual doses were calculated for each animal based on body weight and corrected after each weighing. Administration of the substance was performed individually. The mixture of amitine hydrochloride and the excipient was prepared for intravenous administration to the animals. For this purpose, amitine hydrochloride and the excipient were separately dissolved in distilled water, and then the solutions were mixed. This solution was prepared just before administration to the animals and injected within 10 minutes of preparation. The dose for rats was 0.31-0.42 ml.

[0116] Preheat the rat's tail in a water bath at 43°C for at least 15 minutes. Without anesthesia, collect blood from the lateral caudal vein above the intravenous injection site (1 / 3 to 2 / 3 of the tail length). Using a 23G needle, transfer 0.36 ml of blood into a plastic tube (e.g., Eppendorf, Germany) containing 0.04 ml of 0.11 M sodium citrate solution to a volume of 0.4 ml, achieving a sodium citrate to blood ratio of 1:9. Within 30 minutes of sampling, centrifuge the blood at 8000 rpm (7000 g) for 10 minutes. Transfer the plasma to another tube and centrifuge again at 12000 rpm (15000 g) at 20°C for 10 minutes to obtain platelet-deficient plasma. Pour 110 μl of the resulting plasma into a plastic tube (e.g., Eppendorf, Germany) and freeze at -20°C. Perform six samplings per rat.

[0117] The experiment used water-soluble freeze-dried thromboplastin with added calcium ions, certified by the International Sensitivity Index (ISI) and Regenerant (NPO "RENAM").

[0118] The principle of this method is as follows: When excess tissue thromboplastin and calcium ions are added to citrate plasma, the fibrin clot formation time depends only on the activity of external coagulation pathway factors and general coagulation pathway factors: factors I, II, V, VII, and X. The time from the addition of thromboplastin and plasma to the formation of a fibrin clot is measured.

[0119] Assay: Add 8 ml of distilled water and the lyophilized regenerant to a vial and shake to dissolve. Heat the reagent at 37°C for 30 minutes before assay. Add 50 μl of citrate plasma to the analyzer's cuvette and incubate at 37°C for another 1–2 minutes. Then, add 100 μL of regenerant and record the clotting time in seconds on an ABW Medizin Technology Co., Ltd. Merlin MC-1 coagulation analyzer.

[0120] The obtained result is expressed as the International Normalized Ratio (INR):

[0121] INR=PR ISI ,

[0122] ISI, or International Sensitivity Index, should be stated in the accompanying certificate. PR-Prothrombin Ratio:

[0123] PR = PT B / PT 100％ ,

[0124] Among them PT B It is the prothrombin time (PT) of the test sample plasma, measured in seconds. 100％It is the average prothrombin time of a given animal sample obtained before administration.

[0125] The results of the measured research parameters were averaged across the experimental groups and expressed as M ± m, where M is the group mean and m is the standard deviation. The significance of differences between groups was determined using Student's parametric t-test for the normal sample distribution with p < 0.05, and the non-parametric Mann-Whitney U test for the outlier distribution with p < 0.05.

[0126] The results are shown in Tables 1 and 2, and Figure 3 The table is provided in Table 1. Figure 3 (1) Shows data obtained solely from amidine hydrochloride, as well as Table 2 and Figure 3 (2) Data obtained from a mixture of amidine hydrochloride and excipients are shown.

[0127] Table 1. INR analysis results of amidrine hydrochloride administration

[0128]

[0129]

[0130] Table 2. Analysis results of INR values ​​when amidine hydrochloride is used in combination with excipients.

[0131]

[0132] The results obtained demonstrate the ability of the compositions of the present invention to enhance the efficacy of other therapeutic agents.

[0133] Example 9: Study on the antibacterial activity of a mixture containing moxifloxacin combined with glutathione disulfide and glutathione disulfide S-oxide.

[0134] The formulations obtained according to Examples 3, 4 and 5 of this application were studied.

[0135] The antibacterial activity of this formulation against Gram-negative bacteria was studied: Escherichia coli ATCC 25923, Pseudomonas aeruginosa ATCC 27853, clinically isolated Acinetobacter baumannii, and Gram-positive bacteria: Listeria monocytogenes EGD (ATCC BAA-679), Staphylococcus aureus ATCC 25922, and methicillin-resistant Staphylococcus aureus ATCC33591.

[0136] Microorganisms were incubated overnight (16-18 hours) at 37°C with continuous shaking in 2.1% Müller-Hinton broth M391 (Ossid, Germany). Subsequently, aliquots of the bacterial suspension were taken from the overnight culture and transferred to 15 ml of fresh, sterile 2.1% Müller-Hinton broth, and then incubated at 37°C with shaking for 2.5-3 hours. The optical density (OD) of the resulting suspension was then measured at a wavelength of 620 nm using a DU-50 spectrophotometer (Beckman, USA) in sterile 2.1% Müller-Hinton broth. The colony-forming units (CFU) per milliliter were determined using the formula: 1 × OD620 = 2.5 × 10⁻⁶. 8 cfu / ml [Protocols in antimicrobial peptides. W. Shafer Ed. Sprner-Verlag New York, LLC, 7 / 8 / 1997]. Based on this calculation, the bacterial suspension was diluted to 1×10⁻⁶ with sterile 2.1% Mueller-Hinton broth. 5 The concentration of CFU / ml.

