Pharmaceutical combinations and uses thereof

Abstract

This invention provides a pharmaceutical composition and its application. The pharmaceutical composition of this invention comprises ribavirin and mycophenolate mofetil or a pharmaceutically acceptable salt thereof. The pharmaceutical composition of this invention can be used to prepare an antiviral drug, and the two active components exhibit a synergistic antiviral effect against influenza.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a drug combination and its application. Background Technology

[0002] Influenza virus is a highly contagious seasonal influenza pathogen belonging to the Orthomyxoviridae family. It is cold- and dry-resistant, and is typically active between November and April of the following year, with peak activity in January and February. There are four types of influenza viruses: A, B, C, and D, with types A and B being the main types causing seasonal influenza epidemics in humans. Type A influenza viruses, due to the diversity of their surface hemagglutinin (H) and neuraminidase (N) antigens, possess a strong ability to mutate, enabling them to continuously adapt to their hosts and cause pandemics.

[0003] Currently, the main drugs used clinically to treat influenza include neuraminidase inhibitors (such as oseltamivir and zanamivir) and M2 ion channel blockers (such as amantadine and ribavirin). These drugs inhibit the replication and spread of the influenza virus through different mechanisms, thereby alleviating symptoms and shortening the course of the disease. However, the use of these drugs also has certain limitations. First, they must be started within 48 hours of infection to achieve the best effect, which requires patients to seek medical attention and receive a diagnosis promptly. Second, these drugs may have certain side effects and contraindications, such as gastrointestinal reactions and allergic reactions, and should be used under the guidance of a doctor. In addition, due to the mutability of the influenza virus, these drugs may not be effective against all strains, leading to treatment failure or worsening of the condition.

[0004] Ribavirin is a broad-spectrum nucleoside antiviral drug that has been widely used to treat various viral diseases since its introduction abroad in 1974. In China, ribavirin is also approved for the prevention and treatment of viral pneumonia and bronchitis caused by respiratory syncytial virus (RSV), viral upper respiratory tract infections, and influenza. Ribavirin works by competitively inhibiting viral synthases, thus blocking viral replication and transmission. However, ribavirin also has certain limitations in antiviral treatment. First, while its antiviral spectrum is broad, its effectiveness against certain viruses, such as influenza viruses, is not significant. Second, ribavirin may cause adverse side effects, such as hematologic reactions, requiring close monitoring of the patient. Furthermore, due to its metabolic characteristics, ribavirin is eliminated from the body slowly, which may lead to drug accumulation and adverse reactions.

[0005] Therefore, it is necessary to further enhance the broad-spectrum antiviral activity of ribavirin, especially against influenza virus. Summary of the Invention

[0006] In order to solve one of the aforementioned technical problems in the prior art, the present invention provides a drug combination that has a significantly improved anti-influenza virus effect.

[0007] A first aspect of this application provides a pharmaceutical composition comprising ribavirin and other pharmaceutical components, wherein the other pharmaceutical components include mycophenolate mofetil or a pharmaceutically acceptable salt thereof.

[0008] In some implementations, the drug combination includes ribavirin and mycophenolate mofetil.

[0009] In this invention, "ribavirin" encompasses ribavirin in different crystal forms, as well as ribavirin prodrugs, metabolites, etc.

[0010] The "pharmaceutically acceptable salts" mentioned in this invention include, but are not limited to, pharmaceutically acceptable inorganic acid salts, organic acid salts, and metal salts, such as: hydrochloride, hydrobromide, sulfate, nitrate, hydrogen sulfate, phosphate, hydrogen phosphate, maleate, succinate, citrate, fumarate, salicylate, L-tartrate, fumarate, acetate, oxalate, lactate, lysine, aspartate, sodium salt, potassium salt, magnesium salt, and calcium salt.

[0011] In some embodiments, the concentration of ribavirin in the drug combination is not less than 0.3 μM. In some embodiments, the concentration of ribavirin in the drug combination is 0.3-40 μM, for example, 0.3 μM, 0.35 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 5 μM, 8 μM, 10 μM, 12 μM, 15 μM, 18 μM, 20 μM, 22 μM, 25 μM, 28 μM, 30 μM, 32 μM, 35 μM, 38 μM, 40 μM, or any value between them. In some embodiments, the concentration of ribavirin in the drug combination is 5-30 μM. In some embodiments, the concentration of ribavirin in the drug combination is 10-30 μM. In some embodiments, the concentration of ribavirin in the drug combination is 5-15 μM. In some embodiments, the concentration of ribavirin in the drug combination is 8-12 μM. In some embodiments, the concentration of ribavirin in the drug combination is 0.37 μM, 1.11 μM, 1.25 μM, 2.5 μM, 3.33 μM, 5.0 μM, 10.0 μM, 20.0 μM, 30.0 μM, 40.0 μM, etc.