[0137] 96-well sterile U-bottom plates (Salster, Germany) were used. Serial dilutions of the test formulations were prepared in Müller-Hinton medium (eight dilution gradients per formulation, 50 μl per sample). Additionally, 50 μl of bacterial suspension was added to each well of the plate (the final bacterial concentration in the sample was 0.5 × 10⁻⁶). 5 (cfu / ml). Five replicate samples were prepared for each dilution of each formulation.

[0138] The plates containing the samples were incubated in a thermostat at 37°C for 18 hours.

[0139] Results were recorded the following day. The lowest concentration of the substance at which no microbial growth was observed visually in the corresponding wells of the plate (complete inhibition) was considered the minimum inhibitory concentration (MIC). The final results were calculated based on data from five independent experiments, with five replicates for each dilution of each test sample.

[0140] The antimicrobial activity (AMA) of the formulation was determined by radial diffusion in agarose gels containing the test microorganisms using a method developed by Professor Lehrer of UCLA [Lehrer RI et al. Ultrasensitive assays for endogenous antimicrobial poly-peptides / Journal of Immunological Methods, 1991, V.137, pp.167-173]. The microorganisms were pre-cultured for 16 hours at 37°C in a medium containing 3% soybean trypsin hydrolysate. Then, aliquots of the medium containing the microorganisms were transferred to freshly prepared medium and incubated at 37°C for 2.5 hours to obtain microorganisms in mid-logarithmic growth phase. The cell number of each microorganism was assessed by measuring the optical density of the suspensions on a 620 nm spectrophotometer. A sample containing 4 × 10⁴ microorganisms was used. 6 A suspension of microbial cells was aliquoted and mixed with 10 ml of 1% sterile agarose solution in 10 mM sodium phosphate buffer (pH 7.4) containing 0.15 M NaCl at 42 °C. The resulting mixture was poured into sterile plastic petri dishes with a diameter of 90 mm and left to solidify at room temperature. The analytical sample was a serially diluted formulation in 10 mM pH 7.4 sodium phosphate buffer, 5 μl in volume. The analytical sample was added to wells (3 mm in diameter) prepared by a plating applicator and incubated at 37 °C for 3 hours in an air thermostat. Then, 1% agarose containing 6% TGS was poured into the plates and incubated at 37 °C for 18 hours. The diameter of the growth inhibition zone (the area around the well, free of microorganisms) was determined by taking one standard unit of antimicrobial activity (0.1 mm) and subtracting 30 standard units corresponding to the diameter of the well itself from the measured value. The concentrations of the formulations used were 64 μg / ml, 32 μg / ml, 16 μg / ml, 8 μg / ml, 4 μg / ml, 2 μg / ml, and 1 μg / ml.

[0141] Minimum concentration at which a formulation inhibits microbial growth (MIC) The dependence of antimicrobial activity on peptide concentration was determined by constructing a linear regression: y = a + bx, where y is the antimicrobial activity (cu) and x is the concentration of the formulation. When y = 0, i.e., MIC = -a / b, MIC is considered to be the value of x.

[0142] Table 3 Escherichia coli ATCC 25922 # The minimum inhibitory concentration of the peptide, μg / ml (serial dilution method in liquid culture medium).

[0143]

[0144] #Each displayed value is the mean ± the standard error of the mean (n = 25).

[0145] Table 4 Staphylococcus aureus ATCC 25923 # The minimum inhibitory concentration of the peptide, μg / ml (serial dilution method in liquid culture medium).

[0146]

[0147] # Each displayed value is the mean ± the standard error of the mean (n = 25).

[0148] *Moxifloxacin MIC (No. 13) showed a significant difference in Student's t-test.

[0149] Studies have shown that the compositions of the present invention have high antibacterial activity against both Gram-negative and Gram-positive bacteria.

[0150] Example 10: Use of the composition containing glutathione disulfide and glutathione disulfide S-oxide obtained in Example 3 as an adjuvant in vaccine formulations.

[0151] A mixture of a dried, concentrated, purified, inactivated cell-derived anti-rabies vaccine and the composition in Example 3 of this application was prepared.

[0152] A sterile solution of 0.5 mg / ml aluminum hydroxide in PBS was prepared. 12 g of the composition obtained according to Example 3 of this application was added to 60 ml of the resulting solution. The resulting solution was sterilized by passing it through a filter with a pore size of 0.44 μm. Diluted solutions (ADs) with concentrations of 20 mg / ml, 10 mg / ml, 5 mg / ml, and 1 mg / ml in PBS containing 0.5 mg / ml aluminum hydroxide were prepared from the resulting solution.

[0153] The obtained solution was used to prepare vaccines diluted 1:200 (calculated for 50% protection in mice) and 1:1200 (calculated for 20% protection in mice) to a solution of the substance of Example 3 (this application) at a predetermined concentration in PBS containing 0.5 mg / ml aluminum hydroxide. The resulting solution was incubated on a shaker (approximately 150 rpm) at 4°C for 1 hour, without allowing foaming.

[0154] Dry-concentrated purified inactivated cell-derived anti-rabies vaccines diluted at 1:200 and 1:1200 were used as reference samples.

[0155] BALB / s mice weighing 13-15g from the same batch were used as the subjects of this study.

[0156] Based on the titration results of the CVS test strain of rabies virus (10% brain suspension of rabies virus-infected mice), it was calculated that 0.03 ml contained 20 to 100 LD. 50 The working diluent.