[0012] In some embodiments, the concentration of the other pharmaceutical components in the drug combination is not less than 0.1 μM. In some embodiments, the concentration of the other pharmaceutical components in the drug combination is 0.1-10 μM, for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or any value between them. In some embodiments, the concentration of the other pharmaceutical components in the drug combination is 0.3-5 μM. In some embodiments, the concentration of the other pharmaceutical components in the drug combination is 0.3-1.5 μM. In some embodiments, the concentrations of the other drug components in the drug combination are 0.1 μM, 0.12 μM, 0.3 μM, 0.37 μM, 1.0 μM, 1.11 μM, 3.0 μM, 3.33 μM, 10.0 μM, etc.

[0013] In some embodiments, the concentration of ribavirin in the drug combination is 1.25-30 μM, and the concentration of the other drug components is 0.1-10 μM. In some embodiments, the concentration of ribavirin in the drug combination is 2.5-20 μM, and the concentration of the other drug components is 0.2-5.0 μM. In some embodiments, the concentration of ribavirin in the drug composition is 5-15 μM, and the concentration of the other drug components is 0.3-1.0 μM. In some embodiments, the concentration of ribavirin in the drug composition is 8-12 μM, and the concentration of the other drug components is 0.3-0.5 μM.

[0014] In some embodiments, the concentration of ribavirin in the drug combination is 1-20 μM, and the concentration of the other drug components is 0.3-3 μM. In some embodiments, the concentration of ribavirin in the drug combination is 3-15 μM, and the concentration of the other drug components is 0.3-1.0 μM. In some embodiments, the concentration of ribavirin in the drug combination is 3-10 μM, and the concentration of the other drug components is 0.3-0.5 μM.

[0015] In some embodiments, the concentration of ribavirin in the drug combination is 0.3-30 μM, and the concentration of the other drug components is 0.1-0.2 μM.

[0016] In some embodiments, the concentration of ribavirin in the drug combination is 0.3-0.5 μM, and the concentration of the other drug components is 1.0-3.5 μM.

[0017] In some embodiments, the concentration of ribavirin in the drug combination is 3-30 μM, and the concentration of the other drug components is 1.0-10 μM.

[0018] In some embodiments, the concentration of ribavirin in the drug combination is 10-30 μM, and the concentration of the other drug components is 0.3-0.8 μM.

[0019] In some embodiments, the concentration of ribavirin in the drug combination is 5-15 μM, and the concentration of the other drug components is 1-5 μM. In some embodiments, the concentration of ribavirin in the drug combination is 8-12 μM, and the concentration of the other drug components is 1-3.5 μM.

[0020] In the drug combination of the present invention, ribavirin may be tablets, pills, granules, capsules, powders, solutions, emulsions, suspensions, injections, aerosols, sprays, or powder inhalers; the other drug components may be tablets, pills, granules, capsules, powders, solutions, emulsions, suspensions, injections, aerosols, sprays, or powder inhalers.

[0021] In the drug combinations disclosed herein, ribavirin and the other drug components (e.g., mycophenolate mofetil) can be administered simultaneously or separately at different times. Combined administration can include various enteral or parenteral administration methods, including but not limited to: oral administration in any acceptable form, such as tablets, liquids, capsules, powders, etc.; topical administration in any acceptable form, such as drops, sprays, creams, gels, or ointments; oral, nasal, and / or inhalation administration in any acceptable form; and intravenous administration in any acceptable form, such as intravenous bolus injection. Intravenous infusion, intra-arterial bolus, intra-arterial infusion, and catheter perfusion into the vascular system; administration of drugs to the peri- and intra-tissue systems in any acceptable manner, such as intraperitoneal injection, intramuscular injection, subcutaneous injection, intraocular injection, retinal injection, subretinal injection, or epidural injection; administration of drugs to the intracapsular system in any acceptable manner, such as catheter infusion; administration of drugs by means of delivery devices, such as implants, stents, patches, pellets, catheters, osmotic pumps, suppositories, bioerodible delivery systems, non-bioerodible delivery systems, or other implanted extension or slow-release systems.

[0022] A second aspect of the present invention provides the use of the pharmaceutical combination described in the first aspect of the present invention in the preparation of an anti-influenza virus drug.