[0157] The mice were first immunized by intraperitoneal injection of 0.5 ml of the first 10 dilutions of each composition.

[0158] The mice were given a second immunization 7 days later by intraperitoneal injection of 0.5 ml of the first 10 dilutions of each composition.

[0159] Prepare a working (permissible) dilution of the virus and perform three consecutive 10-fold dilutions in water for injection containing 2% horse serum. Inactivate the virus at 56°C for 30 minutes to determine the actual dose of virus obtained in the experiment.

[0160] The permissible dose of 0.03 ml and its tenfold serial dilutions were administered intracerebrally to control mice and immunized mice, with 6 mice used for each dilution.

[0161] The animals were followed up for 14 days. The evaluation of the experimental results took into account mice that became sick or died between days 5 and 14.

[0162] The results are mathematically processed using the Reed-Muench method.

[0163] Table 5 summarizes the results obtained.

[0164] Table 5 Results of the influence of adjuvants on the efficacy of vaccine formulations

[0165]

[0166] The data provided indicate that animals have high survival rates when given a vaccine containing a combination of glutathione disulfide and glutathione disulfide S-oxide. These values ​​are comparable to, and in most cases even higher than, those of the reference sample (for cell-derived rabies vaccines).

[0167] Example 11 Effect of the composition containing glutathione disulfide and glutathione disulfide S-oxide obtained according to Example 3 on the activity of calcium channel inhibitors.

[0168] The purpose of this study is to test the effects of the composition obtained in Example 3 of this application on cell membrane ion channel activity and calcium channel inhibitor activity.

[0169] The following compounds were used as test compounds: the selective calcium channel inhibitor nifedipine, glutathione disulfide (prepared in Example 2 of this application), and the composition according to Example 3 of this application (comprising glutathione disulfide and glutathione disulfide S-oxide).

[0170] Preparation of the formulation used for research:

[0171] Store the test compounds and compositions at +4°C; dissolve these substances in deionized water (Super Q) before starting the experiment. Store the prepared solutions at +4°C for no more than 5 hours. Add the compounds to the cell culture medium at the final concentration to be studied.

[0172] The formulation is added to the cells once within a specified time period.

[0173] Cell lines and culture conditions used:

[0174] Experiments were performed on cultured rat peritoneal macrophages. Macrophages were isolated from the peritoneal cavity of rats weighing 200–300 g using the previously described method [Conrad R.E. Induction and collection of peritoneal exudate macrophages. Manual of macrophages methodology / New York: Marsell Dekker.–pp.5-11; Randria mampita C. et al. Ionic channels in murine macrophages / Cell Biology, 1987, V.105, pp.761-769]. Immediately after isolation, the cells were spherical with a diameter of 10–20 μm. The cell suspension was placed on a culture dish containing a 10 × 10 mm quartz glass slide. Cells on glass slides were cultured at 37°C for 1-3 days in medium 199 (pH 7.2) supplemented with 20% bovine serum, glutamine solution (3%), penicillin (100 U / ml), and streptomycin (100 mg / ml). α-Naphthaleneacetase staining [Monahan RA et al. Ultrastructural localization of β-specific esterase activity in guinea pig and human monocytes, macrophages and lutes / Blood, 1981, V.58, pp.1089-1099] confirmed that at least 96% of the cell monolayer consisted of macrophages. The experiment was conducted at room temperature (20-22°C) for 2-3 days of cell culture.

[0175] Cell-bearing quartz glass slides were placed in laboratory chambers containing physiological solutions with the following ionic composition (mM): NaCl-140, KCl-5, CaCl2-1, MgCl2-1, HEPES-NaOH-5; pH 7.3-7.4 (Alonso-Torre, Trautmann, 1993). Calcium-free medium contained 0 mM CaCl2 and 1 mM EGTA.

[0176] Reagents from Sigma were used in the experiments. Stock solutions of carotenoid (500 μM) and nifedipine (20 mM) were prepared in dimethyl sulfoxide. Stock solutions of glutathione disulfide, the composition according to Example 3 (0.45 μmol / ml), and ATP (100 mM) were prepared in water.

[0177] Experimental method:

[0178] To measure intracellular calcium concentration ([Ca... 2+ ] i The Fura-2AM fluorescent probe was used. Macrophages were incubated in physiological saline containing 2 μM Fura-2AM for 45 minutes at room temperature (to prevent endocytosis of Fura-2AM microparticles at 37°C) [Alonso-Torre SR et al. Calcium responses elicited by nucleotides in macrophages. Interaction between two receptor subtypes / The Journal of Biological Chemistry, 1993, V.268, pp.18640-18647].