[0023] The drug combination described above in this invention can be used to prepare drugs against influenza A, B, C and D viruses.

[0024] In some embodiments, the influenza virus includes influenza A virus, such as, but not limited to: H1N1, H3N8, H3N2, H5N1, H7N2, H1N7, H7N3, H13N6, H5N9, H11N6, H9N2, H5N2, H4N8, H10N7, H2N2, H8N4, H14N5, H6N5, H12N5, H7N9, H10N8 and their variant strains.

[0025] In some embodiments, the influenza virus includes H1N1, H3N8, H3N2, and their variant strains. In some embodiments, the influenza virus includes H1N1, H3N8, H3N2, and CA07 variants.

[0026] A third aspect of the present invention provides a pharmaceutical formulation for treating influenza viruses, wherein the active components of the pharmaceutical formulation include ribavirin and mycophenolate mofetil or pharmaceutically acceptable salts thereof.

[0027] The pharmaceutical preparations described in this invention can be prepared from raw materials including the pharmaceutical combination described in the first aspect of this invention.

[0028] In some embodiments, the pharmaceutical formulation further includes at least one pharmaceutically acceptable carrier.

[0029] In this invention, the term "pharmaceutically acceptable carrier" refers to a substance that can be used in the preparation or use of a pharmaceutical combination, and includes, for example, suitable fillers, diluents, solvents, solubilizers, dispersion media, surfactants, antioxidants, preservatives, isotonic agents, buffers, wetting agents, emulsifiers, absorption delay agents, salts, pharmaceutical stabilizers, binders, excipients, disintegrants, flow agents, gliding agents, lubricants, wetting agents, sweeteners, colorants, flavoring agents, flavoring agents, buffers, masking agents, antioxidants, dyes, and combinations thereof.

[0030] As non-limiting examples, fillers include: lactose, cellulose, mannitol, sorbitol, calcium phosphate, starch, or derivatives thereof; binders include: cellulose, magnesium aluminum silicate, starch, gelatin, tragacanth gum, methylcellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidone, or derivatives thereof; flow aids include: talc, stearic acid, or salts thereof; fluidizing agents include: calcined silica; diluents include: lactose, glucose, sucrose, mannitol, sorbitol, cellulose, glycerin, etc.; lubricants include: silica, talc, stearic acid and its magnesium and calcium salts, polyethylene glycol, etc.; excipients include: water, trehalose, lactose, leucine, dileucine, trileucine, dextran, cyclodextrin, etc. Maltose, sucrose, glucose, sorbitol, erythritol, mannitol, dextran, maltitol, maltose, raffinose, galactose, xylose, ribose, xylitol, tryptophan, tyrosine, phenylalanine, maltodextrin, glycerol, vegetable oil, lecithin, etc.; disintegrants such as agar, alginate and its sodium salt, effervescent mixtures; antioxidants such as sodium metabisulfite, sodium thiosulfate, acetylcysteine, butylated hydroxyanisole, and butylated hydroxytoluene; preservatives such as benzalkonium chloride, chlorobutanol, phenylmercuric acetate, phenylmercuric nitrate, stable oxychloride compounds, sodium chlorite and chelating agents, DTPA or DTPA-diamide, calcium DTPA and CaNaDTPA-diamide, etc.

[0031] In this invention, the dosage forms of the pharmaceutical preparations include, but are not limited to: tablets, pills, granules, capsules, powders, solutions, emulsions, suspensions, injections, aerosols, sprays, or powder sprays.

[0032] A fourth aspect of the invention provides a method for treating influenza, the method comprising administering to an individual in need a therapeutically effective amount of ribavirin and other pharmaceutical components, wherein the other pharmaceutical components include at least one of mycophenolic acid or a pharmaceutically acceptable salt thereof, mycophenolic ester or a pharmaceutically acceptable salt thereof.

[0033] In this invention, the "therapeutic effective amount" of ribavirin and other pharmaceutical components refers to the amount of ribavirin and other pharmaceutical components that will elicit a biological or medical response in the subject (the individual in need) (e.g., improvement of symptoms, relief of symptoms, slowing or delaying disease progression, or prevention of disease). In some embodiments, the therapeutic effective amount of ribavirin is 0.1-1000 mg / day, for example, 0.1 mg / day, 1 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 50 mg / day, 80 mg / day, 100 mg / day, 200 mg / day, 300 mg / day, 400 mg / day, 500 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, or 1000 mg / day. In some embodiments, the therapeutically effective amount of the other pharmaceutical components is 0.05-500 mg / day, for example, 0.05 mg / day, 0.1 mg / day, 0.5 mg / day, 1 mg / day, 2 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 50 mg / day, 80 mg / day, 100 mg / day, 200 mg / day, 300 mg / day, 400 mg / day, or 500 mg / day.