[0179] Glass slides with stained cells were washed with physiological saline and transferred to a laboratory on a Lyum-KF fluorescence microscope stage. Fura-2 fluorescence was excited at 337 nm using a LGI-503 nitrogen laser. The laser was positioned next to the microscope at a 30° angle to the laboratory to ensure direct beam targeting. Fluorescence intensity was recorded at 510 nm using a SF-10 spectrophotometer. The signal from the FUE-79 was amplified using a specially designed amplifier and recorded on an IBM computer using proprietary software. A 10×0.40 lens was used. At the given magnification, 40–50 cells were placed in the measurement area. To avoid photoburn, measurements were taken every 20 seconds of irradiation for 2.5 seconds. Cells were continuously irradiated after the addition of ATP and UTP until maximum fluorescence was reached. [Ca 2+ ] i The value is calculated using the Grünkiewicz equation [Grünkiewicz G. et al. A new generation of Ca2+ indicators with greatly improved fluorescence properties / The Journal of Biological Chemistry, 1985, V.260, pp.3440-3450]:

[0180] [Ca 2+ ] i =K d ×(FF min ) / (F max -F),

[0181] Where F is the observed fluorescence intensity; F max It is saturated Ca 2+ The fluorescence of the dye; F min It does not contain Ca 2+ The fluorescence of the dye (in calcium-free medium).

[0182] Dissociation constant, K of Fura-2AM d Ca 2+ The complex has a molecular weight of 135 nM at 20 °C and pH 7.1–7.2, and in the presence of Ca... 2+ After adding 10 μM iomycin or 25 μM digoxin to the culture medium, the F-value was measured. max Treatment of cells with digitalis saponins can reduce calcium levels. 2+ Ions can freely penetrate the plasma membrane without affecting the permeability of the mitochondrial membrane and endoplasmic reticulum. After stabilizing the signal, 5 mM MEGTA is added to nominally calcium-free medium (F... minThe fluorescence of the dye was measured in MnCl2. Endogenous fluorescence levels were removed after adding MnCl2 (100 μM) solution to macrophages. 2+ It replaced Ca in the Fura-2 complex 2+ Mn 2+ The fluorescence ratio of the dye complex has Ca 2+ The fluorescence of the Fura-2 complex was 100 times lower. For Fura-2, F... min =F max / 3.

[0183] Two experimental methods were used in the study. First, the effects of pharmacological agents on calcium phosphate (Ca) induced by ATP, UTP, carotenoids, or cyclopyruvate (CPC) in macrophages in physiological saline were investigated. 2+ The effect of the reaction. Reflecting the effect of Ca... 2+ Ca entering from the external culture medium 2+ - The pharmacological agent was administered during the stabilization phase of the signal, either before or after the agonist's action. In a second, different experiment, the following experimental protocol was used (without Ca). 2+ / Ca 2+ -Reintroducing the scheme) Detecting and enhancing Ca 2+ Entering the cell. Macrophages were cultured in a nominally calcium-free medium and then exposed to an agonist, causing intracellular calcium stores to... 2+ Activation. Add 2 mM Ca to the external culture medium. 2+ And restore Ca 2+ After a physiological gradient of concentration, [Ca] was observed 2+ ] i The rapid increase reflects Ca 2+ It enters the cell. Furthermore, when Ca is applied... 2+ Previously or in Ca 2+ During the gradual transition from the external environment, the effects of adding pharmacological agents before administering agonists were investigated.

[0184] The test compound affected the calcium levels induced by ATP and carotenoids in rat macrophages. 2+ and Ca 2+ -Signal cells Results of the effect of internal concentration:

[0185] Adding 200 μM ATP to the culture medium of rat peritoneal macrophages leads to a biphasic Ca2+ pattern. 2+ The signal consists of an initial short-term peak followed by a distinctly prolonged "plateau" phase, the initial short-term peak being primarily associated with stored Ca2+ induced by P2u receptor activation. 2+ This is related to mobilization. The plateau phase is composed of Ca in the external medium. 2+ This was caused by the entry of [something], and it is speculated that it may reflect [something]. 2u and P 2z Simultaneous activation of receptors. Figure 4 (1) Characteristic Ca2+ induced by extracellular ATP (200 μM) in a macrophage population of 40–50 macrophages in physiological saline was shown. 2+ Signal.

[0186] In the response to increased extracellular ATP, [Ca 2+ ] i The baseline level increased from 75 ± 18 nM to a peak of 820 ± 105 nM. This was followed by a slow descent phase at high altitude, during which the average [Ca] level 4 minutes after ATP supplementation... 2+ ] i It is 460±115 nM.

[0187] Endoplasmic Ca 2+ Specific inhibitors of ATPases (0.5 μM) can also induce biphasic calcium channel blockers. 2+ Signal: with Ca from intracellular storage 2+ Activation-related peaks, Ca 2+ The activation is very rapid, and it has a morphology that reflects the Ca from the external environment. 2+ Storage dependencies are entered. Figure 4 (2) It exhibits typical carotenoid-induced calcium deficiency in macrophages in physiological saline. 2+ -Signal.

[0188] Experiments were conducted using calcium-free culture medium to identify and enhance Ca2+. 2+ The cell entry phase. In a nominally calcium-free medium (0 mM CaCl2 and 1 mM EGTA), 200 μM ATP ( Figure 4 (3) or 0.5 μM carotenoids ( Figure 4 (4) After stimulating macrophages, add 2mM Ca to the external environment. 2+ Induced Ca 2+ Enter.

[0189] Figure 5 The effect of glutathione disulfide on [Ca2+] in resting macrophages in physiological saline (1) or nominally calcium-free medium (2), (3) was shown. 2+ ] i The effects of 200 μM ATP (1), (2) and 0.5 μM carotenoid (3) on Ca2+, and the Ca2+ induced by 200 μM ATP (1), (2) and 0.5 μM carotenoid (3). 2+ Signal.

[0190] The data obtained indicate that glutathione disulfide will [Ca] due to the mobilization of calcium from intracellular stores. 2+ ] iIncreased capacity to 180±19 nM. Disruption of intracellular calcium storage reduced ATP utilization. Toxocarotene completely reversed the ability of glutathione disulfide to mobilize calcium from storage.

[0191] Figure 6 The composition (formulation) according to Example 3 of this application shows the effect on intracellular calcium concentration [Ca] at rest. 2+ ] i and ATP-induced Ca 2+ The effect of the signal. This formulation eliminates the inhibitory effect of the selective calcium channel inhibitor nifedipine (1), in which the effect of the formulation itself is inhibited by the reducing agent dithiothreitol (2).

[0192] The data obtained indicate that the combined use of glutathione disulfide and glutathione disulfide S-oxide has an effect on [Ca] due to the mobilization of calcium from intracellular stores. 2+ ] i The ability to increase to 240±28 nM. Disruption of intracellular calcium storage reduces the effectiveness of ATP. Glutathione disulfide, along with glutathione disulfide S-oxide, stabilizes the process of calcium transfer from the culture medium to the cell, thereby inhibiting the effect of the calcium channel inhibitor nifedipine on this process. Dithiothreitol neutralizes the stabilizing effect of glutathione disulfide and glutathione disulfide S-oxide on calcium channel performance.

[0193] Therefore, glutathione disulfide S-oxide can enhance the cellular effects of certain compounds. In the experiments conducted, the compound in question was glutathione disulfide, for which glutathione disulfide S-oxide acted as a synergist. The combination of glutathione disulfide S-oxide and glutathione disulfide can reduce or inhibit the effects of other compounds (nifedipine in the experiments conducted), as the composition acts as an antagonist and a neutralizer of nifedipine toxicity.

[0194] The results of the given examples indicate that the combination of glutathione disulfide and glutathione disulfide S-oxide exhibits high bioactivity, manifested as a 30-50% increase in calcium mobilization activity. Considering the ability of glutathione disulfide to modulate the activity of surface cell receptors and ion channels, it targets the following capabilities: influencing extracellular and intracellular receptors, cytoplasmic and intracellular membrane carrier proteins, extracellular regulation and transport of peptide molecules, cytoskeletal proteins, autoimmune responses; antigen binding and recognition, efflux and endocytosis, chemotaxis, chemomotor activity, cytokinesis; and intercellular, stromal, and humoral cell interactions. It can be hypothesized that S-oxide acts as a synergist in these effects of glutathione disulfide, and could be used to develop new drugs that could potentially be used at lower doses without losing therapeutic efficacy, and reduce various dose-dependent toxicities and side effects.

[0195] Example 12: Effect of the composition obtained in Example 5 on the disulfide bond formation rate.

[0196] 1 g of sample (containing 5% glutathione disulfide S-oxide) obtained according to Example 5 was dissolved in 9 ml of water. 1 ml of reduced L-glutathione sodium salt solution (2.5 mg / ml) was added to the resulting solution with stirring. The reaction mixture was stirred for 5 minutes and analyzed by HPLC. Glutathione disulfide S-oxide was not detected in the resulting solution.

[0197] Example 13: Effect of the composition obtained in Example 6 on the expression of enzymes in the second stage of heterologous biomass detoxification, wherein the composition comprises glutathione disulfide S-oxide and glutathione disulfide and the platinum compound Pt-S cisplatin.

[0198] Test compounds used:

[0199] 1—Glutathione disulfide S-oxide (the compound obtained in Example 1);

[0200] 2-Glutathione disulfide (the compound obtained in Example 2);

[0201] A composition of 3-glutathione disulfide S-oxide and glutathione disulfide (the composition obtained in Example 5);

[0202] 4-A composition of glutathione disulfide S-oxide with glutathione disulfide and the platinum compound pt-S cisplatin (the composition obtained in Example 6).

[0203] The study was conducted on randomly bred white male rats weighing 140–160 g from the RAMS “Rappolovo” breeding farm, whose hepatotoxicity was caused by daily injections of cyclophosphamide (CP) in saline at a dose of 20 mg / kg sc for 10 days.

[0204] Six groups of experimental animals were formed.

[0205] Group 1—Undamaged animals injected with the solvent (saline) of the compound under study (solvent control);

[0206] Group 2 – Animals injected with CP and treated with saline solution (control);

[0207] Experimental group:

[0208] Group 3 – Animals that received an intraperitoneal injection of test compound 1 in saline solution at a dose of 10 mg / kg for 30 minutes, 10 days after administration of the poison CP;

[0209] Group 4 – Animals that received an intraperitoneal injection of test compound 2 in saline solution at a dose of 0.1 mg / kg for 30 minutes 10 days after administration of CP agent.

[0210] Group 5 – Animals that were injected intraperitoneally with test composition 3 in physiological saline at a dose of 10 mg / kg for 30 minutes 10 days after administration of CP agent.

[0211] Group 6 – Animals that were injected intraperitoneally with test composition 4 in physiological saline at a dose of 10 mg / kg for 30 minutes 10 days after administration of CP agent.

[0212] Enzymes involved in the second stage of hepatocyte cytoplasmic detoxification of heterologous biomass: glutathione S-transferase (XE2.5.1.18), glutathione peroxidase (XE 1.11.1.9), glutathione reductase (XE 1.6.4.2), and glucose-6-phosphate dehydrogenase (XE 1.1.1.49).