[0034] In the method of the present invention, ribavirin and the other drug components (e.g., mycophenolate mofetil) can be administered simultaneously or separately at different times.

[0035] In the method of the present invention, the administration route and administration method of ribavirin and the other drug components (e.g., mycophenolate mofetil) may be the same or different.

[0036] As a non-limiting example, ribavirin can be administered orally, by oral or nasal inhalation, or by intravascular, intravenous, intraperitoneal, intramuscular, subcutaneous, or epidural injection; as a non-limiting example, the other drug components (e.g., mycophenolate mofetil) can be administered orally, or by intravascular, intravenous, intraperitoneal, intramuscular, subcutaneous, or epidural injection.

[0037] In the method of the present invention, the influenza includes at least one of influenza A, B, C and D.

[0038] In some implementations, the influenza includes influenza A.

[0039] In some implementations, the influenza includes influenza caused by at least one influenza virus selected from H1N1, H3N8, H3N2, H5N1, H7N2, H1N7, H7N3, H13N6, H5N9, H11N6, H9N2, H5N2, H4N8, H10N7, H2N2, H8N4, H14N5, H6N5, H12N5, H7N9, H10N8, and their variant strains.

[0040] In some embodiments, the influenza includes influenza caused by at least one influenza virus selected from the H1N1, H3N8, H3N2 and CA07 variants.

[0041] In this invention, "treatment" refers to any disease or condition, and in some embodiments, it means improving the disease or condition (i.e., slowing down, stopping, or alleviating the development of the disease or at least one of its clinical symptoms). In other embodiments, "treatment" means alleviating or improving at least one bodily parameter, including bodily parameters that may not be perceived by the patient. In still other embodiments, "treatment" means regulating the disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing bodily parameters) or both. In still other embodiments, "treatment" means preventing or delaying the onset, occurrence, or worsening of the disease or condition.

[0042] In this invention, “administration” means any mechanism of delivery of ribavirin and the other pharmaceutical components (e.g., mycophenolate mofetil) to an individual that may result in clinically, therapeutically, or experimentally beneficial outcomes.

[0043] In this invention, the administration of ribavirin and the other pharmaceutical components (e.g., mycophenolate mofetil) can be a single dose or cumulative (continuous administration). For example, the treatment of influenza may include a single administration of an effective dose of ribavirin and the other pharmaceutical components (e.g., mycophenolate mofetil) or the pharmaceutical formulation of this invention. As a non-limiting example, an effective dose of ribavirin and the other pharmaceutical components (e.g., mycophenolate mofetil) or the pharmaceutical formulation of this invention may be administered to mammals by a single injection or deposition at or near the site of influenza symptoms, or by a single oral administration of ribavirin and the other pharmaceutical components (e.g., mycophenolate mofetil) or the pharmaceutical formulation of this invention.

[0044] A fifth aspect of the present invention provides a method for inhibiting influenza virus in cells or a subject, the method comprising contacting the cells or the subject with a therapeutically effective amount of ribavirin and other pharmaceutical components, said other pharmaceutical components including at least one of mycophenolic acid or a pharmaceutically acceptable salt thereof, mycophenolic ester or a pharmaceutically acceptable salt thereof.

[0045] In some implementations, the cell is a mammalian cell.

[0046] In some implementations, "subject" refers to a mammal or other animal. In some implementations, "subject" refers to a mammal. In some implementations, "subject" refers to a human being (including adults and children).

[0047] In some embodiments, the molar ratio of ribavirin to the other pharmaceutical components is 1:(0.005-2), for example, 1:0.005, 1:0.01, 1:0.015, 1:0.02, 1:0.03, 1:0.05, 1:0.08, 1:0.1, 1:0.11, 1:0.12, 1:0.15, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, or any value between them. In some embodiments, the molar ratio of ribavirin to the other pharmaceutical components is 1:(0.01-0.3). In some embodiments, the molar concentration ratio of ribavirin to other drug components is 1:(0.02-0.15). In some embodiments, the molar concentration ratio of ribavirin to other drug components is 1:(0.03-0.11).