[0213] Research Results

[0214] The results of a series of studies on a range of molecular reactions that provide tolerance to the effects of toxic substances. The study demonstrated the ability of glutathione disulfide S-oxide (1), glutathione disulfide (2), and their combinations (3 and 4) to induce the enzymes glutathione reductase (XE 1.6.4.2), glutathione peroxidase (XE 1.11.1.9), and glutathione-S-transferase (XE 2.5.1.18) in the second stage of heterologous biomass detoxification, as well as the substitution of reduced glutathione associated with them (Table 6).

[0215] Table 6. Changes in enzyme activity during the second stage of heterologous biomass detoxification in hepatocytes of randomly bred white rats after repeated administration of cyclophosphamide at a dose of 20 mg / kg for 10 days.

[0216] Enzyme activity, measured in μmol / (min×g protein, reduced glutathione - μmol / g protein), was determined under the action of the test substances: 1-glutathione disulfide S-oxide; 2-glutathione disulfide; 3-a composition of glutathione disulfide S-oxide and glutathione disulfide (the composition obtained in Example 5); 4-a composition of glutathione disulfide S-oxide, glutathione disulfide, and the platinum compound Pt-S cisplatin (the composition obtained in Example 6).

[0217]

[0218]

[0219] *—Reliability of p<0.05 compared to the control group;

[0220] **—The reliability of the difference compared to the poisoned animal group is p<0.05, uncorrected;

[0221] GR-glutathione reductase (XE 1.6.4.2);

[0222] GP—glutathione peroxidase (XE 1.11.1.9);

[0223] GST—glutathione S-transferase (XE 2.5.1.18);

[0224] G6PDG—glucose-6-phosphate dehydrogenase (XE 1.1.1.49);

[0225] GSH—Reduced glutathione

[0226] Adding a metal compound, particularly the platinum compound Pt-S, to the composition enhances the ability of the combination of glutathione disulfide and glutathione disulfide S-oxide to induce the enzymatic activity of the second stage of heterobiota detoxification, and increases the exchange intensity of key metabolites of reduced glutathione associated with it.

[0227] Therefore, metal compounds, especially platinum Pt compounds with the ability to induce the activity of enzymes in the second stage of heterologous biodetoxification, increase their toxicity and, consequently, the cytoprotective effect produced by the induction of enzymes in the second stage of heterologous biodetoxification through the combination of glutathione disulfide S-oxide and glutathione disulfide.

[0228] Example 14: Effect of glutathione disulfide S-oxide and its composition on the antiviral efficacy of interferon-α.

[0229] The effects of glutathione disulfide S-oxide and its compositions (Examples 1, 2, 5, 6) on the antiviral activity of interferon were studied on cultures of infected cells.

[0230] The method for evaluating the antiviral activity of interferon was based on determining the minimum amount that could protect L-68 cell line cells from viral cytopathic effects. The composition was added to the cell culture medium at concentrations of 0.0015 μmol / ml, 0.015 μmol / ml, and 0.15 μmol / ml before, with, and at 10, 30, and 60 minutes after the addition of interferon. The interferon titer used in the experiments was 4 × 10⁻⁶. -4 U / ml, 8×10 -4 U / ml, 1.6×10 -5 U / ml, 3.2×10 -5 U / ml, 6.4×10 -5 U / ml, 1.28×10-6 U / ml, 2.56×10 -6 U / ml, 5.12×10 -6 U / ml. The antiviral activity of the formulation was assessed by the ability of living cells to absorb crystal violet. After separating the living cell fraction and extracting the dye with methanol, the amount of absorbed crystal violet was measured spectrophotometrically at 595 nm. The amount of dye absorbed was directly proportional to the number of living cells and expressed as optical density.

[0231] Prepare the Л-68 cell line for experiments.

[0232] The L-68 cell line is a diploid cell line derived from the lungs of a human embryo. This cell line was obtained from the Moscow Institute of Viral Agents (MRIVP) of the Russian Academy of Medical Sciences (RAMS). It was derived from the lungs of a 11-week gestational age human embryo from a 28-year-old woman who had experienced a miscarriage. This woman had no tumors, sexually transmitted diseases, hepatitis, tuberculosis, genetic abnormalities, or congenital abnormalities.

[0233] The seed bank of diploid cell strain Л-68 has been certified for use in the preparation of immunobiological agents in MRIVP RAMS and the National Institute for Standardization and Control named after LA Tarasevich (SISC).

[0234] Cells of the L-68 cell line were cultured in complete growth medium 1 at 37°C. Cultures were performed in 250 ml plastic vials (“Costar” type). Cells covered the bottom of the vial, forming a monolayer with typical morphology of diploid fibroblasts. The plated cells were suspended in a special medium consisting of equal volumes of 0.02% EDTA solution and 0.25% trypsin solution. For this purpose, the complete growth medium was gently poured out from the vial containing the formed monolayer of cells, the monolayer was washed twice with the special medium (EDTA solution containing trypsin), and incubated at 37°C for 5 minutes. During this time, the fibroblast monolayer detached from the plastic. The detached cells were diluted with complete growth medium, and the cell aggregates were disrupted by repeated pipetting. The cells were transferred to sterile centrifuge tubes and centrifuged at 1200 rpm for 10 minutes. The supernatant was drained, and the cells were transferred to complete growth medium. Cells were then counted and used in experiments in a Gorjaev chamber.