[0048] In some embodiments, the concentration of ribavirin in the drug combination is not less than 0.3 μM. In some embodiments, the concentration of ribavirin in the drug combination is 0.3-40 μM, for example, 0.3 μM, 0.35 μM, 0.5 μM, 1 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 5 μM, 8 μM, 10 μM, 12 μM, 15 μM, 18 μM, 20 μM, 22 μM, 25 μM, 28 μM, 30 μM, 32 μM, 35 μM, 38 μM, 40 μM, or any value between them. In some embodiments, the concentration of ribavirin in the drug combination is 5-30 μM. In some embodiments, the concentration of ribavirin in the drug combination is 10-30 μM. In some embodiments, the concentration of ribavirin in the drug combination is 5-15 μM. In some embodiments, the concentration of ribavirin in the drug combination is 8-12 μM. In some embodiments, the concentration of ribavirin in the drug combination is 0.37 μM, 1.11 μM, 1.25 μM, 2.5 μM, 3.33 μM, 5.0 μM, 10.0 μM, 20.0 μM, 30.0 μM, 40.0 μM, etc.

[0049] In some embodiments, the concentration of the other pharmaceutical components in the drug combination is not less than 0.1 μM. In some embodiments, the concentration of the other pharmaceutical components in the drug combination is 0.1-10 μM, for example, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.8 μM, 1.0 μM, 1.2 μM, 1.5 μM, 2 μM, 2.5 μM, 3 μM, 3.5 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, or any value between them. In some embodiments, the concentration of the other pharmaceutical components in the drug combination is 0.3-5 μM. In some embodiments, the concentration of the other pharmaceutical components in the drug combination is 0.3-1.5 μM. In some embodiments, the concentrations of the other drug components in the drug combination are 0.1 μM, 0.12 μM, 0.3 μM, 0.37 μM, 1.0 μM, 1.11 μM, 3.0 μM, 3.33 μM, 10.0 μM, etc.

[0050] In some embodiments, the concentration of ribavirin in the drug combination is 1.25-30 μM, and the concentration of the other drug components is 0.1-10 μM. In some embodiments, the concentration of ribavirin in the drug combination is 2.5-20 μM, and the concentration of the other drug components is 0.2-5.0 μM. In some embodiments, the concentration of ribavirin in the drug composition is 5-15 μM, and the concentration of the other drug components is 0.3-1.0 μM. In some embodiments, the concentration of ribavirin in the drug composition is 8-12 μM, and the concentration of the other drug components is 0.3-0.5 μM.

[0051] In some embodiments, the concentration of ribavirin in the drug combination is 1-20 μM, and the concentration of the other drug components is 0.3-3 μM. In some embodiments, the concentration of ribavirin in the drug combination is 3-15 μM, and the concentration of the other drug components is 0.3-1.0 μM. In some embodiments, the concentration of ribavirin in the drug combination is 3-10 μM, and the concentration of the other drug components is 0.3-0.5 μM.

[0052] In some embodiments, the concentration of ribavirin in the drug combination is 0.3-30 μM, and the concentration of the other drug components is 0.1-0.2 μM. In some embodiments, the concentration of ribavirin in the drug combination is 0.3-0.5 μM, and the concentration of the other drug components is 1.0-3.5 μM. In some embodiments, the concentration of ribavirin in the drug combination is 3-30 μM, and the concentration of the other drug components is 1.0-10 μM. In some embodiments, the concentration of ribavirin in the drug combination is 10-30 μM, and the concentration of the other drug components is 0.3-0.8 μM. In some embodiments, the concentration of ribavirin in the drug combination is 5-15 μM, and the concentration of the other drug components is 1-5 μM. In some embodiments, the concentration of ribavirin in the drug combination is 8-12 μM, and the concentration of the other drug components is 1-3.5 μM.

[0053] In some implementations, the influenza virus includes at least one of influenza A, B, C, and D viruses.

[0054] In some implementations, the influenza virus includes influenza A virus.

[0055] In some embodiments, the influenza virus includes at least one of H1N1, H3N8, H3N2, H5N1, H7N2, H1N7, H7N3, H13N6, H5N9, H11N6, H9N2, H5N2, H4N8, H10N7, H2N2, H8N4, H14N5, H6N5, H12N5, H7N9, H10N8 and their variant strains.

[0056] In some embodiments, the influenza virus includes at least one of the H1N1, H3N8, H3N2 and CA07 variants.

[0057] Compared with existing technologies, this invention reveals for the first time the powerful synergistic anti-influenza virus mechanism between mycophenolate mofetil and ribavirin. This discovery not only enriches the research and development strategies for anti-influenza virus drugs but also demonstrates an unprecedentedly high inhibitory effect on influenza viruses in practical applications. Specifically, through carefully designed in vitro experiments, this application confirms that mycophenolate mofetil can significantly enhance the antagonistic efficacy of ribavirin against multiple key human influenza virus strains (including but not limited to the classic PR8 (H1N1) strain, the widely prevalent H3N2 strain, the potentially threatening H3N8 strain, and the recently concerned CA07 variant). This synergistic effect transcends a simple drug additive effect, achieving an extraordinary antiviral efficacy of "1+1>2," marking a significant breakthrough in antiviral drug combination strategies. Particularly noteworthy is that, in in vivo experiments using mouse animal models, we observed that this drug combination not only effectively inhibited the replication and spread of influenza viruses but also significantly reduced the dosage while substantially mitigating the potential toxic side effects of traditional ribavirin used alone. This discovery not only provides an innovative approach to addressing the insufficient efficacy of ribavirin in clinical applications due to dosage limitations, but also opens up new avenues for reducing the treatment burden on patients and improving treatment safety. Therefore, the technical solution presented in this application not only possesses significant scientific research value but also holds broad prospects for clinical application, potentially bringing revolutionary changes to influenza prevention and control and patient treatment. Attached Figure Description

[0058] Figure 1 The matrix shows the inhibition rate matrix of different concentrations of ribavirin and mycophenolate mofetil synergistically antagonizing influenza virus PR8 strain in A549 cells in Example 1.

[0059] Figure 2 The concentration synergistic matrix of different concentrations of ribavirin and mycophenolate mofetil in A549 cells in Example 1 is shown.

[0060] Figure 3 The inhibition rate matrix and synergistic matrix of different concentrations of ribavirin and mycophenolate mofetil synergistically antagonizing influenza virus PR8 strain in Huh7 cells are shown in Example 2.

[0061] Figure 4 The titer results of ribavirin and mycophenolate mofetil synergistic antagonism against influenza virus PR8 strain in Balb / c mice are shown in Example 3.

[0062] Figure 5 The inhibition rate matrix and synergistic matrix of different concentrations of ribavirin and mycophenolate mofetil synergistically antagonizing influenza virus strain H3N8 in Huh7 cells are shown in Example 4. Detailed Implementation

[0063] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0064] Unless otherwise specified, all reagents used in the following experiments of this invention are commercially available products or reagents prepared according to conventional methods. Unless otherwise specified, all methods used in the experiments are conventional experimental methods. Unless otherwise specified, all instruments used in the experiments are commercially available.

[0065] Human influenza viruses mutate rapidly, and existing drug treatments have time limitations. Current single-drug and conventional drug combinations against human influenza viruses generally suffer from low antiviral efficacy and toxic side effects. Furthermore, the development of new drugs is slow and cannot quickly address the drug needs during influenza epidemics. Therefore, it is necessary to find another drug with synergistic broad-spectrum antiviral properties to form a drug combination with broad-spectrum and potent antagonistic effects against influenza viruses.

[0066] Mycophenolate mofetil (MMF) is an IMPDH inhibitor. Mycophenolate mofetil (such as mycophenolate mofetil) is mainly used for immunosuppressive therapy, such as anti-rejection after organ transplantation, and is not a drug directly used for antiviral therapy. However, in some cases, it may indirectly affect the course of viral infection by regulating the immune response. However, there is currently a lack of sufficient research and evidence to support the specific application and efficacy of mycophenolate mofetil in influenza or other viral infections. This invention, through research, creatively discovered that the combination of ribavirin and mycophenolate mofetil can significantly enhance the antiviral effect of ribavirin, exhibiting a synergistic antiviral effect between the two.

[0067] This invention constructs a luciferase reporter virus PR8-NS1-Fluc (recombinant PR8 virus carrying the Fluc reporter gene) and investigates the antiviral efficacy of different concentrations of ribavirin and mycophenolate mofetil used alone and in combination. Antiviral experiments were conducted on lung epithelial-associated cell lines A549 and Huh7, in vivo antiviral experiments were performed in mouse models, and antiviral experiments were also conducted on different subtypes of influenza virus H3N8. Bliss analysis of the concentration matrix confirmed that mycophenolate mofetil synergistically inhibits ribavirin against influenza virus PR8, achieving an antiviral effect far greater than the sum of its parts (1+1>2), which was validated on different subtypes of human influenza virus strains.