[0235] Cell cultures that have been passaged at least 20 times and no more than 30 times can be used to determine activity and toxicity.

[0236] Preparation of vesicular stomatitis virus.

[0237] In the experiment, lyophilized vesicular stomatitis virus (VSV) was used in glass ampoules sealed under sterile conditions.

[0238] The virus was grown on the L-929 cell line. For this purpose, a pre-titrated dose of virus was added to vials containing a cell monolayer formed in complete culture medium (the infectious titer of VSV is the maximum dilution of the virus, which causes complete destruction of the cell monolayer within 1 day at 37°C). The contents of the vials were incubated at 37°C for 1 day, and then the culture medium was poured into 50 ml sterile test tubes and centrifuged at 2000 rpm. Furthermore, under aseptic conditions, the supernatant containing vesicular stomatitis virus in 1 ml aliquots was placed into ampoules and lyophilized.

[0239] Determination of viral infection titer.

[0240] The obtained volume of 0.2 ml was prepared at 5 × 10 4 Cells of the L-68 lineage suspended in complete medium 1 were added to 96-well plates (“Costar” type). The plates were then incubated for one day in a CO2 incubator with a 5% CO2 atmosphere at 37°C. During this time, the cells covered the wells, forming a continuous monolayer. After one day, the medium was gently poured off under aseptic conditions, and a quadruple dilution of the previously prepared virus was added to each well. VSV virus was added to the complete medium at a volume of 0.2 ml. The plates were then incubated under the conditions described above. At the end of incubation (after one day), the medium was poured off, and 0.05 ml of a solution of 0.2% crystal violet in 20% methanol was added to each well. After 10 minutes, the dye was removed, and the plates were washed under running water and dried. Further, 0.1 ml of lysis buffer was added to the plates to elute the dye into the solution. The staining intensity was recorded at 595 nm using a microplate reader.

[0241] Under these conditions, the highest viral dilution that causes complete destruction of the cell monolayer in the well within one day is considered the viral infection titer. The optical density of the solution in these wells will be minimal and close to the background value.

[0242] Activity test.

[0243] Prepare double dilutions (above and below the expected titer) of the standard active sample (42-28-119-96P; LA Talasevich SISC) in fully grown medium, with activity expressed in International Units (IU). Dilute the standard solution in 0.1 mL volumes in 96-well plates (“Costar” type), using at least 4 wells per dilution. Leave one row of plates for control medium (4 wells) and control VSV virus dosage (4 wells). Add 0.1 mL to these wells. After diluting the standard solution, dilute the prepared 0.1 mL volumes at 5 × 10⁻⁶. 4Cells of the Л-68 cell line suspended in complete medium 1 were added to plates at a cell / well density. Subsequently, portions of the study were added at regular time intervals to the portions of the rows containing diluted standards.

[0244] 1—Glutathione disulfide S-oxide (the compound obtained in Example 1);

[0245] 2-Glutathione disulfide (the compound obtained in Example 2);

[0246] A composition of 3-glutathione disulfide and glutathione disulfide S-oxide (the composition obtained in Example 5);

[0247] 4-A composition of glutathione disulfide S-oxide with glutathione disulfide and the platinum compound Pt-S cisplatin (the composition obtained in Example 6).

[0248] At concentrations of 0.0015 μmol / ml, 0.015 μmol / ml, and 0.15 μmol / ml, each plate was incubated for 1 day at 37°C in a CO2 incubator with a 5% CO2 atmosphere. During this period, cells covered the wells, forming a continuous monolayer. After 1 day, the complete growth medium was gently poured off under aseptic conditions, and VSV virus with the predetermined infectious titer was added to the wells of each plate. VSV virus was added to the complete medium at a volume of 0.2 ml. 0.2 ml of the same medium without VSV virus was added to the wells as a control medium. The plates were then incubated under the conditions described above. At the end of the incubation period (after 1 day), the medium was gently poured off, and 0.05 ml of a solution of 0.2% crystal violet in 20% methanol was added to the wells. After 10 minutes, the dye was removed, the plates were washed under running water, and dried. In the control medium wells, the stained monolayer should show no signs of destruction. Further, 0.1 ml of lysis buffer was added to the plate to elute the dye into the solution. The staining intensity was recorded at 595 nm using a microplate reader.

[0249] The reciprocal of the dilution of the formulation that completely protects cell cultures from viral cytopathic effects in 50% of the wells is taken as the interferon titer.

[0250] Experimental results

[0251] The experiment determined that each of the test compounds had no effect on the antiviral activity of interferon during cell pre-culture.

[0252] The results of experiments in which each composition was added after interferon indicated that almost all the tested substances had the ability to increase the efficacy of interferon if the composition was added no earlier than 30 minutes after the cells were exposed to interferon.

[0253] The tested substance had an interferon titer of 6.4 × 10⁻⁶. -5 Up to 1.28×10 -6 The efficacy was improved to varying degrees. In experiments where interferon was used in combination with one or more other substances, a greater increase in optical density was observed compared to experiments where interferon alone was effective.

[0254] To determine more reliable values ​​for the increased efficacy of interferon when used in combination with one or another composition, experiments were conducted to obtain a larger amount of experimental data on interferon dilutions, in which their activity was recorded (Table 7).