[0068] In this invention, the Bliss independent model algorithm is as follows:

[0069] Based on the inhibition rates of the two single drugs, using Formula I AB =IA +I B -I A ×I B Obtain the theoretical additive inhibition rate of the two drugs, where I AB I represents the theoretical additive inhibition rate of the two drugs in the independent model. A and I B The inhibition rates of the two drugs are given.

[0070] Finally, the theoretical superposition effect inhibition rate of the two drugs is compared with the actual antiviral inhibition rate of the two drugs (drug combination experiment) (difference comparison is made), the synergy score is calculated, and it is determined whether there is a synergy effect. A synergy score greater than 0 indicates a synergy effect, and a score less than 0 indicates an antagonistic effect.

[0071] Synergistic score = (actual inhibition rate of the two drugs - theoretical superimposed inhibition rate of the two drugs) × 100;

[0072] The actual inhibition rate of the two drugs was obtained through cell experiments, while the theoretical superposition effect inhibition rate of the two drugs was calculated using the Bliss independent model algorithm mentioned above.

[0073] Example 1

[0074] A549 cells were pretreated with different concentrations of ribavirin and mycophenolate mofetil, and PR8-NS1-Fluc were infected with the drug at a moi of 1 after 2 hours. The inhibition rates of single-drug therapy (when the concentration of the other drug was 0) and the inhibition rates of each concentration combination are shown in the figure. Figure 1 As shown, the collaborative score is calculated simultaneously according to the Bliss independent model algorithm (e.g., Figure 2 The study found that ribavirin and mycophenolate mofetil had a synergistic antiviral effect at different concentration ranges, with the strongest synergistic effect against influenza virus PR8 (H1N1) observed at ribavirin concentrations of 10 μM and mycophenolate mofetil concentrations of 0.3 μM.

[0075] Example 2

[0076] Huh7 cells were pretreated with different concentrations of ribavirin and mycophenolate mofetil, and PR8-NS1-Fluc cells were infected with PR8-NS1-Fluc cells 2 h later at a molar concentration of 1. The inhibition rates of single-drug therapy (when the concentration of the other drug was 0) and the inhibition rates of each concentration combination are shown below. Figure 3 As shown, the collaborative score is calculated simultaneously according to the Bliss independent model algorithm (e.g., Figure 3 The study found that ribavirin and mycophenolate mofetil had synergistic antiviral effects at different concentration ranges, with the strongest synergistic effect against influenza virus PR8 (H1N1) observed at ribavirin concentrations of 10 μM and mycophenolate mofetil concentrations of 0.3 μM.

[0077] Example 3

[0078] Six-week-old Balb / c female mice were administered ribavirin (40 mg / kg / dose) or mycophenolate mofetil (15 mg / kg / dose) via gavage as a control group, while the experimental group received both drugs simultaneously via gavage. A placebo group received PBS via gavage. Administered twice daily, 1000 PFU of PR8 virus solution was inoculated intranasally 2 hours after the first administration. Lung tissue was dissected on the fourth day post-infection, homogenized, and titer was determined by TCID50. The results showed that 40 mg / kg / dose ribavirin alone partially inhibited viral load in lung tissue, while 15 mg / kg / dose mycophenolate mofetil had almost no titer-inhibiting effect. However, in the combined drug group, mycophenolate mofetil, which initially had no inhibitory effect, significantly enhanced the antiviral effect of ribavirin, further reducing the titer and demonstrating a strong synergistic effect against influenza virus PR8 (H1N1). Figure 4 ).

[0079] Example 4

[0080] Huh7 cells were pretreated with different concentrations of ribavirin and mycophenolate mofetil, and then infected with H3N8-GFP (recombinant H3N8 virus carrying the GFP reporter gene) at a moi of 1 after 2 hours. The inhibition rates of single-drug therapy (when the concentration of the other drug was 0) and the inhibition rates of each concentration combination are shown below. Figure 5 As shown, the collaborative score is calculated simultaneously according to the Bliss independent model algorithm (e.g., Figure 5 Ribavirin and mycophenolate mofetil showed the strongest synergistic effect against influenza virus H3N8 at concentrations of 10 μM and 1.1 μM, respectively.

[0081] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A pharmaceutical combination comprising ribavirin and other pharmaceutical components, wherein the other pharmaceutical components include mycophenolate mofetil or a pharmaceutically acceptable salt thereof.

2. The drug combination according to claim 1, characterized in that, In the drug combination, the concentration of ribavirin is not less than 0.3 μM, preferably 0.3-40 μM, more preferably 5-15 μM, and even more preferably 8-12 μM; and / or, The concentration of the other drug components is not less than 0.1 μM, preferably 0.1-10 μM, more preferably 0.3-5 μM, and even more preferably 0.3-1.5 μM.

3. The drug combination according to claim 1 or 2, characterized in that, In the drug combination, the concentration of ribavirin is 1.25-30 μM ribavirin, and the concentration of the other drug components is 0.1-10 μM; Preferably, in the drug combination, the concentration of ribavirin is 2.5-20 μM, and the concentration of the other drug components is 0.2-5.0 μM; More preferably, in the pharmaceutical composition, the concentration of ribavirin is 5-15 μM, and the concentration of the other pharmaceutical components is 0.3-1.0 μM; More preferably, in the pharmaceutical composition, the concentration of ribavirin is 8-12 μM, and the concentration of the other pharmaceutical components is 0.3-0.5 μM.

4. The drug combination according to claim 1 or 2, characterized in that, In the drug combination, the concentration of ribavirin is 1-20 μM, and the concentration of the other drug components is 0.3-3 μM; Preferably, in the drug combination, the concentration of ribavirin is 3-15 μM, and the concentration of the other drug components is 0.3-1.0 μM; More preferably, in the drug combination, the concentration of ribavirin is 3-10 μM, and the concentration of the other drug components is 0.3-0.5 μM.

5. The drug combination according to claim 1 or 2, characterized in that, In the drug combination, the concentration of ribavirin is 0.3-30 μM, and the concentration of the other drug components is 0.1-0.2 μM; or, In the drug combination, the concentration of ribavirin is 0.3-0.5 μM, and the concentration of the other drug components is 1.0-3.5 μM; or, In the drug combination, the concentration of ribavirin is 3-30 μM, and the concentration of the other drug components is 1.0-10 μM; or, In the drug combination, the concentration of ribavirin is 10-30 μM, and the concentration of the other drug components is 0.3-0.8 μM; Preferably, in the drug combination, the concentration of ribavirin is 5-15 μM, and the concentration of the other drug components is 1-5 μM; Preferably, in the drug combination, the concentration of ribavirin is 8-12 μM, and the concentration of the other drug components is 1-3.5 μM.

6. The pharmaceutical combination according to any one of claims 1-5, characterized in that, Ribavirin is available in tablets, pills, granules, capsules, powders, solutions, emulsions, suspensions, injections, aerosols, sprays, or powder formulations; and / or, The other drug components are tablets, pills, granules, capsules, powders, solutions, emulsions, suspensions, injections, aerosols, sprays, or powder sprays.

7. The use of the pharmaceutical combination according to any one of claims 1-6 in the preparation of an antiviral drug for influenza.

8. The application according to claim 7, characterized in that, The influenza viruses include influenza A, B, C and D viruses, preferably influenza A viruses; More preferably, the influenza virus includes H1N1, H3N8, H3N2, H5N1, H7N2, H1N7, H7N3, H13N6, H5N9, H11N6, H9N2, H5N2, H4N8, H10N7, H2N2, H8N4, H14N5, H6N5, H12N5, H7N9, H10N8 and their variant strains; More preferably, the influenza virus includes H1N1, H3N8, H3N2 and CA07 variants.

9. A pharmaceutical preparation for treating influenza virus, the active ingredients of which include ribavirin and mycophenolate mofetil or pharmaceutically acceptable salts thereof; Optionally, the pharmaceutical formulation further includes at least one pharmaceutically acceptable carrier; Preferably, the pharmaceutical preparation is a tablet, pill, granule, capsule, powder, solution, emulsion, suspension, injection, aerosol, spray, or powder spray.

10. A method for inhibiting influenza virus in cells or a subject, comprising contacting the cells or subject with a therapeutically effective amount of ribavirin and other pharmaceutical components, said other pharmaceutical components comprising at least one of mycophenolic acid or a pharmaceutically acceptable salt thereof, mycophenolic ester or a pharmaceutically acceptable salt thereof; Preferably, the influenza virus includes at least one of influenza A, B, C, and D viruses; Preferably, the influenza virus includes influenza A virus; Preferably, the influenza virus includes at least one of H1N1, H3N8, H3N2, H5N1, H7N2, H1N7, H7N3, H13N6, H5N9, H11N6, H9N2, H5N2, H4N8, H10N7, H2N2, H8N4, H14N5, H6N5, H12N5, H7N9, H10N8 and their variant strains; Preferably, the influenza virus includes at least one of the H1N1, H3N8, H3N2 and CA07 variants.

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