[0255] Table 7. Quantitative values ​​of increased interferon efficacy (expressed in units of optical density).

[0256]

[0257] * P<0.1

[0258] ** P<0.05

[0259] The results showed that adding the test composition after interferon-alpha enhanced the antiviral activity of all test compositions within a certain concentration range. The similarity in the compositional properties was due to the lack or relative insufficiency of oxidants in the culture medium when the composition was pre-administered or co-administered with interferon. The complex composition of the culture medium led to a relatively rapid loss of small amounts of active ingredients from the medium into the intracellular space. The action of interferon on tropic cells is accompanied by the production of oxidants; however, this production takes a sufficiently long time, exceeding the time taken by the metal coordination compounds in the culture medium. The initial addition of interferon and its action on tropic cells promotes the production of oxidants in these cells, which are then used to catalyze the thiol groups of various receptors, including the interferon receptor, ultimately contributing to an increase in the number of cells interacting with interferon, which in turn determines the enhancement of its antiviral activity.

[0260] Therefore, all tested substances enhanced the efficacy of interferon α, but the metal coordination compound significantly enhanced its activity in the formulation of this composition, thereby increasing the number of cells capable of receptor-mediated interactions with interferon α. ​​It should be noted that the enhancement of the antiviral activity of interferon α was essentially consistent with that of the substances obtained according to Examples 1 and 2, and was 9-10% at different dilutions. The combined effect of the composition according to Example 5 and the substances according to Examples 1 and 2 resulted in a further enhancement of the antiviral activity of interferon α at lower dilutions (up to 18%), and the enhancement was almost doubled at large dilutions of interferon α, such as lower doses of interferon. A similar pattern was observed when using the composition according to Example 6: the antiviral activity of interferon α was more pronounced, particularly with a 50% increase at low dilutions and a 2.5-fold increase at even lower dilutions. In 72% of patients using interferon α formulations, the antiviral effect of interferon α at therapeutic doses was associated with the development of dose-dependent side effects and toxicity. Flu-like syndrome, symptoms of gastrointestinal and psychiatric disorders, signs of bone marrow suppression, thyroid and parathyroid dysfunction, and the formation of a pool of endogenous interferon-alpha autoantibodies are most commonly observed in treatment-negative outcomes. The possibility of using interferon-alpha at lower therapeutic doses in conjunction with the compositions according to Examples 5 or 6 allows for a significant reduction in the various side effects and dose-dependent toxicities associated with interferon-alpha.

Claims

1. A pharmaceutical composition for eliminating dose-related toxicity of a pharmacologically active compound and enhancing the therapeutic activity of the pharmacologically active compound in the treatment of infectious and non-infectious diseases, said pharmaceutical composition comprising glutathione disulfide or a pharmaceutically acceptable organic or inorganic salt thereof, a metal (Me) acting as a coordination compound containing a Me-S-glutathione bond, and a glutathione disulfide S-oxide with the following structure: Or its pharmaceutically acceptable organic or inorganic salt; in, The amount of glutathione disulfide S-oxide is 0.01-10% of the total weight of the composition, and the metal is platinum, with the metal content in the composition being 1×10⁻⁶ per 1 kg of composition. -10 moles to 1×10 -5 Within the range of molar amounts, the pharmacologically active compound is an antiviral agent with broad-spectrum antiviral activity—α-interferon.

2. A pharmaceutical composition for eliminating dose-related toxicity of a pharmacologically active compound and enhancing the therapeutic activity of the pharmacologically active compound in the treatment of infectious and non-infectious diseases, comprising the pharmaceutical composition of claim 1, wherein the pharmacologically active compound is an antiviral agent—α-interferon—with broad-spectrum antiviral activity.

3. A medicament for eliminating dose-related toxicity of a pharmacologically active compound and enhancing the therapeutic activity of a pharmacologically active compound in the treatment of infectious and non-infectious diseases, the medicament comprising at least one therapeutically effective dose of the pharmaceutical composition of claim 1 and a pharmaceutically acceptable excipient.

4. A medicament for eliminating dose-related toxicity of a pharmacologically active compound and enhancing the therapeutic activity of a pharmacologically active compound in the treatment of infectious and non-infectious diseases, the medicament comprising at least one of the pharmaceutical combinations of claim 2 at a therapeutically effective dose and a pharmaceutically acceptable excipient.

5. The drug according to any one of claims 3-4, characterized in that, The drug can be prepared for external, inhalation, and enteric or parenteral administration.

6. Use of the pharmaceutical composition of claim 1 in the preparation of a medicament for eliminating dose-related toxicity of the pharmacologically active compound and enhancing the therapeutic activity of the pharmacologically active compound in the treatment of infectious and non-infectious diseases; wherein the pharmacologically active compound is an antiviral agent—α-interferon—with broad-spectrum antiviral activity.

7. Use of the pharmaceutical combination of claim 2 in the preparation of a medicament for eliminating dose-related toxicity of the pharmacologically active compound and enhancing the therapeutic activity of the pharmacologically active compound in the treatment of infectious and non-infectious diseases; wherein the pharmacologically active compound is an antiviral agent—α-interferon—with broad-spectrum antiviral activity.

8. The use according to claim 7, wherein, The drug combination is administered via inhalation, enteric, or parenteral administration.

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